JPWO2019241426A5 - - Google Patents

Description

Related Applications This application is a US patent application No. 62 / 648,628 filed on June 13, 2018 and filed on April 12, 2019, the contents of which are incorporated herein by reference in its entirety. Claims priority over US Patent Application No. 62 / 832,991.

Sequence Listing The present application includes a sequence listing electronically submitted in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy made on June 11, 2019 is N2067-7155WO_SL. It is named txt and has a size of 228,604 bytes.

The present invention generally relates to immune effector cells (eg, T cells, etc.) engineered to express a chimeric antigen receptor (CAR) for treating diseases associated with the expression of B cell mature antigen protein (BCMA). Regarding the use of NK cells).

B cell maturation antigens (BCMA) are cells of the B cell lineage expressed by tumor necrosis family receptor (TNFR) members. BCMA expression is highest in terminally differentiated B cells. BCMA is involved in mediating plasma cell survival to maintain long-term humoral immunity. BCMA expression has been associated with many cancers, autoimmune and infectious diseases in recent years. Cancers with increased expression of BCMA include several hematological malignancies such as multiple myeloma, Hodgkin lymphoma and non-Hodgkin lymphoma, various leukemias and gliuloblastoma.

In one aspect, the invention features an isolated nucleic acid molecule that encodes a chimeric antigen receptor (CAR), where CAR is an anti-BCMA binding domain (eg, a human anti-BCMA binding domain, eg, herein. The human anti-BCMA binding domain described), transmembrane domain and intracellular signaling domain are included.

In another embodiment, the invention provides an isolated CAR, where the CAR is an anti-BCMA binding domain (eg, a human anti-BCMA binding domain, eg, a human anti-BCMA binding domain described herein), a membrane. Includes transmembrane domain and intracellular signaling domain.

In some embodiments, the anti-BCMA binding domains (eg, human anti-BCMA binding domains) are the heavy chain complementarity determining regions 1 (HC CDR1s) of the anti BCMA binding domains described herein. One or more (eg, all three) of regions 2 (HC CDR2s) and heavy chain complementarity determining regions 3 (HC CDR3s) and / or light chain complementarity determining regions 1 of the anti-BCMA binding domains described herein. (LC CDR1), one or more (eg, all three) of the light chain complementarity determining regions 2 (LC CDR2) and the light chain complementarity determining regions 3 (LC CDR3). In some embodiments, the anti-BCMA binding domain is described in the heavy chain variable regions described herein (eg, Table 2, 6 or 10) and / or herein (eg, Table 2, 6 or 10). Includes the described light chain variable regions. In some embodiments, the anti-BCMA binding domain comprises scFv comprising the light and heavy chains of the amino acid sequences of Table 2, 6 or 10. In some embodiments, the anti-BCMA binding domain comprises the scFv described herein (eg, Table 2, 6 or 10). In some embodiments, the CAR comprises the CAR sequences disclosed herein (eg, Table 2, 6 or 10).

In some embodiments, the anti-BCMA binding domain is any anti-BMCA heavy chain binding domain amino acid sequence (or at least about 85%, 90%, 95% or 99% sequence identical thereof) set forth in Tables 2-13. A sequence of sex or a sequence having at least one, two or three modifications (eg, substitutions, eg conservative substitutions), but no more than 7, 6 or 5 modifications (eg, substitutions, eg conservative substitutions). Includes any of HC CDR1, HC CDR2 and HC CDR3. In some embodiments, the anti-BCMA binding domain is any of the anti-BMCA light chain binding domain amino acid sequences (or at least about 85%, 90%, 95% or 99% sequence identical thereof) set forth in Tables 2-13. A sequence of sex or a sequence having at least one, two or three modifications (eg, substitutions, eg conservative substitutions), but no more than 7, 6 or 5 modifications (eg, substitutions, eg conservative substitutions). Includes any of LC CDR1, LC CDR2 and LC CDR3.

In some embodiments, HC CDR1, HC CDR2 and HC CDR3 are HC CDR sequences (or at least about 85%, 90 thereof) listed in Tables 3-5 (eg, a single column of Tables 3-5). %, 95% or 99% sequence identity or at least one, two or three modifications (eg, substitutions, eg conservative substitutions), but modifications (eg, substitutions, eg conservative substitutions) of 7, An sequence that does not exceed 6 or 5). In some embodiments, LC CDR1, LC CDR2 and LC CDR3 are LC CDR sequences (or at least about 85%, 90 thereof) listed in Tables 3-5 (eg, a single column of Tables 3-5). %, 95% or 99% sequence identity or at least one, two or three modifications (eg, substitutions, eg conservative substitutions), but modifications (eg, substitutions, eg conservative substitutions) of 7, An sequence that does not exceed 6 or 5). In some embodiments, HC CDR1, HC CDR2 and HC CDR3 have the amino acid sequences of SEQ ID NOs: 44, 45 and 84, respectively (or at least about 85%, 90%, 95% or 99% sequence identity thereof). Contains sequences or sequences having at least one, two or three modifications (eg, substitutions, eg conservative substitutions), but no more than 7, 6 or 5 modifications (eg, substitutions, eg conservative substitutions). In some embodiments, HC CDR1, HC CDR2 and HC CDR3 have (i) the amino acid sequences of SEQ ID NOs: 44, 45 and 46 or at least about 85%, 90%, 95% or 99% sequence identity with them, respectively. Amino acid sequence having or at least 1, 2 or 3 modifications (eg, substitutions, eg conservative substitutions), but no more than 7, 6 or 5 modifications (eg, substitutions, eg conservative substitutions); (Ii) Amino acid sequences of SEQ ID NOs: 44, 45 and 68 or amino acid sequences having at least about 85%, 90%, 95% or 99% sequence identity thereof or at least 1, 2 or 3 modifications (eg, substitutions, Amino acid sequences having (eg, conservative substitutions) but no more than 7, 6 or 5 modifications (eg, substitutions, eg conservative substitutions); or (iii) the amino acid sequences of SEQ ID NOs: 44, 45 and 76, respectively or with it. It has an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence identity or at least one, two or three modifications (eg, substitutions, eg conservative substitutions), but modifications (eg, substitutions, eg. Conservative substitutions) include amino acid sequences that do not exceed 7, 6 or 5.

In some embodiments, LC CDR1, LC CDR2 and LC CDR3 are amino acids having the amino acid sequences of SEQ ID NOs: 54, 55 and 56 or at least about 85%, 90%, 95% or 99% sequence identity thereof, respectively. Includes an amino acid sequence having a sequence or at least one, two or three modifications (eg, substitutions, eg conservative substitutions), but no more than 7, 6 or 5 modifications (eg, substitutions, eg conservative substitutions).

In some embodiments, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR3 are (i) SEQ ID NOs: 44, 45, 46, 54, 55 and 56 or at least about 85% thereof, respectively. Sequences with 90%, 95% or 99% sequence identity or with at least 1, 2 or 3 modifications (eg, substitutions, eg conservative substitutions), but with 7 modifications (eg, substitutions, eg conservative substitutions). , 6 or no more than 5 sequences; (ii) sequences having SEQ ID NOs: 44, 45, 68, 54, 55 and 56 or at least about 85%, 90%, 95% or 99% sequence identity thereof or Sequences having at least one, two or three modifications (eg, substitutions, eg conservative substitutions), but no more than 7, 6 or 5 modifications (eg, substitutions, eg conservative substitutions); or (iii) sequences, respectively. Sequences having sequence identity of numbers 44, 45, 76, 54, 55 and 56 or at least about 85%, 90%, 95% or 99% thereof or at least one, two or three modifications (eg, substitutions, eg conservative). Includes an amino acid sequence having a substitution (substitution) but no more than 7, 6 or 5 modifications (eg, substitution, eg conservative substitution).

In some embodiments, the anti-BCMA binding domain is 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 with it or at least 1. A heavy chain variable region (VH) comprising an amino acid sequence having 2 or 3 modifications (eg, substitutions, such as conservative substitutions), but with modifications (eg, substitutions, such as conservative substitutions) not exceeding 30, 20 or 10. include.

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 is the nucleic acid sequence of SEQ ID NO: 53, 71 or 79 or at least about 85% thereof. , 90%, 95% or 99% contains nucleic acid sequences with sequence identity.

In some embodiments, the anti-BCMA binding domain is 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 with it or at least one, two or three modifications. It comprises a light chain variable region (VL) comprising an amino acid sequence (eg, substitution, eg conservative substitution), but modification (eg, substitution, eg conservative substitution) of no more than 30, 20 or 10.

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 is the nucleic acid sequence of SEQ ID NO: 62 or at least about 85%, 90% thereof. Contains nucleic acid sequences with 95% or 99% sequence identity.

In some embodiments, the anti-BCMA binding domain comprises VH and VL, where VH and VL are (i) sequences of SEQ ID NOs: 52 and 61 or at least about 85%, 90%, 95% or 99% thereof, respectively. A sequence having identity or an amino acid sequence having at least one, two or three modifications (eg, substitutions, eg conservative substitutions) but no more than 30, 20 or 10 modifications (eg, substitutions, eg conservative substitutions). (Ii) Sequences with SEQ ID NOs: 70 and 61 or at least about 85%, 90%, 95% or 99% sequence identity thereof or at least 1, 2 or 3 modifications (eg, substitutions, eg conservative substitutions). Amino acid sequences having but not greater than 30, 20 or 10 modifications (eg, substitutions, eg conservative substitutions), or (iii) SEQ ID NOs: 78 and 61 or at least about 85%, 90%, 95% or 99 thereof, respectively. % Sequence identity or at least one, two or three modifications (eg, substitutions, eg conservative substitutions), but no more than 30, 20 or 10 modifications (eg, substitutions, eg conservative substitutions). Contains the amino acid sequence.

In some embodiments, the anti-BCMA binding domain is an 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 with it or at least 1. A single chain variable fragment (scFv) comprising an amino acid sequence having 2 or 3 modifications (eg, substitutions, such as conservative substitutions), but with no more than 30, 20 or 10 modifications (eg, substitutions, eg conservative substitutions). include. 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 scFv, and the nucleic acid sequence is the nucleic acid sequence of SEQ ID NO: 65, 73 or 81. Or it comprises a nucleic acid sequence having at least about 85%, 90%, 95% or 99% sequence identity.

In some embodiments, CAR is an 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 with it or at least 1, 2 or 3 Contains amino acid sequences that have modifications (eg, substitutions, such as conservative substitutions), but with modifications (eg, substitutions, such as conservative substitutions) no greater than 30, 20 or 10. In some embodiments, the nucleic acid molecule comprises 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 thereof.

In some embodiments, HC CDR1, HC CDR2 and HC CDR3 are HC CDR sequences (or at least about 85%, 90 thereof) listed in Tables 7-9 (eg, a single column of Tables 7-9). %, 95% or 99% sequence identity or at least one, two or three modifications (eg, substitutions, eg conservative substitutions), but modifications (eg, substitutions, eg conservative substitutions) of 7, An sequence that does not exceed 6 or 5). In some embodiments, LC CDR1, LC CDR2 and LC CDR3 are LC CDR sequences (or at least about 85%, 90 thereof) listed in Tables 7-9 (eg, a single column of Tables 7-9). %, 95% or 99% sequence identity or at least one, two or three modifications (eg, substitutions, eg conservative substitutions), but modifications (eg, substitutions, eg conservative substitutions) of 7, An sequence that does not exceed 6 or 5).

In some embodiments, HC CDR1, HC CDR2 and HC CDR3 have the amino acid sequences of SEQ ID NOs: 86, 130 and 88 (or at least about 85%, 90%, 95% or 99% sequence identity thereof, respectively). Contains sequences or sequences having at least one, two or three modifications (eg, substitutions, eg conservative substitutions), but no more than 7, 6 or 5 modifications (eg, substitutions, eg conservative substitutions). In some embodiments, HC CDR1, HC CDR2 and HC CDR3 have (i) the amino acid sequences of SEQ ID NOs: 86, 87 and 88 or at least about 85%, 90%, 95% or 99% sequence identity with them, respectively. Amino acid sequence having or at least 1, 2 or 3 modifications (eg, substitutions, eg conservative substitutions), but no more than 7, 6 or 5 modifications (eg, substitutions, eg conservative substitutions); (Ii) Amino acid sequences of SEQ ID NOs: 86, 109 and 88, or amino acid sequences having at least about 85%, 90%, 95% or 99% sequence identity with them or at least 1, 2 or 3 modifications (eg, substitutions, Amino acid sequences that have (eg, conservative substitutions) but do not have more than 7, 6 or 5 modifications (eg, substitutions, such as conservative substitutions); or (iii) the amino acid sequences of SEQ ID NOs: 86, 109 and 88, respectively, or with it. It has an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence identity or at least one, two or three modifications (eg, substitutions, eg conservative substitutions), but modifications (eg, substitutions, eg. Conservative substitutions) include amino acid sequences that do not exceed 7, 6 or 5.

In some embodiments, LC CDR1, LC CDR2 and LC CDR3 are amino acids having the amino acid sequences of SEQ ID NOs: 95, 131 and 132 or at least about 85%, 90%, 95% or 99% sequence identity thereof, respectively. Includes an amino acid sequence having a sequence or at least one, two or three modifications (eg, substitutions, eg conservative substitutions), but no more than 7, 6 or 5 modifications (eg, substitutions, eg conservative substitutions). In some embodiments, LC CDR1, LC CDR2 and LC CDR3 have (i) the amino acid sequences of SEQ ID NOs: 95, 96 and 97 or at least about 85%, 90%, 95% or 99% sequence identity with them, respectively. Amino acid sequence having or at least 1, 2 or 3 modifications (eg, substitutions, eg conservative substitutions), but no more than 7, 6 or 5 modifications (eg, substitutions, eg conservative substitutions); (Ii) Amino acid sequences of SEQ ID NOs: 95, 114 and 115 or amino acid sequences having at least about 85%, 90%, 95% or 99% sequence identity thereof or at least 1, 2 or 3 modifications (eg, substitutions, Amino acid sequences with (eg, conservative substitutions) but no more than 7, 6 or 5 modifications (eg, substitutions, such as conservative substitutions); or (iii) amino acid sequences of SEQ ID NOs: 95, 114 and 97 or with them, respectively. It has an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence identity or at least one, two or three modifications (eg, substitutions, eg conservative substitutions), but modifications (eg, substitutions, eg. Conservative substitutions) include amino acid sequences that do not exceed 7, 6 or 5.

In some embodiments, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR3 are (i) SEQ ID NOs: 86, 87, 88, 95, 96 and 97 or at least about 85% thereof, respectively. Sequences with 90%, 95% or 99% sequence identity or with at least 1, 2 or 3 modifications (eg, substitutions, eg conservative substitutions), but with 7 modifications (eg, substitutions, eg conservative substitutions). , 6 or no more than 5 sequences; (ii) sequences having SEQ ID NOs: 86, 109, 88, 95, 114 and 115 or at least about 85%, 90%, 95% or 99% sequence identity thereof or Sequences having at least one, two or three modifications (eg, substitutions, eg conservative substitutions), but no more than 7, 6 or 5 modifications (eg, substitutions, eg conservative substitutions); or (iii) sequences, respectively. Sequences having sequence identity of numbers 86, 109, 88, 95, 114 and 97 or at least about 85%, 90%, 95% or 99% thereof or at least 1, 2 or 3 modifications (eg, substitutions, eg conservative). Includes an amino acid sequence having a substitution (substitution) but no more than 7, 6 or 5 modifications (eg, substitution, eg conservative substitution).

In some embodiments, the anti-BCMA binding domain is an 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 with it or at least 1, 2 or It comprises a heavy chain variable region (VH) comprising an amino acid sequence having 3 modifications (eg, substitutions, eg conservative substitutions), but no modification (eg, substitutions, eg conservative substitutions) greater than 30, 20 or 10. 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 is the nucleic acid sequence of SEQ ID NO: 260, 94 or 113 or at least about 85% thereof. , 90%, 95% or 99% of nucleic acid sequences with sequence identity.

In some embodiments, the anti-BCMA binding domain is 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 with it or at least 1. A light chain variable region (VL) comprising an amino acid sequence having 2 or 3 modifications (eg, substitutions, such as conservative substitutions), but with modifications (eg, substitutions, such as conservative substitutions) not exceeding 30, 20 or 10. include. 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 is the nucleic acid sequence of SEQ ID NO: 261, 103, 119 or 125 or at least about about it. Includes nucleic acid sequences with 85%, 90%, 95% or 99% sequence identity.

In some embodiments, the anti-BCMA binding domain comprises VH and VL, where VH and VL are (i) sequences of SEQ ID NOs: 93 and 102 or at least about 85%, 90%, 95% or 99% thereof, respectively. An amino acid sequence having identity or at least one, two or three modifications (eg, substitutions, eg conservative substitutions), but modifications (eg, substitutions, eg conservative substitutions not exceeding 30, 20 or 10). ii) Sequences with SEQ ID NOs: 112 and 118 or at least about 85%, 90%, 95% or 99% sequence identity thereof or at least 1, 2 or 3 modifications (eg, substitutions, eg conservative substitutions). However, modifications (eg, substitutions, eg, amino acid sequences in which the conservative substitution does not exceed 30, 20 or 10, or (iii) SEQ ID NOs: 112 and 124 or at least about 85%, 90%, 95% or 99% thereof, respectively. A sequence having sequence identity or an amino acid sequence having at least one, two or three modifications (eg, substitutions, eg conservative substitutions), but with modifications (eg, substitutions, eg conservative substitutions not exceeding 30, 20 or 10). include.

In some embodiments, the anti-BCMA binding domain is an 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 with it, or at least 1. A single chain variable fragment (scFv) comprising an amino acid sequence having 2 or 3 modifications (eg, substitutions, such as conservative substitutions), but with no more than 30, 20 or 10 modifications (eg, substitutions, eg conservative substitutions). include. 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 scFv, and the nucleic acid sequence is SEQ ID NO: 253, 106, 121 or 127. Includes a nucleic acid sequence or a nucleic acid sequence having at least about 85%, 90%, 95% or 99% sequence identity with it.

In some embodiments, CAR is an 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 with it or at least 1, 2 or 3 Contains amino acid sequences that have modifications (eg, substitutions, such as conservative substitutions), but with modifications (eg, substitutions, such as conservative substitutions) no greater than 30, 20 or 10. In some embodiments, the nucleic acid molecule comprises 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 thereof. In some embodiments, the nucleic acid molecule comprises 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 with it.

In some embodiments, HC CDR1, HC CDR2 and HC CDR3 are HC CDR sequences (or at least about 85%, 90 thereof) listed in Tables 11-13 (eg, a single column of Tables 11-13). %, 95% or 99% sequence identity or at least one, two or three modifications (eg, substitutions, eg conservative substitutions), but modifications (eg, substitutions, eg conservative substitutions) of 7, An sequence that does not exceed 6 or 5). In some embodiments, LC CDR1, LC CDR2 and LC CDR3 are LC CDR sequences (or at least about 85%, 90 thereof) listed in Tables 11-13 (eg, a single column of Tables 11-13). %, 95% or 99% sequence identity or at least one, two or three modifications (eg, substitutions, eg conservative substitutions), but modifications (eg, substitutions, eg conservative substitutions) of 7, An sequence that does not exceed 6 or 5).

In some embodiments, HC CDR1, HC CDR2 and HC CDR3 are the amino acid sequences of SEQ ID NOs: 179, 180 and 181 or sequences having at least about 85%, 90%, 95% or 99% sequence identity with them, respectively. Alternatively, it comprises a sequence having at least one, two or three modifications (eg, substitutions, eg conservative substitutions), but no more than 7, 6 or 5 modifications (eg, substitutions, eg conservative substitutions). In some embodiments, HC CDR1, HC CDR2 and HC CDR3 have (i) the amino acid sequences of SEQ ID NOs: 137, 138 and 139, respectively, or at least about 85%, 90%, 95% or 99% sequence identity with them. Amino acid sequence having or at least 1, 2 or 3 modifications (eg, substitutions, eg conservative substitutions), but no more than 7, 6 or 5 modifications (eg, substitutions, eg conservative substitutions); Or (ii) the amino acid sequence of SEQ ID NOs: 160, 161 and 162, or an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence identity with it or at least 1, 2 or 3 modifications (eg, substitutions). Includes an amino acid sequence having, eg, conservative substitutions, but no more than 7, 6 or 5 modifications (eg, substitutions, eg conservative substitutions).

In some embodiments, LC CDR1, LC CDR2 and LC CDR3 are amino acids having the amino acid sequences of SEQ ID NOs: 147, 182 and 183 or at least about 85%, 90%, 95% or 99% sequence identity thereof, respectively. Includes an amino acid sequence having a sequence or at least one, two or three modifications (eg, substitutions, eg conservative substitutions), but no more than 7, 6 or 5 modifications (eg, substitutions, eg conservative substitutions). In some embodiments, LC CDR1, LC CDR2 and LC CDR3 have (i) the amino acid sequences of SEQ ID NOs: 147, 148 and 149 or at least about 85%, 90%, 95% or 99% sequence identity with them, respectively. Amino acid sequence having or at least 1, 2 or 3 modifications (eg, substitutions, eg conservative substitutions), but no more than 7, 6 or 5 modifications (eg, substitutions, eg conservative substitutions); Or (ii) the amino acid sequence of SEQ ID NOs: 147, 170 and 171 or an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereof or at least 1, 2 or 3 modifications (eg, substitutions). Includes an amino acid sequence having, eg, conservative substitutions, but no more than 7, 6 or 5 modifications (eg, substitutions, eg conservative substitutions).

In some embodiments, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR3 have (i) SEQ ID NOs: 137, 138, 139, 147, 148 and 149 or at least about 85%, respectively. Sequences with 90%, 95% or 99% sequence identity or with at least 1, 2 or 3 modifications (eg, substitutions, eg conservative substitutions), but with 7 modifications (eg, substitutions, eg conservative substitutions). , 6 or no more than 5 sequences; or (ii) sequences having SEQ ID NOs: 160, 161, 162, 147, 170 and 171 or at least about 85%, 90%, 95% or 99% sequence identity thereof. Alternatively, it comprises an amino acid sequence having at least one, two or three modifications (eg, substitutions, eg conservative substitutions) but no more than 7, 6 or 5 modifications (eg, substitutions, eg conservative substitutions).

In some embodiments, the anti-BCMA binding domain is 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 with it or at least 1, 2 or It comprises a heavy chain variable region (VH) comprising an amino acid sequence having 3 modifications (eg, substitutions, eg conservative substitutions), but no modification (eg, substitutions, eg conservative substitutions) greater than 30, 20 or 10. 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 is the nucleic acid sequence of SEQ ID NO: 146 or 169 or at least about 85%, 90 thereof. Includes nucleic acid sequences with%, 95% or 99% sequence identity.

In some embodiments, the anti-BCMA binding domain is 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 with it or at least 1, 2 or It comprises a light chain variable region (VL) comprising an amino acid sequence having three modifications (eg, substitutions, eg conservative substitutions), but no modification (eg, substitutions, eg conservative substitutions) greater than 30, 20 or 10. 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 is the nucleic acid sequence of SEQ ID NO: 155 or 174 or at least about 85%, 90 thereof. Includes nucleic acid sequences with%, 95% or 99% sequence identity.

In some embodiments, the anti-BCMA binding domain comprises VH and VL, where VH and VL are (i) sequences of SEQ ID NOs: 145 and 154 or at least about 85%, 90%, 95% or 99% thereof, respectively. An amino acid sequence having identity or at least one, two or three modifications (eg, substitutions, eg conservative substitutions), but with modifications (eg, substitutions, eg conservative substitutions) not exceeding 30, 20 or 10. Or (ii) a sequence having at least about 85%, 90%, 95% or 99% sequence identity with SEQ ID NOs: 168 and 173, respectively, or at least 1, 2 or 3 modifications (eg, substitutions, eg conservative substitutions). However, it comprises an amino acid sequence in which the modification (eg, substitution, eg conservative substitution) does not exceed 30, 20 or 10.

In some embodiments, the anti-BCMA binding domain is an 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 with it or at least 1, 2 or It comprises a single chain variable fragment (scFv) comprising an amino acid sequence having 3 modifications (eg, substitutions, eg conservative substitutions), but no modification (eg, substitutions, eg conservative substitutions) greater than 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 scFv, and the nucleic acid sequence is the nucleic acid sequence of SEQ ID NO: 157 or 176 or with it. Includes nucleic acid sequences with at least about 85%, 90%, 95% or 99% sequence identity.

In some embodiments, the CAR is an 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 with it or at least one, two or three modifications ( For example, it comprises an amino acid sequence having a substitution, such as a conservative substitution, but with no modification (eg, substitution, eg conservative substitution) greater than 30, 20 or 10. In some embodiments, the nucleic acid molecule comprises 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 thereof.

In some embodiments, the anti-BCMA binding domain comprises VH and VL, the VH and VL are linked by a linker, eg, the linker described herein, and optionally the linker is SEQ ID NO: 63 or 104. Contains an amino acid sequence of or an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence identity with it.

In some embodiments, the transmembrane domain is an alpha chain, beta chain or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64. , CD80, CD86, CD134, CD137 and CD154 include transmembrane domains of proteins selected from. In some embodiments, the transmembrane domain is 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 with it or at least one, two or three modifications ( For example, it comprises an amino acid sequence having a substitution, such as a conservative substitution, but with no modification (eg, substitution, eg conservative substitution) greater than 30, 20 or 10. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding a transmembrane domain, wherein the nucleic acid sequence is the nucleic acid sequence of SEQ ID NO: 17 or at least about 85%, 90%, 95% or 99% sequence identical thereof. Contains a nucleic acid sequence having sex.

In some embodiments, the anti-BCMA binding domain is connected to the transmembrane domain by a hinge region. In some embodiments, the hinge region is 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 with it or at least 1, 2 or It comprises an amino acid sequence having 3 modifications (eg, substitutions, eg conservative substitutions), but no modification (eg, substitutions, eg conservative substitutions) greater than 30, 20 or 10. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding a hinge region, wherein the nucleic acid sequence is the nucleic acid sequence of SEQ ID NO: 13, 14 or 15 or at least about 85%, 90%, 95% or 99% thereof. Includes nucleic acid sequences with sequence identity.

In some embodiments, the transmembrane domain and hinge region are 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 with it or at least 1, 2 or It comprises an amino acid sequence having 3 modifications (eg, substitutions, eg conservative substitutions), but no modification (eg, substitutions, eg conservative substitutions) greater than 30, 20 or 10. 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 thereof.

In some embodiments, the intracellular signaling domain comprises a primary signaling domain, eg, the primary signaling domain described herein, and optionally the primary signaling domain is a CD3 zeta, TCR zeta, FcR. Includes functional signaling domains derived from 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 is 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 with it or at least 1, 2 or It comprises an amino acid sequence having 3 modifications (eg, substitutions, eg conservative substitutions), but no modification (eg, substitutions, eg conservative substitutions) greater than 30, 20 or 10. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding a primary signaling domain, wherein the nucleic acid sequence is the nucleic acid sequence of SEQ ID NO: 20 or 21 or at least about 85%, 90%, 95% or 99% thereof. Includes nucleic acid sequences with sequence identity. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding a primary signaling domain, wherein the nucleic acid sequence is the nucleic acid sequence of SEQ ID NO: 256 or at least about 85%, 90%, 95% or 99% of the sequence thereof. Contains nucleic acid sequences with identity.

In some embodiments, the intracellular signaling domain comprises a co-stimulating signaling domain, eg, the co-stimulating signaling domain described herein, and optionally the co-stimulating signaling domain is an MHC class I molecule. , TNF receptor protein, immunoglobulin-like protein, cytokine receptor, integrin, signaling lymphoid activation molecule (SLAM protein), activated NK cell receptor, BTLA, Toll ligand receptor, OX40, CD2, 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, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), 2LAMF4 (CD24), SLAMF4 (CD24) CD84, CD96 (Protein), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), Functional signal derived from a ligand that specifically binds to BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD28-OX40, CD28-4-1BB or CD83. Includes transmission domain. In some embodiments, the costimulatory signaling domain is 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 with it or at least 1, 2 or 3 Contains amino acid sequences that have modifications (eg, substitutions, such as conservative substitutions), but with modifications (eg, substitutions, such as conservative substitutions) no greater than 30, 20 or 10. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding a costimulatory signaling domain, wherein the nucleic acid sequence is the nucleic acid sequence of SEQ ID NO: 18 or at least about 85%, 90%, 95% or 99% thereof. Includes nucleic acid sequences with sequence identity. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding a costimulatory signaling domain, wherein the nucleic acid sequence is the nucleic acid sequence of SEQ ID NO: 255 or at least about 85%, 90%, 95% or 99% thereof. Includes nucleic acid sequences with sequence identity.

In some embodiments, the intracellular signaling domain comprises a functional signaling domain derived from 4-1BB and a functional signaling domain derived from the CD3 zeta, and optionally the intracellular signaling domain is. It has an amino acid sequence of SEQ ID NO: 7 (or an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence identity with it or at least one, two or three modifications (eg, substitutions, eg conservative substitutions). However, the amino acid sequence in which the modification (eg, substitution, eg conservative substitution) does not exceed 30, 20 or 10) and the amino acid sequence of SEQ ID NO: 9 or 10 (or at least about 85%, 90%, 95% or 99% thereof). Has an amino acid sequence having sequence identity or at least 1, 2 or 3 modifications (eg, substitutions, eg conservative substitutions), but the modifications (eg, substitutions, eg conservative substitutions) do not exceed 30, 20 or 10. Amino acid sequence), and optionally, the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 7 and the amino acid sequence of SEQ ID NO: 9 or 10.

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

In some embodiments, CAR has the following properties: (i) When CAR is expressed in cells (eg, T cells), it is measured, for example, by the JNL screening reporter assay described in Example 1, eg. When evaluated using the method described in Example 1 with respect to FIG. 1A or 1C, activating NFAT signaling in cells in the presence of BCMA-expressing cells; (ii) CAR is a cell (eg, eg). When expressed in (T cells), for example when evaluated using the method described in Example 1 with respect to FIG. 3A, to induce cytotoxicity of BCMA-expressing cells; and (iii) CAR are cells (iii). When expressed in (eg, T cells), eg, cytokines (eg, IFN-γ) in cells in the presence of BCMA expressing cells when evaluated using the method described in Example 1 with respect to FIG. 3C. Includes one or more (eg, one, two, or all) of inducing expression.

In another embodiment, the invention comprises heavy chain complementarity determining regions 1 (HC CDR1), heavy chain complementarity determining regions 2 (HC CDR2) and heavy chain complementarity determining regions 3 (HC CDR3). (VH) and light chain variable regions (VL) comprising light chain complementarity determining regions 1 (LC CDR1), light chain complementarity determining regions 2 (LC CDR2) and light chain complementarity determining regions 3 (LC CDR3). Providing anti-BCMA binding domains, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR3 include the CDR amino acid sequences disclosed herein.

In some embodiments, HC CDR1, HC CDR2 and HC CDR3 are HC CDR sequences (or at least about 85%, 90 thereof) listed in Tables 3-5 (eg, a single column of Tables 3-5). %, 95% or 99% sequence identity or at least one, two or three modifications (eg, substitutions, eg conservative substitutions), but modifications (eg, substitutions, eg conservative substitutions) of 7, An sequence that does not exceed 6 or 5). In some embodiments, LC CDR1, LC CDR2 and LC CDR3 are LC CDR sequences (or at least about 85%, 90 thereof) listed in Tables 3-5 (eg, a single column of Tables 3-5). %, 95% or 99% sequence identity or at least one, two or three modifications (eg, substitutions, eg conservative substitutions), but modifications (eg, substitutions, eg conservative substitutions) of 7, An sequence that does not exceed 6 or 5). In some embodiments, HC CDR1, HC CDR2 and HC CDR3 have the amino acid sequences of SEQ ID NOs: 44, 45 and 84, respectively (or at least about 85%, 90%, 95% or 99% sequence identity thereof). Contains sequences or sequences having at least one, two or three modifications (eg, substitutions, eg conservative substitutions), but no more than 7, 6 or 5 modifications (eg, substitutions, eg conservative substitutions). In some embodiments, HC CDR1, HC CDR2 and HC CDR3 have (i) the amino acid sequences of SEQ ID NOs: 44, 45 and 46 or at least about 85%, 90%, 95% or 99% sequence identity with them, respectively. Amino acid sequence having or at least 1, 2 or 3 modifications (eg, substitutions, eg conservative substitutions), but no more than 7, 6 or 5 modifications (eg, substitutions, eg conservative substitutions); (Ii) Amino acid sequences of SEQ ID NOs: 44, 45 and 68 or amino acid sequences having at least about 85%, 90%, 95% or 99% sequence identity thereof or at least 1, 2 or 3 modifications (eg, substitutions, Amino acid sequences having (eg, conservative substitutions) but no more than 7, 6 or 5 modifications (eg, substitutions, eg conservative substitutions); or (iii) the amino acid sequences of SEQ ID NOs: 44, 45 and 76, respectively or with it. It has an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence identity or at least one, two or three modifications (eg, substitutions, eg conservative substitutions), but modifications (eg, substitutions, eg. Conservative substitutions) include amino acid sequences that do not exceed 7, 6 or 5.

In some embodiments, LC CDR1, LC CDR2 and LC CDR3 are amino acids having the amino acid sequences of SEQ ID NOs: 54, 55 and 56 or at least about 85%, 90%, 95% or 99% sequence identity thereof, respectively. Includes an amino acid sequence having a sequence or at least one, two or three modifications (eg, substitutions, eg conservative substitutions), but no more than 7, 6 or 5 modifications (eg, substitutions, eg conservative substitutions).

In some embodiments, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR3 are (i) SEQ ID NOs: 44, 45, 46, 54, 55 and 56 or at least about 85% thereof, respectively. Sequences with 90%, 95% or 99% sequence identity or with at least 1, 2 or 3 modifications (eg, substitutions, eg conservative substitutions), but with 7 modifications (eg, substitutions, eg conservative substitutions). , 6 or no more than 5 sequences; (ii) sequences having SEQ ID NOs: 44, 45, 68, 54, 55 and 56 or at least about 85%, 90%, 95% or 99% sequence identity thereof or Sequences having at least one, two or three modifications (eg, substitutions, eg conservative substitutions), but no more than 7, 6 or 5 modifications (eg, substitutions, eg conservative substitutions); or (iii) sequences, respectively. Sequences having sequence identity of numbers 44, 45, 76, 54, 55 and 56 or at least about 85%, 90%, 95% or 99% thereof or at least one, two or three modifications (eg, substitutions, eg conservative). Includes an amino acid sequence having a substitution (substitution) but no more than 7, 6 or 5 modifications (eg, substitution, eg conservative substitution).

In some embodiments, the anti-BCMA binding domain is 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 with it or at least 1. A heavy chain variable region (VH) comprising an amino acid sequence having 2 or 3 modifications (eg, substitutions, such as conservative substitutions), but with modifications (eg, substitutions, such as conservative substitutions) not exceeding 30, 20 or 10. include.

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 is the nucleic acid sequence of SEQ ID NO: 53, 71 or 79 or at least about 85% thereof. , 90%, 95% or 99% contains nucleic acid sequences with sequence identity.

In some embodiments, the anti-BCMA binding domain is 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 with it or at least one, two or three modifications. It comprises a light chain variable region (VL) comprising an amino acid sequence (eg, substitution, eg conservative substitution), but modification (eg, substitution, eg conservative substitution) of no more than 30, 20 or 10.

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 is the nucleic acid sequence of SEQ ID NO: 62 or at least about 85%, 90% thereof. Contains nucleic acid sequences with 95% or 99% sequence identity.

In some embodiments, the anti-BCMA binding domain comprises VH and VL, where VH and VL are (i) sequences of SEQ ID NOs: 52 and 61 or at least about 85%, 90%, 95% or 99% thereof, respectively. A sequence having identity or an amino acid sequence having at least one, two or three modifications (eg, substitutions, eg conservative substitutions) but no more than 30, 20 or 10 modifications (eg, substitutions, eg conservative substitutions). (Ii) Sequences with SEQ ID NOs: 70 and 61 or at least about 85%, 90%, 95% or 99% sequence identity thereof or at least 1, 2 or 3 modifications (eg, substitutions, eg conservative substitutions). Amino acid sequences having but not greater than 30, 20 or 10 modifications (eg, substitutions, eg conservative substitutions), or (iii) SEQ ID NOs: 78 and 61 or at least about 85%, 90%, 95% or 99 thereof, respectively. % Sequence identity or at least one, two or three modifications (eg, substitutions, eg conservative substitutions), but no more than 30, 20 or 10 modifications (eg, substitutions, eg conservative substitutions). Includes amino acid sequence.

In some embodiments, HC CDR1, HC CDR2 and HC CDR3 are HC CDR sequences (or at least about 85%, 90 thereof) listed in Tables 7-9 (eg, a single column of Tables 7-9). %, 95% or 99% sequence identity or at least one, two or three modifications (eg, substitutions, eg conservative substitutions), but modifications (eg, substitutions, eg conservative substitutions) of 7, An sequence that does not exceed 6 or 5). In some embodiments, LC CDR1, LC CDR2 and LC CDR3 are LC CDR sequences (or at least about 85%, 90 thereof) listed in Tables 7-9 (eg, a single column of Tables 7-9). %, 95% or 99% sequence identity or at least one, two or three modifications (eg, substitutions, eg conservative substitutions), but modifications (eg, substitutions, eg conservative substitutions) of 7, An sequence that does not exceed 6 or 5).

In some embodiments, HC CDR1, HC CDR2 and HC CDR3 have the amino acid sequences of SEQ ID NOs: 86, 130 and 88 (or at least about 85%, 90%, 95% or 99% sequence identity thereof, respectively). Contains sequences or sequences having at least one, two or three modifications (eg, substitutions, eg conservative substitutions), but no more than 7, 6 or 5 modifications (eg, substitutions, eg conservative substitutions). In some embodiments, HC CDR1, HC CDR2 and HC CDR3 have (i) the amino acid sequences of SEQ ID NOs: 86, 87 and 88 or at least about 85%, 90%, 95% or 99% sequence identity with them, respectively. Amino acid sequence having or at least 1, 2 or 3 modifications (eg, substitutions, eg conservative substitutions), but no more than 7, 6 or 5 modifications (eg, substitutions, eg conservative substitutions); (Ii) Amino acid sequences of SEQ ID NOs: 86, 109 and 88, or amino acid sequences having at least about 85%, 90%, 95% or 99% sequence identity with them or at least 1, 2 or 3 modifications (eg, substitutions, Amino acid sequences that have (eg, conservative substitutions) but do not have more than 7, 6 or 5 modifications (eg, substitutions, such as conservative substitutions); or (iii) the amino acid sequences of SEQ ID NOs: 86, 109 and 88, respectively, or with it. It has an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence identity or at least one, two or three modifications (eg, substitutions, eg conservative substitutions), but modifications (eg, substitutions, eg. Conservative substitutions) include amino acid sequences that do not exceed 7, 6 or 5.

In some embodiments, LC CDR1, LC CDR2 and LC CDR3 are amino acids having the amino acid sequences of SEQ ID NOs: 95, 131 and 132 or at least about 85%, 90%, 95% or 99% sequence identity thereof, respectively. Includes an amino acid sequence having a sequence or at least one, two or three modifications (eg, substitutions, eg conservative substitutions), but no more than 7, 6 or 5 modifications (eg, substitutions, eg conservative substitutions). In some embodiments, LC CDR1, LC CDR2 and LC CDR3 have (i) the amino acid sequences of SEQ ID NOs: 95, 96 and 97 or at least about 85%, 90%, 95% or 99% sequence identity with them, respectively. Amino acid sequence having or at least 1, 2 or 3 modifications (eg, substitutions, eg conservative substitutions), but no more than 7, 6 or 5 modifications (eg, substitutions, eg conservative substitutions); (Ii) Amino acid sequences of SEQ ID NOs: 95, 114 and 115 or amino acid sequences having at least about 85%, 90%, 95% or 99% sequence identity thereof or at least 1, 2 or 3 modifications (eg, substitutions, Amino acid sequences with (eg, conservative substitutions) but no more than 7, 6 or 5 modifications (eg, substitutions, such as conservative substitutions); or (iii) amino acid sequences of SEQ ID NOs: 95, 114 and 97 or with them, respectively. It has an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence identity or at least one, two or three modifications (eg, substitutions, eg conservative substitutions), but modifications (eg, substitutions, eg. Conservative substitutions) include amino acid sequences that do not exceed 7, 6 or 5.

In some embodiments, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR3 are (i) SEQ ID NOs: 86, 87, 88, 95, 96 and 97 or at least about 85% thereof, respectively. Sequences with 90%, 95% or 99% sequence identity or with at least 1, 2 or 3 modifications (eg, substitutions, eg conservative substitutions), but with 7 modifications (eg, substitutions, eg conservative substitutions). , 6 or no more than 5 sequences; (ii) sequences having SEQ ID NOs: 86, 109, 88, 95, 114 and 115 or at least about 85%, 90%, 95% or 99% sequence identity thereof or Sequences having at least one, two or three modifications (eg, substitutions, eg conservative substitutions), but no more than 7, 6 or 5 modifications (eg, substitutions, eg conservative substitutions); or (iii) sequences, respectively. Sequences having sequence identity of numbers 86, 109, 88, 95, 114 and 97 or at least about 85%, 90%, 95% or 99% thereof or at least 1, 2 or 3 modifications (eg, substitutions, eg conservative). Includes an amino acid sequence having a substitution (substitution) but no more than 7, 6 or 5 modifications (eg, substitution, eg conservative substitution).

In some embodiments, the anti-BCMA binding domain is an 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 with it or at least 1, 2 or It comprises a heavy chain variable region (VH) comprising an amino acid sequence having three modifications (eg, substitutions, eg conservative substitutions), but no modification (eg, substitutions, eg conservative substitutions) greater than 30, 20 or 10. 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 is the nucleic acid sequence of SEQ ID NO: 260, 94 or 113 or at least about 85% thereof. , 90%, 95% or 99% of nucleic acid sequences with sequence identity.

In some embodiments, the anti-BCMA binding domain is 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 with it or at least 1. A light chain variable region (VL) comprising an amino acid sequence having 2 or 3 modifications (eg, substitutions, such as conservative substitutions), but with modifications (eg, substitutions, such as conservative substitutions) not exceeding 30, 20 or 10. include. 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 is the nucleic acid sequence of SEQ ID NO: 261, 103, 119 or 125 or at least about about it. Includes nucleic acid sequences with 85%, 90%, 95% or 99% sequence identity.

In some embodiments, the anti-BCMA binding domain comprises VH and VL, where VH and VL are (i) sequences of SEQ ID NOs: 93 and 102 or at least about 85%, 90%, 95% or 99% thereof, respectively. An amino acid sequence having identity or at least one, two or three modifications (eg, substitutions, eg conservative substitutions), but modifications (eg, substitutions, eg conservative substitutions not exceeding 30, 20 or 10). ii) Sequences with SEQ ID NOs: 112 and 118 or at least about 85%, 90%, 95% or 99% sequence identity thereof or at least 1, 2 or 3 modifications (eg, substitutions, eg conservative substitutions). However, modifications (eg, substitutions, eg, amino acid sequences in which the conservative substitution does not exceed 30, 20 or 10, or (iii) SEQ ID NOs: 112 and 124 or at least about 85%, 90%, 95% or 99% thereof, respectively. A sequence having sequence identity or an amino acid sequence having at least one, two or three modifications (eg, substitutions, eg conservative substitutions), but with modifications (eg, substitutions, eg conservative substitutions not exceeding 30, 20 or 10). include.

In some embodiments, HC CDR1, HC CDR2 and HC CDR3 are HC CDR sequences (or at least about 85%, 90 thereof) listed in Tables 11-13 (eg, a single column of Tables 11-13). %, 95% or 99% sequence identity or at least one, two or three modifications (eg, substitutions, eg conservative substitutions), but modifications (eg, substitutions, eg conservative substitutions) of 7, An sequence that does not exceed 6 or 5). In some embodiments, LC CDR1, LC CDR2 and LC CDR3 are LC CDR sequences (or at least about 85%, 90 thereof) listed in Tables 11-13 (eg, a single column of Tables 11-13). %, 95% or 99% sequence identity or at least one, two or three modifications (eg, substitutions, eg conservative substitutions), but modifications (eg, substitutions, eg conservative substitutions) of 7, An sequence that does not exceed 6 or 5).

In some embodiments, HC CDR1, HC CDR2 and HC CDR3 are the amino acid sequences of SEQ ID NOs: 179, 180 and 181 or sequences having at least about 85%, 90%, 95% or 99% sequence identity with them, respectively. Alternatively, it comprises a sequence having at least one, two or three modifications (eg, substitutions, eg conservative substitutions), but no more than 7, 6 or 5 modifications (eg, substitutions, eg conservative substitutions). In some embodiments, HC CDR1, HC CDR2 and HC CDR3 have (i) the amino acid sequences of SEQ ID NOs: 137, 138 and 139, respectively, or at least about 85%, 90%, 95% or 99% sequence identity with them. Amino acid sequence having or at least 1, 2 or 3 modifications (eg, substitutions, eg conservative substitutions), but no more than 7, 6 or 5 modifications (eg, substitutions, eg conservative substitutions); Or (ii) the amino acid sequence of SEQ ID NOs: 160, 161 and 162, or an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence identity with it or at least 1, 2 or 3 modifications (eg, substitutions). Includes an amino acid sequence having, eg, conservative substitutions, but no more than 7, 6 or 5 modifications (eg, substitutions, eg conservative substitutions).

In some embodiments, LC CDR1, LC CDR2 and LC CDR3 are amino acids having the amino acid sequences of SEQ ID NOs: 147, 182 and 183 or at least about 85%, 90%, 95% or 99% sequence identity thereof, respectively. Includes an amino acid sequence having a sequence or at least one, two or three modifications (eg, substitutions, eg conservative substitutions), but no more than 7, 6 or 5 modifications (eg, substitutions, eg conservative substitutions). In some embodiments, LC CDR1, LC CDR2 and LC CDR3 have (i) the amino acid sequences of SEQ ID NOs: 147, 148 and 149 or at least about 85%, 90%, 95% or 99% sequence identity with them, respectively. Amino acid sequence having or at least 1, 2 or 3 modifications (eg, substitutions, eg conservative substitutions), but no more than 7, 6 or 5 modifications (eg, substitutions, eg conservative substitutions); Or (ii) the amino acid sequence of SEQ ID NOs: 147, 170 and 171 or an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereof or at least 1, 2 or 3 modifications (eg, substitutions). Includes an amino acid sequence having, eg, conservative substitutions, but no more than 7, 6 or 5 modifications (eg, substitutions, eg conservative substitutions).

In some embodiments, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR3 have (i) SEQ ID NOs: 137, 138, 139, 147, 148 and 149 or at least about 85%, respectively. Sequences with 90%, 95% or 99% sequence identity or with at least 1, 2 or 3 modifications (eg, substitutions, eg conservative substitutions), but with 7 modifications (eg, substitutions, eg conservative substitutions). , 6 or no more than 5 sequences; or (ii) sequences having SEQ ID NOs: 160, 161, 162, 147, 170 and 171 or at least about 85%, 90%, 95% or 99% sequence identity thereof. Alternatively, it comprises an amino acid sequence having at least one, two or three modifications (eg, substitutions, eg conservative substitutions) but no more than 7, 6 or 5 modifications (eg, substitutions, eg conservative substitutions).

In some embodiments, the anti-BCMA binding domain is 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 with it or at least 1, 2 or It comprises a heavy chain variable region (VH) comprising an amino acid sequence having 3 modifications (eg, substitutions, eg conservative substitutions), but no modification (eg, substitutions, eg conservative substitutions) greater than 30, 20 or 10. 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 is the nucleic acid sequence of SEQ ID NO: 146 or 169 or at least about 85%, 90 thereof. Includes nucleic acid sequences with%, 95% or 99% sequence identity.

In some embodiments, the anti-BCMA binding domain is 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 with it or at least 1, 2 or It comprises a light chain variable region (VL) comprising an amino acid sequence having three modifications (eg, substitutions, eg conservative substitutions), but no modification (eg, substitutions, eg conservative substitutions) greater than 30, 20 or 10. 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 is the nucleic acid sequence of SEQ ID NO: 155 or 174 or at least about 85%, 90 thereof. Includes nucleic acid sequences with%, 95% or 99% sequence identity.

In some embodiments, the anti-BCMA binding domain comprises VH and VL, where VH and VL are (i) sequences of SEQ ID NOs: 145 and 154 or at least about 85%, 90%, 95% or 99% thereof, respectively. An amino acid sequence having identity or at least one, two or three modifications (eg, substitutions, eg conservative substitutions), but with modifications (eg, substitutions, eg conservative substitutions) not exceeding 30, 20 or 10. Or (ii) a sequence having at least about 85%, 90%, 95% or 99% sequence identity with SEQ ID NOs: 168 and 173, respectively, or at least 1, 2 or 3 modifications (eg, substitutions, eg conservative substitutions). However, it comprises an amino acid sequence in which the modification (eg, substitution, eg conservative substitution) does not exceed 30, 20 or 10.

In one aspect, the invention provides an isolated polypeptide molecule encoded by the nucleic acid molecules described herein. In one aspect, the 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 adenoviral 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 aspect, the invention is a cell comprising a nucleic acid molecule described herein, a CAR described herein, a polypeptide molecule described herein or a vector described herein (eg, a T cell). Or NK cells). In some embodiments, the cell further expresses an inhibitor comprising a first polypeptide comprising at least a portion of an inhibitory molecule associated with a second polypeptide comprising a positive signal from the intracellular signaling domain. And, optionally, the inhibitor comprises a first polypeptide containing at least a portion of PD-1 and a second polypeptide containing a co-stimulation domain and a primary signaling domain.

In one aspect, the invention provides a method of producing cells comprising transducing cells (eg, T cells or NK cells) with the vectors described herein. In one aspect, the invention provides a method of making RNA-manipulated cells comprising introducing in vitro transcribed RNA or synthetic RNA into a cell (eg, T cell or NK cell). Here, RNA comprises a nucleic acid molecule described herein or a nucleic acid molecule encoding a CAR described herein.

In one aspect, the invention provides a method of providing antitumor immunity in a subject comprising administering to the subject an effective amount of the cells described herein. In one aspect, the invention provides a method of treating a subject having a disease associated with the expression of BCMA, 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 the expression of BCMA is (i) a precancerous condition selected from one or more of cancer or malignant tumors or myelodysplastic syndrome, myelodysplastic syndrome or preleukemia, or (ii). ) A non-cancer-related indication associated with the expression of BCMA. In some embodiments, the disease is hematological or solid cancer. In some embodiments, the disease is acute leukemia, B-cell acute lymphocytic leukemia (“BALL”), T-cell acute lymphocytic leukemia (“TALL”), acute lymphocytic leukemia (ALL), chronic myeloid leukemia (“BALL”). CML), Chronic lymphocytic leukemia (CLL), pre-B cell lymphocytic leukemia, blast plasma cell-like dendritic cell neoplasm, Berkit 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, myelodystrophy and myelopathy syndrome, non-hodgkin lymphoma, plasma bud Cell lymphoma, plasma cell-like dendritic cell neoplasm, Waldenstrem macroglobulinemia, prostate cancer (eg, castration-resistant or treatment-resistant prostate cancer or metastatic prostate cancer), pancreatic cancer, lung cancer, plasma cells Proliferative disorders (eg, asymptomatic myeloma (smoldering multiple myeloma or painless myeloma), unclear monoclonal hypergamma globulinemia (MGUS), Waldenstrem macroglobulinemia, plasma Cellular tumors (eg, plasma cell overgrowth, solitary myeloma, solitary plasmacytoma, extramedullary plasmacytoma and multiple plasmacytoma), systemic amyloid light chain amyloidosis or POEMS syndrome (Crow-Fukase syndrome, (Also known as high moon disease and PEP syndrome))) or a combination thereof. In some embodiments, the disease is multiple myeloma. In some embodiments, the method further comprises administering to the subject a second therapeutic agent. In some embodiments, the second therapeutic agent is a PD-1 inhibitor and, optionally, the PD-1 inhibitor is PDR001, nivolumab, pembrolizumab, pigilizmab, MEDI0680, REGN2810, TSR-042, PF. It is selected from the group consisting of 60801591 and AMP-224. In some embodiments, the second therapeutic agent is a PD-L1 inhibitor, optionally the PD-L1 inhibitor is selected from the group consisting of FAZ053, atezolizumab, avelumab, durvalumab and BMS-936559. To. In some embodiments, the second therapeutic agent is a LAG-3 inhibitor, optionally consisting of LAG525, BMS-986016, TSR-033, MK-4280 and REGN3767. Selected from the group. In some embodiments, the second therapeutic agent is a TIM-3 inhibitor, optionally the TIM-3 inhibitor is selected from the group consisting of MBG453, TSR-022 and LY3321367. In some embodiments, the second therapeutic agent is a CTLA-4 inhibitor, and optionally the CTLA-4 inhibitor is 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 an IL-15 polypeptide and an IL-15Ra. A combination of both polypeptides, eg 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, optionally the mTOR inhibitor is RAD001 or rapamycin.

CARs comprising anti-BCMA binding domains and such anti-BCMA binding domains disclosed herein have improved properties over previous anti-BCMA binding domains and CARs containing them, eg, increased binding affinity for BCMA. It has sex, increased CAR expression levels in cells (eg, T cells or NK cells) and / or enhanced cytotoxicity and / or the ability to mediate cytokine production in cells (eg, T cells or NK cells).

Unless otherwise defined, all scientific and technological terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. In carrying out or testing the invention, similar or equivalent methods and materials as described herein may be used, but suitable methods and materials are described below. All publications, patent applications, patents and other references referred to herein are incorporated by reference in their entirety. In addition, the materials, methods and examples are merely exemplary and are not intended to be limiting. Headings, subheadings or numbered or lettered elements such as (a), (b), (i), etc. are provided simply for readability. The use of heading or numbered or lettered elements herein does not require that the steps or elements be performed in alphabetical order, or that the steps or elements are not necessarily separate from each other. Other features, objects and advantages of the invention will become apparent from the description and drawings as well as the claims.

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

Definitions Unless otherwise defined, all scientific and technological terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains.

The terms "one (a)" and "one (an)" refer to one or more (ie, at least one) of the grammatical referents of the article. As an example, "element" means one element or more than one element.

The term "about" refers to a measurable value such as quantity, duration of time, etc., ± 20% from the specified value, ± 10% in some cases, or ± 5 in some cases. %, Or ± 1% in some cases, or ± 0.1% in some cases, means that such variations are included as appropriate for the practice of the methods of the present disclosure.

The compositions and methods of the invention are sequences that are at least 85%, 90% or 95% identical to or beyond a particular sequence or a sequence that is substantially identical or similar to that particular sequence. Includes polypeptides and nucleic acids with. In connection with the amino acid sequence, the term "substantially identical" is used herein so that the first and second amino acid sequences can have a common structural domain and / or a common functional activity. , A first amino acid sequence containing a sufficient or minimum number of amino acid residues that are i) identical or ii) a conservative substitution thereof, with the aligned amino acid residues of the second amino acid sequence, eg. At least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of a reference sequence, eg, the sequences provided herein. Used to refer to an amino acid sequence that contains a common structural domain with identity.

In connection with a nucleotide sequence, the term "substantially identical" is used herein where the first and second nucleotide sequences encode or have a common structure for polypeptides having common functional activity. A first nucleic acid sequence, eg, a reference sequence, containing a sufficient or minimal number of nucleotides that are identical to the aligned nucleotides of the second nucleic acid sequence so as to encode a polypeptide domain or common functional polypeptide activity. , For example, at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the sequences provided herein. Used to refer to a nucleotide sequence having.

The term "variant" refers to a polypeptide that has substantially the same amino acid sequence as the reference amino acid sequence or is encoded by substantially the same nucleotide sequence. In some embodiments, the variant is a functional variant.

The term "functional variant" may have an amino acid sequence that is substantially identical to the reference amino acid sequence, or it may be encoded by a nucleotide sequence that is substantially identical and have the activity of one or more of the reference amino acid sequences. Refers to a polypeptide that can.

As used herein, the term "BCMA" refers to a B cell maturation antigen. BCMA (also known as TNFRSF17, BCM or CD269) is a member of the tumor necrosis factor (TNFR) family and is predominantly expressed on terminally differentiated B cells such as memory B cells and plasma cells. The ligands are called B cell activators (BAFFs) and growth-inducing ligands (APRILs) 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 long primary mRNA transcript (NCBI Accession No. NM_001192.2) that encodes 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 the regulation of BCMA expression. (Laabi Y. et al., Nucleic Acids Res., 1994, 22: 1147-1154). Further transcriptional variants have been described for unknown 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 proteins containing variants of wild-type full-length BCMA, such as point mutations, fragments, insertions, deletions and splice variants.

The term "chimeric antigen receptor" or instead "CAR" refers to cytoplasmic signaling including at least an extracellular antigen-binding domain, a transmembrane domain, and a functional signaling domain derived from a stimulator as defined below. Refers to a recombinant polypeptide construct that includes a domain (also referred to herein as an "intracellular signaling domain"). In some embodiments, the domain of the CAR polypeptide construct is in the same polypeptide chain and comprises, for example, a chimeric fusion protein. In some embodiments, the domains of the CAR polypeptide construct are not contiguous with each other and are, for example, in different polypeptide chains, eg, as provided for RCAR as described herein.

In one embodiment, the cytoplasmic signaling domain comprises a primary signaling domain (eg, a CD3-zeta primary signaling domain). In one embodiment, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one co-stimulating molecule as defined below. In one embodiment, the co-stimulator molecule is selected from 41BB (ie, 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 including a functional signaling domain derived from a stimulating molecule. In one embodiment, CAR comprises an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain including a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulating molecule. Contains chimeric fusion proteins. In one aspect, CAR comprises a cell comprising an extracellular antigen recognition domain, a transmembrane domain, two functional signaling domains derived from one or more costimulatory molecules, and a functional signaling domain derived from a stimulatory molecule. Includes a chimeric fusion protein containing an internal signaling domain. In one embodiment, the CAR comprises an extracellular antigen recognition domain, a transmembrane domain, and at least two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from the stimulatory molecule. Includes a chimeric fusion protein containing an intracellular signaling domain. In one embodiment, 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, which is optionally from the antigen recognition domain (eg, scFv) during cell processing and cell membrane localization of CAR. Be disconnected.

An antigen-binding domain (eg, scFv, single-domain antibody or TCR (eg, TCR) that targets a particular tumor marker X, where X can be a tumor marker as described herein). CARs containing alpha binding domains or TCR beta binding domains)) are also referred to as XCARs. For example, a CAR containing an antigen binding domain that targets BCMA is referred to as BCMA CAR. CAR can be expressed in any cell, eg, immune effector cells described herein (eg, T cells or NK cells).

The term "signal transduction domain" conveys information intracellularly to regulate cell activity via signaling pathways defined by producing secondary messengers or acting as effectors in response to such messengers. Refers to a part of the functionality of a protein that functions by.

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 can be polyclonal or monoclonal, multi-chain or single-chain or intact immunoglobulins and can be derived from natural or recombinant sources. The antibody can be a tetramer of an immunoglobulin molecule.

The term "antibody fragment" refers to at least a portion of an intact antibody or recombinant variant thereof and is sufficient to provide recognition and specific binding of the antibody fragment to a target such as an antigen. Refers to a domain, eg, a variable region for antigen determination. 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), camels. Two or more multispecific molecules formed with antibody fragments such as divalent fragments containing two Fab fragments and two or more bound antibodies linked by disulfide crosslinks in the family animal VHH domain and hinge region. For example, two isolated CDRs or other epitope binding fragments can be mentioned. Antibody fragments can also be incorporated into single domain antibodies, maxibody, minibody, nanobody, intrabody, diabody, triabody, tetrabody, v-NAR and bis-scFv (eg, Hollinger and Hoodson, Nature Biotechnology 23). See: 1126-1136, 2005). Antibody fragments can also be grafted onto scaffolds based on polypeptides such as fibronectin type III (Fn3) (see US Pat. No. 6,703,199, which describes the fibronectin polypeptide minibody). ..

The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, the light chain and heavy chain variable regions being short. It is closely linked by a mobile polypeptide linker and can be expressed as a single chain polypeptide, and scFv retains the specificity of the intact antibody from which it is derived. Unless specified, as used herein, scFv can have VL and VH variable regions in any order, eg, with respect to the N-terminal and C-terminal ends of the polypeptide, and scFv is VL-. It may contain a linker-VH or it may contain a VH-linker-VL.

The terms "complementarity determining regions" or "CDRs" as used herein refer to sequences of amino acids within antibody variable regions that confer antigen specificity and binding affinity. For example, there are generally three CDRs in each heavy chain variable region (eg, HCDR1, HCDR2 and HCDR3) and three CDRs in each light chain variable region (LCDR1, LCDR2 and LCDR3). The exact amino acid sequence boundaries for a given CDR are described in 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 combinations thereof, and can be determined using any of a number of well-known schemes. In the combination of Kabat and Chothia numbering schemes, in some embodiments, the CDR corresponds to an amino acid residue that is part of the Kabat CDR, Chothia CDR, or both.

A portion of the CAR composition of the invention comprising an antibody or antibody fragment thereof can be present, for example, in various forms and the antigen binding domain may be, for example, a single domain antibody fragment (sdAb), a single chain antibody (scFv) or eg. Expressed as part of a human or humanized antibody-containing polypeptide chain (Harrow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow antibody, 198. Manual, Cold Spring Harbor, New York; Houseton et al., 1988, Proc. Natl. Acad. Sci. USA 85: 5879-5883; Bird et al., 1988, Science 242: 423-426). In one aspect, the antigen binding domain of the CAR composition of the invention comprises an antibody fragment. In a further embodiment, the CAR comprises an antibody fragment containing scFv.

As used herein, the term "binding domain" or "antibody molecule" (also referred to herein as "antitarget binding domain") is a protein comprising at least one immunoglobulin variable domain sequence, eg. Refers to an immunoglobulin chain or a fragment thereof. The term "binding domain" or "antibody molecule" includes antibodies and antibody fragments. In certain embodiments, the antibody molecule is a multispecific antibody molecule, eg, it comprises a plurality of immunoglobulin variable domain sequences, wherein the first immunoglobulin variable domain sequence of the plurality. The second immunoglobulin variable domain sequence among the plurality has binding specificity for the first epitope and the second immunoglobulin variable domain sequence has binding specificity for the second epitope. In certain embodiments, the multispecific antibody molecule is a bispecific antibody molecule. Bispecific antibodies have specificity for two or less antigens. The bispecific antibody molecule is composed of a first immunoglobulin variable domain sequence having binding specificity for the first epitope and a second immunoglobulin variable domain sequence having binding specificity for the second epitope. Characterized.

The term "antibody heavy chain" refers to the larger of the two polypeptide chains present in its naturally occurring conformating antibody molecule, which usually determines the class to which the antibody belongs.

The term "antibody light chain" refers to the smaller of the two polypeptide chains present in the naturally occurring conformating antibody molecule. Kappa (κ) and lambda (λ) light chains refer to the two major antibody light chain isotypes.

The term "recombinant antibody" refers to an antibody produced using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage or yeast expression system. The term should also be construed to mean a DNA molecule that encodes an antibody and is made by synthesizing a DNA molecule that expresses an antibody protein or an amino acid sequence that specifies the antibody thereof, and DNA or amino acids. The sequence is obtained using a well-known recombinant DNA or amino acid sequence technique available in the art.

The term "antigen" or "Ag" refers to a molecule that triggers an immune response. This immune response may include antibody production or activation of specific immune-qualified cells, or both. One of skill in the art will appreciate that any macromolecule can be an antigen, including virtually any protein or peptide. In addition, the antigen can be derived from recombinant DNA or genomic DNA. Those of skill in the art will therefore appreciate that any DNA containing a nucleotide sequence or partial nucleotide sequence that encodes a protein that gives rise to an immune response encodes an "antigen" as the term is used herein. Will do. Moreover, one of skill in the art will appreciate that the antigen does not need to be encoded only by the full-length nucleotide sequence of a gene. The invention includes, but is not limited to, the use of partial nucleotide sequences of two or more genes and sequences in various combinations such that those nucleotide sequences encode a polypeptide that yields the desired immune response. It is easily clear that it will be done. Moreover, one of ordinary skill in the art will appreciate that the antigen does not need to be encoded by a "gene" at all. It is readily apparent that the antigen can be made synthetically, can be obtained from a biological sample, or can be a macromolecule other than a polypeptide. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells or body fluids, along with other biological components.

The term "antitumor effect" is not limited to, for example, for a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, a decrease in tumor cell proliferation, a decrease in tumor cell survival or a cancerous condition. Refers to biological effects that can be manifested by various means, including improvement of various related physiological symptoms. The "antitumor effect" can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies of the invention to prevent the development of tumors in the first place.

The term "anticancer effect" is not limited to, for example, for a decrease in tumor volume, a decrease in the number of cancer cells, a decrease in the number of metastases, an increase in life expectancy, a decrease in cancer cell proliferation, a decrease in cancer cell survival or a cancerous condition. Refers to biological effects that can be manifested by various means, including improvement of various related physiological symptoms. The "anti-cancer effect" can also be manifested by the ability of peptides, polynucleotides, cells and antibodies to prevent the development of cancer in the first place. The term "antitumor effect" is a biological effect that can be manifested by a variety of means, including, but not limited to, reduction of tumor volume, reduction of tumor cell number, reduction of tumor cell proliferation or reduction of tumor cell survival. Point to. The term "self" refers to any material derived from the same individual that will later be reintroduced into that individual.

The term "homologous" refers to any material derived from a different animal of the same species as the individual into which the material is introduced. Two or more individuals are said to be allogeneic to each other when the genes at one or more loci are not identical. In some embodiments, allogeneic materials from individuals of the same species can be genetically distinct enough to interact antigenically.

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

As used herein, the term "apheresis" refers to the removal of donor or patient blood from the donor or patient and passing it through a device to select one or more selected specific components and the rest. For example, it refers to an in vitro process recognized in the art of returning to the circulation of a donor or patient by a blood return method. Therefore, in relation to the "apheresis sample", it refers to a sample obtained by using apheresis.

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

The terms "tumor" and "cancer" are used interchangeably herein, for example, both terms include solid and humoral, eg diffuse or circulating tumors. As used herein, the term "cancer" or "tumor" includes precancerous as well as malignant cancers and tumors.

"Derived" as used herein refers to the relationship between a first molecule and a second molecule. This generally refers to structural similarities between the first molecule and the second molecule, limiting methods or sources for the first molecule derived from the second molecule. Implications or no inclusion. For example, in the case of an intracellular signaling domain derived from a CD3 zeta molecule, the intracellular signaling domain retains sufficient CD3 zeta structure such that it has the required function, that is, the ability to generate signals under appropriate conditions. ing. This does not imply or include limitation to specific methods of making intracellular signaling domains, eg, starting with a CD3 zeta sequence to provide an intracellular signaling domain and deleting unwanted sequences. , Or does not mean that the mutation must be given to reach the intracellular signaling domain.

The phrase "disease associated with the expression of BCMA" includes, but is not limited to, proliferative diseases such as cancer or malignant tumors or precancerous conditions such as myelodysplasia, myelodysplasia syndrome or pre-leukemia, BCMA ( For example, a disease associated with a cell expressing wild-type or mutant BCMA) or a pathological condition or BCMA associated with a cell expressing BCMA (eg, wild-type or mutant BCMA) or BCMA (eg, wild-type or mutant). Includes non-cancer-related signs associated with cells expressing BCMA). To avoid misunderstanding, diseases associated with BCMA expression are now downregulated because BCMA expression is downregulated, for example, by treatment with molecules that target BCMA, eg, BCMA inhibitors described herein. However, pathological conditions associated with cells that once expressed BCMA may be included. In one embodiment, the cancer associated with the expression of BCMA (eg, wild-type or mutant BCMA) is a hematological cancer. In one embodiment, the hematological cancer is leukemia or lymphoma. In one embodiment, the cancer associated with the expression of BCMA (eg, wild-type or mutant BCMA) is a malignant tumor of differentiated plasma cell B cells. In one embodiment, cancers associated with the expression of BCMA (eg, wild-type or mutant BCMA) are, but are not limited to, eg, B-cell acute lymphocytic leukemia (“BALL”), T-cell acute lymphocytic leukemia (“BALL”). TALL "), one or more acute leukemias including, but not limited to, acute lymphocytic leukemia (ALL), including, but not limited to, chronic myelocytic leukemia (CML), chronic lymphocytic leukemia (CLL), for example. Cancers and malignant tumors including the above chronic leukemia are included. Further cancer or hematological conditions associated with the expression of BMCA (eg, wild-type or mutant BCMA) are not limited, but are, for example, pre-B cell lymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasia. Bioplasm, Berkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small or large cell follicular lymphoma, malignant lymphocytic pathology, MALT lymphoma, mantle cell lymphoma, marginal Of zonal lymphoma, multiple myeloma, myelodystrophy and myelodystrophy syndrome, non-hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic neoplasm, Waldenstrem macroglobulinemia and myeloid hematology Includes "pre-leukemia," which is a group of various hematological conditions summarized by ineffective production (or dysplasia). In some embodiments, the cancer is multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma or glioblastoma. In embodiments, diseases associated with BCMA expression include plasmacytoproliferative disorders such as asymptomatic myeloma (smoldering multiple myeloma or painless myeloma), unclear monoclonal immunoglobulinemia. (MGUS), Waldenstrem macroglobulinemia, plasmacytoma (eg, amyloidosis, solitary myeloma, solitary myeloma, extramedullary plasmacytoma and multiple myeloma), Systemic amyloid light chain amyloidosis and POEMS syndrome (also known as Crow-Fukase syndrome, high moon disease and PEP syndrome) are included. Further diseases associated with the expression of BCMA (eg, wild or mutant BCMA) are not limited to, but are not limited to, for example, atypical and atypical and associated with the expression of BCMA (eg, wild or mutant BCMA). / Or non-classical cancers, malignant tumors, precancerous conditions or proliferative disorders, such as the cancers described herein, such as prostate cancer (eg, castration-resistant or therapy-resistant prostate cancer or metastatic prostate cancer). , Pancreatic cancer or lung cancer.

Non-cancer-related pathologies associated with BCMA (eg, wild-type or mutant BCMA) include viral infections such as HIV, fungal infections such as C.I. Neoformans, autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus (SLE or lupus), tinea vulgaris and Sjogren's syndrome, inflammatory bowel disease, ulcerative colitis, transplants associated with mucosal immunity Includes an undesired immune response to biologics (eg, Factor VIII) where associated allospecific immune disorders and humoral immunity are important. In embodiments, non-cancer-related signs associated with the expression of BCMA include, but are not limited to, for example, autoimmune diseases (eg, lupus), inflammatory disorders (allergies and asthma) and transplants. In some embodiments, the tumor antigen-expressing cells express or at any point in time the mRNA encoding the tumor antigen. In certain embodiments, the tumor antigen expressing cells produce a tumor antigen protein (eg, wild type or mutant), and the tumor antigen protein may be present at normal or reduced levels. In one embodiment, the tumor antigen-expressing cells produced transiently detectable levels of tumor antigen protein, but subsequently substantially stopped producing detectable tumor antigen protein.

The term "conservative sequence modification" refers to an amino acid modification in which the binding properties of the antibody or antibody fragment containing the amino acid sequence are not significantly affected or altered. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the antibodies or antibody fragments of the invention by standard techniques known in the art, such as site-specific mutagenesis and PCR-mediated mutagenesis. Conservative substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. A family of amino acid residues with similar side chains is defined in the art. These families include basic side chains (eg, lysine, arginine, histidine), acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (eg, glycine, asparagine, glutamine, serine, threonine, etc.). Tyrosine, cysteine, tryptophan), non-polar side chains (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (eg, threonine, valine, isoleucine) and aromatic side chains (eg). , Tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the CAR of the present invention can be replaced with other amino acid residues from the same side chain family, and altered CARs are functional assays described herein. Can be tested using.

The term "stimulation" refers to signaling events such as, but not limited to, signaling via the TCR / CD3 complex by which a stimulating molecule (eg, the TCR / CD3 complex) binds to its cognate ligand. Refers to the primary response produced by mediation. Stimulation can mediate changes in the expression of certain molecules and / or recognition of cytoskeletal structures, such as downregulation of TGF-β.

The term "stimulatory molecule" is expressed by T cells that provide one or more primary cytoplasmic signaling sequences that regulate primary activation of the TCR complex in a stimulating manner for at least some aspects of the T cell signaling pathway. Refers to the molecule to be. In some embodiments, the ITAM-containing domain within the CAR reproduces the signaling of the primary TCR independently of the endogenous TCR complex. In one embodiment, the primary signal is triggered, for example, by binding of the TCR / CD3 complex to a peptide-loaded MHC molecule, which includes, but is not limited to, proliferation, activation, differentiation, and the like. Leads to the mediation of. Primary cytoplasmic signaling sequences (also referred to as "primary signaling domains") that function in a stimulating manner can include immunoreceptor-activated tyrosine motifs or signaling motifs known as ITAMs. Examples of ITAM-containing primary cytoplasmic signaling sequences that are particularly useful in the present invention are TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (“ICOS”). , But not limited to those derived from FcεRI and CD66d, DAP10 and DAP12. In a particular CAR of the invention, the intracellular signaling domain in any one or more CARs of the invention comprises an intracellular signaling sequence, eg, a primary signaling sequence of the CD3 zeta. The term "antigen-presenting cell" or "APC" refers to immune system cells such as accessory cells (eg, B cells, dendritic cells) that present foreign antigens complexed with major histocompatibility complex (MHC) on their surface. (Cells, etc.). T cells can recognize these complexes using their T cell receptor (TCR). APC processes the antigen and presents it to T cells.

"Intracellular signaling domain", as the term is used herein, refers to the intracellular portion of a molecule. In embodiments, the intracellular signal domain transmits effector function signals, directing cells to perform specialized functions. The entire intracellular signaling domain can 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, it can be used in place of an intact chain as long as such truncated portion transmits an effector function signal. Thus, the term intracellular signaling domain is intended to include any truncated portion of the intracellular signaling domain sufficient to transmit effector function signals.

The intracellular signaling domain produces signals that promote immune effector function in CAR-containing cells, such as CART cells. Examples of immune effector functions (eg, in CART cells) include cytolytic and helper activities, including the secretion of cytokines.

In certain embodiments, the intracellular signaling domain may include a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from molecules involved in primary or antigen-dependent stimulation. In certain embodiments, the intracellular signaling domain may include a co-stimulating intracellular domain. Exemplary intracellular signaling domains include those derived from molecules involved in co-stimulation signals or antigen-independent stimulation. For example, in the case of CART, the primary intracellular signaling domain can contain the cytoplasmic sequence of the T cell receptor, and the costimulatory intracellular signaling domain can contain the cytoplasmic sequence from the co-receptor or co-stimulator molecule. can.

The primary intracellular signaling domain can include an immunoreceptor-activating tyrosine motif or a signaling motif known as ITAM. Examples of primary cytoplasmic signaling sequences including ITAM include, but are not limited to, CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 beta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also as "ICOS"). Known), those derived from FcεRI, CD66d, DAP10 and DAP12 are included.

The term "zeta" or alternative "zeta chain", "CD3 zeta" or "TCR zeta" refers to CD247. Swiss-Prot accession number P20963 provides an exemplary human CD3 zeta amino acid sequence. A "zeta-stimulated domain" or alternative "CD3 zeta-stimulated domain" or "TCR zeta-stimulated domain" has a CD3 zeta-stimulated domain or a variant thereof (eg, a mutation, eg, a point mutation, fragment, insertion or deletion). Molecules). In some embodiments, the cytoplasmic domain of the zeta comprises residues 52-164 of GenBank Accession No. BAG3666.1 or variants thereof (eg, molecules with mutations such as point mutations, fragments, insertions or deletions). .. In some embodiments, the "zeta-stimulated domain" or "CD3 zeta-stimulated domain" is the sequence provided as SEQ ID NO: 9 or 10 or a variant thereof (eg, mutation, eg, point mutation, fragment, insertion or deletion). (Molecules with).

The term "co-stimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a co-stimulatory ligand, thereby mediating a co-stimulatory response by the T cell, such as, but not limited to, proliferation. Co-stimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an efficient immune response. Co-stimulatory molecules include, but are not limited to, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrin, signaling lymphocyte activation molecules (SLAM proteins), activated NK cell receptors, etc. 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 (LIGHT), 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, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD2) (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP Includes ligands that specifically bind to -76, PAG / Cbp, CD19a, CD28-OX40, CD28-4-1BB and CD83.

The intracellular signaling domain of a co-stimulator refers to the intracellular portion of a co-stimulator molecule.

The intracellular signaling domain may include the entire intracellular portion of the molecule from which it is derived, or the entire natural intracellular signaling domain, or a functional fragment thereof.

The term "4-1BB" refers to CD137 or tumor necrosis factor receptor superfamily member 9. Swiss-Prot Accession No. P20963 provides an exemplary human 4-1BB amino acid sequence. "4-1BB co-stimulation domain" refers to a 4-1BB co-stimulation domain or a variant thereof (eg, a molecule having a mutation, such as a point mutation, fragment, insertion or deletion). In some embodiments, "4-1BB co-stimulation domain" refers to the sequence provided as SEQ ID NO: 7 or a variant thereof (eg, a molecule having a mutation, eg, a point mutation, fragment, insertion or deletion). ..

"Immune effector cell", as the term is used herein, refers to a cell involved in the promotion of an immune response, eg, 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, obesity cells and bone marrow-derived phagocytic cells. Can be mentioned.

"Immune effector function or immune effector response" as used herein refers to, for example, the function or response of an immune effector cell that enhances or promotes the immune attack of the target cell. For example, immune effector function or response refers to the properties of T cells or NK cells that promote the death or growth or growth inhibition of target cells. For T cells, primary and co-stimulation are examples of immune effector function or response.

The term "effector function" refers to the specialized function of a cell. For example, the effector function of T cells can be cytolytic activity or helper activity including the secretion of cytokines.

The term "encoding" is used as a template for the synthesis of other polymers and macromolecules in biological processes having either a defined nucleotide sequence (eg, rRNA, tRNA and mRNA) or a defined amino acid sequence. Refers to the unique properties of specific nucleotide sequences within a polynucleotide, such as genes, cDNAs or mRNAs, and the biological properties resulting thereof. Thus, a gene, cDNA or RNA encodes 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 and the non-coding strand used as a transcription template for the gene or cDNA, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in the sequence table, are proteins or other products of the cDNA of the gene. Can be referred to as coding.

Unless otherwise stated, "nucleotide sequences encoding amino acid sequences" are degenerate versions of each other and include all of the nucleotide sequences encoding the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA may also include introns, as long as the nucleotide sequence encoding the protein may contain one or more introns in any version.

The terms "effective amount" or "therapeutically effective amount" are used interchangeably herein, and a compound, formulation, material or composition as described herein achieves a particular biological result. Refers to an effective amount.

The term "endogenous" refers to any material derived from or within an organism, cell, tissue or system.

The term "exogenous" refers to any material introduced or produced outside an organism, cell, tissue or system.

The term "expression" refers to the transcription and / or translation of a particular nucleotide sequence. In some embodiments, expression comprises translating the mRNA introduced into the cell.

The term "transfer vector" refers to a composition comprising an isolated nucleic acid, which can be used for delivery of the isolated nucleic acid into a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphipathic compounds, plasmids and viruses. Thus, the term "transfer vector" includes self-replicating plasmids or viruses. The term should be construed to further include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids to cells, such as polylysine compounds, liposomes. Examples of virus transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.

The term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to an expression nucleotide sequence. The expression vector contains sufficient cis-acting elements for expression, and other expression elements can be supplied by the host cell or to the in vitro expression system. Expression vectors are known in the art including cosmids incorporating recombinant polynucleotides, plasmids (eg, contained in naked or liposomes) and viruses (eg, lentivirus, retrovirus, adenovirus and adeno-associated virus). All of them are included.

The term "lentivirus" refers to the genus of the family Retrovirus. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells, and lentiviruses can deliver large amounts of genetic information to the DNA of host cells, thus the gene delivery vector. It is one of the most efficient methods. HIV, SIV and FIV are all examples of lentiviruses.

The term "wrench viral vector" is particularly used in Milone et al. , Mol. The. 17 (8): A vector derived from at least a portion of the lentiviral genome, including the self-inactivating lentiviral vector as provided in 1453-1464 (2009). Other examples of clinically usable lentiviral vectors include, but are not limited to, for example, LENTIVECTOR® gene delivery technology from Oxford BioMedica, LENTIMAX ™ vector system from Lentien, and the like. Non-clinical type lentiviral vectors are also available and will be known to those of skill in the art.

The term "homologous" or "identity" refers to subunit sequence identity between two polymer molecules, eg, 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 of the two molecules are occupied by the same monomeric subunit, for example, if the position of each of the two DNA molecules is occupied by adenine, they are homologous or identical at that position. be. Homology between two sequences is a direct function of the number of matching or homologous positions. For example, if half of the positions in the two sequences (eg, five positions in a polymer of 10 subunit length) are homologous, then the two sequences are 50% homologous. If 90% of the positions (eg, 9 out of 10) are matched or homologous, then the two sequences are 90% homologous.

A "humanized" form of a non-human (eg, mouse) antibody is a chimeric immunoglobulin, immunoglobulin chain or fragment thereof (Fv, Fab, Fab', F (ab) containing the smallest sequence derived from a non-human immunoglobulin. ') 2 or other antigen-binding partial sequences, etc.). In most cases, in humanized antibodies and antibody fragments thereof, residues from the complementarity determining regions (CDRs) of the recipient in human immunoglobulins (recipient antibodies or antibody fragments) have the desired specificity, affinity and capacity. It has been replaced with residues from CDRs of non-human species (donor antibodies) such as mice, rats or rabbits. In some examples, the Fv framework region (FR) residue of human immunoglobulin is replaced with the corresponding non-human residue. In addition, humanized antibodies / antibody fragments can contain residues that are not found in either 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. In general, a humanized antibody or antibody fragment thereof will contain substantially all of at least one and typically two variable domains, of which all or substantially all of the CDR regions are non-human immunoglobulins. And all or most of the FR regions are of human immunoglobulin sequences. The humanized antibody or antibody fragment can also include at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin Fc. 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: 593-569, 1992.

"Completely human" refers to an immunoglobulin such as an antibody or antibody fragment that is entirely human in origin or has the same amino acid sequence as an antibody or immunoglobulin in human form.

The term "isolated" means that it has been modified or removed from its natural state. For example, a nucleic acid or peptide that is naturally occurring in a living animal is not "isolated", but the same nucleic acid or peptide that is partially or completely separated from the coexisting material in its natural state. "Isolated". The isolated nucleic acid or protein can be present in a substantially purified form or can be present in a non-natural environment, such as a host cell.

In the context of the present invention, the following abbreviations are used for commonly present nucleobases. "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

The term "operably linked" or "transcriptional control" refers to a functional link that results in the expression of the latter between a regulatory sequence and a heterologous nucleic acid sequence. For example, when the first nucleic acid sequence is placed in a state functionally related to the second nucleic acid sequence, the first nucleic acid sequence is operably linked to the second nucleic acid sequence. For example, if the promoter affects the transcription or expression of the coding sequence, the promoter is operably linked to the coding sequence. The operably linked DNA sequences can be contiguous with each other and are within the same reading frame, for example if two protein coding regions need to be joined together.

The term "parenteral" administration of immunogenic compositions refers to, for example, subcutaneous (s.c.), intravenous (iv), intramuscular (im), or intrasternal injection, intratumor. , Or including infusion techniques.

The terms "nucleic acid," "nucleic acid molecule," "polynucleotide," or "polynucleotide molecule" refer to single-stranded or double-stranded forms of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and their polymers. .. Unless otherwise specified, the term includes nucleic acids containing known analogs of naturally occurring nucleotides that have binding properties similar to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. In some embodiments, the "nucleic acid", "nucleic acid molecule", "polynucleotide" or "polynucleotide molecule" comprises a nucleotide / nucleoside derivative or analog. Unless otherwise stated, a particular nucleic acid sequence is explicitly indicated as its conservatively modified variant (eg, degenerate codon substitution, eg conservative substitution), allele, ortholog, SNP and complementary sequence. Also implicitly includes the sequence. Specifically, degenerate codon substitutions, such as conservative substitutions, produce sequences in which the third position of one or more selected (or all) codons is replaced with a mixed base and / or 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)).

The terms "peptide", "polypeptide" and "protein" are used synonymously to refer to a compound composed of amino acid residues covalently linked by a peptide bond. The protein or peptide must contain at least two amino acids and there is no limit to the maximum number of amino acids that can contain a sequence of proteins or sequences of peptides. Polypeptides include any peptide or protein containing two or more amino acids linked together by peptide bonds. As used herein, the term refers to short chains, commonly referred to in the art as, for example, peptides, oligopeptides and oligomers, and many types, commonly referred to in the art as proteins. Refers to both with long chains. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, etc. Similars, fusion proteins in particular are included. Polypeptides include natural peptides, recombinant peptides or combinations thereof.

The term "promoter" refers to a DNA sequence recognized by a cellular or introduced synthetic mechanism that is required to initiate specific transcription of a polynucleotide sequence.

The term "promoter / regulatory sequence" refers to a nucleic acid sequence required for the expression of a gene product operably linked to that promoter / regulatory sequence. In some examples, this sequence may be a core promoter sequence, in other examples, this sequence may also contain enhancer sequences and other regulatory elements required for expression of the gene product. The promoter / regulatory sequence can be, for example, one that expresses the gene product in a tissue-specific manner.

The term "constitutive" promoter, when operably linked to a polynucleotide encoding or designating a gene product, causes the production of the gene product in the cell under almost or all physiological conditions of the cell. Refers to a nucleotide sequence.

The term "inducible" promoter, when operably linked to a polynucleotide encoding or designating a gene product, is in the cell only if the inducer substantially corresponding to the promoter is present in the cell. Refers to the nucleotide sequence that results in the production of a gene product.

The term "tissue-specific" promoter is used only if the cell is a tissue-type cell corresponding to the promoter when it encodes a gene or is operably linked to a polynucleotide designated by it. Refers to a nucleotide sequence that results in the production of a gene product in a cell.

The terms "cancer-related antigen" or "tumor antigen" are synonymous with molecules (typically proteins, carbohydrates or) expressed as fragments (eg, MHC / peptides) completely or instead on the surface of cancer cells. Lipid), which refers to a molecule useful for preferential targeting of pharmacological agents to cancer cells. In some embodiments, the tumor antigen is a marker expressed by both normal cells and cancer cells, such as a series marker, such as CD19 on B cells. In some embodiments, the tumor antigen is overexpressed in cancer cells as compared to normal cells (eg, 1-fold overexpression, 2-fold overexpression, or 3-fold overexpression as compared to normal cells. It is a cell surface molecule (beyond that). In some embodiments, the tumor antigen is a cell surface molecule that is improperly synthesized in cancer cells, eg, a molecule that contains a deletion, addition, or mutation as compared to a molecule expressed in normal cells. In some embodiments, the tumor antigen will be expressed entirely or instead as a fragment (eg, MHC / peptide) only on the cell surface of the cancer cell and will not be synthesized or expressed on the surface of normal cells. In some embodiments, the CAR of the invention comprises a CAR comprising an antigen binding domain (eg, an antibody or antibody fragment) that binds to an MHC-presenting peptide. Normally, peptides derived from endogenous proteins fit into the pockets of major histocompatibility complex (MHC) class I molecules and are recognized by the T cell receptor (TCR) on CD8 + T lymphocytes. The MHC class I complex is constitutively expressed by all nucleated cells. In cancer, virus-specific and / or tumor-specific peptide / MHC complexes are a unique class of cell surface targets for immunotherapy. TCR-like antibodies targeting peptides derived from viruses or tumor antigens in the context of human leukocyte antigen (HLA) -A1 or HLA-A2 have been described (eg, 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 (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 a library, such as the human scFv phage display library.

The terms "tumor-supporting antigen" or "cancer-supporting antigen" are interchangeably not cancerous in their own right, but support cancer cells, eg, by promoting proliferation or survival, eg resistance to immune cells. Refers to a molecule (typically a protein, carbohydrate or lipid) expressed on the surface of a cell. Exemplary cells of this type include stromal cells and myeloid-derived suppressor cells (MDSCs). The tumor-supporting antigen itself does not have to play a role in supporting tumor cells as long as the antigen is present on the cells that support the cancer cells.

The term "mobile polypeptide linker" or "linker", when used in the context of scFv, is used alone or in combination to link variable heavy chain regions and variable light chain regions together with glycine and / or. Refers to a peptide linker consisting of amino acids such as serine residues. In some embodiments, the mobile polypeptide linker is a Gly / Ser linker and the amino acid sequence (Gly-Gly-Gly-Ser) n (where n is a positive integer greater than or equal to 1 such as 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 another embodiment, the linker comprises multiple iterations of (Gly2Ser), (GlySer) or (Gly3Ser) (SEQ ID NO: 29). Also included within the scope of the invention are the linkers described in WO 2012/138475 (incorporated herein by reference).

As used herein, a 5'cap (also referred to as an RNA cap, RNA7-methylguanosine cap or RNA ^m7G cap) is "before" or 5'of eukaryotic messenger RNA immediately after transcription initiation. It is a modified guanine nucleotide added to the terminal. The 5'cap consists of a terminal group linked to the first transcribed nucleotide. Its presence is important for ribosome recognition and protection from RNases. Cap addition is associated with transcription and occurs simultaneously, with each affecting the other. Immediately after the start of transcription, the cap synthesis complex associated with RNA polymerase binds to the 5'end of the synthesized mRNA. This enzyme complex catalyzes the chemical reactions required for mRNA capping. The synthesis proceeds as a multi-step biochemical reaction. The capping moiety can be modified to regulate functions such as its stability or translation efficiency of mRNA.

As used herein, "in vitro transcribed RNA" refers to RNA synthesized in vitro, preferably mRNA. Generally, in vitro transcribed RNA is made from an in vitro transcribed vector. The in vitro transcription vector contains a template used to generate in vitro transcribed RNA.

As used herein, "poly (A)" is a series of adenosine that is added to mRNA by polyadenylation. In a preferred embodiment of the construct for transient expression, poly A is 50-5000 (SEQ ID NO: 30), preferably more than 64, more preferably more than 100, most preferably more than 300 or 400. The poly (A) sequence can be chemically or enzymatically modified to regulate mRNA function such as localization, stability or translation efficiency.

As used herein, "polyadenylation" refers to the covalent binding of a polyadenylyl moiety or a modified variant thereof to a messenger RNA molecule. In eukaryotes, most messenger RNA (mRNA) molecules are polyadenylated at the 3'end. The 3'poly (A) tail is a long sequence (often hundreds) of adenine nucleotides added to the pre-mRNA by the action of the enzyme polyadenylate polymerase. In higher eukaryotes, the poly (A) tail is added to a transcript containing a specific sequence, a polyadenylation signal. The poly (A) tail and the proteins associated with it help protect the mRNA from degradation by exonucleases. Polyadenylation is also important for transcription termination, transnuclear translocation and translation of mRNA. Polyadenylation occurs in the nucleus immediately after transcription from DNA to RNA, but can also occur later in the cytoplasm. After transcription is terminated, the mRNA strand is cleaved by the action of the endonuclease complex associated with RNA polymerase. The cleavage site is usually characterized by the presence of the nucleotide 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.

As used herein, "transient" refers to the expression of a transgene that is not integrated over a period of hours, days or weeks, which period of expression is integrated into the genome in the host cell. Shorter than the gene expression period if or when contained in a stable plasmid replicon.

As used herein, the terms "treating," "treating," and "treating" refer to the administration of one or more therapies (eg, one or more therapeutic agents, such as CAR of the invention). Refers to the reduction or amelioration of progression, severity and / or duration of proliferative disorders resulting from or improvement of one or more symptoms (preferably one or more recognizable symptoms) of proliferative disorders. In a specific embodiment, the terms "treat", "treat" and "treating" are at least one measurable physical parameter of a proliferative disorder such as tumor growth that is not always recognizable to the patient. Refers to the improvement of. In other embodiments, the terms "treating," "treating," and "treating" are physical, eg, recognizable, recognizable symptom stabilization inhibitions, physiological, eg, physics. Refers to either or both inhibitions due to stabilization of the target parameter. In other embodiments, the terms "treating," "treating," and "treating" refer to reducing or stabilizing tumor size or cancerous cell count.

The term "signal transduction pathway" refers to the biochemical relationship between various signaling molecules that play a role in the transmission of signals from one part of a cell to another. The phrase "cell surface receptor" includes molecules and molecular complexes capable of receiving signals and transmitting signals across cell membranes.

The term "subject" is intended to include living organisms (eg, mammals, humans) capable of producing an immune response.

The term "substantially purified" cell refers to a cell that is essentially free of other cell types. Substantially purified cells also refer to cells that are isolated from other cell types with which they are normally associated in their naturally occurring state. In some examples, a substantially purified cell population refers to a homogeneous cell population. In another example, the term simply refers to cells that are isolated from the cells to which they are naturally associated in their natural state. In some embodiments, these cells are cultured in vitro. In other embodiments, these cells are not cultured in vitro.

The term "therapeutic" as used herein means treatment. Therapeutic effects are achieved by reducing, suppressing, ameliorating or eradicating the disease state.

As used herein, the term "prevention" means prevention of a disease or condition or prophylactic treatment against it.

In the context of the present invention, "tumor antigen", or "hyperproliferative disorder antigen", or "antigen associated with hyperproliferative disorder" refers to an antigen that is common to a particular hyperproliferative disorder. In certain embodiments, the overgrowth disorder antigens of the invention are primary or metastatic melanoma, thoracic adenoma, lymphoma, sarcoma, lung cancer, liver cancer, non-hodgkin lymphoma, hodgkin lymphoma, leukemia, uterine cancer, cervical cancer. , Bladder cancer, kidney cancer and breast cancer, adenocarcinoma such as prostate cancer (eg, castor-resistant or treatment-resistant prostate cancer or metastatic prostate cancer), ovarian cancer, pancreatic cancer, etc., or plasma cell proliferative disorders, such as Asymptomatic myeloma (smoldering multiple myeloma or painless myeloma), unclear monoclonal hypergamma globulinemia (MGUS), Waldenstrem macroglobulinemia, plasmacytoma (eg, traits) Cell overgrowth, solitary myeloma, solitary plasmacytoma, extramedullary plasmacytoma and multiple myeloma), systemic amyloid light chain amyloidosis and POEMS syndrome (Crow-Fukase syndrome, Takatsuki disease and PEP syndrome) (Also known as), but not limited to, derived from cancer.

The term "transfected," or "transformed," or "transduced" refers to the process of transferring or introducing an exogenous nucleic acid into a host cell. A "transfected", "transformed", or "transduced" cell is one that has been transfected, transformed or transduced with an exogenous nucleic acid. The cells include primary target cells and their progeny.

The term "specifically binds" is an antibody or ligand that recognizes and binds to a cognate binding partner (eg, a stimulating and / or co-stimulating molecule present on a T cell) protein present in a sample, but , Other molecules in the sample refer to antibodies or ligands that are substantially unrecognized or unbound.

As used herein, a "regulated chimeric antigen receptor (RCAR)", when located within an immune effector cell, cell specificity for target cells, typically cancer cells, and intracellular signal generation. A set of polypeptides conferred on, typically two polypeptides in the simplest embodiment. In some embodiments, the RCAR is derived from at least the extracellular antigen binding domain, the transmembrane domain, and the stimulatory and / or costimulatory molecule as defined herein in relation to the CAR molecule. Includes a cytoplasmic signaling domain (also referred to herein as the "intracellular signaling domain") that includes a functional signaling domain. In some embodiments, the RPAR polypeptide pairs are not contiguous with each other, eg, in different polypeptide chains. In some embodiments, the RCAR comprises a dimerization switch, which is capable of coupling polypeptides to each other in the presence of a dimerization molecule, eg, an antigen binding domain into the intracellular signaling domain. Can be coupled. In some embodiments, RCAR is expressed on cells as described herein (eg, immune effector cells), such as RPAR-expressing cells (also referred to herein as "RCARX cells"). In certain embodiments, the RCARX cells are T cells and are referred to as RCART cells. In certain embodiments, the RCARX cells are NK cells and are referred to as RCARN cells. RCAR can confer specificity and regulatory intracellular signal generation or proliferation on target cells, typically cancer cells, on RCAR-expressing cells, which can optimize the immune effector properties of RCAR-expressing cells. In embodiments, RCAR cells are at least partially dependent on the antigen binding domain in order to confer specificity for target cells containing the antigen bound by the antigen binding domain.

"Membrane anchor" or "membrane tethered domain" as used herein refers to a polypeptide or moiety, eg, a myristyl group, sufficient to anchor extracellular or intracellular domains to the cell membrane. ..

A "switch domain" is an entity that associates with another switch domain in the presence of a dimer, typically a polypeptide-based entity, when the term is used herein, eg, when referring to RPAR. Point to. The result of this meeting is a functional coupling between a first entity linked to, eg, fused, with a first switch domain and a second entity linked, eg, fused, to a second switch domain. The first and second switch domains are collectively referred to as dimerization switches. In embodiments, the first and second switch domains are identical to each other, eg, they are polypeptides having the same primary amino acid sequence and are collectively referred to as homodimerized switches. In embodiments, the first and second switch domains are different from each other, eg, they are polypeptides having different primary amino acid sequences and are collectively referred to as heterodimerization switches. In embodiments, the switch is intracellular. In embodiments, the switch is extracellular. In embodiments, the switch domain is a polypeptide-based entity, such as FKBP or FRB-based, and the dimer is a small molecule, such as Lapalog. In embodiments, the switch domain is a scFv that binds to a polypeptide-based entity, such as a myc peptide, and the dimerization molecule binds to a polypeptide, a fragment thereof, or a multimer of the polypeptide, such as one or more myc scFv. Is a myc ligand or a multimer of myc ligand. In embodiments, the switch domain is a polypeptide-based entity, such as a myc receptor, and the dimer is an antibody or fragment thereof, such as a myc antibody.

"Dimerization molecule" refers to a molecule that facilitates association between a first switch domain and a second switch domain, when the term is used herein, for example, with reference to RPAR. In embodiments, the dimerization molecule is not naturally present in the subject or is not present at a concentration that can result in significant dimerization. In embodiments, the dimerization molecule is a small molecule, such as rapamycin or rapalog, such as RAD001.

The term "low immunopotentiating dose" refers to mTOR activity when used in conjunction with an mTOR inhibitor, such as an allosteric mTOR inhibitor, such as RAD001 or rapamycin or a catalytic mTOR inhibitor, eg, as measured by inhibition of P70 S6 kinase activity. Refers to the dose of mTOR inhibitor that partially but partially inhibits. Methods for assessing mTOR activity, eg, by inhibition of P70 S6 kinase, are discussed herein. The dose is insufficient to result in complete immunosuppression, but sufficient to enhance the immune response. In certain embodiments, low immunopotentiating doses of mTOR inhibitors reduce the number of PD-1 positive T cells and / or increase the number of PD-1 negative T cells or PD-1 negative T cells / PD-1 positive. It results in an increase in the proportion of T cells. In certain embodiments, low immunopotentiating doses of mTOR inhibitors result in an increase in the number of naive T cells. In certain embodiments, a low immunopotentiating dose of an mTOR inhibitor results in one or more of the following:
For example, on memory T cells, eg, memory T cell precursors, the following markers: CD62L ^high , CD127 ^high , increased expression of one or more of CD27 ⁺ and BCL2,
For example, decreased expression of KLRG1 on memory T cells, such as memory T cell precursors, and memory T cell precursors, eg, the following characteristics: increased CD62L ^high , increased CD127 ^high , increased CD27 ⁺ , KLRG1 Increased number of cells with any one or combination of decreased and increased BCL2.
Here, any of the changes described above occur, for example, at least transiently when compared, for example, to an untreated subject.

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

"Relapsed" or "relapsed", as used herein, is a sign of a disease (eg, cancer) or a disease such as cancer after an improvement or response period, eg, after therapy, eg, pretreatment with cancer therapy. And refers to the recurrence or reappearance of symptoms. The initial response period may be accompanied by levels of cancer cells below a certain threshold, eg, below 20%, 1%, 10%, 5%, 4%, 3%, 2% or 1%. Reappearance may include levels of cancer cells above a certain threshold, eg, 20%, 1%, 10%, 5%, 4%, 3%, 2% or 1%. For example, in the context of B-ALL, for example, reappearance can include, for example, reappearance of blood, bone marrow (> 5%) or blasts at any extramedullary position after complete response. In this regard, a complete response may include <5% BM blasts. More generally, in certain embodiments, the response (eg, complete or partial response) may include the absence of detectable MRD (minimal residual disease). In certain embodiments, the initial response period is 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 It lasts for months or at least 1, 2, 3, 4 or 5 years.

Scope: Throughout the disclosure, various aspects of the invention may be presented in the form of a range. It should be understood that the description in the form of a range is for convenience and brevity only and should not be construed as a firm limitation of the scope of the invention. Therefore, the description of the range should be considered to have all possible subranges specifically disclosed as well as the individual numerical values within that range. For example, the description of the range such as 1 to 6 is included in the specifically disclosed partial range such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6 and the range thereof. It must be considered to have certain individual numbers, such as 1, 2, 2.7, 3, 4, 5, 5.3 and 6. As another example, ranges such as 95-99% identity include those with 95%, 96%, 97%, 98% or 99% identity and 96-99%, 96-98%. , 96-97%, 97-99%, 97-98% and 98-99% identity and the like. This applies regardless of the width of the range.

A "gene editing system", as the term is used herein, guides and implements modification, eg, deletion, of one or more nucleic acids at or near the site of genetic DNA targeted by said system. Refers to a system, eg, one or more molecules. Gene editing systems are well known in the art and are described more fully below.

As used herein, "combined" administration means delivering two (or more) different treatments to a subject during the process in which the subject is suffering from a disorder, eg, two or more treatments. Delivered after the subject has been diagnosed with the disorder and before the disorder is cured or eradicated or the procedure is discontinued for other reasons. In some embodiments, delivery of one treatment is still performed at the start of delivery of the second treatment, as there is an overlap of dosing periods. This may be referred to herein as "simultaneous" or "concurrent delivery." In other embodiments, delivery of one treatment ends before delivery of the other treatment begins. In some embodiments of any case, the treatment is more effective for combination administration. For example, the second treatment is seen in a second treatment that is more effective, eg, less equally effective, or the second treatment administers the second treatment in the absence of the first treatment. Symptoms are alleviated to a greater extent than sometimes seen, or similar situations are seen with the first treatment. In some embodiments, delivery is greater than the reduction of symptoms or other parameters associated with the disorder observed in the delivery of one treatment in the absence of the other treatment. The effect of the two treatments can be partially additive, perfective or greater than additive. Delivery may be such that the effect of the first treatment delivered is still detectable at the time of delivery of the second treatment.

Various aspects of the compositions and methods herein are described in more detail below. Further definitions are given throughout this specification.

Description A composition of a substance and a method of use for the treatment of diseases such as cancer using cells expressing the BCMA chimeric antigen receptor (CAR), such as CART-BCMA, are provided herein.

In one aspect, the invention provides cells engineered to express CAR (eg, immune effector cells such as T cells or NK cells), where CART cells (“CART”) or CAR NK cells are anti-cells. Exhibits tumor characteristics. In one embodiment, the cell is transformed with CAR and CAR is expressed on the cell surface. In some embodiments, cells (eg, immune effector cells, such as 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 (eg, immune effector cells such as T cells or NK cells) are transfected with a nucleic acid encoding CAR, such as mRNA, cDNA, DNA. In some such embodiments, cells may transiently express CAR.

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

Furthermore, the present invention provides BCMA CAR compositions and agents or methods for treating cancers involving cells or tissues expressing BCMA or any malignant tumors or autoimmune diseases, among other diseases.

In one aspect, the CAR of the present invention can be used for eradication of BCMA-expressing normal cells and is therefore applicable for use as a cell conditioning therapy prior to cell transplantation. In one embodiment, the BCMA-expressing normal cell is a BCMA-expressing normal stem cell, and the cell transplantation is a stem cell transplantation.

In one aspect, the invention provides cells (eg, T cells or NK cells) engineered to express a chimeric antigen receptor (CAR), where CAR T cells (“CART”) or CAR. NK cells exhibit antitumor properties. The preferred antigen is BCMA. In one embodiment, the antigen binding domain of CAR comprises a human anti-BCMA antibody fragment. In one embodiment, the antigen binding domain of CAR comprises a human anti-BCMA antibody fragment comprising scFv. Accordingly, the present invention provides a human anti-BCMA binding domain, BCMA-CAR engineered on cells such as T cells or NK cells and its use for adoption therapy.

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

Chimeric antigen receptor (CAR)
The present invention provides CARs (eg, CAR polypeptides) comprising anti-BCMA binding domains (eg, human anti-BCMA binding domains as described herein), transmembrane domains and intracellular signaling domains. Here, the anti-BCMA binding regions are the heavy chain complementarity determining regions 1 (HC CDR1) and the heavy chain complementarity determining regions 2 (HC) of any of the anti-BMCA heavy chain binding domain amino acid sequences listed in Tables 2 to 13. It contains CDR2) and heavy chain complementarity determining regions 3 (HC CDR3). The anti-BCMA binding regions of CAR are the light chain complementarity determining regions 1 (LC CDR1), light chain complementarity determining regions 2 (LC CDR2) of any of the anti-BMCA light chain binding domain amino acid sequences listed in Tables 2-13. And light chain complementarity determining regions 3 (LC CDR3) may be further included.

The invention encodes a CAR as described herein, eg, an anti-BCMA binding domain (eg, a human anti-BCMA binding domain as described herein), a transmembrane domain and intracellular signaling. Nucleic acid molecules encoding CARs comprising domains are also provided, wherein the anti-BCMA binding domains are HC CDR1, HC CDR2 and HC CDR3 of any of the anti-BMCA heavy chain binding domain amino acid sequences listed in Tables 2-13. including. In some embodiments, the anti-BCMA binding domain of the encoded CAR may further comprise LC CDR1, LC CDR2 and LC CDR3 of any of the anti-BMCA light chain binding domain amino acid sequences listed in Tables 2-13.

In one embodiment, the exemplary BCMA CAR construct comprises any leader sequence, extracellular antigen binding domain, hinge, transmembrane domain and intracellular stimulating domain. An exemplary reader sequence is provided as SEQ ID NO: 1. An exemplary nucleic acid sequence encoding a leader sequence is provided as SEQ ID NO: 12. Exemplary hinge / spacer sequences are provided as SEQ ID NOs: 2, 3, 4 or 5. An exemplary transmembrane domain sequence is provided as SEQ ID NO: 6. The sequence of the intracellular signaling domain of the exemplary 4-1BB protein is provided as SEQ ID NO: 7. An exemplary sequence of the intracellular signaling domain of 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 domain is in a separate polypeptide, eg, in a RPAR molecule as described herein.

The CAR construct may include a Gly / Ser linker having one or more of the following sequences: GGGGS (SEQ ID NO: 25); 1-6 "Gly Gly Gly Gly Ser" repeat units, eg, GGGGSGGGGS GGGGGSGGGSG 26); GGGGGSGGGGS GGGGGSGGGGS (SEQ ID NO: 27); GGGGGSGGGGS (SEQ ID NO: 28); GGGS (SEQ ID NO: 29); ).

In embodiments, the CAR construct is a sequence comprising a poly A sequence, eg 50-5000 or 100-5000 adenin (SEQ ID NOs: 30 and 33, respectively) (eg, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 34 or SEQ ID NO: 35). ) Or a sequence containing 50 to 5000 chimin (SEQ ID NO: 32) (eg, SEQ ID NO: 31, SEQ ID NO: 32). Alternatively, the CAR construct may include, for example, a linker containing the sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO: 43).

In certain embodiments, the full length BCMA CAR molecule is R1B6, R1F2, R1G5, PI61, B61-02, B61-10, Hy03 or Hy52 or substantially (eg, 95-99) provided in Tables 2-13. %) Contains an amino acid sequence of the same sequence or is encoded by its nucleotide sequence. In certain embodiments, the BCMA CAR molecule or anti-BCMA antigen binding domain is R1B6, R1F2, R1G5, PI61, B61-02, B61-10, Hy03 or Hy52 or substantially the same provided in Tables 2, 6 and 10. Contains the scFv amino acid sequence of the same sequence (eg, 95-99%). In certain embodiments, the BCMA CAR molecule or anti-BCMA antigen binding domain is R1B6, R1F2, R1G5, PI61, B61-02, B61-10, Hy03 or Hy52 or substantially the same provided in Tables 2, 6 and 10. Includes heavy and / or light chain variable regions of sequences that are identical (eg, 95-99%). In certain embodiments, the BCMA CAR molecule or anti-BCMA antigen binding domain is R1B6, R1F2, R1G5, PI61, B61-02, B61-10, Hy03 or Hy52 or substantially (then) provided in Tables 2-13. For example, one, two or three CDRs and / or light chain variable regions (eg, LCDR1, LCDR1, etc.) from heavy chain variable regions (eg, HCDR1, HCDR2 and / or HCDR3) of sequences that are 95-99% identical. Includes one, two or three CDRs from LCDR2 and / or LCDR3).

A non-limiting example sequence of the various components that can be part of the CAR molecule described herein is listed in Table 1. In the table, "aa" represents an amino acid and "na" represents a nucleic acid encoding the corresponding peptide.

CAR Antigen Binding Domain In one embodiment, a portion of CAR comprising an antigen binding domain comprises a tumor antigen, eg, an antigen binding domain that targets the tumor antigens described herein. In one aspect, the CAR of the invention comprises a binding domain that specifically binds to BCMA (eg, human BCMA).

Antigen-binding domains include, but are not limited to, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies and functional fragments thereof, such as, but not limited to, heavy chain variable domains (VHs), light chain variable domains ( Binds to antigens, including alternative scaffolds known in the art to function as antigen binding domains, such as single domain antibodies such as variable domains (VHH) of nanobodies derived from VL) and camels and recombinant fibronectin domains. It can be any protein.

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 a human antibody fragment. In some embodiments, the human anti-BCMA binding domain is one or more of the LC CDR1, LC CDR2 and LC CDR3 of the human anti-BCMA binding domains described herein (eg, Tables 2-13) (eg, 3). All) and / or include one or more (eg, all three) of the human anti-BCMA binding domains HC CDR1, HC CDR2 and HC CDR3 described herein (eg, Tables 2-13). In some embodiments, the human anti-BCMA binding domain is described in the human VL and / or herein (eg, Tables 2, 6 and 10) described herein (eg, Tables 2, 6 and 10). Includes the human VHs described. In some embodiments, the anti-BCMA binding domain is a scFv containing the VL and VH of the amino acid sequences of Tables 2, 6 and 10. In one embodiment, the anti-BCMA binding domain (eg, scFv) has at least one, two or three modifications (eg, substitutions, eg, conservative substitutions) of the amino acid sequences provided in Tables 2, 6 and 10. A VL; and / or a table comprising an amino acid sequence (eg, substitution, eg conservative substitution) not exceeding 30, 20 or 10 or a sequence having 95-99% identity with the amino acid sequences of Tables 2, 6 and 10. It has at least 1, 2 or 3 modifications (eg, substitutions, such as conservative substitutions) of the amino acid sequences provided in 2, 6 and 10, but modifications (eg, substitutions, such as conservative substitutions) of 30, 20 or 10. Includes VHs containing no excess of amino acid sequences or sequences having 95-99% identity with the amino acid sequences of Tables 2, 6 and 10.

In some embodiments, the human anti-BCMA binding domain comprises HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR3.

In certain embodiments, the CAR molecules described herein or the anti-BCMA binding domains described herein are:
(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 one of the following: :
(I) HC CDR1 of SEQ ID NO: 44, HC CDR2 of SEQ ID NO: 45 and HC CDR3 of SEQ ID NO: 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.
including.

In some embodiments, the CAR molecule described herein or the anti-BCMA binding domain described herein is.
(1) One, two or three light chain (LC) CDRs from one of the following:
(I) LC CDR1 of SEQ ID NO: 95, LC CDR2 of SEQ ID NO: 131 and LC CDR3 of SEQ ID NO: 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 one of the following:
(I) HC CDR1 of SEQ ID NO: 86, HC CDR2 of SEQ ID NO: 130 and HC CDR3 of SEQ ID NO: 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.
including.

In some embodiments, the CAR molecule described herein or the anti-BCMA binding domain described herein is.
(1) One, two or three light chain (LC) CDRs from one of the following:
(I) LC CDR1 of SEQ ID NO: 147, LC CDR2 of SEQ ID NO: 182 and LC CDR3 of SEQ ID NO: 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 one of the following:
(I) HC CDR1 of SEQ ID NO: 179, HC CDR2 of SEQ ID NO: 180 and HC CDR3 of SEQ ID NO: 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.
including.

In some embodiments, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR3 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 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 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 contain the amino acid sequences of SEQ ID NOs: 44, 45, 76, 54, 55 and 56, respectively.

In some embodiments, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR3 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 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 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 contain the amino acid sequences of SEQ ID NOs: 47, 48, 76, 57, 58 and 59, respectively.

In some embodiments, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR3 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 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 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 contain the amino acid sequences of SEQ ID NOs: 49, 50, 77, 60, 58 and 56, respectively.

In some embodiments, the human anti-BCMA binding domain comprises a scFv comprising VH (eg, VH described herein) and VL (eg, VL described herein). In some embodiments, VH is attached to VL via a linker, eg, a linker described herein, eg, a linker set forth in Table 1. In some embodiments, the human anti-BCMA binding domain is a (Gly ₄ -Ser) n linker (where n is 1, 2, 3, 4, 5 or 6, preferably 3 or 4) ( It contains SEQ ID NO: 26). The light chain variable region and heavy chain variable region of scFv can be, for example, any of the following directions: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.

In one embodiment, the anti-BCMA binding domain is a fragment, eg, a single chain variable fragment (scFv). In one embodiment, the anti-BCMA binding domain is Fv, Fab, (Fab') 2 or a bifunctional (eg, bispecific) hybrid antibody (eg, Lanzaveccia et al., Eur. J. Immunol. 17, 105 (eg,). 1987)). In one embodiment, the antibodies of the invention and fragments thereof bind to BCMA proteins with wild-type or enhanced affinity.

In one example, scFv can be produced by methods known in the art (eg, Bird et al., (1988) Science 242: 423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. . USA 85: 5879-5883). The ScFv molecule can be produced by linking the VH region and the VL region together using a mobile polypeptide linker. The scFv molecule comprises a linker having an optimum length and / or amino acid composition (eg, a Ser-Gly linker). The linker length can have a significant effect on how the variable regions of scFv are collapsed and interact. In fact, when short polypeptide linkers are used (eg, 5-10 amino acids), intrachain folding is blocked. Intrachain folding is also required for the two variable regions to unite to form a functional epitope binding site. Examples of linker orientations and sizes are described, for example, in Hollinger et al., Which is incorporated herein by reference. 1993 Proc Natl Acad. Sci. U. S. A. 90: 6444-6448, US Patent Application Publication No. 2005/0100543, 2005/0175606, 2007/0014794 and International Publication No. 2006/20258 and International Publication No. 2007 / Please refer to the pamphlet No. 024715.

The scFv is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, between the VL region and the VH region. It may contain a linker of amino acid residues 19, 20, 25, 30, 35, 40, 45, 50 or more. The linker sequence can include any naturally occurring amino acid. In one embodiment, the linker sequence comprises the amino acids glycine and serine. In another embodiment, the linker sequence comprises a series of glycine and serine iterations such as (Gly ₄ Ser) n, where n is a positive integer greater than or equal to 1 (SEQ ID NO: 25). In some embodiments, the linker can be (Gly ₄ Ser) ₄ (SEQ ID NO: 27) or (Gly ₄ Ser) ₃ (SEQ ID NO: 28). Variations in linker length can maintain or enhance activity and result in excellent efficacy in activity testing.

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 is one or more additional amino acids adjacent to the transmembrane region, eg, one or more amino acids associated with the extracellular region of the protein from which the transmembrane is derived (eg, 1, 2, 3 of the extracellular region). 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids) and / or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived (eg, of the intracellular region). It may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids). In one aspect, the transmembrane domain is one that binds to other domains of CAR. In one example, the transmembrane domain minimizes interaction with, for example, other members of the receptor complex so that such a domain avoids binding to the transmembrane domain of the same or different surface membrane proteins. Can be selected or modified by amino acid substitution as such. In one embodiment, the transmembrane domain can be homodimerized with another CAR on the surface of a CAR-expressing cell (eg, a CART cell). In different embodiments, the amino acid sequence of the transmembrane domain can be modified or substituted to minimize interaction with the binding domain of the naturally binding partner present in the same CAR expressing cell, eg, CART.

The transmembrane domain can be of natural or recombinant source origin. When the source is natural, the domain can be derived from any membrane binding or transmembrane protein. In one embodiment, the transmembrane domain can signal the intracellular domain whenever CAR binds to a target. Transmembrane domains that are particularly useful in the present invention are, for example, alpha chains, beta chains or zeta chains of T cell receptors, CD28, CD3 epsilon, CD45, CD4, CD5, CD8 (eg, CD8 alpha, CD8 beta), CD9. , CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154 may include at least a transmembrane region. In some embodiments, the transmembrane domain is at least a costimulatory molecule (eg, MHC class I molecule, TNF receptor protein, immunoglobulin-like protein, cytokine receptor, integrin, signaling lymphoid activation 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 (LIGHT), 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, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (TacT) , 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 ligands that specifically bind to CD83) can include transmembrane regions.

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

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

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

In one embodiment, the transmembrane domain can be recombinant, in which case it predominantly contains hydrophobic residues such as leucine and valine. In one embodiment, phenylalanine, tryptophan and valine triplets can be found at each end of the recombinant transmembrane domain.

Optionally, a short oligo or polypeptide linker 2-10 amino acids long may form a bond between the transmembrane domain of CAR and the cytoplasmic region. Glycine-serine doublets provide a particularly suitable linker. 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.

In one embodiment, the hinge or spacer comprises a KIR2DS2 hinge.

Cytoplasmic Domain The cytoplasmic domain or region of the CAR of the present invention comprises an intracellular signaling domain. The intracellular signaling domain is generally responsible for the activation of at least one of the normal effector functions of immune cells into which CAR has been introduced.

Examples of intracellular signaling domains used in the CARs of the invention are T cell receptor (TCR) and co-receptor cytoplasmic sequences that function in concert to initiate signal transduction after antigen receptor binding. Includes any derivative or variant and any recombinant sequence with the same functional capacity.

It is known that the signals produced by TCR alone are inadequate for complete activation of T cells and also require secondary and / or co-stimulatory signals. Thus, it can be said that T cell activation is mediated by two different classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary intracellular signaling domain) and secondary. Or those that act in an antigen-independent manner to provide a co-stimulatory signal (secondary cytoplasmic domain, eg, co-stimulatory domain).

The primary signaling domain regulates the primary activation of the TCR complex in either the stimulatory or inhibitory direction. Primary intracellular signaling domains that act in a stimulatory direction may include immunoreceptor tyrosine-based activation motifs or signaling motifs known as ITAM.

Examples of ITAM-containing primary intracellular signaling domains that are particularly useful in the present invention are TCR zetas, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (“ICOS”). Also known as), FcεRI, DAP10, DAP12 and CD66d. In some embodiments, the CAR of the invention comprises an intracellular signaling domain, eg, a primary signaling domain for the CD3 zeta.

In some embodiments, the primary signaling domain comprises a modified ITAM domain having modified (eg, increased or decreased) activity as compared to a native ITAM domain, such as a mutant ITAM domain. In some embodiments, the primary signaling domain comprises 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 comprises 1, 2, 3, 4 or more ITAM motifs.

Further examples of molecules containing the primary intracellular signaling domain that are particularly useful in the present invention include those of DAP10, DAP12 and CD32.

The intracellular signaling domain of CAR can include the primary signaling domain, eg, the CD3 zeta signaling domain, by itself, or any other desired intracellular signal useful in connection with CAR of the present invention. Can be combined with a transmission domain. For example, the intracellular signaling domain of CAR may include a primary signaling domain, such as a CD3 zeta chain moiety and a costimulatory signaling domain. The co-stimulatory signaling domain refers to the portion of CAR that contains the intracellular domain of the co-stimulatory molecule. A co-stimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand required for an efficient response of lymphocytes to an antigen. Examples of such molecules are MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrin, signaling lymphoid activation molecules (SLAM proteins), activated NK cell receptors, BTLA, etc. 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 (LIGHT), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R , 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 , CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD ), 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, CD83 and the like. For example, CD27 co-stimulation has been shown to enhance human CART cell proliferation, effector function and survival in vitro and 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 cytoplasmic portion of the CAR of the present invention can be linked to each other at random or in a particular order. Optionally, a short oligo or polypeptide linker, eg, 2-10 amino acids (eg, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids) long, forms a bond between the intracellular signaling sequences. Can be. In some embodiments, the glycine-serine doublet can be used as a suitable linker. In some embodiments, a single amino acid, such as alanine, glycine, can be used as a suitable linker.

In one embodiment, the intracellular signaling domain is designed to include two or more, eg, 2, 3, 4, 5 or more costimulatory signaling domains. In one embodiment, two or more, eg, 2, 3, 4, 5 or more costimulatory signaling domains are separated by a linker molecule, eg, a linker molecule described herein. In some embodiments, the intracellular signaling domain comprises two costimulatory signaling domains. In some embodiments, the linker molecule is a glycine residue. In some embodiments, the linker is an alanine residue.

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

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

In one embodiment, the intracellular is designed to include the signaling domain of the CD3 zeta and the signaling domain of CD28. In one embodiment, the signaling domain of CD28 comprises the amino acid sequence of SEQ ID NO: 36. In one embodiment, the signaling domain of CD28 is encoded by the nucleic acid sequence of SEQ ID NO: 37.

In one embodiment, the intracellular is designed to include a CD3 zeta signaling domain and an ICOS signaling domain. In one embodiment, the signaling domain of ICOS comprises 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.

Configuration of CAR 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 comprises 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 for the first epitope and a second immunoglobulin variable domain sequence having binding specificity for the second epitope. In one embodiment, the first and second epitopes are the same antigen, eg, the same protein (or 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, eg different proteins (or different subunits of a multimeric protein). In one embodiment, the bispecific antibody molecule is a heavy chain variable domain sequence and a light chain variable domain sequence having a binding specificity to the first epitope and a heavy chain variable domain sequence and a light chain having a binding specificity to the second epitope. Contains variable domain sequences. In one embodiment, the bispecific antibody molecule comprises a semi-antibody having binding specificity to the first epitope and a semi-antibody having binding specificity to the second epitope. In one embodiment, the bispecific antibody molecule comprises a half-antibody or fragment thereof having binding specificity to a first epitope and a half-antibody or fragment thereof having binding specificity to a second epitope. In one embodiment, the bispecific antibody molecule comprises a scFv or fragment thereof having binding specificity to a first epitope and a scFv or fragment thereof having binding specificity to a second epitope.

In certain embodiments, the CARs of the invention comprise an antigen binding domain that is a multispecific (eg, bispecific or trispecific) antibody molecule. Protocols for producing bispecific or heterodimer antibody molecules are known in the art, eg, the "nob-in-a-hole" approach described in US Pat. No. 5,731,168; eg, International Publication No. 1. Electrostatic Steering Fc Pair Formation as described in 09/089004, International Publication 06/106905 and International Publication 2010/129304; eg, as described in International Publication 07/110205. Anti-chain exchange operation domain (SEED) heterodimer formation; for example, Fab arm exchange as described in International Publication No. 08/119353, International Publication No. 2011/131746 and International Publication No. 2013/060867. Antibody cross-linking for producing bispecific structures using, for example, heterobifunctional reagents having, for example, amine-reactive groups and sulfhydryl-reactive groups, as described in US Pat. No. 4,433,059. Dual antibody conjugates by; for example, half-antibodies (heavy-light chain pairs or or) from different antibodies that undergo a cycle of reduction and oxidation of the disulfide bond between the two chains, as described in US Pat. No. 4,444,878. Bispecific antibody determinants produced by recombinant Fab); eg, a trifunctional antibody, a 3Fab'fragment crosslinked with a sulfhydryl reactive group, as described in US Pat. No. 5,273,743; eg, a US patent. A pair of scFv cross-linked by a biosynthetic binding protein such as a C-terminal tail, preferably a disulfide or amine-reactive chemical cross-linking, as described in No. 5534254; eg, described in US Pat. No. 5,582,996. Fab fragments with different binding properties dimerized by leucine zippers (eg, c-fos and c-jun), such as, having a bifunctional antibody, eg, a substituted constant domain; eg, US patent. Bispecific and oligospecific monovalent and oligovalent receptors, such as the CH1 region of one antibody and the VH region of an antibody typically with a light chain, as described in No. 5591828. VH-CH1 region of two antibodies (two Fab fragments) linked via a polypeptide spacer between; for example, a bispecific DNA-antibody conjugate, eg, two, as described in US Pat. No. 5,635,602. Antibodies or F that have passed through the DNA of a double-strand section Cross-linking of ab fragments; expression comprising two scFvs, including, for example, a bispecific fusion protein, eg, a hydrophilic spiral peptide linker, comprising a complete constant region, as described in US Pat. No. 5,637,481. Constructs; eg, binding regions for polyvalent and multispecific binding proteins, such as the first domain having the binding region for the Ig heavy chain variable region and the Ig light chain variable region, as described in US Pat. No. 5,83,242. A dimer of a polypeptide that has a second domain and is commonly referred to as a bispecific antibody (including higher-order structures for making bispecific, trispecific, and tetraspecific molecules); eg, Linked VL chains and ligated VL chains further bound to antibody hinge and CH3 regions with peptide spacers that can be dimerized to form bispecific / polyvalent molecules, as described in US Pat. No. 5,387,821. Minibody constructs with VH chains; linked with or without a short peptide linker (eg, 5 or 10 amino acids) in either direction capable of forming a dimer to form a bispecific antibody. VH and VL domains; eg, trimmers and tetramers as described in US Pat. No. 5,840,494; eg, a series of FVs (or scFv) as described in US Pat. No. 5,64019. A series of VH domains (or VL domains in family members) linked by peptide binding to a crosslinkable group at the C-terminal, further linked to the VL domain so as to form; and described, for example, in US Pat. No. 5,869,620. Both scFV or bispecific antibody forms, such as, are adapted to polyvalent structures by non-covalent or chemical cross-linking, eg, to form homobivalent, heterodivalent, trivalent and tetravalent structures. Examples include, but are not limited to, single-stranded conjugated polypeptides having both a VH domain and a VL domain linked by a peptide linker. Further examples of multispecific and bispecific molecules and their preparation are, for example, US Pat. No. 5,910,573, US Pat. No. 5,932,448, US Pat. No. 5,959,083, US Pat. No. 5,989830. No., US Pat. No. 6,05079, US Pat. No. 6,239,259, US Pat. No. 6,294,353, US Pat. No. 6,333,396, US Pat. No. 6,476,198, US Pat. No. 6,51163 , U.S. Pat. No. 6,670,453, U.S. Pat. No. 6,743896, U.S. Pat. No. 6,809,185, U.S. Pat. No. 6,833,441, U.S. Pat. U.S. Patent No. 7521056, U.S. Patent No. 7527787, U.S. Patent No. 7534866, U.S. Patent No. 7612181, U.S. Patent Application Publication No. 2002004587A1, U.S. Patent Application Publication No. 2002076406A1 , U.S. Patent Application Publication No. 20020133345A1, U.S. Patent Application Publication No. 2003207346A1, U.S. Patent Application Publication No. 200321178A1, U.S. Patent Application Publication No. 2004219643A1, U.S. Patent Application Publication No. 2002420388A1 , US Patent Application Publication No. 2004424847A1, US Patent Application Publication No. 2005003403A1, US Patent Application Publication No. 2005004352A1, US Patent Application Publication No. 2005069552A1, US Patent Application Publication No. 2005079170A1 , US Patent Application Publication No. 2005005433A1, US Patent Application Publication No. 200513649A1, US Patent Application Publication No. 2005136501A1, US Patent Application Publication No. 2005163782A1, US Patent Application Publication No. 2005266425A1. , US Patent Application Publication No. 2006083747A1, US Patent Application Publication No. 2006120960A1, US Patent Application Publication No. 2006204493A1, US Patent Application Publication No. 2006263637A1, US Patent Application Publication No. 2007004909A1. Book, U.S. Patent Application Publication No. 2007087381A1, U.S. Patent Application Publication No. 2007128150A1, U.S.A. Japanese Patent Application Publication No. 200714149A1, US Patent Application Publication No. 20071754901A1, US Patent Application Publication No. 2007274985A1, US Patent Application Publication No. 2008050370A1, US Patent Application Publication No. 2008069820A1, US Patent Application Publication No. 2008152645A1, US Patent Application Publication No. 2008171855A1, US Patent Application Publication No. 2008241884A1, US Patent Application Publication No. 2008254512A1, US Patent Application Publication No. 200826038A1, US Patent Application Publication No. 2009130106A1, US Patent Application Publication No. 2009448905A1, US Patent Application Publication No. 2009155275A1, US Patent Application Publication No. 2009162359A1, US Patent Application Publication No. 2009162360A1, US Patent Application Publication No. 2009175851A1, US Patent Application Publication No. 2009175867A1, US Patent Application Publication No. 20095232811A1, US Patent Application Publication No. 2009234105A1, US Patent Application Publication No. 2009263392A1, US Patent Application Publication No. 2009274649A1, European Patent No. 346087A2, International Publication No. 0006605A2, International Publication No. 020762635A2, International Publication No. 040810551A1, International Publication No. 06020258A2, International Publication No. 2007044887A2 No. pamphlet, International Publication No. 2007095338A2 Pamphlet, International Publication No. 2007137760A2 Pamphlet, International Publication No. 2008119353A1 Pamphlet, International Publication No. 2009021754A2 Pamphlet, International Publication No. 2009068630A1 Pamphlet, International Publication No. 9103493A1 Pamphlet, International Publication No. 9323537A1 It can be found in the pamphlet, International Publication No. 9409131A1, International Publication No. 9412625A2, International Publication No. 95099717A1, International Publication No. 9637621A2, and International Publication No. 9964460A1. The contents of the application cited above are incorporated herein by reference in their entirety.

Within each antibody or antibody fragment (eg, scFv) of the bispecific antibody molecule, the VH can be upstream or downstream of the VL. In one embodiment, an upstream antibody or antibody fragment (eg, scFv) is placed with its VH (VH ₁ ) upstream of its VL (VL ₁ ) and a downstream antibody or antibody fragment (eg, scFv) is located in its VL. It is located upstream of (VL ₂ ) with its VH (VH ₂ ) so that the overall bispecific antibody molecule has the configuration VH ₁ -VL ₁ -VL ₂ -VH ₂ . In other embodiments, the upstream antibody or antibody fragment (eg, scFv) is placed with its VL (VL ₁ ) upstream of its VH (VH ₁ ) and the downstream antibody or antibody fragment (eg, scFv) is its. It is located upstream of VH (VH ₂ ) with its VL (VL ₂ ) so that the overall bispecific antibody molecule has the arrangement VL ₁ -VH ₁ -VH ₂ -VL ₂ . Optionally, the linker may be between two antibodies or antibody fragments (eg, scFv), eg, between VL ₁ and VL ₂ or constructs when the construct is sequenced as VH ₁ -VL ₁ -VL ₂ -VH ₂ . Is placed between VH ₁ and VH ₂ when is placed as VL ₁ -VH ₁ -VH ₂ -VL ₂ . The linker may be a linker described herein, eg, a (Gly ₄ -Ser) n linker (SEQ ID NO: 26) (where n is 1, 2, 3, 4, 5 or 6, preferably 4). Can be. In general, the linker between two scFvs must be long enough to avoid mismatching between the domains of the two scFvs. Optionally, the linker is placed between the VL and VH of the first scFv. Optionally, the linker is placed between the VL and VH of the second scFv. In constructs with multiple linkers, any two or more of the linkers may be the same or different. Thus, in one embodiment, the bispecific CAR comprises VL, VH and optionally one or more linkers in an arrangement as described herein.

In one embodiment, the bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence, eg scFv, which has binding specificity to BCMA, eg, as described herein, eg, table. A second immunoglobulin variable domain that contains scFv as described in 2, 6 and 10 or has binding specificity for light chain CDRs and / or heavy chain CDRs and second epitopes from BCMA scFv described herein. Includes sequences in different antigens. In some embodiments, the second immunoglobulin variable domain sequence has binding specificity for an antigen expressed on AML cells, such as an antigen other than BCMA. For example, the second immunoglobulin variable domain sequence has binding specificity for CD123. As another example, the second immunoglobulin variable domain sequence has binding specificity for CLL-1. As another example, the second immunoglobulin variable domain sequence has binding specificity for CD34. As another example, the second immunoglobulin variable domain sequence has binding specificity for FLT3. For example, the second immunoglobulin variable domain sequence has binding specificity for folic acid receptor β. In some embodiments, the second immunoglobulin variable domain sequence is specific for binding to an antigen expressed on B cells, such as CD10, CD19, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b or CD79a. Has sex.

Chimera TCR
In one embodiment, the T cell receptor (". It can be transplanted into one or more constant domains of the TCR ") chain, such as the TCR alpha or TCR beta chain. Without being bound by theory, chimeric TCRs are thought to signal via the TCR complex by antigen binding. For example, BCMA scFv as disclosed herein can be transplanted into a constant domain of a TCR chain, such as a TCR alpha chain and / or a TCR beta chain, such as at least an extracellular constant domain, a transmembrane domain and a cytoplasmic domain. As another example, a BCMA antibody fragment, eg, a VL domain as described herein, can be transplanted into the constant domain of the TCR alpha chain, and a BCMA antibody fragment, eg, a VH domain as described herein, is TCR. It can be transplanted into the constant domain of the beta chain (or instead the VL domain can be transplanted into the constant domain of the TCR beta chain and the VH domain can be transplanted into the TCR alpha chain). As another example, the CDRs of an anti-BCMA antibody or antibody fragment, eg, the CDRs of an anti-BCMA antibody or antibody fragment as set forth in Tables 2-13, are TCRs to create a chimeric TCR that specifically binds to BCMA. It can be transplanted into alpha and / or beta chains. For example, the LCDR disclosed herein can be transplanted into the variable domain of the TCR alpha chain, the HCDR disclosed herein can be transplanted into the variable domain of the TCR beta chain, and vice versa. Such chimeric TCRs can be produced by methods known in the art (eg, Willemsen RA et al, Gene Therapy 2000; 7: 1369-1377; Zhang T et al, Cancer Gene Ther 2004; 11: 487- 496; Agen et al, Gene Ther. 2012 Apr; 19 (4): 365-74).

Further Embodiment In one embodiment, the CAR-expressing cells described herein are a second CAR, eg, the same target (BCMA) or a different target (eg, CD19, CD20 or CS-1 or other multiple myeloma). Further comprising a different antigen binding domain of, for example, a second CAR to a target such as Kappa light chain, CD138, Lewis Y antigen or CD38 (Garfall et al., Discovery Medicine, 2014, 17 (91): 37-46)). obtain. In some embodiments, CAR-expressing cells target a first antigen and include a first CAR and a first intracellular signaling domain having a costimulatory signaling domain but no primary signaling domain. Includes a second CAR that targets two different antigens and contains an intracellular signaling domain that has a primary signaling domain but no costimulatory signaling domain. Although not bound by theory, the second of the co-stimulatory signaling domains to the first CAR, such as 4-1BB, CD28, CD27 ICOS or OX-40 and the primary signaling domain, such as the CD3 zeta. Placement in CAR may limit CAR activity to cells in which both targets are expressed. In some embodiments, the CAR expressing cell is a first BCMA CAR containing a BCMA binding domain, a transmembrane domain and a costimulatory domain and an antigen other than BCMA (eg, an antigen expressed on leukemia or lymphoma cells, eg CD19. , CD20, CS-1, Kappa light chain, CD139, Lewis Y antigen or CD38), including a second CAR containing an antigen binding domain, a transmembrane domain and a primary signaling domain. In another embodiment, the CAR expressing cell is a first BCMA CAR containing a BCMA binding domain, a transmembrane domain and a primary signaling domain and an antigen other than BCMA (eg, an antigen expressed on leukemia or lymphoma cells, eg CD19. , CD20, CS-1, Kappa light chain, CD139, Lewis Y antigen or CD38) and comprises a second CAR containing an antigen binding domain, a transmembrane domain and a costimulatory signaling domain for the antigen. In some embodiments, CAR-expressing cells include BCMA CARs as described herein and CARs targeting CD19 (CD19 CARs).

In some embodiments, CAR expressing cells include BCMA CAR and inhibitory CAR described herein. In some embodiments, the inhibitory CAR comprises an antigen binding domain that binds to an antigen that is found in normal cells but not in cancer cells. In some embodiments, the inhibitory CAR comprises an antigen binding domain, a transmembrane domain and an intracellular domain of the inhibitory molecule. For example, the intracellular domains of inhibitory CAR are PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (eg, 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. It can be the intracellular domain of beta.

In some embodiments, when CAR-expressing cells contain two or more different CARs, the antigen-binding domains of the various CARs are such that the antigen-binding domains do not interact with each other. For example, cells expressing the first and second CARs do not form a binding with the antigen binding domain of the second CAR, eg, the antigen binding domain of the second CAR, which is VHH, eg, a fragment, eg, scFv. It may have one CAR antigen binding domain.

In one embodiment, the antigen binding domain comprises a single domain antigen binding (SDAB) molecule, comprising a molecule whose complementary determination region is part of a single domain polypeptide. Examples include heavy chain variable domains, binding molecules that naturally lack a light chain, single domains from conventional 4-chain antibodies, engineered domains and single domain scaffolds other than those derived from antibodies. Not limited. The SDAB molecule can be a single domain molecule in the art or in the future. SDAB molecules can be derived from any species including, but not limited to, mice, humans, camels, llamas, lampreys, fish, sharks, goats, rabbits and cows. The term also includes naturally occurring single domain antibody molecules from species other than Camelidae and sharks.

In one embodiment, the SDAB molecule can be derived from a variable region of immunoglobulin found in fish, such as, for example, from 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 NAR (“IgNAR”) are described in International Publication No. 03/014161 Pamphlet and Streltsov (2005) Protein Sci. 14: 2901-2909.

According to another aspect, the SDAB molecule is a naturally occurring single domain antigen binding molecule known as a heavy chain lacking a light chain. Such single domain molecules are described, for example, in International Publication No. 9404678 and Hamers-Casterman, C.I. et al. (1993) Nature 363: 446-448. For clarity, this variable domain from a heavy chain molecule that naturally lacks a light chain is known here as VHH or Nanobody to distinguish it from the conventional VH of 4-chain immunoglobulins. Such VHH molecules can be derived from Camelidaceae species such as camelids, llamas, dromedaries, alpaca and guanaco. Other species than Camelidae can naturally produce heavy chain molecules lacking a light chain, such VHHs are within the scope of the invention.

SDAB molecules can be recombinant, CDR transplanted, humanized, camelized, deimmunized and / or produced in vitro (eg, selected by phage display).

Cells with multiple chimeric membrane-embedded receptors containing antigen-binding domains that interact between the antigen-binding domains of the receptor, for example, to prevent one or more of the antigen-binding domains from binding to their cognate antigens. It also turned out to be potentially undesirable. Accordingly, disclosed herein are cells with first and second non-naturally occurring chimeric membrane-embedded receptors containing antigen-binding domains that minimize such interactions. Also disclosed herein are nucleic acids encoding first and second non-naturally occurring chimeric membrane-embedded receptors, including antigen-binding domains that minimize such interactions, as well as such. A method for producing and using cells and nucleic acids. In one embodiment, one of the antigen-binding domains of the first and second non-naturally occurring chimeric membrane-embedded receptors comprises scFv and the other is a single VH domain, eg, camels, sharks or eels. Includes a single VH domain or a single VH domain from a human or mouse sequence.

In one embodiment, the invention comprises a first and second CAR, wherein one of the first CAR and the second CAR antigen-binding domain comprises a variable light domain and a variable heavy domain. do not have. In one embodiment, one antigen-binding domain of the first CAR and the second CAR is scFv and the other is not scFv. In one embodiment, one of the first CAR and the second CAR antigen-binding domain is a single VH domain, eg, a single VH domain from a camelid, shark or lamprey, or a single VH from a human or mouse sequence. Includes domain. In one embodiment, one of the antigen binding domains of the first CAR and the second CAR comprises Nanobodies. In one embodiment, the antigen binding domain of one of the first CAR and the second CAR comprises a Camelid VHH domain.

In one embodiment, one antigen-binding domain of the first CAR and the second CAR comprises scFv and the other is a single VH domain, such as a camelid, shark or lamprey single VH domain or human or Contains a single VH domain from the mouse sequence. In one embodiment, one antigen binding domain of the first CAR and the second CAR comprises scFv and the other comprises Nanobodies. In one embodiment, one of the first CAR and the second CAR antigen-binding domain comprises scFv and the other comprises a Camelid VHH domain.

In one embodiment, when present on the surface of a cell, the binding of the antigen-binding domain of the first CAR to its cognate 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 cognate antigen in the presence of the second CAR is the antigen binding of the first CAR in the absence of the second CAR. 85%, 90%, 95%, 96%, 97%, 98% or 99% of the binding of the domain to its cognate antigen.

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

In another embodiment, the CAR-expressing cells described herein can further express another agent, eg, an agent that enhances the activity of the CAR-expressing cells. For example, in some embodiments, the agent can be an agent that inhibits an inhibitory molecule, eg, an agent described herein. Inhibitors, such as PD1, can reduce the ability of CAR-expressing cells to initiate an immune effector response. Examples of inhibitory molecules are PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (eg 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 that inhibits the inhibitory molecule is a second polypeptide that provides a positive signal to the cell, eg, a first polypeptide bound to the intracellular signaling domain described herein. For example, it contains an inhibitory molecule. In some embodiments, the agents are, for example, PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (eg, 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 A first polypeptide of an inhibitory molecule, such as TGFβ or a fragment thereof (eg, at least a portion of any of these extracellular domains) and the intracellular signaling domain described herein (eg, co-stimulation). A second, which is a domain (eg, eg 41BB, CD27, ICOS or CD28 as described herein) and / or a primary signaling domain (eg, including the CD3 zeta signaling domain described herein). Includes Polypeptides. In some embodiments, the agent is a first polypeptide of PD1 or a fragment thereof (eg, at least a portion of the extracellular domain of PD1) as well as the intracellular signaling domain described herein (eg, a portion of the extracellular domain of PD1). For example, it comprises a second polypeptide of the CD28 signaling domain described herein and / or the CD3 zeta signaling domain described herein). In embodiments, the CAR-expressing cells described herein. For example, the switch costimulatory receptors as described in WO 2013/019615, which is incorporated herein by reference in its entirety. PD1 also includes CD28, CTLA-4, ICOS and BTLA. An inhibitory member of the CD28 family of receptors, PD-1 is expressed on activated B cells, T cells and bone marrow cells (Agata et al. 1996 Int. Immunol 8: 765-75). Against PD1. Two ligands, 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; Ratchman et al. 2001 Nat Immunol 2: 261-8; Carter et al. 2002 Eur J Immunol ol 32: 634-43). PD-L1 is abundant in human cancer (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 Res10. : 5094). Immunosuppression can be reversed by inhibiting the local interaction between PD1 and PD-L1.

In some embodiments, agents comprising an inhibitory molecule, eg, the extracellular domain (ECD) of Programmed Cell Death 1 (PD1), can be fused to the transmembrane domain and the intracellular signaling domains such as 41BB and the CD3 zeta (the present specification). In the book, it is called PD1 CAR). In some embodiments, PD1 CAR improves the persistence of CAR-expressing cells, such as T cells or NK cells, when used in combination with the BCMA CARs described herein. In some embodiments, the CAR is a PD1 CAR comprising the extracellular domain of PD1 underlined in SEQ ID NO: 24. In some embodiments, PD1 CAR comprises the amino acid sequence of SEQ ID NO: 24.

In some embodiments, PD1 CAR comprises the following amino acid sequence (SEQ ID NO: 22).

In some embodiments, the agent comprises a PD1 CAR, eg, a nucleic acid sequence encoding the PD1 CAR described herein. In some embodiments, the nucleic acid sequence of PD1 CAR is provided as SEQ ID NO: 23 and PD1 ECD is underlined.

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

In another embodiment, the invention provides a population of cells, wherein at least one cell in the population expresses CAR having the anti-BCMA domain described herein, the second cell is separate. Drugs, such as drugs that enhance the activity of CAR-expressing cells. For example, in some embodiments, the agent can be an agent that inhibits an inhibitory molecule. Inhibitor molecules can, for example, reduce the ability of CAR-expressing cells to initiate an immune effector response, in some embodiments. Examples of inhibitory molecules are PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (eg 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 that inhibits the inhibitory molecule is a second polypeptide that provides a positive signal to the cell, eg, a first polypeptide bound to the intracellular signaling domain described herein. For example, it contains an inhibitory molecule. In some embodiments, the agents are, for example, PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (eg, 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 A first polypeptide of an inhibitory molecule, such as TGFβ or a fragment thereof (eg, at least a portion of any of these extracellular domains) and the intracellular signaling domains described herein (eg, co-stimulation). A second, which is a domain (eg, eg 41BB, CD27, ICOS or CD28 as described herein) and / or a primary signaling domain (eg, including the CD3 zeta signaling domain described herein). Including polypeptides. In some embodiments, the agent is a first polypeptide of PD1 or a fragment thereof (eg, at least a portion of the extracellular domain of PD1) and the intracellular signaling domain described herein. A second polypeptide includes, for example, the CD28 signaling domain described herein and / or the CD3 zeta signaling domain described herein.

In one aspect, the invention is combined with another agent, eg, a kinase inhibitor such as the kinase inhibitor described herein, in combination with a CAR-expressing cell population (eg, CART cells or CAR-expressing NK cells), eg, various. Provided are methods comprising administering a mixture of cells expressing CAR. In another embodiment, the invention expresses a CAR in which at least one cell in the population has an anticancer-related kinase binding domain as described herein and a second cell expresses another agent, eg, CAR. Provided are methods comprising administering a cell population expressing an agent that enhances cell activity in combination with another agent, eg, a kinase inhibitor such as a kinase inhibitor described herein.

Natural Killer Cell Receptor (NKR) CAR
In one embodiment, the CAR molecule described herein comprises one or more components of a natural killer cell receptor (NKR), thereby forming an NKR-CAR. The NKR component can be a transmembrane domain, hinge domain or cytoplasmic domain derived from any of the following natural killer cell receptors: killer cell immunoglobulin-like receptors (KIR) such as KIR2DL1, KIR2DL2 / L3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, DIR2DS5, KIR3DL1 / S1, KIR3DL2, KIR3DL3, KIR2DP1 and KIR3DP1; The lymphocyte activation molecule (SLAM) family, such as CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME and CD2F-10; Fc receptors (FcR), such as CD16 and CD64; and Ly49 receptors, such as LY49A. , LY49C. The NKR-CAR molecules described herein can interact with adapter molecules or intracellular signaling domains such as DAP12. Exemplary arrangements and sequences of CAR molecules containing NKR elements are described in WO 2014/145252, the contents of which are incorporated herein by reference.

Strategies for Controlling Chimeric Antigen Receptors There are many ways in which CAR activity can be controlled. In one embodiment, controllable CAR (RCAR) with controllable CAR activity is desirable for optimizing the safety and efficacy of CAR treatment. See, for example, induction of apoptosis using caspase fused to the dimerization domain (eg, Di et al., N Engl. J. Med. 2011 Nov. 3; 365 (18): 1673-1683. Can be used as a safety switch in the CAR treatment of the present invention. In another example, CAR-expressing cells can also express an inducible caspase-9 (iCaspase-9) molecule, which can also be expressed with a dimerization factor drug (eg, rimiducid (AP1903 (Bellicum Pharmaceuticals) or AP20187 (Arid)). Administration of (called) leads to cellular caspase-9 activation and apoptosis. The iCaspace-9 molecule contains a chemical inducer of the dimerization (CID) binding domain, which is dimeric in the presence of CID. Intervenes in somatization. This leads to inducible and selective depletion of CAR-expressing cells. In some cases, the iCaspase-9 molecule is encoded by a different nucleic acid molecule from the CAR coding vector. In some cases, iCaspase-9. The molecule is encoded by the same nucleic acid molecule as the CAR coding vector. ICaspase-9 can provide a safety switch to avoid any toxicity of CAR-expressing cells, eg, Song et al. Cancer Gene Ther. 2008; 15 ( 10): 667-75; Clinical Trial Id. No. NCT02107963; and Di Stasi et al. N. Engl. J. Med. 2011; 365: 1673-83.

An alternative strategy for controlling CAR treatment of the present invention is a small molecule that deactivates or blocks CAR activity, eg, by inducing antibody-dependent cellular cytotoxicity (ADCC), eg, by eliminating CAR-expressing cells. Or include the use of antibodies. For example, the CAR-expressing cells described herein can also express antigens recognized by molecules capable of inducing cell death, such as ADCC or complement-induced cell death. For example, the CAR-expressing cells described herein can also express receptors that can be targeted by an antibody or antibody fragment. Examples of such receptors are EpCAM, VEGFR, integrins (eg, integrins ανβ3, α4, αΙ3 / 4β3, α4β7, α5β1, ανβ3, αν), members of the TNF receptor superfamily (eg, TRAIL-R1, TRAIL). -R2), PDGF receptor, interferon receptor, folic acid 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, CD15, CD18 / ITGB2, CD19, CD20, CD22, CD23 / lgE 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 cleavage versions thereof (eg, one or more extracellular epitopes). However, it contains a version that lacks one or more regions within the cytoplasmic domain). For example, the CAR-expressing cells described herein are cleavage-type epidermal growth factor receptors that lack signaling capacity but retain an epitope recognized by a molecule capable of inducing ADCC, such as cetuximab (Arbitux®). The body (EGFR) is also expressed, so that administration of cetuximab induces depletion of ADCC and subsequent CAR-expressing cells (eg, WO 2011/056894 and Jonnalagada et al., Gene Ther. 2013; 20 (eg). 8) See 853-860). Another strategy is to combine target epitopes from both the CD32 and CD20 antigens in the CAR-expressing cells described herein, which bind to rituximab, which results in selective depletion of CAR-expressing cells, eg, by ADCC, and are highly compact. Includes expression of the marker / suicide gene (see, eg, Philippet al., Blood. 2014; 124 (8) 1277-1287). Another method of depleting CAR-expressing cells described herein is, for example, with a monoclonal anti-CD52 antibody that selectively binds to and targets mature lymphocytes, such as CAR-expressing cells, for destruction by ADCC induction. Includes administration of certain CAMPATH®. In other embodiments, CAR-expressing cells can be selectively targeted using CAR ligands, such as anti-idiotype antibodies. In one embodiment, the anti-idiotype antibody can induce effector cell activity, such as ADCC or ADC activity, thereby reducing the number of CAR expressing cells. In other embodiments, CAR ligands, such as anti-idiotype antibodies, can be attached to cell lethal agents, such as toxins, thereby reducing the number of CAR-expressing cells. Alternatively, the CAR molecule itself can be arranged such that its activity can be controlled, eg, activated or blocked, as described below.

In some embodiments, the RCAR is typically a series of elements of the standard CAR described herein, eg, an antigen binding domain and an intracellular signaling domain located in separate polypeptides or members. In the simplest embodiment it comprises two polypeptides. In some embodiments, the set of polypeptides comprises a dimerization switch capable of binding the polypeptides to each other in the presence of a dimerization molecule, eg, an antigen binding domain to an intracellular signaling domain. Further descriptions and exemplary arrangements of such controllable CARs are provided herein and in reference to International Publication No. 2015/090229, which is incorporated herein by reference in its entirety.

In one embodiment, RCAR comprises two polypeptides or members: 1) for example, an intracellular signaling domain comprising a primary intracellular signaling domain and a first switch domain as described herein; 2) eg. , An antigen-binding member comprising an antigen-binding domain and a second switch domain that target tumor antigens as described herein. Optionally, the RCAR comprises the transmembrane domain described herein. In one embodiment, the transmembrane domain can be located on an intracellular signaling member, an antigen binding member, or both. (Unless otherwise specified, when a member or element of a RPAR is 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, eg, the order of the elements at one end of the transmembrane region may be different, eg, the intracellular signaling domain. The placement of the switch domain for is different, for example the opposite).

In one embodiment, the first and second switch domains can form an intracellular or extracellular dimerization switch. In one embodiment, the dimerization switch is, for example, a homodimerization switch in which the first and second switch domains are the same, or, for example, heterodimerization in which the first and second switch domains are different from each other. Can be a switch.

In embodiments, the RCAR may include a "multi-switch". The multiswitch may include a heterodimerized switch domain or a homodimerized switch domain. A multi-switch is an independent first member of a plurality of, eg, 2, 3, 4, 5, 5, 6, 7, 8, 9 or 10 switch domains and, for example, an antigen binding member and Included on a second member, eg, an intracellular signaling member. In one embodiment, the first member may comprise a plurality of first switch domains, eg, FKBP-based switch domains, and the second member may include a plurality of second switch domains, eg, FRB-based switch domains. Can include. In one embodiment, the first member may include first and second switch domains, such as FKBP-based switch domains and FRB-based switch domains, and the second member may include first and second switch domains. For example, FKBP-based switch domains and FRB-based switch domains may be included.

In one embodiment, the intracellular signaling member comprises one or more intracellular signaling domains, such as a primary intracellular signaling domain and one or more costimulatory signaling domains.

In one embodiment, the antigen binding member may comprise one or more intracellular signaling domains, eg, one or more costimulatory signaling domains. In one embodiment, the antigen binding member comprises a co-stimulatory signaling domain described herein selected from multiple, eg, 2 or 3 eg, 4-1BB, CD28, CD27, ICOS and OX40. , In one embodiment, does not include the primary intracellular signaling domain. In one embodiment, the antigen binding member comprises the following costimulatory signaling domains from extracellular to intracellular: 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 an embodiment, the intracellular binding member comprises the CD3 zeta domain. In such embodiments, the RCAR is an antigen binding member comprising (1) an antigen binding domain, a transmembrane domain and two costimulatory domains and a first switch domain; and (2) a transmembrane domain or a transmembrane domain and It comprises an intracellular signaling domain including at least one primary intracellular signaling domain and a second switch domain.

One embodiment provides RCAR in which the antigen binding member is not tethered to the CAR cell surface. This allows cells with intracellular signaling members 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 is 1) an intracellular signaling member comprising a first switch domain, a transmembrane domain, an intracellular signaling domain such as a primary intracellular signaling domain and a first switching domain; 2) Includes an antigen-binding member, including an antigen-binding domain and a second switch domain, where the antigen-binding member does not include a transmembrane domain or a transmembrane domain, and optionally does not include an intracellular signaling domain. In one embodiment, RCAR comprises 3) a second antigen-binding domain, eg, a second antigen-binding domain that binds to a different antigen than that bound by the antigen-binding domain; and a second switch domain. It may further include antigen binding members.

RCRs in which antigen-binding members include bispecific activation and targeting ability are also provided herein. In this embodiment, the antigen binding member may comprise a plurality of, eg, 2, 3, 4 or 5 antigen binding domains, eg scFv, where each original binding domain is a target antigen, eg, a different antigen. Or bind to the same antigen, eg, the same or different epitopes of the same antigen. In one embodiment, the plurality of antigen binding domains is tandem and optionally a linker or hinge region is located between each of the antigen binding domains. Suitable linkers and hinge regions are described herein.

One embodiment provides a RCAR with an arrangement capable of switching proliferation. In this embodiment, the RCAR is 1) one or more costimulatory signals optionally selected from a transmembrane domain or a transmembrane domain; eg, 4-1BB, CD28, CD27, ICOS and OX40 and a switch domain. Intracellular signaling members comprising a transduction domain; and 2) including an antigen-binding domain, a transmembrane domain and a primary intracellular signaling domain, eg, an antigen-binding member comprising a CD3 zeta domain, where the antigen-binding member is a switch domain. Or does not contain a switch domain that dimerizes with a switch domain on an 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 the heterodimerization switch and the RPAR contains a second intracellular signal comprising a second switch domain of the heterodimerization switch. Including transmission members. In such an embodiment, 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.

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

Also provided herein are cells containing the RCARs described herein. Any cell engineered to express RPAR can be used as a RPARX cell. 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.

Nucleic acids and vectors containing RPAR-encoded sequences are also provided herein. The sequences encoding the various elements of RPAR can be placed on the same nucleic acid molecule, eg, the same plasmid or vector, eg, a viral vector, eg, a lentiviral vector. In one embodiment, (i) the sequence encoding the antigen-binding member and (ii) the sequence encoding the intracellular signaling member can be present in the same nucleic acid, eg, a vector. The production of the corresponding protein is, for example, by the use of separate promoters or by bicistronic transcript (which can result in the production of two proteins by cleavage of a single translation product or translation of two separate protein products). Can be achieved by using. In one embodiment, a sequence encoding a cleaving peptide, such as a P2A or F2A sequence, is placed between (i) and (ii). In one embodiment, an IRES, eg, an EMCV or EV71 IRES-encoding sequence, 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 linked to (i) and the second promoter is operably linked to (ii) so that (i) and (ii) are transcribed as separate mRNAs. connect.

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

Dimerization switch The dimerization switch can be non-shared or shared. In a non-shared dimerization switch, the dimerization molecule promotes non-covalent interaction between switch domains. In a shared dimerization switch, the dimerization molecule promotes shared interactions between switch domains.

In one embodiment, the RCAR comprises an FKBP / FRAP or FKBP / FRB based dimerization switch. FKBP12 (FKBP or FK506 binding protein) is a rich cytoplasmic protein that serves as the first intracellular target for the natural product immunosuppressant rapamycin. Rapamycin binds to FKBP and the large PI3K homolog FRAP (RAFT, mTOR). The Fed is the 93 amino acid portion of FRAP that is sufficient for binding 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 Ｕ Ｓ Ａ ９２：４９４７－５１．）。

In embodiments, FKBP / FRAP, eg FKBP / FRB based switches, can use dimerized molecules such as rapamycin or rapamycin analogs.

An exemplary amino acid sequence of FKBP is:

In embodiments, the FKBP switch domain may comprise a fragment of FKBP capable of binding to the Fed or a fragment thereof or an analog in the presence of rapamycin or rapalog. In some embodiments, the FKBP switch domain comprises the following amino acid sequence:

The amino acid sequence of the FRB is as follows.

As used herein, the term "FKBP / FRAP, eg FKBP / FRB-based switch" is an FKBP fragment or similar capable of binding to FRB or a fragment or analog thereof in the presence of rapamycin or rapalog, eg RAD001. Containing the body 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 a first switch domain that does not differ beyond 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 amino acid residues; and FRB or its in the presence of rapamycin or rapalog. Contains at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% with the FRB sequence of SEQ ID NO: 277, including FRB fragments or analogs thereof capable of binding to the fragment or analog. , 98% or 99% identity, or a second switch that does not differ beyond 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 amino acid residues. Refers to a dimerization switch containing a domain. In one embodiment, the RCAR described herein comprises one switch domain comprising the amino acid residues disclosed in SEQ ID NO: 275 (or SEQ ID NO: 589) and one switch domain comprising the amino acid residues disclosed in SEQ ID NO: 277. include.

In embodiments, the FKBP / FRB dimerization switch is a complex formation between FRB-based switch domains such as modified FRB switch domains, FKBP-based switch domains and dimerization molecules such as rapamycin or rapalog, such as RAD001. Includes modified, eg enhanced, modified Fed switch domains. In one embodiment, the modified FRB switch domain is one or more selected from amino acid positions L2031, E2032, S2035, R2036, F2039, G2040, T2098, W2101, D2102, Y2105 and F2108, eg, two, three, four. It comprises, 5, 5, 7, 8, 9, 10 or more mutations, wherein the wild-type amino acid is mutated to any other naturally occurring amino acid. In one embodiment, the variant FRB comprises a mutation in E2032, where E2032 is phenylalanine (E2032F), methionine (E2032M), arginine (E2032R), valine (E2032V), tyrosine (E2032Y), isoleucine (E2032I). ), For example SEQ ID NO: 278 or leucine (E2032L), eg, SEQ ID NO: 279. In one embodiment, the variant FRB comprises a mutation in T2098, where T2098 is mutated to phenylalanine (T2098F) or leucine (T2098L), eg, SEQ ID NO: 280. In one embodiment, the variant FRB comprises a mutation in E2032 and T2098, where E2032 is mutated to any amino acid and T2098 is mutated to any amino acid, eg, SEQ ID NO: 281. In one embodiment, the variant FRB comprises the E2032I and T2098L mutations, eg, SEQ ID NO: 282. In one embodiment, the variant FRB comprises the E2032L and T2098L mutations, eg, SEQ ID NO: 283.

Other suitable dimerization switches include GyrB-GyrB-based dimerization switches, gibberellin-based dimerization switches, tag / binder dimerization switches and halotag / snap tag dimerization switches. include. According to the guidance provided herein, such switches and related dimerization molecules will be apparent to those of skill in the art.

Dimericization Molecules Binding between switch domains is facilitated by dimerization molecules. Interactions or bindings between switch domains in the presence of a dimerized molecule are between a polypeptide that binds to a first switch domain, eg, fused, and a polypeptide that binds, eg, fused to a second switch domain. Enables signal transmission. As measured in the systems described herein in the presence of non-rate-determining levels of dimerized molecules, signal transduction is 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1. It increases by 5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times, 5 times, 10 times, 50 times, and 100 times.

Rapamycin and rapamycin analogs (sometimes referred to as rapalogs), such as RAD001, can be used as dimerization molecules in the FKBP / FRB-based dimerization switches described herein. In one embodiment, the dimerization molecule can be selected from rapamycin (sirolimus), RAD001 (everolimus), zotarolimus, temsirolimus, AP-23573 (ridaphorolimus), biolimus and AP21967. Further rapamycin analogs suitable for use with FKBP / FRB-based dimerization switches are entitled "Combination Therapy" or "Combination with Low Immunoboosting Dose of mTOR Inhibitor". Further described in the section.

Split CAR
In one embodiment, CAR expressing cells use split CAR. The split CAR approach is described in more detail in International Publication No. 2014/054542 and International Publication No. 2014/505657, which are incorporated herein by reference. Briefly, the split CAR system comprises cells expressing the first CAR having a first antigen binding domain and a costimulatory domain (eg, 41BB), where the cells are the second antigen binding domain and the intracellular signal. A second CAR with a transduction domain (eg, CD3 zeta) is also expressed. When the cell encounters the first antigen, the co-stimulation domain is activated and the cell proliferates. When a cell encounters a second antigen, the intracellular signaling domain is activated and cell-killing activity is initiated. Therefore, CAR-expressing cells are fully activated only in the presence of both antigens. In embodiments, the first antigen binding domain recognizes BCMA and comprises, for example, the antigen binding domains described herein, the second antigen binding domain is an antigen expressed in acute myeloid leukemia cells, eg CD123. , CLL-1, CD34, FLT3 or folic acid receptor beta. In embodiments, the first antigen binding domain recognizes BCMA and comprises, for example, the antigen binding domains described herein, the second antigen binding domain is an antigen expressed on B cells, such as CD10, CD19. , CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b or CD79a.

Stability and variation The stability of an anti-BCMA binding domain, eg, a scFv molecule (eg, soluble scFv), is assessed with reference to the biophysical properties (eg, thermal stability) of a conventional control scFv molecule or full-length antibody. can.

The improved thermal stability of the anti-BCMA binding domain, eg scFv, subsequently contributes to the entire CART-BCMA construct, leading to improved therapeutic properties of the CART-BCMA construct. The thermal stability of the anti-BCMA binding domain, eg scFv, can be improved by at least about 2 ° C or 3 ° C as compared to conventional antibodies. In some embodiments, the anti-BCMA binding domain, eg scFv, has 1 ° C. improved thermal stability to conventional antibodies. In another embodiment, the anti-BCMA binding domain, eg scFv, has improved thermal stability by 2 ° C. against conventional antibodies. In another embodiment, scFv is 4 ° C., 5 ° C., 6 ° C., 7 ° C., 8 ° C., 9 ° C., 10 ° C., 11 ° C., 12 ° C., 13 ° C., 14 ° C., 15 ° C. with respect to conventional antibodies. Has improved thermal stability. Comparisons can be made, for example, between the scFv molecule disclosed herein and the scFv molecule or Fab fragment of the antibody from which the scFv VH and VL are derived. Thermal stability can be measured using methods known in the art. For example, in some embodiments, Tm can be measured. Methods for measuring Tm and other methods for determining protein stability are described in more detail below.

Mutations in scFv (caused by humanization or direct mutagenesis of soluble scFv) alter the stability of scFv and improve the overall stability of scFv and CART33 constructs. The stability of human scFv can be compared to mouse scFv using measures such as Tm, denaturation temperature and aggregation temperature.

The binding capacity of the mutant scFv can be determined using the assay described in the Examples.

In some embodiments, the anti-BCMA binding domain, eg, scFv, comprises 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, eg scFv, is at least 1, 2, 3, 4, 5, 6 derived from the humanization process such that the mutant scFv provides improved stability of the CART-BCMA construct. , 7, 8, 9, and 10 mutations are included.

Methods for Assessing Protein Stability The stability of an antigen-binding domain can be assessed, for example, using the methods described below. Such methods limit the overall stability threshold of multi-domain units (eg, multi-domain proteins exhibiting a single denaturing transition) in which the least stable domain is initially denatured or coordinatedly denatured. Allows determination of degenerative transitions. The least stable domain can be identified in a number of additional ways. Mutagenesis can be performed to explore which domains limit overall stability. In addition, protease resistance of multidomain proteins can be performed by DSC or other spectroscopic methods under conditions known to essentially denature the least stable domain (Fontana, et al., (1997) Fold. Des., 2: R17-26; Dimasi et al. (2009) J. Mol. Biol. 393: 672-692). Once the least stable domain has been identified, the sequence (or portion thereof) encoding this domain can be used as the test sequence in this method.

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

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, eg, Sanchez-Riz, et al., Biochemistry, 27: 1648-52, 1988). ). To determine the thermal stability of the protein, place the protein sample in a calorimeter and raise the temperature until Fab or scFv is denatured. The temperature at which a protein denatures is an indicator of overall protein stability.

Another example of analytical spectroscopy is circular dichroism (CD) spectroscopy. CD spectroscopy measures the optical activity of a composition as a function of temperature rise. Circular dichroism (CD) spectroscopy measures the difference in absorption between left-handed and right-handed polarization due to structural asymmetry. The damaged or denatured structure results in a CD spectrum that is very different from the regular or folded structure. The CD spectrum reflects the sensitivity of the protein to the denaturing effect of elevated temperature and is therefore an indicator of protein thermal stability (see van Mierlo and Steemsma, J. Biotechnol., 79 (3): 281-98, 2000). sea bream).

Another example of analytical spectroscopy for measuring thermal stability is fluorescence emission spectroscopy (see van Mierlo and Steemsma, supra). Yet another example of analytical spectroscopy for measuring thermal stability is nuclear magnetic resonance (NMR) spectroscopy (see, eg, van Mierlo and Steemsma, supra).

The thermal stability of the composition can be measured biochemically. An example of a biochemical method for assessing thermal stability is a heat loading assay. In a "heat load assay", the composition is exposed to a range of high temperatures for a set period of time. For example, in some embodiments, the test scFv molecule or a molecule containing the scFv molecule is exposed to a range of temperature increases, eg, 1-1.5 hours. The activity of the protein is then assayed by the relevant biochemical assay. For example, if the protein is a binding protein (eg, a scFv or scFv-containing polypeptide), the binding activity of the binding protein can be determined by functional or quantitative ELISA.

Such assays can be performed in a high-throughput format and are disclosed in Examples using E. coli and high-throughput screening. A library of anti-BCMA binding domains (eg, scFv variants) can be created using methods known in the art. Expression of an anti-BCMA binding domain, such as scFv, can be induced, and an anti-BCMA binding domain, such as scFv, can be subjected to heat loading. Load test samples can be assayed for binding and stable anti-BCMA binding domains such as scFv can be scaled up and further characterized.

Thermal stability is evaluated by measuring the melting temperature (Tm) of the composition using any of the above techniques (eg, analytical spectroscopic techniques). The melting temperature is the temperature at the midpoint of the temperature transition curve, where 50% of the molecules of the composition are folded (eg, Dimasi et al. (2009) J. Mol Biol. 393: 672-692. Please refer to). In some embodiments, the Tm value of the anti-BCMA binding domain, eg scFv, is about 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 ℃, 68 ℃, 69 ℃, 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 ℃, It is 100 ° C. In some embodiments, the Tm value of IgG is about 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, 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., 100 ° C. In some embodiments, the Tm value of the polyvalent antibody is about 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 ℃, 53 ℃, 54 ℃, 55 ℃, 56 ℃, 57 ℃, 58 ℃, 59 ℃, 60 ℃, 61 ℃, 62 ℃, 63 ℃, 64 ℃, 65 ℃, 66 ℃, 67 ℃, 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, 100 ° C. ..

Thermal stability is also assessed by measuring the specific heat or heat capacity (Cp) of the composition using analytical calorimetric techniques (eg, DSC). The specific heat of the composition is the energy required to raise the temperature of 1 mol of water by 1 ° C. (eg, at kcal / mol). The large Cp is characteristic of the denatured or inert protein composition. The change in heat capacity (ΔCp) of the composition is measured by determining the specific heat of the composition before and after the temperature transition. Thermal stability can also be assessed by measuring or determining other parameters of thermodynamic stability, including the Gibbs free energy of modification (ΔG), the enthalpy of modification (ΔH) or the entropy of modification (ΔS). One or more of the above biochemical assays (eg, heat loading assays) are used to determine the temperature (ie, _TC value) at which 50% of the composition retains its activity (eg, binding activity).

In addition, mutations to anti-BCMA binding domains, such as scFv, alter the thermal stability of anti-BCMA binding domains, such as scFv, as compared to unmutated anti-BCMA binding domains, such as scFv. When a human or humanized anti-BCMA binding domain, eg scFv, is integrated into a BCMA construct, the anti-BCMA binding domain, eg humanized scFv, provides thermal stability to the entire anti-BCMA CART construct. In some embodiments, the anti-BCMA binding domain, eg scFv, comprises a single mutation that imparts thermal stability to the anti-BCMA binding domain, eg scFv. In another embodiment, the anti-BCMA binding domain, eg scFv, comprises a plurality of mutations that confer thermal stability to the anti-BCMA binding domain, eg scFv. In some embodiments, multiple mutations in the anti-BCMA binding domain, eg scFv, have an additive effect on the thermal stability of the anti-BCMA binding domain, eg scFv.

b) Aggregation%
The stability of the 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, agglutination of the composition can be evaluated using chromatography, such as size exclusion chromatography (SEC). SEC separates molecules based on size. The column is filled with semi-solid beads of polymer gel that contain ions and small molecules inside but not large ones. When the protein composition is applied to the top of the column, the small folded proteins (ie, non-aggregating proteins) are dispersed in a larger amount of solvent for which large protein aggregation is available. As a result, large aggregates can move faster in 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 (eg, by light scattering). Therefore, the% aggregation of the composition can be determined by comparing the concentration of fractions with the total concentration of protein applied to the gel. The stable composition elutes from the column essentially as a single fraction and appears as essentially a single peak in the elution profile or chromatogram.

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

In one embodiment, the antigen-binding domain of CAR comprises an amino acid sequence that is homologous to the antigen-binding domain amino acid sequence described herein, and the antigen-binding domain is the desired function of the anti-BCMA antibody fragment described herein. Retains the characteristic characteristics. In a particular embodiment, the CAR composition of the invention comprises an antibody fragment. In a further embodiment, the antibody fragment comprises scFv.

In various embodiments, the antigen-binding domain of CAR is one of one or both variable regions (eg, VH and / or VL), eg, one or more CDR regions and / or one or more framework regions. It is manipulated by the above amino acid modifications. In a particular embodiment, the CAR composition of the invention comprises an antibody fragment. In a further embodiment, the antibody fragment comprises scFv.

It will be appreciated by those skilled in the art that the antibodies or antibody fragments of the invention may be further modified to differ in amino acid sequence (eg, from wild type) but not the desired activity. For example, further nucleotide substitutions, such as conservative substitutions that result in amino acid substitutions, such as conservative substitutions at "non-essential" amino acid residues, can be made on the protein. For example, a non-essential amino acid residue in a molecule can be replaced with another amino acid residue from the same side chain family. In another embodiment, the set of amino acids can be replaced with a series of structurally similar amino acids with different order and / or composition of side chain family members, eg, amino acid residues in which the amino acid residues have similar side chains. Can be a conservative replacement replaced by.

Basic side chains (eg, lysine, arginine, histidine), acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (eg, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non- Polar side chains (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta branched side chains (eg, threonine, valine, isoleucine) and aromatic side chains (eg, tyrosine, phenylalanine, tryptophan). , Histidine), a family of amino acid residues with similar side chains is defined in the art.

In connection with more than one nucleic acid or polypeptide sequence, percent identity refers to more than one sequence that is the same. The two sequences are the same amino acid residues or nucleotides when measured using one of the following sequence comparison algorithms or when the maximum correspondence is compared and aligned across the comparison window or designated region by manual alignment and visual inspection. If they have a particular percentage of, they are "substantially identical" (eg 60% identity, optionally 70%, 71%, 72%, 73% over the entire sequence, when not specified or in a particular region. , 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). Optionally, the identity is a region of at least about 50 nucleotides (or 10 amino acids) long or more preferably a region of 100-500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) long. There is identity across.

For sequence comparison, typically one sequence serves as a control sequence against which the test sequences are compared. When using the sequence comparison algorithm, the test and control sequences are input to the computer, the partial sequence coordinates are specified as needed, and the 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 percent sequence identity of the test sequence relative to the control sequence based on the program parameters. Methods of aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison is described, for example, in Smith and Waterman, (1970) Adv. Apple. Math. 2: 482c Local Homology Algorithm, Needleman and Wunsch, (1970) J. Mol. Mol. Biol. 48: 443 Homology Alignment Algorithm, Pearson and Lipman, (1988) Proc. Nat'l. Acad. Sci. Similar methods for searching USA 85: 2444, computerized execution of these algorithms (Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, and GAP, BESTAST, FA in WI). It can be performed visually (see, eg, Brent et al., (2003) Currant Algorithms in Molecular Biology).

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

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

In one aspect, the invention contemplates modification of a starting antibody or fragment (eg, scFv) amino acid sequence that produces a functionally equivalent molecule. For example, the VH or VL of the anti-BCMA binding domain (eg, scFv) contained in the CAR is at least about 70%, 71% of the starting VH or VL framework region of the anti-BCMA binding domain (eg, scFv). 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% can be modified to retain identity. The present invention contemplates modifications of the entire CAR construct to produce functionally uniform molecules, eg, modifications in one or more amino acid sequences of various domains in the CAR construct. CAR constructs are at least about 70%, 71% of the departure CAR constructs. 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% can be modified to retain identity.

Nucleic Acid Constructs Encoding CARs 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.

Thus, in one embodiment, the invention relates to an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR is an anti-BCMA binding domain (eg, a human anti-BCMA binding domain), a transmembrane domain and as well. Includes stimulating domains such as co-stimulating signaling domains and / or primary signaling domains such as intracellular signaling domains containing zeta chains. In some embodiments, the anti-BCMA binding domain is the anti-BCMA binding domain described herein or a sequence that identifies 95-99% thereof. In some embodiments, the transmembrane domain is an alpha chain, beta chain or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64. , CD80, CD86, CD134, CD137 and CD154 are transmembrane domains of proteins selected from the group. In some embodiments, the anti-BCMA binding domain is connected to the transmembrane domain by a hinge region, eg, 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 domains are 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, which comprises a functional signaling domain. In some embodiments, the isolated nucleic acid molecule further comprises a sequence encoding a co-stimulation domain. In some embodiments, the costimulatory domain is, for example, an MHC class I molecule, a TNF receptor protein, an immunoglobulin-like protein, a cytokine receptor, an integulin, a signaling lymphoid activation molecule (SLAM protein), activation. 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 (LIGHT), 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, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEMAC1 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, A functional signaling domain of a protein selected from the group consisting of ligands that specifically bind to LAT, GADS, SLP-76, PAG / Cbp, CD19a and CD83.

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

In another aspect, the invention relates to an isolated polypeptide molecule encoded by a nucleic acid molecule.

Nucleic acid sequences encoding the desired molecule can be obtained by using standard techniques, eg, by screening a library from cells expressing the gene, or by inducing a gene from a vector known to contain it. Recombinations can be obtained using methods known in the art, such as by isolating directly from cells and tissues known to contain. Alternatively, the gene of interest can be produced synthetically rather than cloned.

The present invention also provides a vector into which the DNA of the present invention is inserted. Vectors derived from retroviruses such as lentivirus are suitable tools for achieving long-term gene transfer because they allow long-term stable integration of transgenes and transmission to daughter cells. Lentiviral vectors have additional advantages over onco-retrovirus-derived vectors such as murine leukemia virus in that non-proliferative cells such as hepatocytes can be transduced. It also has the additional advantage of being hypoimmunogenic. The retroviral vector can also be, for example, a gamma retroviral vector. The gamma retroviral vector encodes, for example, a promoter, a packaging signal (ψ), a primer binding site (PBS), one or more (eg, 2) terminal repeat sequences (LTRs) and a transgene of interest, eg, CAR. May contain a gene. Gammaretrovirus vectors can lack viral structural genes such as gag, pol and env. Exemplary gammaretrovirus vectors include murine leukemia virus (MLV), splenic focus-forming virus (SFFV) and myeloid proliferative sarcoma virus (MPSV) and vectors derived thereof. Other gammaretrovirus vectors are described, for example, in Tobias Matzig et al. , "Gammaretrolial Vectors: Biology, Technology and Application" Viruses. 2011 Jun; 3 (6): 677-713.

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

In general, expression of a natural or synthetic nucleic acid encoding a CAR is typically achieved by operably linking the nucleic acid encoding a CAR polypeptide or a portion thereof to a promoter and incorporating its construct into an expression vector. Will be done. Vectors may be suitable for replication and integration in eukaryotes. A typical cloning vector contains a transcription and translation terminator, initiation sequence and promoter useful for controlling the expression of the desired nucleic acid sequence.

Expression of constructs of the invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods for gene delivery are known in the art. See, for example, US Pat. Nos. 5,399,346, 5,580,859, and 5,589,466, which are incorporated herein by reference in their entirety. sea bream. In another embodiment, the invention provides a gene therapy vector.

Nucleic acids can be cloned into many 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. Particularly interesting vectors include expression vectors, replication vectors, probe production vectors and sequencing vectors.

In addition, expression vectors can be provided to cells in the form of viral vectors. Viral vector techniques are well known in the art and are 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. In general, a suitable vector comprises a functional origin of replication in at least one organism, a promoter sequence, a convenient restriction endonuclease site and one or more selectable markers (eg, WO 01/96584; WO). No. 01/29058; and US Pat. No. 6,326,193).

Numerous virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered in vivo or ex vivo to the cells of interest. 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, lentiviral vectors are used.

Additional promoter elements, such as enhancers, control the frequency of transcription initiation. Typically, they are located in the region 30-110 bp upstream of the initiation site, but it has been shown that many promoters also contain functional elements downstream of the initiation site. The space between promoter elements is often mobile, so that promoter function is retained when the elements are reversed or moved from each other. In the thymidine kinase (tk) promoter, the space between promoter elements can increase up to 50 bp apart before activity begins to decline. By the promoter, the individual elements appear to be able to function cooperatively or independently to activate transcription.

An example of a promoter capable of expressing the CAR transgene in mammalian T cells is the EF1a promoter. The native EF1a promoter drives the expression of the alpha subunit of the elongation factor-1 complex responsible for the enzymatic delivery of aminoacyl-tRNA to the ribosome. The EF1a promoter has been widely used in mammalian expression plasmids and has been shown to be effective in driving CAR expression from transgenes cloned into lentiviral vectors. For example, Milone et al. , Mol. The. 17 (8): 1453-1464 (2009). In one embodiment, the EF1a promoter comprises the sequence provided as SEQ ID NO: 11.

Another example of a promoter is the earliest 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, the monkey virus 40 (SV40) early promoter, mouse breast tumor virus (MMTV), human immunodeficiency virus (HIV) terminal repeat sequence (LTR) promoter, MoMuLV promoter, trileukemia virus promoter, Epstein-Barvirus earliest promoter, Other constitutive promoter sequences including, but not limited to, the Raus sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the elongation factor-1α promoter, the hemoglobin promoter and the creatin kinase promoter are also used. Can be. Furthermore, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also intended as part of the present invention. The use of an inducible promoter 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 inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters and tetracycline promoters.

例示的な切断型ＰＧＫプロモーター：
ＰＧＫ１００：

ＰＧＫ２００：

ＰＧＫ３００：

ＰＧＫ４００：

Another example of a promoter is the phosphoglycerate kinase (PGK) promoter. In embodiments, a PGK having a cleaved PGK promoter (eg, one or more, eg, one, two, five, 10, 100, 200, 300 or 400 nucleotide deletions when compared to a wild-type PGK promoter sequence). Promoter) may be desirable. The nucleotide sequence of an example PGK promoter is provided below.
WT PGK promoter

An exemplary truncated PGK promoter:
PGK100:

PGK200:

PGK300:

PGK400:

Vectors are, for example, signal sequences for promoting secretion, polyadenylation signals and transcription terminators (eg, from the bovine growth hormone (BGH) gene), elements that allow episomal replication and replication in prokaryotes (eg, SV40 origin). And ColE1 or others known in the art) and / or elements that allow selection (eg, ampicillin resistance genes and / or zeosin markers) may also be included.

To facilitate the identification and selection of expressed cells from expressed cells from a cell population for which an expression vector gene transfer or viral vector to be introduced into a cell is sought to evaluate the expression of the CAR polypeptide or a portion thereof. , Selectable marker gene and / or reporter gene. In another embodiment, the selectable marker is carried on another cross section of DNA and is used in co-transfection methods. Both the selectable marker and the reporter gene may be flanked by suitable regulatory sequences for expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes such as neo.

Reporter genes are probably used to identify transgenic cells and evaluate the functionality of regulatory sequences. In general, a reporter gene is a gene that encodes a polypeptide that is not present or expressed in a recipient organism or tissue and is expressed with readily detectable properties, eg, manifested by enzymatic activity. Expression of the reporter gene is assayed after a suitable time after the DNA is introduced into the recipient cells. Suitable reporter genes can include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase or the green fluorescent protein gene (eg, 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. In general, constructs with a minimum of 5'flanking regions showing the highest levels of expression of the reporter gene are identified as promoters. Such promoter regions can be used to ligate reporter genes and evaluate drugs for their ability to regulate promoter-driven transcription.

In some embodiments, the vector may further comprise a nucleic acid encoding a second CAR. In some embodiments, the second CAR is a target expressed on acute myeloid leukemia cells such as, for example, CD123, CD34, CLL-1, folic acid receptor beta or FLT3; or a target expressed on B cells. For example, it comprises an antigen binding domain for CD10, CD19, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b or CD79a. In some embodiments, the vector specifically binds to a first antigen and comprises a first CAR that comprises an intracellular signaling domain having a costimulatory signaling domain but no primary signaling domain. Encodes a second CAR that contains an intracellular signaling domain that contains a nucleic acid sequence encoding a, specifically binds to a second different antigen, has a primary signaling domain, but does not have a costimulatory signaling domain. Contains the nucleic acid sequence to be used. In some embodiments, the vector is a nucleic acid encoding a first BCMA CAR, including a BCMA binding domain, a transmembrane domain and a costimulatory domain, and an antigen other than BCMA (eg, an antigen expressed on AML cells, eg CD123). , CD34, CLL-1, folic acid receptor beta or FLT3; or antigens expressed on B cells such as CD10, CD19, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b or CD79a). The target comprises a nucleic acid encoding a second CAR, including an antigen binding domain, a transmembrane domain and a primary signaling domain. In another embodiment, the vector is a nucleic acid encoding a first BCMA CAR including a BCMA binding domain, a transmembrane domain and a primary signaling domain and an antigen other than BCMA (eg, an antigen expressed on AML cells, eg CD123). , CD34, CLL-1, folic acid receptor beta or FLT3; or antigens expressed on B cells such as CD10, CD19, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b or CD79a). A nucleic acid that specifically binds and encodes a second CAR, including an antigen-binding domain, a transmembrane domain and a costimulatory signaling domain for an antigen.

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 comprises an antigen binding domain that binds to an antigen found in normal cells, eg, normal cells expressing BCMA, but not in cancer cells. In some embodiments, the inhibitory CAR comprises an antigen binding domain, a transmembrane domain and an intracellular domain of the inhibitory molecule. For example, the intracellular domains of inhibitory CAR are PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (eg, 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. Can be the intracellular domain of.

In embodiments, the vector is expressed on two or more nucleic acid sequences encoding CARs, eg, BCMA CARs described herein and a second CAR, eg, an inhibitory CAR or an antigen other than BCMA (eg, AML cells). On antigens such as CD123, CLL-1, CD34, FLT3 or folic acid receptor beta; or antigen-expressing B cells such as CD10, CD19, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b or CD79a). It may include CARs that specifically bind. In such an embodiment, the nucleic acid sequences encoding 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 can be separated, for example, by one or more peptide cleavage sites (eg, self-cleaving sites or substrates for intracellular proteases). Examples of peptide cleavage sites include the following, where the GSG residue is optional.
T2A: (GSG) E G R G S L L T C G D V E E N P GP (SEQ ID NO: 194)
P2A: (GSG) A T N F S L L K Q A G D V E E N P GP (SEQ ID NO: 195)
E2A: (GSG) QC T N Y A L L K L A G D V E S N P GP (SEQ ID NO: 196)
F2A: (GSG) V K Q T L N F D L L K L A G D V E S N P GP (SEQ ID NO: 197)

Methods of introducing and expressing genes into cells are known in the art. In connection with the expression vector, the vector can be readily introduced into host cells such as mammalian, bacterial, yeast or insect cells by any method in the art. For example, the expression vector can be transferred to a host cell by physical, chemical or biological means.

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

Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, especially retroviral vectors, have become the most widely used method for gene insertion into mammals, such as human cells. Other viral vectors can be derived from lentivirus, poxvirus, herpes simplex virus I, adenovirus and adeno-related viruses and the like. See, for example, US Pat. Nos. 5,350,674 and 5,585,362.

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

When utilizing a non-viral delivery system, a typical delivery medium is a liposome. 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, the nucleic acid can be bound to a lipid. Lipid-bound nucleic acids are encapsulated in the aqueous interior of the liposome, dispersed in the lipid bilayer of the liposome, bound to the liposome via a linking molecule that binds to both the liposome and the oligonucleotide, encapsulation in the liposome, and the liposome. Complexing with, dispersion in lipid-containing solutions, mixing with lipids, combination with lipids, inclusion as suspensions in lipids, inclusion in micelles or complexing or otherwise binding to lipids. do. The lipid, lipid / DNA or lipid / expression vector binding composition is not limited to any particular structure in solution. For example, they may exist as micelles or with a "collapse" structure in a double layer structure. They are simply dispersed in solution and may also form aggregates that are probably non-uniform in size or shape. Lipids are fatty substances that can be naturally occurring or synthetic lipids. For example, lipids include lipid droplets naturally occurring in the cytoplasm and compounds and derivatives thereof containing long chain aliphatic hydrocarbons such as fatty acids, alcohols, amines, amino alcohols and aldehydes.

Lipids suitable for use can be obtained from commercial sources. For example, dimyristylphosphatidylcholine (“DMPC”) can be found in Sigma, St. Can be obtained from Louis, MO, disetyl phosphate (“DCP”) can be obtained from K & K Laboratories (Plainview, NY); cholesterol (“Choi”) can be obtained from Calbiochem-Behring, di Myristylphosphatidylglycerol (“DMPG”) and other lipids are described in Avanti Polar Lipids, Inc. It can be obtained from (Birmingham, AL.). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about −20 ° C. Chloroform is used as the only solvent because it volatilizes more easily than methanol. "Liposome" is a general term that includes a variety of mono and multilayer lipid media formed by the production of encapsulated lipid bilayers or aggregates. Liposomes can be characterized by having a vesicle structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilayer liposomes have multiple lipid layers separated by an aqueous medium. They form naturally when phospholipids are suspended in excess aqueous solution. Lipid elements self-aggregate prior to closed structure formation and encapsulate water and dissolved solutes between lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions having a solution in a structure different from the usual vesicle structure are also included. For example, lipids can be thought of as micellar structures or simply as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also considered.

A variety of assays can be performed to confirm the presence of recombinant DNA sequences in a host cell, regardless of how the foreign nucleic acid is introduced into the host cell or otherwise the cell is exposed to the inhibitors of the invention. Such assays are, for example, "molecular biological" assays well known to those of skill in the art such as Southern and Northern Blotting, RT-PCR and PCR, eg immunological means (ELISA and) for use in drug identification. Western blots) include "biochemical" assays that detect the presence or absence of a particular peptide or assays described herein that fall within the scope of the invention.

The present invention further provides a vector containing a CAR-encoding nucleic acid molecule. In one embodiment, the CAR vector can be transduced directly into cells such as T cells or NK cells. In one embodiment, the vector comprises a cloning or expression vector, eg, one or more plasmids (eg, an expression plasmid, a cloning vector, a minicircle, a minivector, a double microchromatium), a retrovirus and a lentivirus vector construct. It is a vector not limited to these. In one embodiment, the vector is capable of expressing CAR constructs in mammalian T cells or NK cells. In one embodiment, the mammalian T cell is a human T cell. In one embodiment, the mammalian NK cell is a human NK cell.

RNA Transfection Disclosed herein is a method of producing transcribed RNA CAR in vitro. The invention also includes CAR encoding an RNA construct that can be directly transfected into cells. Methods of producing mRNA for use in transfection include in vitro transcription (IVT) of a template with specifically designed primers, followed by poly A addition, 3'and 5'untranslated sequences ("UTR"). A construct containing a 5'cap and / or an internal ribosome entry site (IRES), expressed nucleic acid and polyA tail, typically 50-2000 base length (SEQ ID NO: 35) can be produced. RNA thus produced can be efficiently transfected in cells of different species. In one embodiment, the template comprises a sequence of CAR.

In one embodiment, the anti-BCMA CAR is encoded by messenger RNA (mRNA). In one embodiment, the 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 (eg, CART cells or CAR-expressing NK cells).

In some embodiments, in vitro transcribed RNA CAR can be introduced into cells in the form of transient transfection. RNA is produced by in vitro transcription using a polymerase chain reaction (PCR) production template. 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 source of DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequences or any other suitable source of DNA. The desired template for in vitro transcription is the CAR of the present invention. For example, the template for RNA CAR is an extracellular region containing a single chain variable domain of an antitumor antibody; a hinge region, a transmembrane domain (eg, the transmembrane domain of CD8a); and an intracellular signaling domain, such as the CD3-zeta. Includes a cytoplasmic region containing a signaling domain and one containing a 4-1BB signaling domain.

In some embodiments, the DNA used for PCR comprises an open reading frame. DNA can be derived from naturally occurring DNA sequences from the genome of an organism. In some embodiments, the nucleic acid may contain some or all of the 5'and / or 3'untranslated regions (UTRs). Nucleic acids 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 containing 5'and 3'UTR. The DNA may instead be an artificial DNA sequence that is not normally expressed in naturally occurring organisms. An exemplary artificial DNA sequence comprises a portion of a gene co-ligated to form an open reading frame encoding a fusion protein. The portions of DNA ligated together can be of single organism origin or of two or more organisms.

PCR is used to produce a template for in vitro transcription of mRNA used for transfection. Methods of performing PCR are well known in the art. Primers used in PCR are designed to have regions that are substantially complementary to the region of DNA used as a template for PCR. As used herein, "substantially complementary" refers to a sequence of nucleotides in which most or all of the residues in the primer sequence are complementary or one or more bases are non-complementary or mismatched. .. Substantially complementary sequences can be annealed or hybridized to the intended DNA target under the annealing conditions used for PCR. Primers can be designed to be substantially complementary to any portion of the DNA template. For example, the primer can be designed to amplify the portion of the nucleic acid normally transcribed (open reading frame) in the cell, including the 5'and 3'UTRs. Primers can also be designed to amplify the portion of nucleic acid that encodes a particular domain of interest. In some embodiments, the primer is designed to amplify the coding region of the human cDNA containing all or part of the 5'and 3'UTR. 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 a nucleotide that is substantially complementary to the nucleotide on a DNA template that is upstream of the DNA sequence to be amplified. "Upstream" is used herein to refer to the 5th position with respect to the DNA sequence to be amplified with respect to the coding strand. A "reverse primer" is a primer containing a region of nucleotides that is substantially complementary to a double-stranded DNA template, downstream of the DNA sequence to be amplified. "Downstream" is used herein to refer to the 3'position with respect to the DNA sequence to be amplified with respect to the coding strand.

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

Chemical structures capable of promoting stability and / or translation efficiency may also be used. RNA preferably has 5'and 3'UTRs. In some embodiments, the 5'UTR is 1 to 3000 nucleotides in length. The length of the 5'and 3'UTR sequences added to the coding region may vary by different methods, including but not limited to the design of primers for PCR to anneal different regions of the UTR. Using this approach, one of skill in the art can modify the 5'and 3'UTR lengths required to achieve optimal translation efficiency after transfection of the transcribed RNA.

The 5'and 3'UTRs can be naturally occurring endogenous 5'and 3'UTRs for the nucleic acid of interest. Alternatively, a UTR sequence that is not endogenous to 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 UTR sequences that are not endogenous to the nucleic acid of interest may be useful for modifying RNA stability and / or translation efficiency. For example, the AU-rich element of the 3'UTR sequence is known to reduce the stability of mRNA. Therefore, the 3'UTR can be selected and designed to increase the stability of the transcribed RNA based on the characteristics of the UTR that are well known in the art.

In some embodiments, the 5'UTR may comprise the Kozak sequence of the endogenous nucleic acid. Alternatively, when a 5'UTR that is not endogenous to the nucleic acid of interest is added by PCR as described above, the consensus Kozak sequence can be redesigned by adding the 5'UTR sequence. Kozak sequences can increase the translation efficiency of certain RNA transcripts, but do not appear to be required for total RNA to allow efficient translation. The need for Kozak sequences for many mRNAs is known in the art. In other embodiments, the 5'UTR can be the 5'UTR of an RNA virus in which the RNA genome is stable in cells. In other embodiments, various nucleotide analogs can be used in the 3'or 5'UTR to interfere with the exonuclease degradation of mRNA.

To allow the synthesis of RNA from the DNA template without the need for gene cloning, the transcriptional promoter should bind to the DNA template upstream of the sequence to be transcribed. When a sequence that acts as an RNA polymerase promoter is added to the 5'end of the forward primer, the RNA polymerase promoter is incorporated into the PCR product upstream of the transcribed open reading frame. In a preferred embodiment, the promoter is a T7 polymerase promoter as described elsewhere herein. Other useful promoters include, but are not limited to, the T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences of the T7, T3 and SP6 promoters are known in the art.

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

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 Verzal-Herranz, Eur. J. Biochem., 270: 1485-65 (2003)).

A conventional method for incorporating poly A / T elongation into a DNA template is molecular cloning. However, poly A / T sequences integrated into plasmid DNA can result in plasmid instability, which is that plasmid DNA templates obtained from bacterial cells are often highly contaminated with deletions and other abnormalities. That's why. This not only makes the cloning process difficult and time consuming, but also makes it unreliable in many cases. This is why a method that allows the production of DNA templates with poly A / T 3'stretch without cloning is highly desirable.

The poly A / T segment of the transcribed DNA template can be in PCR (SEQ ID NO: 31) (size can be 50-5000T (SEQ ID NO: 32)) or DNA ligation using a reverse primer containing a poly T tail such as 100T tail. Alternatively, it can be produced after PCR by any other method including, but not limited to, in vitro recombination. The poly (A) tail also provides stability to RNA and reduces its degradation. In general, the length of the poly (A) tail is positively correlated with the stability of the transcribed RNA. In some embodiments, the poly (A) tail is 100-5000 adenosine (SEQ ID NO: 33).

The poly (A) tail of RNA can be further extended after in vitro transcription by the use of a poly (A) polymerase such as E. coli poly A polymerase (E-PAP). In some embodiments, an extension of the length of the poly (A) tail from 100 nucleotides to 300-400 nucleotides (SEQ ID NO: 34) results in a approximately 2-fold increase in RNA translation efficiency. In addition, the addition of different chemical groups to the 3'end can increase mRNA stability. Such bonds may include modified / artificial nucleotides, aptamers and other compounds. For example, ATP analogs can be incorporated into the poly (A) tail using a poly (A) polymerase. ATP analogs can further enhance RNA stability.

The 5'cap also provides stability to the RNA molecule. In a preferred embodiment, RNA produced by the methods disclosed herein comprises a 5'cap. 5'caps are known in the art and are 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. Biophyss. Res. Commun., 330: 958-966 (2005)).

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

RNA can be introduced into target cells using any of a number of different methods, eg, commercially available methods, which include electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)),. (ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) Or Gene Pulser II (BioRad, Denver, Colo.), Multiportor (Eppendort, Hambulg Germany), transfection-mediated, transfection, transfection, transfection, transfection, transfection, transfection, transfection, transfection, These include, but include peptide-mediated transfection or microparticulate gun particle delivery systems such as "gene guns" (see, eg, Nishikawa, et al. Hum Gene Ther., 12 (8): 861-70 (2001)). Not limited to.

Non-viral delivery method In some embodiments, the non-viral delivery method can be used to deliver the CAR-encoding nucleic acid described herein to a cell or tissue or subject.

In one embodiment, the non-viral method comprises the use of a transposon (also referred to as a transposable element). In some embodiments, the transposon is spliced out of a piece of DNA that can itself be inserted into a position in the genome, eg, a piece of DNA that is self-replicating and a copy of which can be inserted into the genome, or a long nucleic acid, and the genome. A piece of DNA that can be inserted elsewhere in the. For example, a transposon contains a DNA sequence consisting of an inverted repeating flanking gene for transposition.

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

SBTS contains two components: 1) a transposon containing a transgene and 2) a source of transposase enzyme. Transposases can transpose transposons from carrier plasmids (or other donor DNAs) to target DNAs such as host cell chromosomes / genomes. For example, the transposase binds to the carrier plasmid / donor DNA, excises the transposon (containing the transgene) out of the plasmid, and puts it into the genome of the host cell. For example, Aronovic et al. See above.

Exemplary transposons include pT2-based transposons. For example, Grabundzija et al., Which is incorporated herein by reference in its entirety. Nucleic Acids Res. 41.3 (2013): 1829-47; and Singh et al. Cancer Res. 68.8 (2008): 2961-2971. Exemplary transposases include Tc1 / mariner type transposases such as SB10 transposase or SB11 transposase (eg, hyperactive transposases that can be expressed from the cytomegalovirus promoter). For example, Aronovic et al., All incorporated herein by reference. Kebriaei et al. And Grabundzija et al. Please refer to.

The use of SBTS allows for efficient integration and expression of transgenes, eg, nucleic acids encoding the CARs described herein. For example, a method for producing cells stably expressing the CAR described herein, such as T cells or NK cells, using a transposon system such as SBTS is provided herein.

By the methods described herein, in certain embodiments, one or more nucleic acids containing SBTS components, such as plasmids, are delivered to the cells (eg, T cells or NK cells). For example, nucleic acids are delivered by standard methods of nucleic acid (eg, plasmid DNA) delivery, such as the methods described herein, such as electroporation, transfection or lipofection. In some embodiments, the nucleic acid comprises a transposon containing a transgene, eg, a nucleic acid encoding the CAR described herein. In some embodiments, the nucleic acid comprises a transposon containing a transgene (eg, a nucleic acid encoding the CAR described herein) as well as a nucleic acid sequence encoding a transposase enzyme. In other embodiments, there is provided, for example, a dual plasmid system, eg, a two-nucleic acid system in which the first plasmid contains a transposon containing a transgene and the second plasmid contains a nucleic acid sequence encoding a transposase enzyme. For example, the first and second nucleic acids are co-delivered to the host cell.

In some embodiments, the CAR-expressing cells described herein, such as T cells or NK cells, are nucleases (eg, zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), CRISPR /. It is produced using a combination of SBTS and gene insertion with gene editing, using a Cas system or an engineered meganuclease re-engineered homing endonuclease).

In some embodiments, the use of non-viral delivery methods allows for reprogramming of cells, such as T cells or NK cells, and direct injection of cells into a subject. Advantages of non-viral vectors include, but are not limited to, easy and relatively inexpensive production of sufficient amounts required to fit the patient population, lack of stability and immunogenicity during storage.

Sources of cells Sources of cells, such as immune effector cells (eg, T cells or NK cells), can be obtained from the subject prior to proliferation and genetic modification or other alterations. The term "subject" is intended to include living organisms (eg, mammals) capable of eliciting an immune response. Examples of subjects include humans, dogs, cats, mice, rats and transgenic species thereof. T cells can be obtained from a number of sources including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymic tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue and tumors.

In one aspect of the invention, any number of immune effector cell (eg, T cell or NK cell) systems available in the art can be used. In one aspect of the invention, T cells can be obtained from a unit of blood taken from a subject using any technique known to those of skill in the art, such as Ficoll ™ isolation. In certain preferred embodiments, cells from the individual's circulating blood are obtained by apheresis. Apheresis products typically include T cells, monocytes, granulocytes, B cells, lymphocytes including other nucleated leukocytes, erythrocytes and platelets. In one embodiment, cells harvested by apheresis are washed to remove plasma fractions and the cells are placed in a suitable buffer or medium for subsequent processing processes. In one embodiment of the invention, cells are washed with phosphate buffered saline (PBS). In another embodiment, the wash solution may lack calcium, magnesium, or many, if not all, divalent cations.

The initial activation process in the absence of calcium can lead to enhanced activation. As will be readily appreciated by those of skill in the art, the cleaning process can be accomplished by semi-automated "flow-through" centrifugation according to the manufacturer's instructions (eg, Cobe 2991 cell processor, Baxter CytoMate or Haemonetics Cell Saver 5). After washing, cells can be resuspended in a variety of biocompatible buffers, such as aqueous saline with or without Ca, Mg PBS, PlasmaLite A or other buffers. Alternatively, the unwanted elements of the apheresis sample can be removed and the cells can be resuspended directly in the culture medium.

The methods of the present application can utilize culture medium conditions containing 5% or less, eg 2%, human AB serum, and known culture medium conditions and compositions such as Smith et al. , "Ex vivo expansion of human T cells for adaptive immunotherapy using the novel Xeno-free CTS Immuno Cell Serum Replethement4, It is recognized that those described in 2014.31 can be used.

In one embodiment, T cells are isolated from peripheral blood lymphocytes by lysing erythrocytes and depleting monocytes, for example by centrifugation with a PERCOLTM gradient or by countercurrent 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, the T cells are subjected to anti-CD3 / anti-CD28 (eg, 3x28) conjugated beads such as DYNABEADS® M-450 CD3 / CD28 T for a sufficient time for positive selection of the desired T cells. Isolate by incubation. In one aspect, 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. Long incubation time to isolate T cells in all situations where there are fewer T cells compared to other cell types, such as isolating tumor infiltrating lymphocytes (TIL) from tumor tissue or immunocompromised individuals. Can be used. In addition, the use of long incubation times can increase the efficiency of CD8 + T cell capture. Therefore, T cells by simply shortening or lengthening the time to bind T cells to CD3 / CD28 beads and / or increasing or decreasing the bead-to-T cell ratio (as described herein). Subpopulations can be preferentially selected at the start of culture or at other times during the process. In addition, by increasing or decreasing the anti-CD3 and / or anti-CD28 antibody ratio on the beads or other surface, the T cell subpopulation may be preferentially selected at the start of culture or at other times during the process. .. Those skilled in the art will recognize that multiple selections can 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 subject to further selection.

T cell population enrichment by negative selection can be achieved by a combination of antibodies that direct a surface marker unique to the negatively selected cells. One method is cell selection and / or selection by negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies directed at cell surface markers present in cells selected negatively. For example, to enrich CD4 + cells by negative selection, monoclonal antibody cocktails typically include 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 +. Instead, in one embodiment, T-regulatory cells are depleted by anti-C25 conjugated beads or other similar selection methods.

The methods described herein use, for example, a specific subpopulation of immune effector cells, eg, a T-regulatory cell depleted population, CD25 + depleted cells, using, for example, the negative selection technique described herein. It may include selection of T cells. Preferably, the T-regulatory depleted cell population comprises less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, and less than 1% CD25 + cells.

In some embodiments, T-regulatory cells, such as CD25 + T cells, are removed from the population using an anti-CD25 antibody or fragment thereof or a CD25 binding ligand, IL-2. In some embodiments, an anti-CD25 antibody or fragment thereof or a CD25 binding ligand is conjugated to a substrate, such as beads, or otherwise coated on a substrate, such as beads. In some embodiments, the anti-CD25 antibody or fragment thereof is conjugated to a substrate described herein.

In some embodiments, T-regulatory cells, such as CD25 + T cells, are removed from the population using a CD25 depleting agent from Miltenyi ™. In some embodiments, the cell-to-CD25 depleting agent ratio is 1e7 cells vs. 20 μL, or 1e7 cells vs. 15 μL, or 1e7 cells vs. 10 μL, or 1e7 cells vs. 5 μL, or 1e7 cells vs. 2.5 μL, or 1e7 cells. It is 1.25 μL. In some embodiments, for example T-regulatory cells, such as CD25 + 500 million cells / ml for depletion are used. In a further embodiment, a cell concentration of 600 million, 700 million, 800 million or 900 million cells / ml is used.

In some embodiments, the immune effector cell population to be depleted comprises approximately 6 × 10 ⁹ CD25 + T cells. In another embodiment, the immune effector cell population to be depleted comprises about 1 × 10 ⁹ to 1 × 10 ¹⁰ (and any integer value in between) CD25 + T cells. In some embodiments, the resulting T-regulatory depleted cell population is 2 × 10 ⁹ T-regulatory cells, such as CD25 + cells or less (eg, 1 × 10 ⁹ , 5 × 10 ⁸ , 1 × 10 ⁸ ). 5 × 10 ⁷ , 1 × 10 ⁷ or less CD25 + cells).

In some embodiments, T-regulatory cells, such as CD25 + cells, are removed from the population using the CliniMAC system with a depleted tubing set, such as tubing 162-01. In some embodiments, the CliniMAC system is run in a depletion setting such as DEPLETION 2.1.

We do not want to be bound by a particular theory, but a decrease in the level of negative regulators of immune cells in the subject before affelesis or during the production of CAR-expressing cell products (eg, the number of unwanted immune cells, eg _TREG cells). (Reduction of) may reduce the subject's risk of recurrence. For example, methods of depleting _TREG cells are known in the art. For example, methods of reducing _TREG cells include, but are not limited to, cyclophosphamide, anti-GITR antibodies (anti-GITR antibodies described herein), CD25 depletion and combinations thereof.

In one embodiment, the production method comprises reducing the number of _TREG cells (eg, depletion) prior to the production of CAR-expressing cells. For example, the process involves contacting a sample, eg, an afferesis sample, with an anti-GITR antibody and / or an anti-CD25 antibody (or fragment thereof or CD25 binding ligand) to produce CAR-expressing cells (eg, T cells, NK cells). Includes depleting T _REG cells prior to production.

In one embodiment, the subject is pretreated with one or more treatments that reduce _TREG cells prior to cell harvesting for the production of CAR-expressing cell products, whereby the subject again requires CAR-expressing cell treatment. Reduce risk. In one embodiment, methods of reducing _TREG cells include, but are not limited to, administration of one or more of cyclophosphamide, anti-GITR antibody, CD25 depletion or a combination thereof to a subject. Administration of one or more of cyclophosphamide, anti-GITR antibody, CD25 depletion or a combination thereof can be performed before, during or after infusion of CAR-expressing cellular products.

In one embodiment, the subject is pretreated with cyclophosphamide prior to harvesting cells for the production of CAR-expressing cell products, thereby reducing the risk of the subject relapsed to CAR-expressing cell treatment. In one embodiment, the subject is pretreated with an anti-GITR antibody prior to harvesting cells for the production of CAR-expressing cell products, thereby reducing the risk of the subject relapsed to CAR-expressing cell treatment.

In some embodiments, the cell population to be removed is not regulatory T cells or tumor cells, but cells that have other negative effects on the proliferation and / or function of CART cells, such as CD14, CD11b, CD33, CD15 or Cells that express other markers expressed by potential immunosuppressive cells. In some embodiments, such cells are intended to be removed simultaneously with regulatory T cells and / or tumor cells, or after said depletion, or in another order.

The methods described herein may include two or more selection steps, eg, two or more depletion steps. Enrichment of the T cell population by negative selection can be achieved, for example, by a combination of antibodies that direct a surface marker unique to the negatively selected cells. One method is cell selection and / or selection by negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies directed at cell surface markers present in cells selected negatively. For example, to enrich CD4 + cells by negative selection, the monoclonal antibody cocktail may include antibodies against CD14, CD20, CD11b, CD16, HLA-DR and CD8.

The method described herein further comprises removal from a cell population expressing a tumor antigen, eg, a tumor antigen free of CD25, such as CD19, CD30, CD38, CD123, CD20, CD14 or CD11b, thereby CAR. For example, T-regulated depletion suitable for the expression of CAR described herein, such as CD25 + depletion and tumor antigen depleted cell populations is provided. In some embodiments, tumor antigen-expressing cells are removed simultaneously with T-regulatory, eg, CD25 + cells. For example, an anti-CD25 antibody or fragment thereof and an anti-tumor antigen antibody or fragment thereof can be bound to the same substrate, eg, beads, and can be used for cell removal, or an anti-CD25 antibody or fragment thereof and an anti-tumor antigen antibody or its fragment. The fragment can be attached to another bead and the mixture can be used for cell removal. In other embodiments, removal of T-regulatory cells such as CD25 + cells and removal of tumor antigen-expressing cells is sequential and can occur, for example, in any order.

It involves removal from one or more populations of checkpoint inhibitors, such as cells expressing the checkpoint inhibitors described herein, such as PD1 + cells, LAG3 + cells and TIM3 + cells, thereby T-regulated depletion, eg. Also provided are methods of providing CD25 + depleted cells and checkpoint inhibitor depleted cells such as PD1 +, LAG3 + and / or TIM3 + depleted cell populations. Exemplary checkpoint inhibitors are PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (eg 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. include. In embodiments, the checkpoint inhibitor is PD1 or PD-L1. In some embodiments, checkpoint inhibitor-expressing cells are eliminated simultaneously with T regulatory, eg, CD25 + cells. For example, an anti-CD25 antibody or fragment thereof and an anti-checkpoint inhibitor antibody or fragment thereof can be bound to the same beads and used for cell removal, or an anti-CD25 antibody or fragment thereof and an anti-checkpoint inhibitor antibody or The fragment can be attached to another bead and the mixture can be used for cell removal. In other embodiments, removal of T-regulatory cells such as CD25 + cells and removal of checkpoint inhibitor-expressing cells are sequential and can occur, eg, in any order.

In some embodiments, IFN-γ, TNFα, IL-17A, IL-2, IL-3, IL-4, GM-CSF, IL-10, IL-13, Granzyme B and perforin or other suitable. You can select a T cell population that expresses a molecule, eg, one or more of other cytokines. The method for screening for cell expression can be determined, for example, by the method described in WO 2013/126712.

The concentration of cells and surfaces (eg, particles such as beads) can vary in order to isolate the desired cell population by positive or negative selection. In one embodiment, it may be desired to significantly reduce the volume at which the beads and the cells are mixed together (eg, increase the cell concentration) to ensure maximum contact between the cells and the beads. For example, in one embodiment, a concentration of 2 billion cells / ml is used. In one embodiment, a concentration of 1 billion cells / ml is used. In a further embodiment, over 100 million cells / ml are used. In a further embodiment, a cell concentration of 10 million, 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 45 million or 50 million cells / ml is used. In a further embodiment, a concentration of 75 million, 80 million, 85 million, 90 million, 95 million or 100 million cells / ml of cells is used. In a further embodiment, a concentration of 125 million or 150 million cells / ml can be used. High concentrations can be used to increase cell yield, cell activation and cell proliferation. In addition, the use of high cell concentrations is more efficient from cells that can weakly express the target antigen of interest, such as CD28-negative T cells, or from samples in which many tumor cells are present (eg, leukemic blood, tumor tissue, etc.). Enables targeted capture. Such cell populations may have therapeutic value and are desirable to obtain. For example, the use of high concentration cells allows for more efficient selection of CD8 + T cells, which normally have weak CD28 expression.

In related embodiments, it may be desirable to use low concentrations of cells. Significant dilution of the mixture of T cells with the surface (eg, particles such as beads) minimizes the interaction of the particles with the cells. It selects cells that express high amounts of the desired antigen that binds to the particles. For example, CD4 + T cells express high levels of CD28 and are more efficiently captured than CD8 + T cells at diluted concentrations. In one embodiment, the concentration of cells used is 5 × 10e6 / ml. In other embodiments, the concentration used can be from about 1 × 10 ⁵ / ml to 1 × 10 ⁶ / ml and any integer value in between.

In other embodiments, cells can be incubated in a rotator at various lengths of time, at various rates, at 2-10 ° C. or at room temperature.

T cells for irritation can also be frozen after the wash step. Without wishing to be bound by theory, the freezing and subsequent thawing steps provide a more uniform product by removing granulocytes and to some extent monocytes from the cell population. After a wash step to remove plasma and platelets, the cells can be suspended in a frozen solution. Many frozen solutions and parameters are known in the art and are useful in this context, but one method is PBS containing 20% DMSO and 8% human glucose albumin or 10% Dextran 40 and 5% dextrose. 20% human serum albumin and 7.5% DMSO or 31.25% Plasmalyte-A, 31.25% dextrose 5%, 0.45% NaCl, 10% Dextran 40 and 5% dextrose, 20% human glucose albumin and 7 Use a culture medium containing .5% DMSO or other suitable cell freezing medium containing, for example, Hespan and PlasmaLite A, then freeze the cells to −80 ° C. at a rate of 1 ° / min and store the liquid nitrogen in the gas phase. Store in tank. Other methods of controlled freezing as well as direct -20 ° C or uncontrolled freezing of liquid nitrogen can also be used.

In one embodiment, cryopreserved cells are thawed and washed as described herein and rested at room temperature for 1 hour prior to activation using the methods of the invention.

In connection with the present invention, it is also contemplated to collect blood samples or apheresis products from the subject in the period prior to the time when the proliferating cells described herein may be needed. Thus, a source of proliferating cells can be harvested at any time required and the desired cells, such as immune effector cells, eg T cells or NK cells, can be obtained from cell therapies such as those described herein, eg T cells. Any number of diseases and conditions that may benefit from treatment can be isolated and frozen for subsequent use in cell therapy, eg, T cell therapy. In one embodiment, a blood sample or apheresis is generally taken from a healthy subject. In one embodiment, a blood sample or apheresis is generally taken from a healthy subject at risk of developing the disease but not yet developing the disease, and the cells of interest are isolated and frozen for subsequent use. In one embodiment, immune effector cells (eg, T cells or NK cells) can be subsequently proliferated, frozen and used. In one embodiment, a sample is taken from the patient immediately after the diagnosis of the particular disease described herein, but prior to any treatment. In a further embodiment, immunosuppressive agents such as natalismab, efarizumab, antiviral agents, chemotherapeutic agents, radiation, cyclosporine, azathioprine, methotrexate, mycophenolate and FK506, CAMPATH, anti-CD3 antibodies, cytoxan, fludalabine, cyclosporine, FK506, From blood samples or aferesis from subjects prior to any number of relevant treatment modalities, including but not limited to treatment with antibodies or other immunosuppressive agents such as rapamycin, mycophenolic acid, steroids, FR901228 and irradiation. Isolate cells.

In a further aspect of the invention, T cells are obtained directly from the patient after treatment leaving functional T cells in the subject. The quality of the resulting T cells is optimal for the ability of Exvivo to proliferate in certain cancer treatments, especially after treatment with drugs that damage the immune system, immediately after treatment, and usually between the time the patient recovers from the treatment. It has been observed that it can be obtained or improved. Similarly, after ex vivo manipulation using the methods described herein, these cells may be in a favorable state for engraftment and in vivo proliferation enhancement. Therefore, in connection with the present invention, it is contemplated to collect blood cells, including T cells, dendritic cells or other cells of the hematopoietic cell lineage, during this recovery phase. Further, in one embodiment, mobilization (eg, mobilization in GM-CSF) and conditioning regimens are used to condition conditions that are favorable for the regrowth, recirculation, regeneration and / or proliferation of a particular cell type. In particular, it can be formed on the subject within a defined time frame after treatment. Examples of cell types include T cells, B cells, dendritic cells and other cells of the immune system.

In some embodiments, cells expressing the immune effector CAR molecule, eg, the CAR molecule described herein, are obtained from a subject receiving a low immunopotentiating dose of mTOR inhibitor. In one embodiment, a population of immune effector cells engineered to express CAR, such as T cells, is the subject or PD1-negative immune effector cells harvested from the subject, such as T cell levels or PD1-negative immune effector cells, such as T. Collect after sufficient time or after sufficient dose of low immunopotentiating dose mTOR inhibitor so that the ratio of cell / PD1-positive immune effector cells, eg T cells, increases at least transiently.

In other embodiments, immune effector cells that are engineered or engineered to express CAR, such as T cell populations, are subjected to PD1-negative immune effector cells, such as increasing the number of T cells or PD1-negative immune effector cells. It can be treated with Exvivo, for example by contacting with T cells / PD1-positive immune effector cells, eg, an amount of mTOR inhibitor that increases the ratio of T cells.

In some embodiments, the T cell population is deficient in diacylglycerol kinase (DGK). DGK-deficient cells are cells that do not express DGK RNA or protein, or have reduced or inhibited DGK activity. DGK-deficient cells can be produced by genetic methods for reducing or blocking DGK expression, such as administration of RNA interfering agents such as siRNA, shRNA, miRNA. Alternatively, DGK-deficient cells can be produced by treatment with the DGK inhibitors described herein.

In some embodiments, the T cell population is Icarus deficient. Icarus-deficient cells include cells that do not express Icarus RNA or protein, or have reduced or inhibited Icarus activity, and Icarus-deficient cells are genetic methods for reducing or blocking Icarus expression, such as RNA interfering agents. For example, it can be produced by administration of siRNA, shRNA, or miRNA. Alternatively, Icarus-deficient cells can be produced by treatment with an Icarus inhibitor, such as lenalidomide.

In embodiments, the T cell population is DGK deficient and Icarus deficient, eg, does not express DGK and Icarus, or has reduced or inhibited DGK and Icarus activity. Such DGK and Icarus-deficient cells can be produced by any of the methods described herein.

In one embodiment, NK cells are obtained from the subject. In another embodiment, the NK cell is an NK cell line, eg, an NK-92 cell line (Conkwest).

Homogeneous CAR
In the embodiments described herein, the immune effector cells can be alloimmunity effector cells, such as T cells or NK cells. For example, the cell is an allogeneic T cell that lacks expression of, for example, a functional T cell receptor (TCR) and / or a human leukocyte antigen (HLA), such as HLA class I and / or HLA class II.

T cells lacking a functional TCR are, for example, engineered to not express any functional TCR on their surface and not to express one or more subunits containing the functional TCR (eg, TCRα, TCRβ). , TCRγ, TCRdelta, TCR epsilon and / or TCR zeta expression is absent (or is engineered to show reduced expression), or can be engineered to produce minimal functional TCR on its surface. Alternatively, T cells can express a substantially impaired TCR, for example by expression of one or more mutated or truncated forms of subunits of the TCR. The term "substantially impaired TCR" means that this TCR does not elicit a harmful immune response in the host.

The T cells described herein can be engineered, for example, to not express functional HLA on their surface. For example, the T cells described herein can be engineered so that cell surface expressed HLA, such as HLA class 1 and / or HLA class II, is downregulated. In some embodiments, downregulation of HLA may be accompanied by diminished or eliminated expression of β-2 microglobulin (B2M). In one real embodiment, T cells may lack functional TCR and functional HLA, such as HLA class I and / or HLA class II.

Modified T cells lacking expression of functional TCR and / or HLA can be obtained by any suitable means, including knockout or knockdown of one or more subunits of TCR or HLA. For example, T cells are knocked down of TCR and / or HLA using siRNA, shRNA, clustered evenly spaced short repetitive sequences (CRISPR) transcriptional activator-like effector nucleases (TALENs) or zinc finger endonucleases (ZFNs). May include.

In some embodiments, the allogeneic cell can be a cell that does not or expresses at a low level an inhibitory molecule, eg, a cell engineered by any of the methods described herein. For example, the cell can be, for example, a cell that does not express an inhibitory molecule or expresses an inhibitory molecule at a low level, which can reduce the ability of CAR expressing cells to initiate an immune effector response. Examples of inhibitory molecules are PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (eg 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. Inhibition of inhibitory molecules by inhibition at the DNA, RNA or protein level can optimize CAR-expressing cell performance. In embodiments, eg, inhibitory nucleic acids described herein, such as inhibitory nucleic acids such as dsRNAs such as siRNA or shRNA, clustered evenly spaced short repetitive sequences (CRISPRs), transcriptional activator-like effector nucleases (TALENs). Alternatively, zinc finger endonuclease (ZFN) can be used.

SiRNA and shRNA for inhibiting TCR or HLA
In one embodiment, TCR expression and / or HLA expression is expressed in cells, such as TCR and / or HLA in T cells, and / or inhibitory molecules described herein (eg, PD1, PD-L1, PD-L2). , CTLA4, TIM3, CEACAM (eg CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7 -Can be inhibited using siRNA or shRNA targeting nucleic acids encoding H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine and TGFRβ).

Expression of siRNA and shRNA in T cells can be achieved using any conventional expression system, such as, for example, a lentivirus expression system.

Representative shRNAs that downregulate the expression of elements of TCR are described, for example, in US Patent Application Publication No. 2012/0321667. Representative siRNAs and shRNAs that downregulate the expression of HLA class I and / or HLA class II genes are disclosed, for example, in US Patent Application Publication No. 2007/0036773.

CRISPR to inhibit TCR or HLA
As used herein, "CRISPR", or "CRISPR for TCR and / or HLA", or "CRISPR for inhibiting TCR and / or HLA" is a series of clustered evenly spaced short palindromic repeats or this. Refers to a system containing a series of iterations such as. As used herein, "Cas" refers to a CRISPR-related protein. The "CRISPR / Cas" system is a TCR and / or HLA gene and / or an inhibitory molecule described herein (eg, PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (eg, CEACAM-1, etc.). 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), Refers to a system derived from CRISPR and Cas that can be used for expression suppression or mutation of KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine and TGFRβ).

The naturally occurring CRISPR / Cas system is found in approximately 40% of sequenced eubacterial genomes and 90% of sequenced archaea. Grissa et al. (2007) BMC Bioinformatics 8: 172. This system is a prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and phage, providing a form of acquired immunity. Barrangou et al. (2007) Science 315: 1709-1712; Marragini et al. (2008) Science 322: 1843-1845.

The CRISPR / Cas system has been modified for use in gene editing (silencing, enhancing or altering specific genes) in eukaryotes such as mice or primates. Widenheft et al. (2012) Nature 482: 331-8. This is achieved by introducing into eukaryotic cells a plasmid containing a specially designed CRISPR and one or more suitable Cass.

The CRISPR sequence, sometimes referred to as the CRISPR locus, contains alternating repeats and spacers. In naturally occurring CRISPR / spacers contain sequences such as plasmids or phage sequences that are normally foreign to bacteria, and in TCR and / or HLA CRISPR / Cas systems, spacers are derived from the TCR or HLA gene sequence. ..

RNA from the CRISPR locus is constitutively expressed and processed into small RNA by the Cas protein. These include spacers with adjacent repeat sequences. RNA leads to suppression of expression of other Cas proteins against exogenous genetic elements at the RNA or DNA level. Horvast et al. (2010) Science 327: 167-170; Makarova et al. (2006) Biology Direct 1: 7. Spacers therefore serve as templates for RNA molecules, similar to siRNA. Pennisi (2013) Science 341: 833-836.

As naturally occurring in many different types of bacteria, the exact arrangement and structure of CRISPR, the function and number of Cas genes and their products vary somewhat from species to species. Haft et al. (2005) PLoS Comput. Biol. 1: e60; Kunin et al. (2007) Genome Biol. 8: R61; Mojica et al. (2005) J.M. Mol. Evol. 60: 174-182; Volotin et al. (2005) Microbiol. 151: 2551-2561; Paulcel et al. (2005) Microbiol. 151: 653-663; and Stern et al. (2010) Trends. Genet. 28: 335-340. For example, the Cse (Cas subtype, E. coli) protein (eg, CasA) forms a functional complex, Cascade, which processes the CRISPR RNA transcript into spacer-repetition units held by Cascade. Brons et al. (2008) Science 321: 960-964. In other prokaryotes, Cas6 processes CRISPR transcripts. CRISPR-based phage inactivation in E. coli requires Cascade and Cas3, but not Cas1 or Cas2. The Cmr (Cas RAMP module) protein in Pyrococcus furiosus and other prokaryotes forms a functional complex with small CRISPR RNA, which recognizes and cleaves complementary target RNA. The simpler CRISPR system relies on the protein Cas9, a nuclease with a biactive cleavage site for each strand of the double helix. A combination of Cas9 and modified CRISPR locus RNA can be used in the system for gene editing. Pennisi (2013) Science 341: 833-836.

The CRISPR / Cas system can be used to edit the TCR and / or HLA gene and introduce (base pair addition or deletion) or immature termination, thus expressing TCR and / or HLA. Reduce. Alternatively, the CRISPR / Cas system can be used like RNA interference to turn off the TCR and / or HLA genes in a reversible manner. In mammalian cells, for example, RNA can direct the Cas protein to the TCR and / or HLA promoter, sterically blocking RNA polymerase.

Artificial CRISPR / Cas that inhibits TCR and / or HLA using techniques known in the art, such as those described in US Patent Application Publication No. 201400689779 and Kong (2013) Science 339: 819-823. Can produce a system. For example, Tsai (2014) Nature Biotechnology. , 32: 6 569-576, US Pat. No. 8,871,445; No. 8,856,406; No. 8,795,965; No. 8,771,945. Other artificial CRISPR / Cas systems known in the art that inhibit TCR and / or HLA, such as those described in the specification; and the same No. 8,697,359, can also be produced.

TALEN to inhibit TCR and / or HLA
"TALEN", or "TALEN against HLA and / or TCR", or "TALEN for inhibiting HLA and / or TCR" is the HLA and / or TCR gene, and / or the inhibitory molecule described herein. (For example, PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (eg, CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, Artificial nucleases that can be used to edit CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine and TGFRβ). Refers to a transcriptional activator-like effector nuclease.

TALEN is artificially produced by fusing a TAL effector DNA binding domain and a DNA cleavage domain. The transcriptional activator-like effect (TALE) can be engineered to bind to any desired DNA sequence, including a portion of the HLA or TCR gene. By combining the engineered TALE with the DNA cleavage domain, restriction enzymes specific for any desired DNA sequence, including HLA or TCR sequences, can be produced. They can then be introduced into cells where they can be used for genomic editing. Boch (2011) Nature Biotech. 29: 135-6; and Boch et al. (2009) Science 326: 1509-12; Moscow et al. (2009) Science 326: 3501.

TALE is a protein secreted by the Zantomonas bacterium. The DNA binding domain contains repeated highly conservative 33-34 amino acid sequences, except for the 12th and 13th amino acids. These two locations are highly variable and show a strong correlation with specific nucleotide recognition. They can therefore be engineered to bind the desired DNA sequence.

To produce TALEN, the TALE protein is fused with a wild-type or mutant FokI endonuclease, nuclease (N). Several mutations have been made to FokI for use in TALEN, including, for example, improved cleavage specificity or activity. Cermak et al. (2011) Nucl. Acids Res. 39: e82; Miller et al. (2011) Nature Biotechnology. 29: 143-8; Hockemeyer et al. (2011) Nature Biotechnology. 29: 731-734; Wood et al. (2011) Science 333: 307; Doyon et al. (2010) Nature Methods 8: 74-79; Szczepek et al. (2007) Nature Biotechnology. 25: 786-793; and Guo et al. (2010) J.M. Mol. Biol. 200: 96.

The FokI domain functions as a dimer and requires two constructs with unique DNA binding domains for sites in the target genome with proper orientation and spacing. Both the number of amino acid residues between the TALE DNA binding domain and the FokI cleavage domain and the number of bases between the two individual TALEN binding sites are believed to be important parameters for achieving high levels of activity. Miller et al. (2011) Nature Biotechnology. 29: 143-8.

HLA or TCR TALEN can be used intracellularly to produce double chain cleavage (DSB). Mutations can be introduced at the site of cleavage if the repair mechanism improperly repairs the cleavage by non-homologous end binding. For example, improper repair can introduce frameshift mutations. Alternatively, foreign DNA can be introduced into cells with TALEN, and the sequence and chromosomal sequence of the foreign DNA allows this method to correct for defects in the HLA or TCR gene, or to introduce such defects in the wt HLA, or TCR gene. It can be used, thus reducing the expression of HLA or TCR.

Sequence-specific TALENs in HLA or TCR can be constructed using any method known in the art, including various schemes using modular elements. Zhang et al. (2011) Nature Biotechnology. 29: 149-53; Gibler et al. (2011) PLoS ONE 6: e19509.

Zinc finger nuclease "ZFN" or "zinc finger nuclease" to inhibit HLA and / or TCR, or "ZFN to HLA and / or TCR", or "ZFN to inhibit HLA and / or TCR" , HLA and / or TCR genes and / or inhibitory molecules described herein (eg, PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (eg, 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), which is an artificial nuclease that can be used to edit zinc finger nucleases.

Like TALENs, ZFNs include FokI nuclease domains (or derivatives thereof) fused to DNA binding domains. For ZFNs, the DNA binding domain comprises one or more zinc fingers. Carroll et al. (2011) Genetics Society of America 188: 773-782; and Kim et al. (1996) Proc. Natl. Acad. Sci. USA 93: 1156-1160.

Zinc fingers are small protein structural motifs that are stabilized by one or more zinc ions. The zinc finger can include, for example, Cys2His2 and can recognize an approximately 3bp sequence. A variety of zinc fingers of known specificity can be combined to produce a multi-finger polypeptide that recognizes sequences of approximately 6bp, 9bp, 12bp, 15bp or 18bp. A variety of selection and modular assembly techniques for producing specific sequence-recognizing zinc fingers (and combinations thereof), including phage display, yeast one-hybrid, bacterial one-hybrid and two-hybrid, and mammalian cells. It is available.

Like TALENs, ZFNs must be dimerized to cleave DNA. Therefore, it is necessary for ZFN pairs to target non-palindromic DNA sites. Each ZFN in 2 must have its nuclease properly separated and attached to the reverse strand of DNA. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA 95: 10570-5.

Also, like TALENs, ZFNs can create double-strand breaks in DNA, which can create frameshift mutations when improperly repaired, resulting in reduced expression and amount of HLA and / or TCR in cells. To reach. ZFNs can also be used with homologous recombination due to mutations in the HLA or TCR genes.

Sequence-specific ZFNs in HLA and / or TCR can be constructed using any method known in the art. For example, Provide (2011) Nature Med. 18: 807-815; Torikai (2013) Blood 122: 1341-1349; Cathomen et al. (2008) Mol. The. 16: 1200-7; Guo et al. (2010) J.M. Mol. Biol. 400: 96; US Patent Application Publication No. 2011/015897; and US Patent Application Publication No. 2012/0060230.

Telomerase expression Although not bound by any particular theory, in one embodiment therapeutic T cells have short persistence in patients due to shortened telomeres in T cells and thus use the telomerase gene. Transfection can prolong T cell telomeres and improve T cell persistence in patients. See Carl June, "Adaptive T cell therapy for cancer in the clinic", Journal of Clinical Investigation, 117: 1466-1476 (2007). Thus, in one embodiment, immune effector cells, such as T cells, ectopically express a telomerase subunit, such as a catalytic subunit of telomerase, such as TERT, such as hTERT. In some embodiments, the present disclosure provides a method of producing CAR-expressing cells comprising contacting the cell with a telomerase subunit, eg, a catalytic subunit of telomerase, eg, TERT, eg, a nucleic acid encoding hTERT. .. The cells may be contacted with the nucleic acid at the same time or after contact with the construct encoding the CAR.

In one aspect, the present disclosure relates to a method of producing an immune effector cell population (eg, T cells, NK cells). In one embodiment, the method provides an immune effector cell population (eg, T cells or NK cells), the immune effector cell population is contacted with a nucleic acid encoding CAR, and the immune effector cell population is telomerase subunit. For example, it involves contacting a nucleic acid encoding hTERT under conditions that allow expression of CAR and telomerase.

In one embodiment, the nucleic acid encoding the telomerase subunit is DNA. In one embodiment, the nucleic acid encoding the telomerase subunit comprises a promoter capable of driving the expression of the telomerase subunit.

In one embodiment, the hTERT has the amino acid sequence of GenBank Protein ID AAC5124.1 (Meyerson et al., "hEST2, the Putative Human Telomerase Catalitic Subunition "Cell Volume 90, Issue 4,22 August 1997, Pages 785-795).

In one embodiment, hTERT has at least 80%, 85%, 90%, 95%, 96 ^, 97%, 98% or 99% identical sequence to the sequence of SEQ ID NO: 284. In one embodiment, hTERT has the sequence of SEQ ID NO: 284. In one embodiment, hTERT comprises a deletion at the N-terminus, C-terminus or both (eg, 5, 10, 15, 20 or 30 amino acids or less). In one embodiment, hTERT comprises a transgenic amino acid sequence (eg, 5, 10, 15, 20 or 30 amino acids or less) at the N-terminus, C-terminus or both.

Activation and Proliferation of T Cells T cells are described, for example, in US Pat. Nos. 6,352,694; 6,534,055; 6,905,680; , 692,964; 5,858,358; 6,888,466; 6,905,681; 7,144,575; No. 7,067,318; No. 7,172,869; No. 7,232,566; No. 7,175,843; No. 5, 883,223; 6,905,874; 6,797,514; 6,867,041; and US Patent Application Publication No. 20060121005. Can generally be activated and propagated using the methods described in.

In general, T cells of the present invention can be proliferated by contacting the surface of the T cell to which a drug that stimulates a CD3 / TCR complex binding signal is bound with a ligand that stimulates a co-stimulatory molecule on the surface of the T cell. In particular, the T cell population is exposed to contact with an anti-CD3 antibody or an antigen-binding fragment thereof or an anti-CD2 antibody immobilized on the surface or with a protein kinase C activator (eg, bryostatin) in combination with a calcium ionophore. It can be irritating as described herein. A ligand that binds to the accessory molecule is used for co-stimulation of the accessory molecule on the surface of the T cell. For example, a T cell population can be contacted with anti-CD3 and anti-CD28 antibodies under conditions suitable for stimulating T cell proliferation. Anti-CD3 and anti-CD28 antibodies can be used to stimulate the proliferation of CD4 + T cells or CD8 + T cells. Examples of anti-CD28 antibodies include 9.3, BT3, XR-CD28 (Diaclone, Besancon, France) and can be used like other methods commonly known in the art (Berg et al., Transrant). Proc. 30 (8): 3975-3977, 1998; Haanen et al., J. Exp. Med. 190 (9): 13191328, 1999; Garland et al., J. Immunol Meth. 227 (1-2) :. 53-63, 1999).

In one embodiment, the primary and co-stimulating signals for T cells can be provided by different protocols. For example, the agent providing each signal can bind to the surface even in solution. When attached to a surface, the agent may bind to the same surface (ie, "cis" form) or another surface (ie, "trans" form). Instead, one drug may bind to the surface and the other may be in solution. In one embodiment, the agent providing the co-stimulation signal binds to the cell surface and the agent providing the primary activation signal is in solution or binds to the surface. In one embodiment, both agents can be in solution. In one embodiment, the agent is in an insoluble form and can be crosslinked to a surface such as a cell expressing an Fc receptor or an antibody or other binder that binds to the agent. In this regard, see, for example, US Patent Application Publication Nos. 20040101519 and 20060034810 for artificial antigen presenting cells (aAPC) intended for T cell activation and proliferation in the present invention. ..

In one embodiment, the two agents are immobilized on the beads with the same beads, i.e. "cis" or another bead, i.e. "trans". As an example, the agent providing the primary activation signal is an anti-CD3 antibody or an antigen-binding fragment thereof, the agent providing a co-stimulation signal is an anti-CD28 antibody or an antigen-binding fragment thereof, and both agents have equal molecular weights. Is co-immobilized to the same bead. In one embodiment, 1: 1 ratio antibodies bound to beads for CD4 + T cell proliferation and T cell proliferation are used. In one aspect of the invention, the ratio of anti-CD3: CD28 antibody bound to the beads is used so that growth of T cell growth is observed as compared to growth observed using a 1: 1 ratio. .. In one particular embodiment, an increase of about 1 to about 3 times is observed compared to the growth observed using the 1: 1 ratio. In one embodiment, the CD3: CD28 antibody ratio bound to the beads ranges from 100: 1 to 1: 100 and all integer values in between. In one aspect of the invention, more anti-CD28 antibodies are bound to the particles than anti-CD3 antibodies, i.e., the CD3: CD28 ratio is less than 1. In one aspect of the invention, the anti-CD28 antibody-to-CD3 antibody ratio bound to the beads is greater than 2: 1. In one particular embodiment, a 1: 100 CD3: CD28 ratio antibody bound to the beads is used. In one embodiment, a 1:75 CD3: CD28 ratio antibody bound to the beads is used. In a further embodiment, a 1:50 CD3: CD28 ratio antibody bound to the beads is used. In one embodiment, a 1:30 CD3: CD28 ratio antibody bound to the beads is used. In certain preferred embodiments, a 1:10 CD3: CD28 ratio antibody bound to the beads is used. In one embodiment, a 1: 3 CD3: CD28 ratio antibody bound to the beads is used. In a further embodiment, a 3: 1 CD3: CD28 ratio antibody bound to the beads is used.

Particle-to-cell ratios of 1: 500 to 500: 1 and any integer value in between can be used for stimulation of T cells or other target cells. As will be readily recognized by those of skill in the art, the particle-to-cell ratio may depend on the particle size relative to the target cell. For example, small size beads can bind only to a small number of cells, and large beads can bind to many. In one embodiment, the cell-to-particle ratio ranges from 1: 100 to 100: 1 and any integer value in between, and in a further embodiment a ratio consisting of 1: 9 to 9: 1 and any integer value in between. Can be used for T cell stimulation. The ratio of anti-CD3 and anti-CD28 binding particles to T cells that results in T cell stimulation can vary as described above, however, certain preferred values are 1: 100, 1:50, 1:40, 1:30, 1:20, 1:10, 1: 9, 1: 8, 1: 7, 1: 6, 1: 5, 1: 4, 1: 3, 1: 2, 1: 1, 2: 1, 3: Containing 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, and 15: 1, one preferred ratio is at least 1: 1 particle to T cell. be. In one embodiment, a particle-to-cell ratio of 1: 1 or less is used. In one particular embodiment, the preferred particle: cell ratio is 1: 5. In a further embodiment, the particle-to-cell ratio can vary from day to day of stimulation. For example, in one embodiment, the particle-to-cell ratio is 1: 1 to 10: 1 on day 1 and additional particles are added to the cells daily or every other day until day 10 with a final ratio of 1: 1 to 10. 1:10 (based on the number of cells in the ratio of addition). In one particular embodiment, the particle-to-cell ratio is 1: 1 on day 1 of stimulation and adjusted to 1: 5 on days 3 and 5 of stimulation. In one embodiment, the particles are added daily or every other day based on a final ratio of 1: 1 on day 1 and 1: 5 on days 3 and 5 of stimulation. In one embodiment, the particle-to-cell ratio is 2: 1 on day 1 of stimulation and adjusted to 1:10 on days 3 and 5 of stimulation. In one embodiment, the particles are added daily or every other day based on a final ratio of 1: 1 on day 1 and 1:10 on days 3 and 5. Those skilled in the art will recognize that a variety of other ratios may be suitable for use in the present invention. In particular, the ratio depends on the particle size as well as the cell size and type. In one embodiment, the most typical ratio for use is around 1: 1, 2: 1 and 3: 1 on day 1.

In a further aspect of the invention, cells such as T cells are combined with drug coated beads, the beads and cells are subsequently separated, and then the cells are cultured. In different embodiments, the drug-coated beads and cells are separated prior to culturing, but cultured together. In a further embodiment, the beads and cells are first concentrated by application of a force such as a magnetic force to increase the ligation of the cell surface marker, thereby inducing cell stimulation.

As an example, cell surface proteins can be ligated by contacting T cells with paramagnetic beads (3x28 beads) to which anti-CD3 and anti-CD28 are bound. ^In one embodiment, cells (eg, ^104-109 T cells) and beads (eg, DYNA beads S® M-450 CD3 / CD28 T paramagnetic beads 1: 1 ratio) are buffered, eg PBS. Combine in (without divalent cations such as calcium and magnesium). Again, it can be readily appreciated by those of skill in the art that any cell concentration can be used. For example, target cells are extremely rare in a sample and may contain only 0.01% of the sample or the entire sample (ie 100%) may be composed of the target cells of interest. Therefore, all cell numbers are integrated in the context of the present invention. In one embodiment, it may be desirable to significantly reduce the volume of the particles and the mixture of cells (ie, increase the concentration of the cells) to ensure maximum contact between the cells. For example, in one embodiment, concentrations of about 10 billion cells / ml, 9 billion / ml, 8 billion / ml, 7 billion / ml, 6 billion / ml, 5 billion / ml or 2 billion cells / ml are used. In one embodiment, over 100 million cells / ml are used. In a further embodiment, a cell concentration of 10 million, 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 45 million or 50 million cells / ml is used. In a further embodiment, a concentration of 75 million, 80 million, 85 million, 90 million, 95 million or 100 million cells / ml of cells is used. In a further embodiment, a concentration of 125 million or 150 million cells / ml can be used. High concentrations can be used to increase cell yield, cell activation and cell proliferation. Moreover, the use of high cell concentrations allows for more efficient capture of cells that can weakly express the target antigen of interest, such as CD28-negative T cells. Such cell populations may have therapeutic value and may be desirable in certain embodiments. For example, the use of high concentration cells usually allows for more efficient selection of CD8 + T cells with weak CD28 expression.

In some embodiments, cells transduced with CAR, eg, the nucleic acid encoding CAR described herein, are propagated, eg, by the methods described herein. In some embodiments, the cells are allowed to grow for several hours (eg, about 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 15 hours, 18 hours, 21 hours. ) ~ About 14 days (for example, 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th or 14th) Proliferate by culturing for the period of. In some embodiments, cells are grown for a period of 4-9 days. In some embodiments, cells are grown for a period of 8 days or less, eg, 7 days, 6 days or 5 days. In some embodiments, cells, such as BCMA CAR cells described herein, are grown in 5 days of culture and the resulting cells are more than the same cells grown in 9 days of culture under the same culture conditions. It's powerful. Efficacy can be defined, for example, by a variety of T cell functions such as proliferation, target cell death, cytokine production, activation, migration or combinations thereof. In some embodiments, cells grown for 5 days, eg, BCMA CAR cells described herein, are antigen-stimulated cell doubling as compared to the same cells grown in 9 days culture under the same culture conditions. Shows at least a one-fold, two-fold, three-fold or four-fold increase in. In some embodiments, cells, eg, cells expressing BCMA CAR described herein, were grown in 5 days of culture and the resulting cells were grown in 9 days of culture under the same culture conditions. Compared to the same cells, they exhibit high pro-inflammatory cytokine production, eg IFN-γ and / or GM-CSF levels. In some embodiments, cells grown for 5 days, eg, BCMA CAR cells described herein, produce pro-inflammatory cytokines as compared to the same cells grown in 9 days culture under the same culture conditions. For example, at pg / ml of IFN-γ and / or GM-CSF levels, it increases at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more.

In one embodiment of the invention, the mixture can be cultured for a few hours (about 3 hours) to about 14 days or any temporal integer value in between. In one embodiment, the mixture can be cultured for 21 days. In one embodiment of the invention, beads and T cells are cultured together for about 8 days. In one embodiment, the beads and T cells are cultured together for 2-3 days. Several cycles of stimulation may also be desired such that the T cell culture period can be 60 days or longer. Suitable conditions for T cell culture are serum (eg, fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10. , IL-12, IL-15, TGFβ and TNF-α or any other additive for cell proliferation known to those of skill in the art, which may contain factors necessary for proliferation and viability. For example, the minimum essential medium or RPMI medium 1640 or X-vivo 15 (Lonza)) is included. Other additives for cell proliferation include, but are not limited to, detergents, plasmanates, and reducing agents such as N-acetyl-cysteine, and 2-mercaptoethanol. The medium is RPMI 1640, AIM, supplemented with serum-free or appropriate amount of serum (or plasma), or supplemented with a defined set of hormones and / or cytokines sufficient for T cell growth and expansion. -V, DMEM, MEM, α-MEM, F-12, X-Vivo15 and X-Vivo20, Optimizer may be included. Antibiotics such as penicillin and streptomycin are included only in experimental cultures and not in cultures of cells to be injected into the subject. Target cells are maintained under the conditions necessary to support proliferation, such as the appropriate temperature (eg, 37 ° C.) and atmosphere (eg, air + 5% CO ₂ ).

In some embodiments, cells are at least 200-fold (eg, 200-fold, 250-fold,, for example, 200-fold, 250-fold, over a 14-day growth period, as measured by methods described herein, such as flow cytometry. It is grown on a suitable medium containing one or more interleukins (eg, the medium described herein) that results in a 300-fold, 350-fold increase. In some embodiments, cells are grown in the presence of IL-15 and / or IL-7 (eg, IL-15 and IL-7).

In embodiments, the methods described herein, eg, CAR-expressing cell production methods, use T-regulatory cells, such as CD25 + T cells, using, for example, an anti-CD25 antibody or fragment thereof or a CD25 binding ligand, IL-2. Includes removal from the population. Methods for removing T-regulatory cells, such as CD25 + T cells, from a cell population are described herein. In embodiments, the method, eg, the production method, is a cell population (eg, a cell population depleted of T-regulatory cells such as CD25 + T cells; or cells previously contacted with an anti-CD25 antibody, fragment thereof or CD25 binding ligand. Population) further includes contact with IL-15 and / or IL-7. For example, a cell population (eg, previously contacted with an anti-CD25 antibody, fragment thereof or CD25 binding ligand) is grown in the presence of IL-15 and / or IL-7.

In one embodiment, the CAR-expressing cells described herein are the interleukin-15 (IL-15) polypeptide, the interleukin-15 receptor alpha (IL-15Ra) polypeptide or the IL-15 polypeptide and IL. It comprises contacting a composition comprising a -15Ra polypeptide, eg, hetIL-15, with, for example, Exvivo during the production of CAR-expressing cells. In embodiments, the CAR-expressing cells described herein are contacted with a composition comprising an IL-15 polypeptide, eg, Exvivo, during the production of CAR-expressing cells. In embodiments, the CAR-expressing cells described herein are contacted with a composition comprising a combination of both IL-15 and IL-15Ra polypeptides, eg, in Exvivo, during the production of CAR-expressing cells. In embodiments, the CAR-expressing cells described herein are contacted with a composition containing hetIL-15, eg, at Exvivo, during the production of CAR-expressing cells.

In some embodiments, the CAR-expressing cells described herein are contacted with a composition comprising he IL-15 during Exvivo proliferation. In one embodiment, the CAR-expressing cells described herein are contacted with a composition comprising an IL-15 polypeptide during Exvivo proliferation. In one embodiment, CAR-expressing cells described herein are contacted with a composition comprising both IL-15 and IL-15Ra polypeptides during Exvivo proliferation. In some embodiments, contact results in survival and proliferation of lymphocyte subpopulations such as CD8 + T cells.

T cells exposed to different stimulation times can exhibit different characteristics. For example, a typical blood or apheretic peripheral blood mononuclear cell product has a helper T cell population (TH, CD4 +) that is larger than the cytotoxic or suppressor T cell population (TC, CD8 +). Exvibo proliferation of T cells stimulated with CD3 and CD28 receptors produces a T cell population consisting predominantly TH cells until about 8-9 days, but after about 8-9 days, the T cell population Includes a gradually growing TC cell population. Therefore, depending on the purpose of treatment, it may be advantageous to inject a T cell population predominantly containing TH cells into the subject. Similarly, if an antigen-specific subset of TC cells has been isolated, it may be advantageous to grow this subset to a large extent.

Moreover, in addition to the CD4 and CD8 markers, other phenotypic markers change significantly, but predominantly, reproducibly during the cell proliferation process. Thus, such reproducibility enables the ability to deliver activated T cell products for specific purposes.

Once BCMA CAR is constructed, the ability to proliferate T cells after antigen stimulation in appropriate in vitro and animal models, sustained T cell proliferation and anticancer activity in the absence of restimulation, but not limited to these. Various assays can be used to assess the activity of the molecule. Assays for assessing the effects of BCMA CAR are described in more detail below.

Western blot analysis of CAR expression in primary T cells can be used to detect the presence of monomers and dimers. For example, Milone et al. , Molecular Therapy 17 (8): 1453-1464 (2009). Very briefly, CAR-expressing T cells (a 1: 1 mixture of CD4 ⁺ and CD8 ⁺ T cells) are grown in vitro for more than 10 days, followed by SDS-PAGE under lysis and reduction conditions. CAR containing the full-length TCR-zeta cytoplasmic domain and the endogenous TCR-zeta chain is detected by Western blotting using an antibody against the TCR-zeta chain. The same T cell subset is used for SDS-PAGE analysis under non-reducing conditions to allow evaluation of covalent dimer formation.

In vitro proliferation of CAR ⁺ T cells after antigen stimulation can be measured by flow cytometry. For example, a mixture of CD4 ⁺ and CD8 ⁺ T cells is stimulated with αCD3 / αCD28 aAPC and then transduced with a GFP-expressing lentiviral vector under the control of a promoter to be analyzed. Representative promoters include the CMVIE gene, EF-1α, ubiquitin C or phosphoglycerokinase (PGK) promoters. GFP fluorescence is evaluated by flow cytometry on day 6 of culture of CD4 ⁺ and / or CD8 ⁺ T cell subsets. For example, Milone et al. , Molecular Therapy 17 (8): 1453-1464 (2009). Instead, a mixture of CD4 ⁺ and CD8 ⁺ T cells was stimulated with αCD3 / αCD28 coated magnetic beads on day 0 and a bicistronic lentiviral vector expressing CAR and eGFP using a 2A ribosome skipping sequence was used. CAR is transduced on day 1. The culture is washed and then restimulated with multiple myeloma cell lines or BCMA expressing cells such as K562-BCMA. Alien IL-2 is added to the culture at 100 IU / ml every other day. GFP + T cells are counted by flow cytometry using bead-based counting. For example, Milone et al. , Molecular Therapy 17 (8): 1453-1464 (2009).

Persistent CAR ⁺ T cell proliferation in the absence of restimulation can also be measured. For example, Milone et al. , Molecular Therapy 17 (8): 1453-1464 (2009). Briefly, the average T cell volume (fl) was measured on day 0 of stimulation with αCD3 / αCD28 coated magnetic beads and day 1 of the described CAR transduction, and then on day 8 of culture, the Collector Multisizer III particle counter. Measurements are made using the Nexus Cellometer Vision or Millipore Septer.

Animal models can also be used to measure CART activity. For example, a xenograft model using human BCMA-specific CAR ⁺ T cells can be used to treat primary human multiple myeloma in immunodeficient mice. For example, Milone et al. , Molecular Therapy 17 (8): 1453-1464 (2009). Very simply, after the MM is established, the mice are randomized to the treatment group. Different numbers of BCMA CART cells can be injected into immunodeficient mice carrying MM. Animals are assessed for disease progression and tumor loading at weekly intervals. The logrank test is used to compare the survival curves of the groups. In addition, absolute peripheral blood CD4 ⁺ and CD8 ⁺ T cell counts 4 weeks after T cell injection into immunodeficient mice can also be analyzed. Mice are injected with multiple myeloma cells and 3 weeks later injected with T cells engineered to express BCMA CAR, eg, with a bicistronic lentiviral vector encoding CAR linked to eGFP. T cells are standardized by mixing 45-50% of infused GFP ⁺ T cells with pseudotransduced cells prior to injection and confirmed by flow cytometry. Animals are evaluated for leukemia at weekly intervals. Survival curves of CAR ⁺ T cell populations are compared using a logrank test.

Evaluation of cell proliferation and cytokine production is described, for example, in Milone et al. , Molecular Therapy 17 (8): 1453-1464 (2009). Briefly, evaluation of growth via CAR is performed by mixing washed T cells with BCMA-expressing K562 cells in a microtiter plate, or other BCMA-expressing myeloma cells. Irradiated with gamma rays before use. Anti-CD3 (clone OKT3) and anti-CD28 (clone 9.3) monoclonal antibodies, KT32-BBL cells that serve as positive controls for stimulated T cell proliferation because these signals support long-term CD8 ⁺ T cell proliferation in Exvivo. Add to the culture of. T cells are counted in culture using CountBright ™ fluorescent beads (Invitrogen, Carlsbad, CA) and flow cytometry as described by the manufacturer. CAR ⁺ T cells are identified by GFP expression using T cells engineered with the eGFP-2A linked CAR expression lentiviral vector. For CAR + T cells that do not express GFP, CAR + T cells are detected by biotinylated recombinant BCMA protein and secondary avidin-PE conjugate. CD4 + and CD8 ⁺ expression in T cells is also detected simultaneously using specific monoclonal antibodies (BD Biosciences). Cytokine measurements are performed on the supernatant collected after 24 hours of restimulation using a human TH1 / TH2 cytokine cell numbering bead array kit (BD Biosciences, San Diego, CA) according to the manufacturer's instructions. Fluorescence is assessed using a FACScalibur flow cytometer and the data is analyzed according to the manufacturer's instructions.

Cytotoxicity can be assessed by a standard 51Cr release assay. For example, Milone et al. , Molecular Therapy 17 (8): 1453-1464 (2009). Briefly, target cells (eg, BCMA-expressing strain K562 and primary multiple myeloma cells) with 51Cr ( _NaCrO4 , New England Nuclear, Boston, MA) at 37 ° C. for 2 hours with frequent agitation. Fill, wash twice with complete RPMI and seed on microtiter plates. Effector T cells are mixed with target cells in complete RPMI in wells in various effector cell: target cell (E: T) ratios. Wells containing additional medium only (spontaneous release, SR) or triton-X100 detergent 1% solution (total release, TR) are also prepared. After incubation at 37 ° C. for 4 hours, the supernatant is collected from each well. The released 51Cr is then measured using a gamma particle counter (Packard Instrument Co., Waltham, MA). Each condition was performed at least 3 times and the percentage of dissolution was given using the formula: dissolution% = (ER-SR) / (TR-SR) (in the formula, ER indicates an average of 51Cr released under each experimental condition). To calculate.

Contrast techniques can be used to assess the specific transport and proliferation of CAR in tumor-carrying animal models. Such assays are described, for example, in Barrett et al. , Human Gene Therapy 22: 1575-1586 (2011). Briefly, NOD / SCID / γc ^{− / −} (NSG) mice or other immunodeficiencies are injected IV with multiple myeloma cells, and 7 days later, BCMA CART cells are injected with CAR constructs 4 hours after electroporation. do. T cells are stably transfected with a lentiviral construct to express firefly luciferase and the mice are imaged for bioluminescence. Alternatively, the therapeutic efficacy and specificity of a single injection of CAR ⁺ T cells in a multiple myeloma xenograft model can be measured as follows: NSG mice are injected with multiple myeloma cells transduced to stably express firefly luciferase, followed by a single tail vein injection of T cells electrically perforated with the BCMA CAR construct 7 days later. Animals are imaged at various time points after injection. For example, photon density heatmaps of firefly luciferase-positive tumors in representative mice can be created on day 5 (2 days before treatment) and day 8 (24 hours after CAR ⁺ PBL).

CAR-expressing cells (eg, in vitro or in vivo (eg, clinical monitoring)), immune cell proliferation and / or activation and / or CAR, comprising the use of CAR ligands, in place of or in combination with the methods disclosed herein. Methods and compositions for the detection and / or quantification of one or more specific selections are disclosed. In one exemplary embodiment, the CAR ligand is an antibody that binds to a CAR molecule, eg, an antibody that binds to the extracellular antigen binding domain of CAR (eg, an antibody that binds to the antigen binding domain, eg, an anti-idiotype antibody; or Antibodies that bind to the constant region of the extracellular binding domain). In other embodiments, the CAR ligand is a CAR antigen molecule (eg, a CAR antigen molecule as described herein).

In one embodiment, a method for detecting and / or quantifying CAR-expressing cells is disclosed. For example, CAR ligands can be used for detection and / or quantification of CAR-expressing cells in vitro or in vivo (eg, clinical monitoring of CAR-expressing cells in a patient or dosing to a patient). The method is to provide a CAR ligand (optionally a labeled CAR ligand, eg, a CAR ligand containing a tag, beads, radioactive or fluorescent label); to obtain CAR expressing cells (eg, a production sample or a clinical sample, etc.). Acquisition of CAR-expressing cell-containing samples); comprising contacting CAR-expressing cells with a CAR ligand under conditions in which binding occurs, thereby detecting the level (eg, amount) of CAR-expressing cells present. Binding of CAR-expressing cells to CAR ligand can be detected using standard techniques such as FACS, ELISA and the like.

In another embodiment, a method of proliferating and / or activating cells (eg, immune effector cells) is disclosed. The method is to provide CAR-expressing cells (eg, first CAR-expressing cells or transiently expressed CAR cells); said CAR-expressing cells and CAR ligands, eg CAR ligands as described herein. And / or contact under conditions where immune cell proliferation and / or proliferation occurs, thereby producing an activated and / or proliferating population.

In one embodiment, a CAR ligand is present (eg, immobilized or bound to a substrate, eg, a non-naturally occurring substrate). In one embodiment, the substrate is a non-cellular substrate. The non-cellular substrate can be, for example, a solid support selected from plates (eg, microtiter plates), membranes (eg, nitrocellulose membranes), matrices, chips or beads. In embodiments, the CAR ligand is present on the substrate (eg, on the surface of the substrate). The CAR ligand can be immobilized, bound or associated with the substrate by covalent non-sharing (eg, cross-linking). In some embodiments, the CAR ligand binds to the beads (eg, covalently). In the embodiment, the immune cell population can be grown in vitro or exvivo. The method may further comprise culturing an immune cell population in the presence of a ligand for the CAR molecule, eg, using any of the methods described herein.

In other embodiments, the method of proliferating and / or activating cells further comprises the addition of a second stimulating molecule, such as CD28. For example, a CAR ligand and a second stimulating molecule can be immobilized on a substrate, eg, one or more beads, thereby increasing cell proliferation and / or activation.

In yet another embodiment, a method for selecting or enriching CAR-expressing cells is provided. The method comprises contacting a CAR-expressing cell with a CAR ligand as described herein and selecting the cell based on the binding of the CAR ligand.

In yet another embodiment, a method of depleting, reducing and / or killing CAR-expressing cells is provided. The method is to contact CAR-expressing cells with a CAR ligand as described herein and target the cells based on the binding of the CAR ligand, thereby reducing and / or killing the number of CAR-expressing cells. including. In some embodiments, the CAR ligand binds to a toxic agent (eg, a toxin or cell scavenger). In another embodiment, the anti-idiotype antibody can give rise to effector cell activity, such as ADCC or ADC activity.

Exemplary anti-CAR antibodies that can be used in the methods disclosed herein are, for example, WO 2014/190273 and Jena et al. , "Chimeric Antigen Receptor (CAR) -Special Monoclonal Antibodies to Detector CD19-Specific T cells in Clinical Trials", PLOS March 2013, p. 38: PLOS March 2013, p. In some embodiments, the anti-idiotype antibody molecule recognizes an anti-CD19 antibody molecule, such as an anti-CD19 scFv. For example, anti-idiotype antibody molecules are described in Jena et al. , PLOS March 2013 8: 3 e57838 can compete for binding with the CD19-specific CAR mAb clone number 136.20.1; the same CDR as the CD19-specific CAR mAb clone number 136.20.1 (eg, Kabat definition, It may have one or more, eg, all VH CDR1, VH CDR2, CH CDR3, VL CDR1, VL CDR2 and VL CDR3) using a Chothia-defined or a combination of Kabat and Chothia-defined; CD19-specific CAR mAb clone numbers. It may have one or more (eg, 2) variable regions such as 136.20.1 or may contain a CD19-specific CAR mAb clone number 136.20.1. In one embodiment, the anti-idiotype antibody is described in Jena et al. Manufactured by the method described in. In another embodiment, the anti-idiotype antibody molecule is the anti-idiotype antibody molecule described in International Publication No. 2014/190273. In one embodiment, the anti-idiotype antibody molecule has the same CDR as the antibody molecule described in WO 2014/190273, such as 136.20.1 (eg, one or more, eg, all VH CDR1, VH CDR2). , CH CDR3, VL CDR1, VL CDR2 and VL CDR3); can have one or more (eg, 2) variable regions of the antibody molecule of the antibody molecule of WO 2014/190273 or 136.20. 1 and the like described in International Publication No. 2014/190273 pamphlet may include antibody molecules. In other embodiments, the anti-CAR antibody binds to the constant region of the extracellular binding domain of the CAR molecule, eg, as described in WO 2014/190273. In one embodiment, the anti-CAR antibody binds to a constant region of the extracellular binding domain of a CAR molecule, eg, a heavy chain constant region (eg, CH2-CH3 hinge region) or a light chain constant region. For example, in one embodiment, the anti-CAR antibody competes for binding with the 2D3 monoclonal antibody described in WO 2014/190273 and has the same CDR as 2D3 (eg, one or more, eg, all VH CDR1, VH). Have a CDR2, CH CDR3, VL CDR1, VL CDR2 and VL CDR3), or have one or more (eg, 2) variable regions of 2D3, or as described in WO 2014/190273. Includes 2D3.

In some embodiments and embodiments, the compositions and methods herein are incorporated herein by reference in their entirety, eg, US Patent Application No. 62/031, filed July 31, 2014. Optimized for a particular subset of T cells as described in 699. In one embodiment, an optimized subset of T cells exhibits enhanced persistence compared to control T cells, eg, T cells of different types (eg, CD8 ⁺ or CD4 ⁺ ) expressing the same construct.

In one embodiment, the CD4 ⁺ T cell comprises the CAR described herein, which CAR is an intracellular signaling domain suitable for CD4 ⁺ T cells (eg, leading to optimization, eg, enhancement of persistence). For example, the ICOS domain is included. In one embodiment, CD8 ⁺ T cells include the CARs described herein, which CARs are suitable for CD8 ⁺ T cells (eg, optimizing, eg, enhancing persistence) intracellular signaling domains. Includes, for example, co-stimulation domains other than the 4-1BB domain, CD28 domain or ICOS domain. In one embodiment, the CAR described herein is a CAR comprising the antigen binding domain described herein, eg, a CAR comprising an antigen binding domain targeting BCMA, eg, a CAR in Table 2, 6 or 10. ..

In one aspect, described herein is a method of treating a subject, eg, a subject having cancer. The method is to apply an effective amount to the subject.
1) CD4 ⁺ T cells containing CAR (CAR ^{CD4 +} )
Antigen-binding domains, eg, antigen-binding domains described herein, eg, antigen-binding domains that target BCMA, eg, antigen-binding domains in Table 2, 6 or 10.
A transmembrane domain and an intracellular signaling domain, eg, a first costimulatory domain, eg, a CD4 ⁺ T cell (CAR ^{CD4 +} ) containing an ICOS domain, and 2) a CD8 ⁺ T cell containing CAR (CAR ^{CD8 +} ). ,
Antigen-binding domains, eg, antigen-binding domains described herein, eg, antigen-binding domains that target BCMA, eg, antigen-binding domains in Table 2, 6 or 10.
CD8 ⁺ T cells (CAR ^{CD8 +} ) containing a transmembrane domain and an intracellular signaling domain, such as a second co-stimulation domain, such as a 4-1BB domain, a CD28 domain, or another co-stimulation domain other than the ICOS domain.
Where the CAR ^{CD4 +} and CAR ^{CD8 +} are different from each other.

By optional method,
3) A second CD8 ⁺ T cell containing CAR (second CAR ^{CD8 +} ).
Antigen-binding domains, such as those described herein, antigen-binding domains that target BCMA, such as the antigen-binding domains of Table 2, 6 or 10.
An intracellular signaling domain in which the transmembrane domain and the second CAR ^{CD8 +} include an intracellular signaling domain that is not present on CAR ^{CD8 +} , such as a co-stimulation signaling domain, and optionally no ICS signaling domain. Contains the administration of CD8 ⁺ T cells containing (second CAR ^{CD8 +} ).

Other assays, including those described in the Examples section herein as well as those known in the art, can also be used to evaluate the BCMA CAR constructs of the invention.

Therapeutic Application BCMA-Related Diseases and / or Disorders In one aspect, the invention provides a method of treating a disease associated with the expression of BCMA. In one aspect, the invention provides a method of treating a disease, wherein some of the tumors are negative for BCMA and some of the tumors are positive for BCMA. For example, the CARs of the present invention are useful in treating subjects who are being treated for diseases associated with elevated expression of BCMA, and subjects who are being treated for elevated levels of BCMA are those who are being treated for elevated levels of BCMA. Indicates a disease associated with. In embodiments, the CARs of the invention are useful in treating subjects who are being treated for a disease associated with BCMA expression, and for subjects who are being treated for BCMA expression, a disease associated with BCMA expression. Is shown.

In some embodiments, the invention provides a method of treating a disease, where BCMA is expressed in both normal and cancer cells, but at lower levels in normal cells. In some embodiments, the method further comprises selecting a CAR of the invention in which BCMA CAR binds with an affinity capable of binding to and killing BCMA expressing cancer cells, but expressing BCMA. 30%, 25%, 20%, 15%, 10%, less than 5% or less than normal cells (eg, as measured by the assays described herein) die. For example, a mortality assay such as flow cytometry based on Cr51 CTL can be used. In some embodiments, BCMA CAR has a binding affinity of 10 ^-4 M to ^10-8 M, such as ^10-5 M to ^10-7 M, such as ^10-6 M or ^10-7 M, for the target antigen. It has an antigen-binding domain with KD. In some embodiments, the BCMA antigen binding domain binds at least 5-fold, 10-fold, 20-fold, 30-fold, 50-fold, 100-fold or 1,000-fold smaller than a reference antibody, eg, an antibody described herein. Has affinity.

In one aspect, the invention relates to a vector comprising BCMA CAR operably linked to a promoter for expression in mammalian immune effector cells such as T cells or NK cells. In one aspect, the invention provides recombinant immune effector cells expressing BCMA CAR for use in the treatment of BCMA expressing tumors, such as T cells or NK cells, wherein the recombinant immunity expresses BCMA CAR. Effector cells (eg, T cells or NK cells) are referred to as BCMA CAR-expressing cells (eg, BCMA CART or BCMA CAR-expressing NK cells). In one embodiment, the BCMA CAR-expressing cells of the invention (eg, BCMA CART or BCMA CAR-expressing NK cells) are those in which the BCMA CAR-expressing cells (eg, BCMA CART or BCMA CAR-expressing NK cells) target the tumor cells and of the tumor. Tumor cells can be contacted with at least one BCMA CAR of the invention expressed on their surface so that proliferation is inhibited.

In one aspect, the invention is a method of inhibiting the growth of BCMA-expressing tumor cells, in which BCMA CAR-expressing cells (eg, BCMA CART or BCMA CAR-expressing NK cells) are activated in response to an antigen to generate cancer cells. The present invention relates to a method comprising contacting a tumor cell with a BCMA CAR expressing cell (eg, BCMA CART or BCMA CAR expressing NK cell) of the present invention so as to be targeted and inhibit tumor growth.

In one aspect, the invention relates to a method of treating cancer in a subject. The method comprises administering to the subject the BCMA CAR-expressing cells of the invention (eg, BCMA CART or BCMA CAR-expressing NK cells) such that the cancer is treated in the subject. An example of a cancer that can be treated with BCMA CAR-expressing cells of the invention (eg, BCMA CART or BCMA CAR-expressing NK cells) is a cancer associated with BCMA expression.

The invention includes certain cell therapies, wherein immune effector cells (eg, T cells or NK cells) are genetically modified to express chimeric antigen receptors (CARs) and express BCMA CARs. The cells (eg, BCMA CART or BCMA CAR expressing NK cells) are injected into the recipient in need thereof. The injected cells can kill the tumor cells at the recipient. Unlike antibody therapy, CAR-modified cells, such as T cells or NK cells, can replicate in vivo and provide long-term persistence that can result in sustained tumor control. In various embodiments, the cells administered to the patient (eg, T cells or NK cells) or their progeny are at least 4 months and 5 months after administering the cells (eg, T cells or NK cells) to the patient in the patient. , 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 Lasts for months, 23 months, 2 years, 3 years, 4 years or 5 years.

The invention also includes certain cell therapies, wherein immune effector cells (eg, T cells or NK cells) transiently express a chimeric antigen receptor (CAR), eg, in vitro transcribed RNA. Modified by, immune effector cells (eg, T cells or NK cells) are injected into the recipient in need of it. The injected cells can kill the tumor cells at the recipient. Therefore, in various embodiments, the immune effector cells (eg, T cells or NK cells) administered to the patient are one month, eg, three weeks, after the administration of the immune effector cells (eg, T cells or NK cells) to the patient. Lasts for less than 2 weeks and 1 week.

Without being bound by any particular theory, the antitumor immune response elicited by CAR-modified immune effector cells (eg, T cells or NK cells) can be an active or passive immune response or instead a direct pair. It can be due to an indirect immune response. In one embodiment, CAR transfecting immune effector cells (eg, T cells or NK cells) exhibit specific pro-inflammatory cytokine secretion and potent cytolytic activity in response to BCMA-expressing human cancer cells and are soluble. It resists BCMA inhibition, mediates bystander cell death, and mediates established human tumor regression. For example, antigen-low tumor cells in a heterogeneous field of BCMA-expressing tumors are BCMA redirected immune effector cells (eg, T cells or NK cells) of cancer-related antigens that are previously reacting to nearby antigen-positive cancer cells. ) Can be indirectly destroyed.

In one aspect, the fully human CAR-modified immune effector cells of the invention (eg, T cells or NK cells) are a vaccine for exvivo immunization and / or in vivo therapy in mammals. In one embodiment, the mammal is a human.

With respect to exvivo immunization, at least one of the following occurs in vitro prior to administration of cells to the mammal. i) Cell proliferation, ii) Introduction of nucleic acid encoding CAR into cells or iii) Cryopreservation of cells.

The Exvivo process is well known in the art and is described in more detail below. Briefly, cells are isolated from mammals (eg, humans) and genetically modified (ie, transduced or transfected in vitro) with the CAR-expressing vectors described herein. CAR-modified cells can be administered to a mammalian recipient to provide a therapeutic effect. The mammalian recipient can be human and the CAR modified cell can be self to the recipient. Alternatively, the cells can be allogeneic, allogeneic or heterologous to the recipient.

The methods for exvivo proliferation of hematopoietic stem cells and progenitor cells described in US Pat. No. 5,199,942, which is incorporated herein by reference, can be applied to the cells of the invention. Other suitable methods are known in the art and therefore the invention is not limited to specific methods of cell exvivo proliferation. Briefly, exvivo culture and proliferation of T cells involves (1) recovery of CD34 + hematopoietic stem cells and progenitor cells from peripheral blood or extramedullary implants collected from mammals, and (2) such cells in exvivo. Including proliferation of. In addition to the cell growth factors described in US Pat. No. 5,199,942, other factors such as flt3L, IL-1, IL-3 and c-kit ligands can be used for cell culture and proliferation. ..

In addition to using cell-based vaccines in terms of exvivo immunization, the invention also provides compositions and methods for in vivo immunization to elicit an immune response against an antigen in a patient.

In general, cell activation and proliferation as described herein can be utilized for the treatment and prevention of diseases caused in immunocompromised individuals. In particular, CAR-modified immune effector cells of the invention (eg, T cells or NK cells) are used to treat diseases, disorders and conditions associated with BCMA expression. In certain embodiments, the cells of the invention are used to treat patients at risk of developing diseases, disorders and conditions associated with BCMA expression. Accordingly, the present invention is a method for the treatment or prevention of diseases, disorders and conditions associated with the expression of BCMA, the CAR-modified immune effector cells of the present invention (eg, T cells or, etc.) for subjects in need thereof. Provided are methods comprising administering a therapeutically effective amount of NK cells).

In one embodiment, CAR-expressing cells of the invention (eg, CART cells or CAR-expressing NK cells) can be used in the treatment of proliferative disorders such as cancer or malignant tumors or spinal cord dysplasia, myelodysplasia syndrome or prior. It is a precancerous condition such as leukemia. In one embodiment, the cancer is a blood cancer. Hematological cancer states are types of cancer such as leukemia and malignant lymphoproliferative states that affect the blood, bone marrow and lymphatic system. In one embodiment, the hematological cancer is leukemia or hematological. An example of a disease or disorder associated with BCMA is multiple myeloma (also known as MM) (Claudio et al., Blood. 2002, 100 (6): 2175-86; and Novak et al., Blood. . 2004, 103 (2): 689-94). Multiple myeloma, also known as plasma cell myeloma or Kahler's disease, is a cancer characterized by the accumulation of abnormal or malignant trait B cells in the bone marrow. Often, cancer cells invade adjacent bones, destroying skeletal structures and causing bone pain and fractures. The majority of myeloma cases are also characterized by the production of paraprotein (also known as M protein or myeloma protein), an abnormal immunoglobulin that is overproduced by clone proliferation of malignant plasma cells. Serum paraprotein levels above 30 g / L are diagnostic criteria for multiple myeloma according to the International Myeloma Working Group (IMWG) diagnostic criteria (Kyle et al. (2009), Leukemia. 23: 3-9. Please refer to). Other symptoms or signs of multiple myeloma include decreased renal function or renal failure, bone lesions, anemia, hypercalcemia and neurological symptoms.

Criteria for distinguishing multiple myeloma from other plasma cell proliferative disorders have been established by the International Myeloma Working Group (see Kyle et al. (2009), Leukemia. 23: 3-9). All three criteria must be met:
-Clone bone marrow plasma cells ≧ 10%
-Presence of serum and / or urinary monoclonal protein (excluding patients with true non-secretory multiple myeloma)
-Evidence of terminal organ damage due to impaired basal plasma cell proliferation, specifically
○ Hypercalcemia: Serum calcium ≧ 11.5 mg / 100 ml
○ Renal failure: Serum creatinine> 1.73 mmol / l
○ Anemia: Hemoglobin value is below the lower limit of normal value by more than 2 g / 100 ml, or hemoglobin value is <10 g / 100 ml, orthochromic, orthospherical ○ Bone lesion: soluble lesion, severe osteopenia Or a pathological fracture.

Other plasmacytoproliferative disorders that can be treated by the compositions and methods described herein include asymptomatic myeloma (smoldering multiple myeloma or painless myeloma), a single clone of unknown significance. Sexual hypergamma globulinemia (MGUS), Waldenstraem macroglobulinemia, plasmacytoma (eg, plasmacytoproliferative disorder, isolated myeloma, isolated plasmacytoma, extramedullary plasmacytoma and multiple) Myeloma), systemic amyloid light chain amyloidosis and POEMS syndrome (also known as Crow-Fukase syndrome, high moon disease and PEP syndrome), but not limited to these.

For staging of multiple myeloma, two staging systems: International Staging System (ISS) (Gripp et al. (2005), J. Clin. Oncol. 23 (15): 3412-3420). (See) and the Disease-Salmon staging system (DSS) (see Disease et al. (1975), Cancer 36 (3): 842-854). The two staging are summarized in the table below.

* The Disease-Salmon staging system also includes subclassifications that specify the state of renal function. The designation "A" or "B" is added after the stage number, where "A" indicates relatively normal renal function (serum creatinine level <2.0 mg / dL) and B is abnormal. It shows good renal function (serum creatinine level> 2.0 mg / dL).

The third staging system for multiple myeloma is referred to as the Revised International Staging System (R-ISS) (Palumbo A, Avet-Loiseau H, Oliva S, et al. Journal of Clinical oncology: offical). Please refer to journal of the American Society of Clinical Oncology 2015; 33: 2863-9, which is incorporated herein by reference in its entirety). R-ISS stage I includes ISS stage I (serum β2-microglobulin level <3.5 mg / L and serum albumin level ≥3.5 g / dL), no high-risk CA [del (17p) and / or t ( 4; 14) and / or t (14; 16)] and normal LDH levels (below the upper limit of the normal range) are included. R-ISS stage III includes ISS stage III (serum β2-microglobulin level> 5.5 mg / L) and high-risk CA or high LDH levels. R-ISS Stage II includes all other possible combinations.

Patient responses are incorporated herein by reference in their entirety, Kumar S, Paiva B, Anderson KC, et al. International Myeloma Working Group consensus criteria for reaction and minimal residual disorder assembly in multiple myeloma. The Lancet Oncology; 17 (8): can be determined based on the criteria of IMWG2016 disclosed in e328-e346. Table 16 provides IMWG 2016 criteria for response evaluation.

Standard treatments for multiple myeloma and related diseases include chemotherapy, stem cell transplantation (self or allogeneic), radiation therapy and other medications. Frequently used anti-myeloma drugs include alkylating agents (eg, bendamustine, cyclophosphamide, melphalan), proteasome inhibitors (eg, bortezomib), corticosteroids (eg, dexamethasone, prednisone), immunity. Modifiers (eg, salidamide, lenalidomide, Revlimid®) or any combination thereof are included. Biphosphonate drugs are often also given in combination with standard anti-MM treatment to prevent bone loss. Patients over the age of 65-70 are less likely to be candidates for stem cell transplantation. In some cases, double autologous stem cell transplantation is an option for patients under the age of 60 who have a suboptimal response to the initial transplantation. The compositions and methods of the invention can be administered in combination with any of the currently prescribed treatments for multiple myeloma.

Another example of a disease or disorder associated with BCMA is Hodgkin lymphoma and non-Hodgkin lymphoma (Chiu et al., Blood. 2007, 109 (2): 729-39; He et al., J Immunol. 20044. 172 (5): 3268-79).

Hodgkin lymphoma (HL), also known as Hodgkin's disease, is a cancer of the lymphatic system derived from white blood cells or lymphocytes. The abnormal cells that make up the lymphoma are called Reed-Sternberg cells. In Hodgkin lymphoma, the cancer spreads from one lymph node group to another. Hodgkin lymphoma has four pathological subtypes: nodular sclerosing HL, mixed-cell sub It can be subdivided into type, lymphocyte-rich or lymphocyte-dominant, and lymphocyte-depleted. Some Hodgkin lymphomas may be nodular lymphocyte-dominant Hodgkin lymphomas or may not have been identified. Symptoms and signs of Hodgkin lymphoma include painless swelling of the lymph nodes in the neck, axilla or inguinal region, fever, night sweats, weight loss, fatigue, pruritus or abdominal pain.

Non-Hodgkin's lymphoma (NHL) includes a diverse group of hematological cancers, including any type of lymphoma other than Hodgkin's lymphoma. The subtypes of non-Hodgkin's lymphoma are mainly classified by cell morphology, chromosomal abnormalities and surface markers. NHL subtypes (or NHL-related cancers) are, but are not limited to, Burkit's lymphoma, B-cell chronic lymphocytic leukemia (B-CLL), pre-B-cell lymphocytic leukemia (B-PLL), chronic lymphocytic leukemia (CLL). ), Diffuse large cell type B cell lymphoma (DLBCL) (eg, intravascular large cell type B cell lymphoma and primary mediastinal B cell lymphoma), follicular lymphoma (eg, follicular central lymphoma, follicular small fissure cell) , Hairy cell leukemia, high-grade B-cell lymphoma (Berkit-like), lymphoplasmic cell lymphoma (Waldenstrem macroglobulinemia), mantle cell lymphoma, marginal zone B-cell lymphoma (eg extranodal) Marginal B-cell lymphoma or mucosal-related lymphoma (MALT) lymphoma, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma), plasmacytoma / myeloma, precursor B lymphoblastic leukemia / lymphoma B-cell lymphomas such as (PB-LBL / L), primary central nervous system (CNS) lymphoma, primary intraocular lymphoma, small lymphocytic lymphoma (SLL); and, but not limited to, undifferentiated large cell lymphoma (ALCL). ), Adult T-cell lymphoma / leukemia (eg, smoldering, chronic, acute and lymphomatous), angiocentralized lymphoma, angioimmunoblastic T-cell lymphoma, cutaneous T-cell lymphoma (bacteriolytic sarcoma, cesarly syndrome, etc.) , Extranodal natural killer / T-cell lymphoma (nasal type), intestinal disease-type intestinal T-cell lymphoma, large granular lymphocytic leukemia, precursor T lymphoblastic lymphoma / leukemia (T-LBL / L), T-cell chronic lymphoma Includes T-cell lymphomas such as sex leukemia / prelymphoma leukemia (T-CLL / PLL) and unspecified peripheral T-cell lymphoma. Symptoms and signs of Hodgkin lymphoma include painless swelling of the lymph nodes in the neck, axilla or inguinal region, fever, night sweats, weight loss, fatigue, pruritus, abdominal pain, cough or chest pain.

The staging is the same for both Hodgkin and non-Hodgkin's lymphomas and refers to the extent of the spread of cancer cells in the body. At stage I, lymphoma cells are present in one lymph node group. In stage II, lymphoma cells are present in at least two lymph node groups, both in the same side of the diaphragm or in parts of a tissue or organ and in lymph nodes near that organ on the same side of the diaphragm. exist. In stage III, lymphoma cells are present in the lymph nodes on either side of the diaphragm or in part of a tissue or organ near these lymph node groups or in the spleen. At stage IV, lymphoma cells are found in some part of at least one organ or tissue, or lymphoma cells are found in organs and lymph nodes on the opposite side of the diaphragm. In addition to the designation of the Roman number staging, the stage can also be represented by the letters A, B, E and S, where A refers to the asymptomatic patient and B refers to the symptom. E refers to patients with lymphoma in tissues outside the lymphatic system, and S refers to patients with lymphoma in the spleen.

Hodgkin lymphoma is usually treated with radiation therapy, chemotherapy or hematopoietic stem cell transplantation. The most common therapy for non-Hodgkin's lymphoma is R-CHOP, which consists of four different chemotherapies (cyclophosphamide, doxorubicin, vincristine and prenizolone) and rituximab (Rituxan®). Other therapies commonly used to treat NHL include other chemotherapeutic agents, radiation therapy, and biological therapies such as stem cell transplantation (self or allogeneic bone marrow transplantation) or immunotherapy. Other examples of biotherapeutic agents include rituximab (Rituxan®), tositumomab (Bexxar®), eprazumab (LymphoCide®) and alemtuzumab (MabCampath®). However, it is not limited to these. The compositions and methods of the invention can be administered in combination with any of the currently prescribed treatments for Hodgkin lymphoma or non-Hodgkin lymphoma.

Expression of BCMA is also associated with Waldenström macroglobulinemia (WM), also known as lymphoplasmacytic lymphoma (LPL). (See Elsawa et al., Blood. 2006, 107 (7): 2882-8). Waldenström macroglobulinemia, previously thought to be associated with multiple myeloma, has recently been classified as a subtype of non-Hodgkin's lymphoma. WM is characterized by uncontrolled B-cell lymphocyte proliferation, causing anemia and the production of excess paraprotein or immunoglobulin M (IgM), which increases blood concentration and causes hyperviscosity syndrome. Other symptoms or signs of WM include fever, night sweats, fatigue, anemia, weight loss, lymphadenopathy or splenomegaly, blurred vision, dizziness, nosebleeds, bleeding of the gums, abnormal bruising, renal dysfunction or insufficiency, amyloidosis or Includes peripheral neuropathy.

Standard treatment for WM consists specifically of chemotherapy with rituximab (Rituxan®). Other chemotherapies such as chlorambucil (Leukeran®), cyclophosphamide (Neosar®), fludarabine (Fladara®), cladribine (Leustatin®), vincristine and / or salidamide. Can be used in combination with drugs. Corticosteroids such as prednisone can also be given in combination with chemotherapy. Plasmapheresis or plasmapheresis is commonly used throughout the patient's procedure to relieve some symptoms by removing paraprotein from the blood. In some cases, stem cell transplantation is an option for some patients.

Another example of a disease or disorder associated with BCMA is a brain tumor. Specifically, expression of BCMA has been associated with astrocytoma or glioblastoma (Deshayes et al, Oncogene. 2004, 23 (17): 3005-12, Pelekanou et al., PLoS One. 2013, 8 (12): see e83250). Astrocytomas are tumors that arise from astrocytes, a type of glial cell in the brain. Glioblastoma (also known as polymorphic glioblastoma or GBM) is the most malignant form of astrocytoma and is considered to be the most advanced stage (stage IV) of brain tumors. There are two variants of glioblastoma: giant cell glioblastoma and glioma. Other astrocytomas include juvenile hairy astrocytomas (JPA), fibrous astrocytomas, polymorphic astrocytomas (PXA), and germ dysplastic neuroepithelial tumors (JPA). DNET) and undifferentiated astrocytoma (AA) are included.

Symptoms or signs associated with glioblastoma or stellate cell tumor include elevated brain pressure, headache, seizures, memory loss, behavioral changes, loss of movement or sensation on one side of the body, speech dysfunction, cognitive impairment. , Visual impairment, nausea, vomiting and weakness of the arm or leg.

Surgical removal (or resection) of the tumor is the standard procedure for removing as much glioma as possible without causing damage to the normal surrounding brain or with minimal damage. Radiation therapy and / or chemotherapy are often used to control and delay recurrent disease from residual cancer cells or satellite lesions after surgery. Radiation therapy includes whole-brain radiation therapy (conventional external beam radiation), targeted three-dimensional proto-radiation therapy and targeted radioactive nuclei. Chemotherapeutic agents commonly used to treat glioblastoma include temozolomide, gefitinib or erlotinib and cisplatin. Angiogenesis inhibitors such as bevacizumab (Avastin®) are also commonly used in combination with chemotherapy and / or radiation therapy.

Supportive treatments are also frequently used to relieve neurological symptoms and improve neurological function and are administered in combination with any of the cancer therapies described herein. The main supporters include anticonvulsants and corticosteroids. Accordingly, the compositions and methods of the invention can be used in combination with either standard or supportive treatments for treating glioblastoma or astrocytoma.

Non-cancer-related diseases and disorders associated with BCMA expression can also be treated by the compositions and methods disclosed herein. Examples of non-cancer-related diseases and disorders associated with BCMA expression include viral infections; eg HIV, fungal infections such as cryptococcal neoformans; hypersensitive bowel disease; ulcerative colitis. And disorders associated with, but not limited to, mucosal immunity.

CAR-modified immune effector cells of the invention (eg, T cells or NK cells) alone or in combination with diluents and / or IL-2 or other components such as IL-2 or other cytokines or cell populations as pharmaceutical compositions. Can be administered.

The present invention provides compositions and methods for treating cancer. In one embodiment, the cancer is a hematological cancer, including, but not limited to, the hematological cancer being leukemia or lymphoma. In one embodiment, the CAR-expressing cells of the invention (eg, CART cells or CAR-expressing NK cells) are used, including, but not limited to, B-cell acute lymphocytic leukemia (“BALL”). ”), T-cell acute lymphocytic leukemia (“TALL”), acute leukemia including acute lymphocytic leukemia (ALL); but not limited to, for example, chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL). One or more chronic leukemias including, but not limited to, preB-cell lymphocytic leukemia, neoplasms of blastic plasmacytoid dendritic cells, Berkit lymphoma, diffuse large B-cell lymphoma, etc. Follicular lymphoma, hairy cell leukemia, small or large cell follicular lymphoma, malignant lymphoproliferative state, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myelogenous tumors, spinal cord dysplasia and myelogenous dysplasia Hematological with syndrome, non-hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstreme-type macroglobulinemia and ineffective production (or dysplasia) of blood cells in the bone marrow Further cancers and malignant tumors such as hematological conditions or hematological conditions can be treated, including "pre-leukemic conditions" which are diverse aggregates of conditions. Furthermore, diseases associated with BCMA expression include, but are not limited to, for example, atypical and / or non-classical cancers, malignant tumors, precancerous conditions or proliferative diseases expressing BCMA.

In embodiments, the compositions described herein are plasma cell proliferative disorders such as asymptomatic myeloma (smoldering multiple myeloma or painless myeloma), unclear monoclonal hypergammaglobulin blood. Disease (MGUS), Waldenstrem macroglobulinemia, plasmacytoma (eg, plasmacytoproliferative disorder, isolated myeloma, isolated plasmacytoma, extramedullary plasmacytoma and multiple myeloma), It can be used to treat diseases including, but not limited to, systemic amyloid light chain amyloidosis and POEMS syndrome (also known as Crow-Fukase syndrome, high moon disease and PEP syndrome).

In embodiments, the compositions described herein are cancers, such as cancers described herein, such as prostate cancer (eg, castration-resistant or treatment-resistant prostate cancer or metastatic prostate cancer), pancreatic cancer. Alternatively, it can be used to treat diseases including, but not limited to, lung cancer.

The present invention also provides a method of inhibiting or reducing the proliferation of a BCMA-expressing cell population, wherein the anti-BCMA CAR expression of the present invention binds a cell population containing a BCCA-expressing cell to a BCMA-expressing cell. Includes contact with cells (eg, BCMA CART cells or BCMA CAR expressing NK cells). In certain embodiments, the present invention provides a method of inhibiting or reducing the growth of a population of BCMA-expressing cancer cells, which method binds the BCMA-expressing cancer cell population to BCMA-expressing cells. It comprises contacting with the anti-BCMA CAR expressing cells of the present invention (eg, BCMA CART cells or BCMA CAR expressing NK cells). In one aspect, the invention provides a method of inhibiting or reducing the growth of a population of BCMA-expressing cancer cells, wherein the method binds a BCMA-expressing cancer cell population to BCMA-expressing cells. It comprises contacting with the anti-BCMA CAR expressing cells of the invention (eg, BCMA CART cells or BCMA CAR expressing NK cells). In certain embodiments, the anti-BCMA CAR-expressing cells of the invention (eg, BCMA CART cells or BCMA CAR-expressing NK cells) compare the content, number, amount or percentage of cells and / or cancer cells to a negative control. At least 25%, at least 30%, at least 40%, at least 50%, at least 65%, at least 75%, at least 85%, at least in a subject or animal model with myeloid leukemia or another cancer associated with BCMA expressing cells. Reduce by 95% or at least 99%. In one embodiment, the subject is a human.

The present invention also provides a method for preventing, treating and / or managing a disease associated with a BCMA expressing cell (eg, a blood cancer expressing BCMA or an atypical cancer), wherein the method binds to BCMA expressing cells. The present invention comprises administering the anti-BCMA CAR expressing cells (eg, BCMA CART cells or BCMA CAR expressing NK cells) to a subject in need. In one embodiment, the subject is a human. Non-limiting examples of disorders associated with BCMA-expressing cells include disorders associated with viral or fungal infections and mucosal immunity.

The invention also provides a method of preventing, treating and / or managing a disease associated with BCMA expressing cells, wherein the method is an anti-BCMA CAR expressing cell of the invention that binds to BCMA expressing cells (eg, BCMA CART cells or BCMA CAR expressing NK cells), which comprises administering to a subject in need. In one aspect, the subject is a human.

The present invention provides a method of preventing recurrence of cancer associated with BCMA expressing cells, wherein the method comprises anti-BCMA CAR expressing cells of the invention that bind to BCMA expressing cells (eg, BCMA CART cells or BCMA CAR expressing NK). Includes administration of cells) to a subject in need of it. In one embodiment, the method provides an effective amount of an anti-BCMA CAR-expressing cell described herein (eg, BCMA CART cell or BCMA CAR-expressing NK cell) to a subject in need thereof. Includes administration in combination with different doses of treatment.

Combination Therapies The CAR-expressing cells described herein can be used in combination with other known agents and therapies.

The CAR-expressing cells described herein and at least one additional therapeutic agent can be administered simultaneously, in the same or different compositions, or sequentially. For sequential administration, the CAR-expressing cells described herein may be administered first, then additional agents may be administered, or the order of administration may be reversed.

CAR treatment and / or other therapeutic agents, methods or modality can be administered during the period of active disorder or in remission or underactive disease. CAR treatment can be administered before, at the same time as, after, or during remission of the disorder of other treatments.

When administered in combination, CAR treatment and additional agents (eg, second or third agent) or all, individually, eg, higher, lower, or the same amount as each drug used as monotherapy. Alternatively, it can be administered in a dose. In one embodiment, the amount or dose of CAR treatment, additional agents (eg, second or third agent) or all administered is lower than the amount or dose of each agent used individually, eg, as monotherapy. (For example, at least 20%, at least 30%, at least 40% or at least 50%). In other embodiments, CAR treatments that produce the desired effect (eg, treatment of cancer), additional agents (eg, second or third agents) or all administered amounts or doses achieve the same therapeutic effect. Individually, for example, lower than the amount or dose of each drug used as monotherapy (eg, at least 20%, at least 30%, at least 40%, or at least 50% lower).

In a further embodiment, the CAR-expressing cells described herein are used in a treatment regimen for immunosuppressive agents such as surgery, chemotherapy, radiation, cyclosporine, azathiopurine, methotrexate, mycophenorate, FK506, antibodies or CAMPATH, anti-CD3. Other immunosuppressive agents such as antibodies or other antibody therapies, cytoxin, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines and irradiation, Izumoto et al. It can be used in combination with peptide vaccines such as those described in 2008 J Neurosurg 108: 963-971.

In certain examples, the compounds of the invention are combined with other therapeutic agents such as other anti-cancer agents, anti-allergic agents, antiemetics (or antiemetics), analgesics, cytoprotectants and combinations thereof.

In some embodiments, the first CAR-expressing cell described herein, eg, the BCMA CAR-expressing cell described herein, can be used in combination with a second CAR-expressing cell. In some embodiments, the second CAR expressing cell is different from the anti-BCMA binding domain of CAR expressed by a different anti-BMCA binding domain, eg, the first CAR expressing cell, as described herein. Express CAR with a domain. In some embodiments, the second CAR expressing cell has an antigen binding domain that targets an antigen other than BCMA (eg, CD19, CD20, CS-1, kappa light chain, CD139, Lewis Y antigen or CD38). Express the including CAR. In some embodiments, the first CAR-expressing cells described herein, eg, BCMA CAR-expressing cells described herein, are used in combination with a second CAR-expressing cell, including CD19 CAR. In some embodiments, the BCMA CAR-expressing cells described herein are used in combination with CD19 CAR-expressing cells to treat the BCMA-related cancers described herein, such as multiple myeloma. .. In some embodiments, the multiple myeloma is CD19 negative, eg, the majority of neoplastic plasma cells having a CD19 negative phenotype (eg, at least 98%, 99%, 99.5%, 99.9). % Or 99.95%), which is detected, for example, by flow cytometry, RT-PCR or both flow cytometry and RT-PCR. CD19 CAR is also effective against CD19-negative multiple myeloma. Without wishing to be bound by theory, CD19 CAR is non-neoplastic by targeting cells expressing CD19 at levels below the detection threshold of the assays described herein or in support of neoplastic cells. By targeting cells, it can act on a small but important CD19-positive population of neoplastic cells. In embodiments, the CD19 CAR is capable of removing B cells, such as B regulatory B cells.

For example, in some embodiments, the first CAR-expressing cells described herein, such as BCMA CAR-expressing cells, and the second CAR-expressing cells described herein, such as CD19 CAR-expressing cells, have the same composition. It is prepared in a substance and administered at the same time. In another embodiment, the first CAR-expressing cells described herein, eg, BCMA CAR-expressing cells and the second CAR-expressing cells described herein, eg, CD19 CAR-expressing cells, are in separate compositions. The separate compositions are administered simultaneously or sequentially. When BCMA CAR-expressing cells and a second CAR-expressing cell are prepared in separate compositions, the BCMA CAR-expressing cells can be administered first, the second CAR-expressing cells can be administered second, or the order of administration. Can be reversed.

In some embodiments, the CD19 CAR is the human described in the CD19 CAR, eg, Pamphlet International Publication No. 2014/153270 filed on March 15, 2014, which is incorporated herein by reference in its entirety. CD19 CAR or at least 95% identical sequence thereof, eg 95-99%. In some embodiments, the CD19 CAR construct is the CAR19 construct provided in PCT International Publication No. 2012/079000, which is incorporated herein by reference in its entirety, or at least 95% thereof, eg 95-99. % Same sequence. In some embodiments, the anti-CD19 binding domain is the scFv described in WO 2012/079000 or at least 95%, eg, 95-99% identical to the sequence.

In an embodiment, a first CAR-expressing cell is administered to a subject and a second CAR-expressing cell is administered to the subject. In embodiments, the first CAR expressing cell comprises a CAR (eg, BCMA or CD19 CAR) comprising a CD27 co-stimulating domain and a CD3 zeta (mutant or wild-type) primary signaling domain. In embodiments, the second CAR expressing cell comprises a CAR (eg, BCMA CAR) comprising a 4-1BB co-stimulating domain and a CD3 zeta (mutant or wild-type) primary signaling domain. Without wishing to be bound by theory, in embodiments, the first CAR-expressing cells are less toxic than the second CAR-expressing cells and can be used to reduce tumor weight.

In some embodiments, the CAR-expressing cells described herein can be used in combination with a chemotherapeutic agent. Exemplary chemotherapeutic agents include anthracyclines (eg, doxorubicin (eg, liposome doxorubicin)), binca alkaloids (eg, vincristine, vincristine, bindesin, binorerbin), alkylating agents (eg, cyclophosphamide, dacarbazine, mel). Faran, phosphamide, temozolomid), immune cell antibodies (eg, alemtuzumab, gemtuzumab, rituximab, toshitsumomab), metabolic antagonists (eg, folic acid antagonists, pyrimidine analogs, purine analogs and adenocindeaminase inhibitors (eg, fludarabin)), Includes immunomodulators such as mTOR inhibitors, TNFR glucocorticoid-induced TNFR-related protein (GITR) agonists, proteasome inhibitors (eg, acracinomycin A, gliotoxin or bortezomib), salidamide or salidamide derivatives (eg, renalidemide).

Common chemotherapeutic agents intended for use in combination therapy are anastrozole (Arimidex®), bicartamide (Casodex®), bleomycin sulfate (Brenoxane®), and busulfan (Myrelan (registered trademark)). (Trademark)), Busulfan Injection (Busulfex®), Capesitarabine (Xeloda®), N4-Pentoxycarbonyl-5-deoxy-5-fluorocitidine, Carboplatin (Paraplatin®), Carmustin (BiCNU) (Registered Trademark)), Chlorambusil (Lukelan®), Sisplatin (Platinol®), Fludarabine (Lyostatin®), Cyclophosphamide (Cytoxan® or Neosar®) , Cytarabine, Cytocin arabinoside (Cytosar-U®), Cytarabine liposome injection (DepoCyt®), Daunorubicin (DTIC-Dome®), Dactinomycin (Actinomycin D, Cosmegan), Daunorubicin hydrochloride (Cerubidine®), daunorubicin citrate liposome injection (DaunoXome®), dexamesazone, docetaxel (taxotere®), doxorubicin hydrochloride (adriamycin®, rubex®), Etoposide (Vepsid®), Fludarabine Phosphate (Fludara®), 5-Fluorouracil (Adolcil®, Efdex®), Flutamide (Eulexin®), Tezacitibin, Gemcytarabine (Difluorodeoxycytidine), hydroxyurea (Hydrea®), idarubicin (Idamicin®), phosphamide (IFEX®), irinotecan (Camptosar®), L-asparaginase (ELSPAR®) Trademarks)), leucovorin calcium, merphalan (Alkeran®), 6-mercaptopurine (Purinesol®), methotrexate (Folex®), mitoxanthron (Novantron®), Myrotarg , Paclitaxel (Taxol®), phoenix (Ittrium 90 / MX-DTPA), pentostatin, polyfeprozan 20 and calm Stin implant (Gliadel®), tamoxifen citrate (Nolvadex®), teniposide (Vumon®), 6-thioguanine, thiotepa, tirapazamin (Tirazone®), topotecan hydrochloride for injection (Tirazone®) Includes high camtin®), vinblastine (Velban®), vincristine (oncobin®) and vinorelbine (navellbine®).

Particularly interesting anticancer agents for combination with the compounds of the invention are anthracyclins; alkylating agents; metabolic antagonists; calcium-dependent phosphatase calcinuulin or agents that inhibit p70S6 kinase FK506); mTOR inhibitors; Immunomodulators; anthraciclin; binca alkaloids; proteosome inhibitors; GITR agonist protein tyrosine phosphatase inhibitors; CDK4 kinase inhibitors; BTK inhibitors; MKN kinase inhibitors; DGK kinase inhibitors; or tumor lytic viruses.

Exemplary alkylating agents are nitrogen mustard, ethyleneimine derivatives, alkylsulfonates, nitrosophreas and triazenes): uracil mustard (aminouracil mustard®, Chlorethaminecil®, Demethyldopan®, Demethyldopan (registered trademark). (Registered Trademark), Haemanthamine (Registered Trademark), Nordopan (Registered Trademark), Uracil nitrogen Mustard (Registered Trademark), Uracillost (Registered Trademark), Uracilmostaza (Registered Trademark), Uramstin (Registered Trademark), Uramstin (Registered Trademark) (Mustargen®), cyclophosphamide (Citoxan®, Neosar®, Clafen®, Endoxan®, Procytox®, Revimmune®), Iphosphamide (Mitoxana®), Melfaran (Alkeran®), Chlorambusil (Lukelan®), Pipobroman (Amedel®, Vercyte®), Triethylenemelamine (Hemel®) ), Hexalen®, Hexastat®), Triethylenethiophosphoroamine, Temozoromide (Temodar®), Thiotepa (Thioplex®), Busilvex®, Myrelan ( Includes, but is limited to, carmustin (BiCNU®), romustin (CeeNU®), streptosocin (Zanosar®) and dacarbazine (DTIC-Dome®). Not done. Further examples of alkylating agents are oxaliplatin (eroxatin®); temozoromide (temodar® and temodar®); dactinomycin (also known as actinomycin-D, Cosmegen®). Melfaran (also known as L-PAM, L-sarcolicin and phenylalanine mustard, Alkeran®); Altretamine (also known as hexamethylmelamine (HMM), Hexalen®); Calmustin (BiCNU®); Bendamustine (Tranda®); Busulfan (Busulfex® and Milelan®); Carboplatin (Paraplatin®); Romustin (also known as CCNU, CeeNU®); Sisplatin (also known as CDDP, Platinol) (Registered Trademarks) and Platinol®-AQ); Chlorumbusyl (Lukelan®); Cyclophosphamide (Citoxane® and Neosar®); Dacarbazine (also known as DTIC, DIC and Imidazole Carboxamide) , DTIC-Dome®); Altretamine (also known as hexamethylmelamine (HMM), Hexalen®); Ifphosphamide (Ifex®); Prednumustine; Procarbazine (Matullane®); Mechloretamine (also known as). Nitrogen mustard, mustine and mechloroethamine hydrochloride, Mustargen®; streptozosine (Zanosar®); thiotepa (also known as thiophosphoamide, TESPA and TSPA, Thioplex®); cyclophosphamide (endoxan®). ), Citoxane®, Neosar®, Procytox®, Revimmine®); and Bendamustine HCl (Tranda®), but not limited to these.

Exemplary mTOR inhibitors are, for example, temsirolimus; lidaphorolimus (formerly known as deferolimus, (1R, 2R, 4S) -4-[(2R) -2-[(1R, 9S, 12S, 15R, 16E). , 18R, 19R, 21R, 23S, 24E, 26E, 28Z, 30S, 32S, 35R) -1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2, 3,10,14,20-pentaoxo-11,36-dioxa-4-azatricyclo [ ^30.3.1.0 4.9] hexatriaconta-16,24,26,28-tetraene-12-yl] propyl ] -2-Methoxycyclohexyldimethylphosphinate, also known as AP23573 and MK8669, described in International Publication No. 03/064383); Everolimus (Affinitol® or RAD001); Rapamycin (AY22989, Sirolimus®). ); Shimapimod (CAS 164301-51-3); everolimus, (5- {2,4-bis [(3S) -3-methylmorpholin-4-yl] pyrido [2,3-d] pyrimidin-7- Il} -2-methoxyphenyl) methanol (AZD8055); 2-amino-8- [trans-4- (2-hydroxyethoxy) cyclohexyl] -6- (6-methoxy-3-pyridinyl) -4-methyl-pyrido [2,3-d] pyrimidin-7 (8H) -on (PF04691502, CAS 1013101-36-4); and N2- [1,4-dioxo- ⁴ -[[4- (4-oxo-8-) Phenyl-4H-1-benzopyran-2-yl) morpholinium-4-yl] methoxy] butyl] -L-arginylglycyl-L-α-aspartyl L-serine- (SEQ ID NO: 285), intramolecular salt (SF1126) , CAS 936487-67-1) and XL765.

Exemplary immunomodulators are, for example, aftuzumab (available from Roche®); pegfilgrastim (Neulasta®); renalidemide (CC-5013, revrimid®); thalidomide (thalidomide). (Registered Trademarks)), Actimid (CC4047); and IRX-2 (a human cytokine mixture containing interleukin 1, interleukin 2 and interferon gamma, available from CAS 951209-71-5, IRX Therapeutics).

Exemplary anthracicins are, for example, doxorubicin (adriamycin® and rubex®); bleomycin (lenoxane®); daunorubicin (daunorubicin hydrochloride, daunorubicin and ruvidyne hydrochloride, Cerubide®); Daunorubicin liposomes (Daunorubicin liposomes, DaunoXome®); Mitoxantron (DHAD, Novantron®); Epirubicin (Ellence®); Idalbisin (Idamicin®, Idamycin PFS®) ); Mitomycin C (Mutamycin®); geldanamycin; harbimycin; ravidomycin; and desacetylrabidomycin.

Exemplary vinca alkaloids are, for example, vinorelbine tartrate (navelbine®, vincristine (oncovin®) and vindesine (Eldisine®)); vinblastine (also known as vinblastine sulfate, vinblastine and VLB). , Alkaban-AQ® and Velban®); and vinorelbine (Navelbine®).

Exemplary proteosome inhibitors are voltezomib (Velcade®); carfilzomib (PX-171-007, (S) -4-methyl-N-((S) -1-(((S) -4-). Methyl-1-((R) -2-methyloxylan-2-yl) -1-oxopentan-2-yl) amino) -1-oxo-3-phenylpropane-2-yl) -2-((S) ) -2- (2-Molholinoacetamide) -4-phenylbutaneamide) -pentanamide); marizomib (NPI-0052); ixazomibu citrate ester (MLN-9708); delanzomib (CEP-18770); and O -Methyl-N-[(2-Methyl-5-thiazolyl) carbonyl] -L-ceryl-O-methyl-N-[(1S) -2-[(2R) -2-methyl-2-oxylanyl] -2 -Oxo-1- (phenylmethyl) ethyl] -L-serine amide (ONX-0912) is included.

In embodiments, CAR-expressing cells described herein are administered to a subject in combination with fludarabine, cyclophosphamide and / or rituximab. In embodiments, CAR-expressing cells described herein are administered to a subject in combination with fludarabine, cyclophosphamide and rituximab (FCR). In embodiments, the subject has a CLL. For example, the subject has a deletion in the short arm of chromosome 17 (eg, del (17p) in leukemic cells). In another example, the subject does not have a del (17p). In embodiments, the subject comprises leukemic cells containing a mutation in the immunoglobulin heavy chain variable region ( _IgVH ) gene. In other embodiments, the subject is free of leukemic cells containing a mutation in the immunoglobulin heavy chain variable region ( _IgVH ) gene. In embodiments, fludarabine is about 10-50 mg / m ² (eg, about 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45 or 45-50 mg / m). It is administered intravenously, for example, at the dose of ² ). In embodiments, cyclophosphamide is administered, for example, intravenously at a dose of about 200-300 mg / m ² (eg, about 200-225, 225-250, 250-275 or 275-300 mg / m ² ). In embodiments, rituximab is administered, for example, intravenously at a dose of about 400-600 mg / m2 (eg, 400-450, 450-500, 500-550 or 550-600 mg / m ² ).

In embodiments, CAR-expressing cells described herein are administered to a subject in combination with bendamustine and rituximab. In embodiments, the subject has a CLL. For example, the subject has a deletion in the short arm of chromosome 17 (eg, del (17p) in leukemic cells). In another example, the subject does not have a del (17p). In embodiments, the subject comprises leukemic cells containing a mutation in the immunoglobulin heavy chain variable region ( _IgVH ) gene. In other embodiments, the subject is free of leukemic cells containing a mutation in the immunoglobulin heavy chain variable region ( _IgVH ) gene. In embodiments, bendamustine is administered, for example, intravenously at a dose of about 70-110 mg / m2 (eg, 70-80, 80-90, 90-100 or 100-110 mg / m2). In embodiments, rituximab is administered, for example, intravenously at a dose of about 400-600 mg / m2 (eg, 400-450, 450-500, 500-550 or 550-600 mg / m ² ).

In embodiments, CAR-expressing cells described herein are administered to a subject in combination with rituximab, cyclophosphamide, doxorubicin, vincristine and / or a corticosteroid (eg, prednisone). In embodiments, CAR-expressing cells described herein are administered to a subject in combination with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP). In embodiments, the subject has diffuse large B-cell lymphoma (DLBCL). In embodiments, the subject has a localized stage DLBCL (eg, including a tumor with a size / diameter less than 7 cm) without a giant mass. In embodiments, the subject is treated with radiation in combination with R-CHOP. For example, the subject is administered R-CHOP (eg, 1-6 cycles, eg 1, 2, 3, 4, 5 or 6 cycles of R-CHOP) followed by radiation. If there is, the subject is administered R-CHOP (eg, 1-6 cycles, eg 1, 2, 3, 4, 5 or 6 cycles of R-CHOP) after radiation.

In embodiments, CAR-expressing cells described herein are administered to a subject in combination with etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin and / or rituximab. In embodiments, CAR-expressing cells described herein are administered to a subject in combination with etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin and rituximab (EPOCH-R). In embodiments, CAR-expressing cells described herein are administered to a subject in combination with dose-controlled EPOCH-R (DA-EPOCH-R). In embodiments, the subject has a B-cell lymphoma, such as a Myc rearranged invasive B-cell lymphoma.

In embodiments, CAR-expressing cells described herein are administered to a subject in combination with rituximab and / or lenalidomide. Lenalidomide ((RS) -3- (4-amino-1-oxo-1,3-dihydro-2H-isoindole-2-yl) piperidine-2,6-dione) is an immunomodulator. In embodiments, CAR-expressing cells described herein are administered to a subject in combination with rituximab and lenalidomide. In embodiments, the subject has follicular lymphoma (FL) or mantle cell lymphoma (MCL). In embodiments, the subject has FL and has not been previously treated with a cancer therapeutic agent. In embodiments, lenalidomide is administered, for example, daily at a dose of about 10-20 mg (eg, 10-15 mg or 15-20 mg). In embodiments, rituximab is administered intravenously, for example, at a dose of about 350-550 mg / m ² (eg, 350-375, 375-400, 400-425, 425-450, 450-475 or 475-500 mg / m ² ). do.

In embodiments, CAR-expressing cells described herein are administered to a subject in combination with brentuximab. Brentuximab is an antibody-drug conjugate of anti-CD30 antibody and monomethyloristatin E. In embodiments, the subject has Hodgkin lymphoma (HL), such as relapsed or refractory HL. In embodiments, the subject comprises CD30 + HL. In embodiments, the subject is undergoing an autologous stem cell transplant (ASCT). In embodiments, the subject has not undergone an ASCT. In embodiments, brentuximab is about 1-3 mg / kg (eg, about 1-1.5 mg / kg, 1.5-2 mg / kg, 2-2.5 mg / kg or 2.5-3 mg / kg). For example, it is administered intravenously, for example, every 3 weeks.

In embodiments, CAR-expressing cells described herein are administered to a subject in combination with brentuximab and dacarbazine or a combination of brentuximab and bendamustine. Dacarbazine is an alkylating agent having the chemical name 5- (3,3-dimethyl-1-triazenyl) imidazole-4-carboxamide. Bendamustine is an alkylating agent having the chemical name 4- [5- [bis (2-chloroethyl) amino] -1-methylbenzimidazol-2-yl] butanoic acid. In embodiments, the subject has Hodgkin lymphoma (HL). In embodiments, the subject has not been previously treated with a cancer therapeutic agent. In embodiments, the subject is at least 60 years old, eg, 60 years old, 65 years old, 70 years old, 75 years old, 80 years old, 85 years old or beyond. In embodiments, dacarbazine is about 300-450 mg / m ² (eg, about 300-325 mg / m ² , 325-350 mg / m ² , 350-375 mg / m ² , 375-400 mg / m ² , 400-425 mg / m). Administer, for example, intravenously at a dose of ² or 425-450 mg / m ² ). In embodiments, bendamustine is administered, for example, intravenously at a dose of about 75-100 mg / m ² (eg, 75-100 mg / m ² or 100-125 mg / m ² , eg, about 90 mg / m ² ). In embodiments, brentuximab is about 1-3 mg / kg (eg, about 1-1.5 mg / kg, 1.5-2 mg / kg, 2-2.5 mg / kg or 2.5-3 mg / kg). For example, it is administered intravenously, for example, every 3 weeks.

In one embodiment, CAR-expressing cells described herein are administered in combination with a CD20 inhibitor, such as an anti-CD20 antibody (eg, anti-CD20 mono or bispecific antibody) or a fragment thereof. Exemplary anti-CD20 antibodies include, but are not limited to, rituximab, ofatumumab, ocrelizumab, beltzzumab, obinutuzumab, TRU-015 (Trubion Pharmaceuticals), ocrelizumab and Pro131921 (Genentech). For example, Lim et al. Haematologica. 95.1 (2010): 135-43.

In one embodiment, the anti-CD20 antibody comprises rituximab. Rituximab is available, for example, at www. accessdata. fda. gov / drugsattfda_docs / label / 2010/103705s5311lbl. As described in pdf, it is a chimeric mouse / human monoclonal antibody IgG1 kappa that binds to CD20 and causes cytolysis of CD20-expressing cells. In embodiments, CAR-expressing cells described herein are administered to a subject in combination with rituximab. In embodiments, the subject has a CLL or SLL.

In one embodiment, rituximab is administered intravenously, eg, as an intravenous drip. For example, each infusion is about 500-2000 mg (eg, about 500-550 mg, 550-600 mg, 600-650 mg, 650-700 mg, 700-750 mg, 750-800 mg, 800-850 mg, 850-900 mg, 900-950 mg, Rituximab (950 to 1000 mg, 1000 to 1100 mg, 1100 to 1200 mg, 1200 to 1300 mg, 1300 to 1400 mg, 1400 to 1500 mg, 1500 to 1600 mg, 1600 to 1700 mg, 1700 to 1800 mg, 1800 to 1900 mg or 1900 to 2000 mg) is provided. In one embodiment, rituximab is 150 mg / m 2-750 mg / m ² , for example about 150-175 mg / m ² , 175-200 mg / m ² , 200-225 mg / m ² , 225-250 mg / ^{m 2 ,} 250-300 mg. / M ² , 300-325 mg / m ² , 325-350 mg / m ² , 350-375 mg / m ² , 375-400 mg / m ² , 400-425 mg / m ² , 425-450 mg / m ² , 450-475 mg / m ² , 475 to 500 mg / m ² , 500 to 525 mg / m ² , 525 to 550 mg / m ² , 550 to 575 mg / m ² , 575 to 600 mg / m ² , 600 to 625 mg / m ² , 625 to 650 mg / m ² , 650-675 mg / m ² or 675-700 mg / m ² doses, where m ² indicates the body surface area of the subject. In one embodiment, rituximab is administered at a dosing interval of at least 4 days, eg, 4 days, 7 days, 14 days, 21 days, 28 days, 35 days or more. For example, rituximab is administered at a dosing interval of at least 0.5 weeks, such as 0.5 weeks, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks or more. In one embodiment, rituximab is given for a period of time, eg at least 2 weeks, eg at least 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks. Administer at 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks or longer at the doses and dosing intervals described herein. For example, rituximab is administered at a total of at least 4 doses per treatment cycle at the doses and dosing intervals described herein (eg, at least 4, 5, 6, 7, 8, 9, 10, per treatment cycle). 11, 12, 13, 14, 15, 16 doses or more).

In one embodiment, the anti-CD20 antibody comprises ofatumumab. Ofatumumab is an anti-CD20 IgG1κ human monoclonal antibody with a molecular weight of approximately 149 kDa. For example, ofatumumab is produced using transgenic mouse and hybridoma technology and is expressed and purified from the recombinant mouse cell line (NS0). For example, www. accessdata. fda. gov / drugsattfda_docs / label / 2009/125326lbl. See pdf; and Clinical Trial Identifier number NCT01363128, NCT01515176, NCT01626352 and NCT01397591. In embodiments, CAR-expressing cells described herein are administered to a subject in combination with ofatumumab. In embodiments, the subject has a CLL or SLL.

In one embodiment, ofatumumab is administered as an intravenous drip. For example, each infusion is about 150-3000 mg (eg, about 150-200 mg, 200-250 mg, 250-300 mg, 300-350 mg, 350-400 mg, 400-450 mg, 450-500 mg, 500-550 mg, 550-600 mg, 600-650 mg, 650-700 mg, 700-750 mg, 750-800 mg, 800-850 mg, 850-900 mg, 900-950 mg, 950-1000 mg, 1000-1200 mg, 1200-1400 mg, 1400-1600 mg, 1600-1800 mg, 1800- Ofatumumab (2000 mg, 2000-2200 mg, 2200-2400 mg, 2400-2600 mg, 2600-2800 mg or 2800-3000 mg) is administered. In embodiments, ofatumumab is administered at a starting dose of about 300 mg followed by 2000 mg, such as about 11 doses, for example for 24 weeks. In one embodiment, ofatumumab is administered at a dosing interval of at least 4 days, eg, 4 days, 7 days, 14 days, 21 days, 28 days, 35 days or more. For example, offatumumab for at least 1 week, eg 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 24 weeks, 26 weeks , 28 weeks, 20 weeks, 22 weeks, 24 weeks, 26 weeks, 28 weeks, 30 weeks or more at dosing intervals. In one embodiment, ofatumumab is applied for a period of time, eg at least 1 week, eg 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 Week, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 22 weeks, 24 weeks, 26 weeks, 28 weeks, 30 weeks, 40 weeks, 50 weeks, 60 weeks or More than that, or 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months or more, or 1 year Administer at the doses and dosing intervals described herein for 2, 2, 3, 4, 5, or more. For example, ofatumumab is administered at a total of at least 2 doses per treatment cycle at the doses and dosing intervals described herein (eg, at least 2, 3, 4, 5, 6, 7, 8, 9 per treatment cycle, 10, 11, 12, 13, 14, 15, 16, 18, 20 or higher doses).

In some cases, the anti-CD20 antibody comprises ocrelizumab. Ocrelizumab is described, for example, in Clinical Trials Identifier Nos. NCT00077870, NCT01412333, NCT00779220, NCT00673920, NCT01194570 and Kappos et al. Lancet. 19.378 (2011): Humanized anti-CD20 monoclonal antibody as described in 1779-87.

In some cases, the anti-CD20 antibody comprises beltszumab. Bertzzumab is a humanized monoclonal antibody against CD20. For example, Clinical Trial Identifier No. NCT00547066, NCT00546793, NCT01101581 and Goldenberg et al. Leuk Lymphoma. 51 (5) (2010): See 747-55.

In some cases, the anti-CD20 antibody comprises GA101. GA101 (also referred to as obinutuzumab or RO5072759) is a humanized and sugar chain modified anti-CD20 monoclonal antibody. For example, Robak. Curr. Opin. Investig. Drugs. 10.6 (2009): 588-96; Clinical Trial Identifier Numbers: NCT01895669, NCT01889797, NCT02229422 and NCT01414205; and www. accessdata. fda. gov / drugsattfda_docs / label / 2013/125486s000lbl. See pdf.

In some cases, the anti-CD20 antibody comprises AME-133v. AME-133v (also referred to as LY2469298 or okalatuzumab) is a humanized IgG1 monoclonal antibody against CD20 with increased affinity for the FcγRIIIa receptor and enhanced antibody-dependent cellular cytotoxicity (ADCC) activity compared to rituximab. .. For example, Robak et al. BioDrugs 25.1 (2011): 13-25; and Forero-Torres et al. Clin Cancer Res. See 18.5 (2012): 1395-403.

In some cases, the anti-CD20 antibody comprises PRO131921. PRO131921 is a humanized anti-CD20 monoclonal antibody that has better binding to FcγRIIIa compared to rituximab and has been engineered to enhance ADCC. For example, Robak et al. BioDrugs 25.1 (2011): 13-25; and Casulo et al. Clin Immunol. 154.1 (2014): 37-46; and Clinical Trial Identifier No. See NCT00452127.

In some cases, the anti-CD20 antibody comprises TRU-015. TRU-015 is an anti-CD20 fusion protein derived from the domain of an antibody against CD20. TRU-015 is smaller than a monoclonal antibody but retains Fc-mediated effector function. For example, Robak et al. See BioDrugs 25.1 (2011): 13-25. TRU-015 contains an anti-CD20 single chain variable fragment (scFv) linked to the human IgG1 hinge, CH2 and CH3 domains, but lacks the CH1 and CL domains.

In one embodiment, the anti-CD20 antibody described herein is used as a therapeutic agent, eg, a chemotherapeutic agent described herein (eg, cytoxane, fludalabine, histon deacetylase inhibitor, demethylating agent, peptide vaccine, anti). Tumor antibiotics, tyrosine kinase inhibitors, alkylating agents, antimicrotubes or thread division inhibitors), antiallergic agents, antiemetics (or antiemetics), analgesics or cytoprotective agents conjugated or other methods Combine with.

In embodiments, CAR-expressing cells described herein are targeted in combination with a B-cell lymphoma 2 (BCL-2) inhibitor (eg, venetoclax, also referred to as ABT-199 or GDC-0199) and / or rituximab. Administer. In embodiments, CAR-expressing cells described herein are administered to a subject in combination with venetoclax and rituximab. Venetoclax is a small molecule that inhibits the anti-apoptotic protein BCL-2. Venetoclax (4- (4-{[2- (4-chlorophenyl) -4,4-dimethylcyclohex-1-en-1-yl] methyl} piperazine-1-yl) -N- ({3-nitro-) The structure of 4-[(tetrahydro-2H-pyran-4-ylmethyl) amino] phenyl} sulfonyl) -2- (1H-pyrrolo [2,3-b] pyridin-5-yloxy) benzamide) is shown below.

In embodiments, the subject has a CLL. In embodiments, the subject has relapsed CLL, for example, the subject has previously been administered cancer treatment. In embodiments, venetoclax is at about 15-600 mg (eg, 15-20 mg, 20-50 mg, 50-75 mg, 75-100 mg, 100-200 mg, 200-300 mg, 300-400 mg, 400-500 mg or 500-600 mg). For example, administer daily. In embodiments, rituximab is about 350-550 mg / m2 (eg, 350-375 mg / m2, 375-400 mg / m2, 400-425 mg / m2, 425-450 mg / m2, 450-475 mg / m2 or 475-500 mg / m2). ), For example, intravenously, for example, every month.

Without being bound by theory, it is believed that B cells (eg, B regulatory cells) can suppress T cells in some cancers. Furthermore, it is believed that the combination of oxyplatin and B cell depleting agents can reduce tumor size and / or eliminate tumors in the subject. In some embodiments, the CAR-expressing cells described herein (eg, BCMA CAR) are B cell depleting agents (eg, CD19 CAR-expressing cells, CD20 CAR-expressing cells, rituximab, ocrelizumab, epratumab or belimumab) and It is given in combination with oxyplatin. In embodiments, the cancer cells can be CD19 negative or CD19 positive or BCMA negative or BMCA positive. In embodiments, the CAR-expressing cells described herein (eg, BCMA CAR) are B for treating a cancer, eg, a cancer described herein, eg, solid cancer, eg, prostate cancer, pancreatic cancer or lung cancer. It is administered in combination with a cell depleting agent and oxyplatin.

In embodiments, the CAR-expressing cells described herein (eg, BCMA CAR) express B cells in the subject (eg, B cells with a plasma cell-like phenotype, such as BCMA, CD19 and / or CD20). Things) can be depleted. In embodiments, B cells can be CD19 negative or CD19 positive or BCMA negative or BMCA positive. In some embodiments, the CAR-expressing cells described herein (eg, BCMA CAR) are administered in combination with oxyplatin. In embodiments, the CAR-expressing cells described herein, administered in combination with oxyplatin, are used to treat cancers such as solid cancers such as prostate cancer, pancreatic cancer or lung cancer. In some embodiments, the CAR-expressing cells described herein are administered in combination with an oncolytic virus. In embodiments, the oncolytic virus can selectively replicate within cancer cells and induce their death or delay their growth. In some cases, oncolytic viruses have no or minimal effect on non-cancerous cells. Oncolytic viruses include oncolytic adenovirus, oncolytic simple herpesvirus, oncolytic retrovirus, oncolytic parvovirus, oncolytic vaccinia virus, oncolytic sindobis virus, oncolytic influenza virus or It includes, but is not limited to, oncolytic RNA viruses such as, but not limited to, oncolytic leovirus, oncolytic Newcastle disease virus (NDV), oncolytic measles virus or oncolytic vesicular stomatitis virus (VSV).

In one embodiment, the oncolytic virus is the virus described in US Patent Application Publication No. 2010/01768684A1, which is incorporated herein by reference in its entirety, eg, a recombinant oncolytic virus. In one embodiment, the recombinant oncolytic virus is an inhibition of an immune or inflammatory response, as described, for example, in US Patent Application Publication No. 2010/0178684A1, which is incorporated herein by reference in its entirety. Includes a nucleic acid sequence encoding an agent (eg, a heterologous nucleic acid sequence). In embodiments, recombinant tumor-dissolving viruses, such as tumor-soluble NDVs, are anti-proliferative proteins (eg, apoptin), cytokines (eg, GM-CSF, interferon-γ, interleukin-2 (IL-2), tumors. Necrosis factor-α), immunoglobulins (eg, antibodies to ED-B fibronectin), tumor-related antigens, bispecific adapter proteins (eg, NDV HN protein and bispecific to T-cell costimulatory receptors such as CD3 or CD28). Sex antibodies or antibody fragments; or fusion proteins of single-stranded antibodies against human IL-2 and NDV HN proteins). For example, Zamarin et al., Which is incorporated herein by reference in its entirety. Future Microbiol. See 7.3 (2012): 347-67. In one embodiment, the oncolytic virus is the US Patent No. 8591881B2, US Patent Application Publication No. 2012/0122185A1 or US Patent Application Publication No. 2014, each of which is incorporated herein by reference in its entirety. / 0271677A1 is a chimeric oncolytic NDV described in the specification.

In one embodiment, the oncolytic virus is a conditional replication adenovirus (CRAd) designed to replicate exclusively in cancer cells. For example, Germany et al. Nature Biotechnology. 18 (2000): 723-27. In one embodiment, the oncolytic adenovirus is incorporated herein by reference in its entirety. Includes those listed in Table 1 on page 725 of.

Exemplary oncolytic viruses include, but are not limited to:
Group B Oncolytic Adenovirus (ColoAd1) (PsiOxus Therapeutics Ltd.) (see, for example, Clinical Trial Identifier: NCT02053220);
ONCOS-102 (formerly known as CGTG-102), an adenovirus containing granulocyte-macrophage colony stimulating factor (GM-CSF) (see, for example, Clinical Trial Identifier: NCT01598129);
VCN-01 (VCN Biosciences, SL), a genetically modified oncolytic human adenovirus encoding human PH20 hyaluronidase (see, eg, Clinical Trial Ideas: NCT02045602 and NCT02045589);
Conditional replication adenovirus ICOVIR-5, a virus derived from wild-type human adenovirus serotype 5 (Had5) that has been modified to selectively replicate in cancer cells with deregulation of the retinoblastoma / E2F pathway. (Institut Catala d'Oncology) (see, for example, Clinical Trial Identity: NCT018647759);
ICOVIR5, Celyvir (Hospital Infantil Universal Nino Jesus, Madrid, Spanish / Ramon Alimany) containing bone marrow-derived autologous mesenchymal stem cells (MSC) infected with oncolytic adenovirus (see, eg, Clinsil );
CG0070, a conditioned replicative tumor-soluble serotype 5 adenovirus (Ad5), in which the human E2F-1 promoter drives the expression of the essential E1a virus gene, thereby limiting viral replication and cytotoxicity to Rb pathway-deficient tumor cells. Cold Genesis, Inc. (see, eg, Clinical Trial Identity: NCT02143804); or selectively replicates in retinoblastoma (Rb) pathway-deficient cells to more efficiently express certain RGD-binding integrans. DNX-2401 (formerly referred to as Delta-24-RGD) (Clinica Universalsidad de Navarra, Universalsidad de Navarra / DNAtrix, Inc.) (eg, Clinical), which is an adenovirus engineered to infect cells that Trial Identityr: See NCT01956734).

In one embodiment, the oncolytic viruses described herein are administered by injection, eg, subcutaneous, intraarterial, intravenous, intramuscular, intrathecal or intraperitoneal injection. In embodiments, the oncolytic viruses described herein are administered intratumorally, transdermally, transmucosally, orally, intranasally or by pulmonary administration.

In one embodiment, cells expressing the CAR described herein are administered to a subject in combination with a molecule that reduces the Treg cell population. Methods of reducing (eg, depleting) the number of Treg cells are known in the art and include, for example, CD25 depletion, cyclophosphamide administration, GITR functional modification. Although not bound by theory, reducing the number of Treg cells in a subject before administration of CAR-expressing cells described herein or before administration of CAR-expressing cells is an unwanted immune cell in the tumor microenvironment (eg, Treg). It is thought to reduce the number and reduce the risk of recurrence of the subject. In some embodiments, the CAR-expressing cells described in the invention are molecules that target and / or regulate GITR function, such as GITR agonists and / or GITR antibodies that deplete regulatory T cells (Tregs). Is administered to the subject in combination with. In embodiments, cells expressing the CARs described herein are administered in combination with cyclophosphamide. In some embodiments, a GITR-binding molecule and / or a molecule that regulates GITR function (eg, a GITR agonist and / or a Treg-depleted GITR antibody) is administered prior to CAR-expressing cells. For example, in some embodiments, the GITR agonist can be administered prior to cell apheresis. In embodiments, cyclophosphamide is administered to the subject prior to administration (eg, infusion or reinjection) of CAR-expressing cells or prior to cell apheresis. In embodiments, cyclophosphamide and anti-GITR antibodies are administered to the subject prior to administration (eg, infusion or reinjection) of CAR-expressing cells or prior to cell apheresis. In some embodiments, the subject has a cancer (eg, solid cancer or multiple myeloma, blood cancer such as ALL or CLL). In one embodiment, the subject has a CLL. In embodiments, the subject has multiple myeloma. In embodiments, the subject has a solid cancer, eg, the solid cancer described herein. Exemplary GITR agonists are, for example, US Pat. No. 6,111,090, European Patent No. 090505B1, US Pat. No. 8,586,023, International Publication No. 2010/003118 and The GITR fusion protein described in the 2011/090754 pamphlet or, for example, US Pat. No. 7,025,962, European Patent No. 1947183B1, US Pat. No. 7,812,135, US Pat. No. 8, , 388,967, US Pat. No. 8,591,886, European Patent No. 1866339, International Publication No. 2011/028683, International Publication No. 2013/039954, International Publication No. 2005 / 00790, International Publication No. 2007/133822, International Publication No. 2005/055808, International Publication No. 99/40196, International Publication No. 2001/03720, International Publication No. 99/20758, Anti-GITR antibodies as described in WO 2006/083289, WO 2005/115451, US Pat. Nos. 7,618,632 and WO 2011/015726, eg, GITR. Includes fusion proteins and anti-GITR antibodies (eg, bivalent anti-GITR antibodies).

In some embodiments, CAR-expressing cells described herein are administered to a subject in combination with an mTOR inhibitor, eg, an mTOR inhibitor described herein, such as everolimus. In some embodiments, the mTOR inhibitor is administered prior to CAR-expressing cells. For example, in some embodiments, the mTOR inhibitor can be administered prior to cell apheresis.

In some embodiments, CAR-expressing cells described herein are administered to a subject in combination with a GITR agonist, eg, a GITR agonist described herein. In some embodiments, the GITR agonist is administered prior to CAR-expressing cells. For example, in some embodiments, the GITR agonist can be administered prior to cell apheresis.

In some embodiments, CAR-expressing cells described herein are administered to a subject in combination with a protein tyrosine phosphatase inhibitor, eg, a protein tyrosine phosphatase inhibitor described herein. In some embodiments, the protein tyrosine phosphatase inhibitor is an SHP-1 inhibitor, such as sodium stibogluconate, eg, the SHP-1 inhibitor described herein. In some embodiments, the protein tyrosine phosphatase inhibitor is a SHP-2 inhibitor.

In some embodiments, the CAR-expressing cells described herein can be used in combination with a kinase inhibitor. In some embodiments, the kinase inhibitor is a CDK4 inhibitor, eg, a CDK4 inhibitor described herein, eg, 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl). -Ppyridine-2-ylamino) -8H-pyrido [2,3-d] pyrimidin-7-one, a hydroxychloride (also called palbociclib or PD0332991) and the like, for example CDK4 / 6 inhibitors. In some embodiments, the kinase inhibitor is a BTK inhibitor, such as ibrutinib, eg, the BTK inhibitor described herein. In some embodiments, the kinase inhibitor is an mTOR inhibitor, such as rapamycin, rapamycin analog, OSI-027, etc., eg, the mTOR inhibitor described herein. The mTOR inhibitor can be, for example, an mTORC1 inhibitor and / or an mTORC2 inhibitor, such as the mTORC1 inhibitor and / or mTORC2 inhibitor described herein. In some embodiments, the kinase inhibitor is an MNK inhibitor, such as a 4-amino-5- (4-fluoroanilino) -pyrazolo [3,4-d] pyrimidine, eg, the MNK described herein. It is an inhibitor. The MNK inhibitor can be, for example, an MNK1a, MNK1b, MNK2a and / or MNK2b inhibitor. In some embodiments, the kinase inhibitor is a dual PI3K / mTOR inhibitor as described herein, such as, for example, PF-04695102. In some embodiments, the kinase inhibitor is a DGK inhibitor, such as a DGKinh1 (D5919) or DGKinh2 (D5794), eg, a DGK inhibitor described herein.

In some embodiments, the kinase inhibitor is alloyin A; flavopyridol or HMR-1275, 2- (2-chlorophenyl) -5,7-dihydroxy-8-[(3S, 4R) -3-hydroxy-. 1-Methyl-4-piperidinyl] -4-chromenone; crizotinib (PF-02341066; 2- (2-chlorophenyl) -5,7-dihydroxy-8-[(2R, 3S) -2- (hydroxymethyl) -1 -Methyl-3-pyrrolidinyl] -4H-1-benzopyran-4-one, hydrochloride (P276-00); 1-methyl-5-[[2- [5- (trifluoromethyl) -1H-imidazole-2) -Il] -4-pyridinyl] oxy] -N- [4- (trifluoromethyl) phenyl] -1H-benzimidazole-2-amine (RAF265); indislam (E7070); roscobitin (CYC202); parvocyclib (PD0332991) Dinacyclib (SCH72765); N- [5-[[(5-tert-butyloxazol-2-yl) methyl] thio] thiazole-2-yl] piperidine-4-carboxamide (BMS 387032); 4-[[9 -Chloro-7- (2,6-difluorophenyl) -5H-pyrimid [5,4-d] [2] benzazepine-2-yl] amino] -benzoic acid (MLN8054); 5- [3- (4,4) 6-Difluoro-1H-benzimidazole-2-yl) -1H-indazole-5-yl] -N-ethyl-4-methyl-3-pyridinemethaneamine (AG-024322); 4- (2,6-dichloro) Benzoylamino) -1H-pyrazole-3-carboxylic acid N- (piperidine-4-yl) amide (AT7519); 4- [2-methyl-1- (1-methylethyl) -1H-imidazole-5-yl] A CDK4 inhibitor selected from -N- [4- (methylsulfonyl) phenyl] -2-pyrimidineamine (AZD5438); and XL281 (BMS908662).

In some embodiments, the kinase inhibitor is a CDK4 inhibitor, such as palbociclib (PD0332991), which is palbociclib for a period of time approximately 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, 100 mg, 105 mg, 110 mg, per day. Doses at doses of 115 mg, 120 mg, 125 mg, 130 mg, 135 mg (eg, 75 mg, 100 mg or 125 mg) are administered daily, for example 14-21 days in a 28-day cycle or 7-12 days daily in a 21-day cycle. In some embodiments, palbociclib is administered in 1 cycle, 2 cycles, 3 cycles, 4 cycles, 5 cycles, 6 cycles, 7 cycles, 8 cycles, 9 cycles, 10 cycles, 11 cycles, 12 cycles or more. ..

In embodiments, CAR-expressing cells described herein are administered to a subject in combination with a cyclin-dependent kinase (CDK) 4 or 6 inhibitor, such as a CDK4 or CDK6 inhibitor described herein. In embodiments, CAR-expressing cells described herein are combined with a CDK4 / 6 inhibitor (eg, an inhibitor that targets both CDK4 and CDK6), eg, a CDK4 / 6 inhibitor described herein. Administer to the subject. In one embodiment, the subject has an MCL. MCL is an essentially incurable invasive cancer that responds poorly to currently available therapies. In many cases of MCL, cyclin D1 (a regulator of CDK4 / 6) is expressed in MCL cells (eg, due to chromosomal translocations involving immunoglobulins and the cyclin D1 gene). Therefore, without being bound by theory, MCL cells are considered to be highly specific (ie, minimal effect on normal immune cells) and highly sensitive to CDK4 / 6 inhibition. The CDK4 / 6 inhibitor alone has some efficacy in treating MCL, but only partial remission is achieved and the rate of recurrence is high. An exemplary CDK4 / 6 inhibitor is LEE011 (ribociclib), the structure of which is shown below.

Without being bound by theory, administration of CAR-expressing cells and CDK4 / 6 inhibitors described herein (eg, LEE011 or other CDK4 / 6 inhibitors described herein) may, for example, inhibit CDK4 / 6. It is believed to achieve higher responsiveness, eg, higher remission rate and / or lower recurrence rate, compared to the agent alone.

In some embodiments, the kinase inhibitor is ibrutinib (PCI-32765); GDC-0834; RN-486; CGI-560; CGI-1764; HM-17224; CC-292; ONO-4059; CNX-774. A BTK inhibitor selected from LFM-A13. In a preferred embodiment, the BTK inhibitor does not reduce or inhibit the kinase activity of interleukin-2-induced kinase (ITK), GDC-0834; RN-486; CGI-560; CGI-1764; HM-17124. CC-292; ONO-4059; CNX-774; and LFM-A13.

In some embodiments, the kinase inhibitor is a BTK inhibitor, such as ibrutinib (PCI-32765). In embodiments, CAR-expressing cells described herein are administered to a subject in combination with a BTK inhibitor (eg, ibrutinib). In embodiments, CAR-expressing cells described herein are administered to a subject in combination with ibrutinib (also referred to as PCI-32765). Ibrutinib (1-[(3R) -3- [4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d] pyrimidin-1-yl] piperidine-1-yl] prop-2 The structure of -en-1-on) is shown below.

In embodiments, the subject has CLL, mantle cell lymphoma (MCL) or small lymphocytic lymphoma (SLL). For example, the subject has a deletion in the short arm of chromosome 17 (eg, del (17p) in leukemic cells). In another example, the subject does not have a del (17p). In embodiments, the subject has a relapsed CLL or SLL, eg, the subject has been previously administered cancer treatment (eg, previously received 1, 2, 3 or 4 prior cancer treatments). ing). In embodiments, the subject has refractory CLL or SLL. In other embodiments, the subject has follicular lymphoma, such as relapsed or refractory follicular lymphoma. In some embodiments, ibrutinib is about 300-600 mg / day (eg, about 300-350, 350-400, 400-450, 450-500, 500-550 or 550-600 mg / day, eg, about 420 mg / day. Alternatively, it is administered, for example, orally at a dose of about 560 mg / day). In embodiments, ibrutinib is administered at doses of about 250 mg, 300 mg, 350 mg, 400 mg, 420 mg, 440 mg, 460 mg, 480 mg, 500 mg, 520 mg, 540 mg, 560 mg, 580 mg, 600 mg (eg, 250 mg, 420 mg or 560 mg) daily for a period of time. For example, it is administered daily in a 21-day cycle or daily in a 28-day cycle. In some embodiments, ibrutinib is administered in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 cycles or more. In one embodiment, ibrutinib is administered in combination with rituximab. For example, Burger et al. （２０１３）Ｉｂｒｕｔｉｎｉｂ Ｉｎ Ｃｏｍｂｉｎａｔｉｏｎ Ｗｉｔｈ Ｒｉｔｕｘｉｍａｂ（ｉＲ）Ｉｓ Ｗｅｌｌ Ｔｏｌｅｒａｔｅｄ ａｎｄ Ｉｎｄｕｃｅｓ ａ Ｈｉｇｈ Ｒａｔｅ Ｏｆ Ｄｕｒａｂｌｅ Ｒｅｍｉｓｓｉｏｎｓ Ｉｎ Ｐａｔｉｅｎｔｓ Ｗｉｔｈ Ｈｉｇｈ－Ｒｉｓｋ Ｃｈｒｏｎｉｃ Ｌｙｍｐｈｏｃｙｔｉｃ Ｌｅｕｋｅｍｉａ（ＣＬＬ）：Ｎｅｗ，Ｕｐｄａｔｅｄ Ｒｅｓｕｌｔｓ Ｏｆ ａ Ｐｈａｓｅ ＩＩ Ｔｒｉａｌ Ｉｎ ４０ Ｐａｔｉｅｎｔｓ，Ａｂｓｔｒａｃｔ ６７５ See presented at 55th ASH Annual Meeting and Expression, New Orleans, LA 7-10 Dec. Although not theorized, it is believed that the addition of ibrutinib can enhance the T cell proliferative response and shift T cells from the T-helper-2 (Th2) to the T-helper-1 (Th1) phenotype. Be done. Th1 and Th2 are phenotypes of helper T cells and direct different immune response pathways between Th1 and Th2. The Th1 phenotype is associated with perpetuating an pro-inflammatory or autoimmune response to kill or injure cells such as intracellular pathogens / viruses or cancerous cells. The Th2 phenotype is associated with eosinophil accumulation and anti-inflammatory response.

（式中、
Ｒ１は水素、任意選択によりヒドロキシで置換され得るＣ１－Ｃ６アルキルであり；
Ｒ２は水素又はハロゲンであり；
Ｒ３は水素又はハロゲンであり；
Ｒ４は水素であり、
Ｒ５は水素又はハロゲンであり；
又はＲ４とＲ５は互いに結合し、結合、－ＣＨ２－、－ＣＨ２－ＣＨ２－、－ＣＨ＝ＣＨ－、－ＣＨ＝ＣＨ－ＣＨ２－；－ＣＨ２－ＣＨ＝ＣＨ－；又は－ＣＨ２－ＣＨ２－ＣＨ２－を意味し；
Ｒ６及びＲ７は互いに独立してＨ、任意選択によりヒドロキシで置換され得るＣ１－Ｃ６アルキル、任意選択によりハロゲン又はヒドロキシ又はハロゲンで置換され得るＣ３－Ｃ６シクロアルキルを表し；
Ｒ８、Ｒ９、Ｒ、Ｒ’、Ｒ１０及びＲ１１は互いに独立してＨ又は任意選択によりＣ１－Ｃ６アルコキシで置換され得るＣ１－Ｃ６アルキルを表すか；又はＲ８、Ｒ９、Ｒ、Ｒ’、Ｒ１０及びＲ１１の任意の２個は、それらが結合している炭素原子と一体となって３～６員飽和炭素環式環を形成し得；
Ｒ１２は水素又は任意選択によりハロゲン若しくはＣ１－Ｃ６アルコキシで置換され得るＣ１－Ｃ６アルキルであり；
又はＲ１２とＲ８、Ｒ９、Ｒ、Ｒ’、Ｒ１０又はＲ１１のいずれか１つは、それらが結合している原子と一体となって、４員、５員、６員又は７員アザシクロ環を形成し得、その環は、任意選択によりハロゲン、シアノ、ヒドロキシル、Ｃ１－Ｃ６アルキル又はＣ１－Ｃ６アルコキシで置換され得；
ｎは０又は１であり；
Ｒ１３は任意選択によりＣ１－Ｃ６アルキルで置換され得るＣ２－Ｃ６アルケニル、Ｃ１－Ｃ６アルコキシ又は任意選択によりＣ１－Ｃ６アルキル若しくはＣ１－Ｃ６アルコキシで置換され得るＮ，Ｎ－ジ－Ｃ１－Ｃ６アルキルアミノＣ２－Ｃ６アルキニル；又は任意選択によりＣ１－Ｃ６アルキルで置換され得るＣ２－Ｃ６アルキレニルオキシドである）。 In one embodiment of the methods, uses and compositions herein, a BTK inhibitor is the BTK inhibitor described in International Publication No. 2015/079417, which is incorporated herein by reference in its entirety. For example, in one embodiment, the BTK inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt thereof.

(During the ceremony,
R1 is hydrogen, a C1-C6 alkyl that can be optionally substituted with hydroxy;
R2 is hydrogen or halogen;
R3 is hydrogen or halogen;
R4 is hydrogen,
R5 is hydrogen or halogen;
Or R4 and R5 bind to each other and bind, -CH2-, -CH2-CH2-, -CH = CH-, -CH = CH-CH2-; -CH2-CH = CH-; or -CH2-CH2-CH2. -Meaning;
R6 and R7 represent H independently of each other, C1-C6 alkyl which can be optionally substituted with hydroxy, and optionally halogen or hydroxy or C6 cycloalkyl which can be substituted with hydroxy or halogen;
Do R8, R9, R, R', R10 and R11 represent C1-C6 alkyls that can be independently substituted with H or optionally C1-C6 alkoxy; or R8, R9, R, R', R10 and Any two of R11 may be integrated with the carbon atom to which they are bonded to form a 3- to 6-membered saturated carbocyclic ring;
R12 is a C1-C6 alkyl that can be substituted with hydrogen or optionally a halogen or C1-C6 alkoxy;
Alternatively, any one of R12 and R8, R9, R, R', R10 or R11 is integrated with the atom to which they are bonded to form a 4-membered, 5-membered, 6-membered or 7-membered azacyclo ring. The ring may optionally be substituted with halogen, cyano, hydroxyl, C1-C6 alkyl or C1-C6 alkoxy;
n is 0 or 1;
R13 is optionally substituted with C2-C6 alkoxy, C1-C6 alkoxy or optionally C1-C6 alkyl or C1-C6 alkoxy N, N-di-C1-C6 alkylamino. C2-C6 Alkoxyl; or C2-C6 Alkoxyloxide which can optionally be substituted with C1-C6 Alkoxy).

In some embodiments, the BTK inhibitor of formula I is N- (3-(5-((1-acryloyl azetidine-3-yl) oxy) -6-aminopyrimidine-4-yl) -5-. Fluoro-2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide; (E) -N- (3- (6-amino-5-((1- (but-2-enoyl) azetidine-3-yl) ) Oxy) Pyrimidine-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide; N- (3- (6-amino-5-) ((1-propioloyl azeti)) Din-3-yl) oxy) Pyrimidine-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide; N- (3- (6-amino-5-((1) -(Buta-2-inoyl) azetidine-3-yl) oxy) pyrimidine-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide; N- (3- (5) -((1-Acryloylpiperidin-4-yl) oxy) -6-aminopyrimidine-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide; N- (3- (3-yl) (6-Amino-5- (2- (N-methylacrylamide) ethoxy) pyrimidine-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide; (E) -N -(3- (6-Amino-5- (2- (N-methylbut-2-enamide) ethoxy) pyrimidine-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2-fluoro Benzamide; N- (3- (6-amino-5- (2- (N-methylpropiolamide) ethoxy) pyrimidine-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2 -Fluorobenzamide; (E) -N- (3- (6-amino-5- (2- (4-methoxy-N-methylbut-2-enamide) ethoxy) pyrimidine-4-yl) -5-fluoro-2 -Methylphenyl) -4-cyclopropyl-2-fluorobenzamide; N- (3- (6-amino-5- (2- (N-methylbut-2-inamide) ethoxy) pyrimidine-4-yl) -5 Fluoro-2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide; N-(2-((4-amino-6- (3- (4-cyclopropyl-2) -) -Fluorobenzamide) -5-fluoro-2-methylphenyl) pyrimidin-5-yl) oxy) ethyl) -N-methyloxylan-2-carboxamide; N- (2-((4-Amino-6- (3-amino-6- (3-) (6-Cyclopropyl-8-fluoro-1-oxoisoquinolin-2 (1H) -yl) phenyl) pyrimidin-5-yl) oxy) ethyl) -N-methylacrylamide; N- (3- (5- (2) -Acrylamideethoxy) -6-aminopyrimidin-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide; N- (3- (6-amino-5- (2-) (N-Ethylacrylamide) ethoxy) pyrimidin-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide; N- (3- (6-amino-5- (2-) (N- (2-fluoroethyl) acrylamide) ethoxy) pyrimidin-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide; N- (3- (5-(((() 1-acrylamide cyclopropyl) methoxy) -6-aminopyrimidine-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide; (S) -N- (3- (5) -(2-acrylamide propoxy) -6-aminopyrimidine-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide; (S) -N- (3- (6- (6-) Amino-5- (2- (but-2-inamide) propoxy) pyrimidin-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide; (S) -N- ( 3- (6-Amino-5- (2- (N-methylacrylamide) propoxy) pyrimidin-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide; (S) -N- (3- (6-Amino-5- (2- (N-methylbut-2-inamide) propoxy) pyrimidin-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2 -Fluorobenzamide; N- (3- (6-amino-5- (3- (N-methylacrylamide) propoxy) pyrimidin-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2 -Fluorobenzamide; (S) -N- (3- (5-((1) -Acryloylpyrrolidin-2-yl) methoxy) -6-aminopyrimidine-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide; (S) -N- (3-) (6-Amino-5-((1- (Buta-2-inoyl) pyrrolidine-2-yl) methoxy) pyrimidin-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2- Fluorobenzamide; (S) -2- (3-(5-((1-acryloylpyrrolidin-2-yl) methoxy) -6-aminopyrimidine-4-yl) -5-fluoro-2- (hydroxymethyl) phenyl ) -6-Cyclopropyl-3,4-dihydroisoquinolin-1 (2H) -on; N-(2-((4-Amino-6- (3- (6-cyclopropyl-1-oxo-3,4) -Dihydroisoquinolin-2 (1H) -yl) -5-fluoro-2- (hydroxymethyl) phenyl) pyrimidin-5-yl) oxy) ethyl) -N-methylacrylamide; N- (3- (5-(((((- (2S, 4R) -1-acryloyl-4-methoxypyrrolidin-2-yl) methoxy) -6-aminopyrimidine-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2-fluoro Benzamide; N- (3- (6-amino-5-(((2S, 4R) -1- (buta-2-inoyl) -4-methoxypyrrolidin-2-yl) methoxy) pyrimidin-4-yl)- 5-Fluoro-2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide; 2-(3-(5-((((2S, 4R) -1-acryloyl-4-methoxypyrrolidin-2-yl) methoxy) ) -6-Aminopyrimidine-4-yl) -5-Fluoro-2- (hydroxymethyl) phenyl) -6-cyclopropyl-3,4-dihydroisoquinolin-1 (2H) -one; N- (3- (3-( 5-(((2S, 4S) -1-acryloyl-4-methoxypyrrolidin-2-yl) methoxy) -6-aminopyrimidine-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl -2-Fluorobenzamide; N- (3- (6-amino-5-(((2S, 4S) -1- (buta-2-inoyl) -4-methoxypyrrolidin-2-yl) methoxy) pyrimidin-4 -Il) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide; N- (3-(5-(((2S, 4R)) ) -1-Acryloyl-4-fluoropyrrolidin-2-yl) methoxy) -6-aminopyrimidine-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide; N- (3- (6-Amino-5-(((2S, 4R) -1- (Buta-2-inoyl) -4-fluoropyrrolidin-2-yl) methoxy) pyrimidin-4-yl) -5-fluoro- 2-Methylphenyl) -4-cyclopropyl-2-fluorobenzamide; (S) -N- (3-(5-((1-acryloyl azetidine-2-yl) methoxy) -6-aminopyrimidine-4- Il) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide; (S) -N- (3- (6-amino-5-((1-propioloyl azetidine-2)) -Il) methoxy) pyrimidin-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide; (S) -2- (3-(5-((1-acryloyl azeti)) Din-2-yl) methoxy) -6-aminopyrimidine-4-yl) -5-fluoro-2- (hydroxymethyl) phenyl) -6-cyclopropyl-3,4-dihydroisoquinolin-1 (2H) -one (R) -N- (3- (5-((1-Acryloyl azetidine-2-yl) methoxy) -6-aminopyrimidine-4-yl) -5-fluoro-2-methylphenyl) -4- Cyclopropyl-2-fluorobenzamide; (R) -N- (3-(5-((1-acryloylpiperidin-3-yl) methoxy) -6-aminopyrimidine-4-yl) -5-fluoro-2- Methylphenyl) -4-cyclopropyl-2-fluorobenzamide; N-(5-((((2R, 3S) -1-acryloyl-3-methoxypyrrolidin-2-yl) methoxy) -6-aminopyrimidine) -4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide; N-(3-(5-(((2S, 4R) -1-acryloyl-4-cyanopyrimidine) -2-yl) methoxy) -6-aminopyrimidine-4-yl) -5-fluoro-2-methylphenyl) -4-cyclopropyl-2-fluorobenzamide; 2S, 4S) -1-acryloyl-4-cyanopyrrolidin-2-yl) methoxy) -6-aminopyrimidine-4-yl) -5-fluoro-2 -Methylphenyl) -4-cyclopropyl-2-fluorobenzamide is selected.

Unless otherwise stated, the chemical terms used in the BTK inhibitors of Formula I above are used in their entirety in accordance with the meanings set forth in WO 2015/079417, incorporated herein by reference in their entirety.

In some embodiments, the kinase inhibitor is temsilolimus; lidaforolimus (1R, 2R, 4S) -4-[(2R) -2-[(1R, 9S, 12S, 15R, 16E, 18R, 19R,), 21R, 23S, 24E, 26E, 28Z, 30S, 32S, 35R) -1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14 , 20-Pentaoxo-11,36-dioxa-4-azatricyclo [ ^30.3.1.0 4.9] hexatriacenta-16,24,26,28-tetraene-12-yl] propyl] -2-methoxy Also known as cyclohexyldimethylphosphinate, AP23573 and MK8669; everolimus (RAD001); rapamycin (AY22989); semapimod; (5- {2,4-bis [(3S) -3-methylmorpholin-4-yl] pyrido] 2,3-d] pyrimidin-7-yl} -2-methoxyphenyl) methanol (AZD8055); 2-amino-8- [trans-4- (2-hydroxyethoxy) cyclohexyl] -6- (6-methoxy- 3-Pyridinyl) -4-methyl-pyrido [2,3-d] pyrimidin-7 (8H) -on (PF04691502); and N2- [1,4-dioxo- ⁴ -[[4- (4-oxo) -8-Phenyl-4H-1-benzopyran-2-yl) morpholinium-4-yl] methoxy] butyl] -L-arginylglycyl-L-α-aspartyl L-serine- (SEQ ID NO: 285), intramolecular A salt (SF1126); and an mTOR inhibitor selected from XL765.

In some embodiments, the kinase inhibitor is an mTOR inhibitor, eg rapamycin, which is constant daily at doses of about 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg (eg, 6 mg). Administer daily for a period of time, eg, a 21-day cycle or a 28-day cycle. In some embodiments, rapamycin is administered in 1 cycle, 2 cycles, 3 cycles, 4 cycles, 5 cycles, 6 cycles, 7 cycles, 8 cycles, 9 cycles, 10 cycles, 11 cycles, 12 cycles or more. .. In some embodiments, the kinase inhibitor is an mTOR inhibitor, such as everolimus, which is an everolimus of about 2 mg, 2.5 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg daily over a period of time. , 11 mg, 12 mg, 13 mg, 14 mg, 15 mg (eg, 10 mg) doses daily, eg in a 28-day cycle. In some embodiments, 1 cycle, 2 cycles, 3 cycles, 4 cycles, 5 cycles, 6 cycles, 7 cycles, 8 cycles, 9 cycles, 10 cycles, 11 cycles, 12 cycles or more is administered everolimus. ..

In some embodiments, the kinase inhibitor is CGP052088; 4-amino-3- (p-fluorophenylamino) -pyrazolo [3,4-d] pyrimidine (CGP57380); cercosporamide; ETC-1780445-2; and A MNK inhibitor selected from 4-amino-5- (4-fluoroanilino) -pyrazolo [3,4-d] pyrimidines.

In embodiments, CAR-expressing cells described herein are targeted in combination with a phosphoinositide 3-kinase (PI3K) inhibitor (eg, a PI3K inhibitor described herein, eg, idelalisib or duvericib) and / or rituximab. Administer. In embodiments, CAR-expressing cells described herein are administered to a subject in combination with idelalisib and rituximab. In embodiments, the CAR-expressing cells described herein are administered to a subject in combination with duverisive and rituximab. Idelalisib (also called GS-1011 or CAL-101; Gilead) is a small molecule that blocks the delta isoform of PI3K. The structure of idelalisib (5-fluoro-3-phenyl-2-[(1S) -1- (7H-purine-6-ylamino) propyl] -4 (3H) -quinazolinone) is shown below.

Duvellisive (also called IPI-145; Infinity Pharmaceuticals and Abbvie) is a small molecule that blocks PI3K-δ, γ. In one embodiment, the structure of duvericive (8-chloro-2-phenyl-3-[(1S) -1- (9H-purine-6-ylamino) ethyl) -1 (2H) -isoquinolinone) is shown below.

In embodiments, the subject has a CLL. In embodiments, the subject has relapsed CLL, eg, the subject has been previously administered cancer treatment (eg, previously anti-CD20 antibody or previously ibrutinib). ). For example, the subject has a deletion in the short arm of chromosome 17 (eg, del (17p) in leukemic cells). In another example, the subject does not have a del (17p). In embodiments, the subject comprises leukemic cells containing a mutation in the immunoglobulin heavy chain variable region ( _IgVH ) gene. In other embodiments, the subject is free of leukemic cells containing a mutation in the immunoglobulin heavy chain variable region ( _IgVH ). In embodiments, the subject has a deletion in the long arm of chromosome 11 (del (11q)). In other embodiments, the subject does not have a del (11q). In embodiments, about 100-400 mg of idelalisib (eg, 100-125 mg, 125-150 mg, 150-175 mg, 175-200 mg, 200-225 mg, 225-250 mg, 250-275 mg, 275-300 mg, 325-350 mg, 350). -375 mg or 375-400 mg), for example, BID is administered. In embodiments, devericive is administered at about 15-100 mg (eg, about 15-25, 25-50, 50-75 or 75-100 mg), eg, twice daily. In embodiments, rituximab is about 350-550 mg / m ² (eg, 350-375 mg / m ² , 375-400 mg / m ² , 400-425 mg / m ² , 425-450 mg / m ² , 450-475 mg / m ² ). Alternatively, it is administered intravenously, for example, at 475 to 500 mg / m ² ).

In embodiments, CAR-expressing cells described herein are administered to a subject in combination with an anaplastic lymphoma kinase (ALK) inhibitor. Exemplary ALK kinases are crizotinib (Pfizer), ceritinib (Novartis), alectinib (middle and outer), brigatinib (also called AP26113; Arid), entrectinib (Ignyta), PF-06463922 (Pfizer), TSR-01. See, for example, Clinical Trial Ideal Pfizer No. NCT02048488), CEP-37440 (Teva) and X-396 (Xcovery). In one embodiment, the subject has a solid cancer, eg, the solid cancer described herein, eg, lung cancer.

The chemical name for crizotinib is 3-[(1R) -1- (2,6-dichloro-3-fluorophenyl) ethoxy] -5- (1-piperidin-4-ylpyrazole-4-yl) pyridine-2- Amine. The chemical name of ceritinib is 5-chloro-N ^2- [2-isopropoxy-5-methyl-4- ( ⁴ -piperidinyl) phenyl] -N4- [2- (isopropylsulfonyl) phenyl] -2,4- Pyrimidine diamine. The chemical name for alectinib is 9-ethyl-6,6-dimethyl-8- (4-morpholinopiperidin-1-yl) -11-oxo-6,11-dihydro-5H-benzo [b] carbazole-3-carboline. It is nitrile. The chemical name of brigatinib is 5-chloro-N ^2- {4- [4- (dimethylamino) -1-piperidinyl] -2-methoxyphenyl} -N ^4- [2- (dimethylphosphoryl) phenyl] -2, 4-Pyrimidine diamine. The chemical name of enrectinib is N- (5- (3,5-difluorobenzyl) -1H-indazole-3-yl) -4- (4-methylpiperazin-1-yl) -2-((tetrahydro-2H-). Pyran-4-yl) amino) benzamide. The chemical name for PF-06463922 is (10R) -7-amino-12-fluoro-2,10,16-trimethyl-15-oxo-10,15,16,17-tetrahydro-2H-8,4- (Meteno). ) Pyrazolo [4,3-h] [2,5,11] -benzoxadiazacyclotetradecine-3-carbonitrile. The chemical name of CEP-37440 is (S) -2-((5-chloro-2-((6- (4- (2-hydroxyethyl) piperazine-1-yl) -1-methoxy-6,7, 8,9-Tetrahydro-5H-benzo [7] annulene-2-yl) amino) pyrimidine-4-yl) amino) -N-methylbenzamide. The chemical name of X-396 is (R) -6-amino-5- (1- (2,6-dichloro-3-fluorophenyl) ethoxy) -N- (4- (4-methylpiperazine-1-carbonyl). ) Phenyl) Pyridazine-3-carboxamide.

In some embodiments, the kinase inhibitor is 2-amino-8- [trans-4- (2-hydroxyethoxy) cyclohexyl] -6- (6-methoxy-3-pyridinyl) -4-methyl-pyrido [ 2,3-d] pyrimidin-7 (8H) -one (PF-04691502); N- [4-[[4- (dimethylamino) -1-piperidinyl] carbonyl] phenyl] -N'-[4-( 4,6-di-4-morpholinyl-1,3,5-triazine-2-yl) phenyl] urea (PF-05212384, PKI-587); 2-methyl-2- {4- [3-methyl-2) -Oxo-8- (quinoline-3-yl) -2,3-dihydro-1H-imidazole [4,5-c] quinoline-1-yl] phenyl} propanenitrile (BEZ-235); apitrisive (GDC-0980) , RG7422); 2,4-difluoro-N- {2- (methyloxy) -5- [4- (4-pyridazinyl) -6-quinolinyl] -3-pyridinyl} benzenesulfonamide (GSK2126458); 8-( 6-methoxypyridin-3-yl) -3-methyl-1- (4- (piperazine-1-yl) -3- (trifluoromethyl) phenyl) -1H-imidazole [4,5-c] quinoline-2 (3H) -Onmaleic acid (NVP-BGT226); 3- [4- (4-morpholinyl pyrido [3', 2': 4,5] flo [3,2-d] pyrimidin-2-yl ] Phenyl (PI-103); 5- (9-isopropyl-8-methyl-2-morpholino-9H-purin-6-yl) pyrimidin-2-amine (VS-5584, SB2343); and N- [2- Dual phosphatidylinositol 3-kinase selected from [(3,5-dimethoxyphenyl) amino] quinoxalin-3-yl] -4-[(4-methyl-3-methoxyphenyl) carbonyl] aminophenyl sulfonamide (XL765) (PI3K) and mTOR inhibitor.

A drug that inhibits calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or p70S6 kinase, which is important for proliferative factor-induced signaling (rapamycin) (Liu et al., Cell 66: 807-815, 1991; Henderson et al. , Immun. 73: 316-321, 1991; Bierer et al., Curr. Opin. Immuno. 5: 763-773, 1993) can also be used. In a further embodiment, the cell composition of the invention is used for bone marrow transplantation, chemotherapeutic agents such as fludarabine, in vitro irradiation therapy (XRT), cyclophosphamide and / or T cell depletion therapy using antibodies such as OKT3 or CAMPATH. Can be administered to the patient in combination with (eg, before, at the same time, or after). In one embodiment, the cell composition of the invention is administered after a B cell depletion therapy such as a drug that reacts with CD20, such as Rituxan. For example, in some embodiments, the subject is subject to standard treatment with high-dose chemotherapeutic agents followed by peripheral blood stem cell transplantation. In one embodiment, after transplantation, the subject receives an infusion of proliferated immune cells of the invention. In a further embodiment, the proliferating cells are administered before or after surgery.

In some embodiments, the CAR-expressing cells described herein are biphosphonates, such as pamidronate (Aredia®); zoledronic acid or zoledronic acid (Zoledronic acid, Zomera®, Acclasta®. Trademark) or Reclast®); Allendronate (Fosamax®); Lysedronate (Actonel®); Zoledronic Acid (Boniva®); Clodronicate (Bonefos®); Zoledronic (Didronel®); Childronate (SKelid®); Pamidronate (Aredia®); Neridronate (Nerixia®); Strongin lanelate (Proteros®) or Protos®); and teriparatide (Forteo®) to administer to the subject.

In some embodiments, the CAR-expressing cells described herein are corticosteroids such as dexamesazone (eg, Prednisolone®), bechrometazone (eg, Beclovent®), hydrocortisone (cortisone, hydrocortisone sodium). Succinate, also known as hydrocortisone sodium phosphate, trade names Ala-Cort®, Hydrocortisone Phosphate, Solu-Cortef®, Hydrocort Actate® and Lanacort®, prednisolone. (Sold under trade names Delta-Cortel®, Orapred®, Prednisolone® and Prednisolone®), Prednisolone (trade names Deltasone®, LiquidRed®, Methylprednisolone) (Registered Trademark) and Sold as Orazone®), Methylprednisolone (also known as 6-methylprednisolone, Methylprednisolone acetate, Methylprednisolone sodium succinate, trade names Duralone®, Medralone®) , Sold as Medrol®, M-Prednisolone® and Solu-Medrol®); antihistamines such as diphenhydramine (eg, Benderlyl®), hydroxydine and cyproheptazine; and beta-adrenaline acceptance. It is administered to a subject in combination with a bronchial dilator such as a body agonist, albuterol (eg, Proventil®) and terbutalin (Brethine®).

In some embodiments, the CAR-expressing cells described herein are immunomodulators such as aftuzumab (available from Roche®); pegfilgrastim (Neulasta®); thalidomide (CC-). 5013, Revlimid®); thalidomide (Thalidomide®), actimid (CC4047); and IRX-2 (interleukin 1, interleukin 2 and human cytokine mixture containing interferon gamma, CAS 951209-71-5). , Available from IRX Therapeutics).

In some embodiments, the CAR-expressing cells described herein are proteasome inhibitors such as bortezomib (Velcade®); ixazomib citrate (MLN9708, CAS 1201902-80-8); danoprevir (RG7227, CAS). 850876-88-9); ixazomib (MLN2238, CAS 1072833-77-2); and (S) -N-[(phenylmethoxy) carbonyl] -L-leucine-N- (1-formyl-3-methylbutyl)- It is administered to the subject in combination with L-leucine amide (MG-132, CAS133407-82-6).

In some embodiments, the CAR-expressing cells described herein are vascular endothelial growth factor (VEGF) receptors such as bevasizumab (Avastin®), axitinib (Inlyta®); brivanib alaninate. (BMS-582664, (S)-((R) -1-(4- (4-fluoro-2-methyl-1H-indole-5-yloxy) -5-methylpyrrolo [2,1-f] [1, 2,4] Triazine-6-yloxy) Propan-2-yl) 2-aminopropanoate); Sorafenib (Nexavar®); Pazopanib (Votrient®); Sunitinib apple acid (Registered trademark). )); Sedilanib (AZD2171, CAS 288383-20-1); Vargatef (BIBF1120, CAS 928326-83-4); Foretinib (GSK1363089); Terratinib (BAY57-9352, CAS 332012-40-5); Apatinib (YN96) CAS 811803-05-1); imatinib (Gleevec®); pazopanib (AP24534, CAS 943319-70-8); tibozanib (AV951, CAS 475108-18-0); legorafenib (BAY73-4506, CAS 755037-). 03-7); batalanib dihydrochloride (PTK787, CAS 212141-51-0); brivanib (BMS-540215, CAS 649735-46-6); bandetanib (Caprelsa® or AZD6474); motesanib diphosphate (AMG706, CAS 857876-30-3, N- (2,3-dihydro-3,3-dimethyl-1H-indole-6-yl) -2-[(4-pyridinylmethyl) amino] -3-pyridinecarboxamide, PCT International Publication No. 02/066470); Dobitinib dihydrate (TKI258, CAS 852433-84-2); Linfanib (ABT869, CAS 769667-16-3); Cabozantinib (XL184, CAS 849217-681) ); Restaultinib (CAS 111358-88-4); N- [5-[[[5- (1,1-dimethylethyl) -2-oxazolyl] methyl] thio] -2-thiazolyl] -4-piperidincarboxamide ( BMS3870 3, CAS 345627-80-7); (3R, 4R) -4-amino-1-((4-((3-methoxyphenyl) amino) pyrolo [2,1-f] [1,2,4]] Triazine-5-yl) Methyl) piperidine-3-ol (BMS690514); N- (3,4-dichloro-2-fluorophenyl) -6-methoxy-7-[[(3aα, 5β, 6aα) -octahydro- 2-Methylcyclopenta [c] pyrrol-5-yl] methoxy] -4-quinazolineamine (XL647, CAS 781613-23-8); 4-methyl-3-[[1-methyl-6- (3-pyridinyl) ) -1H-pyrazolo [3,4-d] pyrimidin-4-yl] amino] -N- [3- (trifluoromethyl) phenyl] -benzamide (BHG712, CAS 940310-85-0); and afribercept (Eylea®) to administer to the subject in combination.

In some embodiments, the CAR-expressing cells described herein are the CD20 antibody or conjugate thereof, such as rituximab (Rituxan® and MabThera®); and tositumomab (Bexxar®); And ofatumumab (Arzerra®), ibritumomab tiuxetan (Zevalin®); and tositumomab in combination with the subject.

In some embodiments, the CAR-expressing cells described herein are anticonvulsants, such as anticonvulsants (antiepileptic or antiepileptic drugs): aldehydes, eg paraaldehydes; aromatic allyl alcohols, eg stiripentol (Diacomit). (Registered Trademarks)); valbiturates such as phenovalbital (Luminal®), methylphenovalbital (Mebaral®), carbexacron (Mariasin®), benzodiazepines such as clovasam (Onfi®). , Klonopine (registered trademark), Chlorazepate (Tranxene (registered trademark) and Novo-Clopate (registered trademark)), diazepine (Valium (registered trademark), Lembrol (registered trademark), Diastat (registered trademark)), Midazolam ( Versed (registered trademark)), lorazepam (Ativan (registered trademark) and Orfidal (registered trademark)), nitrazepam (Alodom (registered trademark), Arem (registered trademark), Insoma (registered trademark)), temazepam (restoril (registered trademark)) , Normison®), Nimetzepam (Erimin®), bromide, eg potassium bromide; carbamate, eg Felbamate (Felbatol®); carbomazepine, eg carbamazepine (Regetol®, Equitro®). Trademarks)), oxycarbamazepine (Trilptal®, Oxcarb®), eslicarbamazepine acetate (Aptiom®); fatty acids such as valproate (valproic acid, sodium valproate, di). Valprox sodium), bigabatrin (Sabril®), progavid (Gabren®), thiagabin (Gabitril®); fructose derivatives such as topiramate (Topamax®); GABA analogs, eg. Gabapentin (Neurontin®), Pregavalin (Lyrica®); Hydantin, eg Etotoin (Peganone®), Fenitin (Dilantin®), Mefentoin (Mesantoin®), Foss Phenitoin (Cerebyx®, Prodilantin®); Xazozolidindione, such as Parameterzion (Paradione®), Trimethadione (Tridione®); Propionates, such as Beclamid (Choracon®, Hibicon®, Posedline®); Pyrimidin. Dione, eg, Primidone (Mysoline®); pyrrolidine, eg bribalacetam, levetylacetam, celetrasetam (Keppra®); succinimide, eg ethosuccimid (Zarontin®), fence ximid (Milontin®). , Mescusmidin (Celontin®, Petinutin®); sulfonamides such as acetazolamide (Diamox®), Sultium (Osport®), Metazolamide (Neptazane®), Zonegran. (Registered Trademark)); Triazine, such as Lamotrigin (Lamictal®); Urea, such as phenethylide, phenacemide (Phenuron®); Valproylamide (amide derivative of valproic acid), such as Valpromid (Registered Trademark). )), Valnoctamide; administered to the subject in combination with an AMPA receptor antagonist, eg, Perampa® (Fycompa®).

In embodiments, CAR-expressing cells described herein are administered to a subject in combination with an indoleamine 2,3-dioxygenase (IDO) inhibitor. IDO is an enzyme that catalyzes the decomposition of the amino acid L-tryptophan into kynurenine. Many cancers, such as prostate cancer, colorectal cancer, pancreatic cancer, cervical cancer, gastric cancer, ovarian cancer, head cancer and lung cancer, overexpress IDO. pDCs, macrophages and dendritic cells (DCs) can express IDO. Without being bound by theory, a decrease in L-tryptophan (eg, catalyzed by IDO) is thought to result in an immunosuppressive environment by inducing T cell anergy and apoptosis. Therefore, without being bound by theory, it is believed that IDO inhibitors can enhance the effects of CAR-expressing cells described herein, for example, by suppressing or reducing the death of CAR-expressing immune cells. In embodiments, the subject has a solid tumor, such as a solid tumor described herein, such as prostate cancer, colorectal cancer, pancreatic cancer, cervical cancer, gastric cancer, ovarian cancer, head cancer or lung cancer. Exemplary IDO inhibitors are 1-methyl-tryptophan, NewLink Genetics (see, eg, Clinical Trial Identifier Nos. NCT01191216; see NCT017792050) and INCB024360 (Incyte Corp.) (eg, Clin. NCT01604889; see NCT01685255), but is not limited to these.

In embodiments, CAR-expressing cells described herein are administered to a subject in combination with a modulator of myeloid-derived suppressor cells (MDSC). MDSC accumulates at the tumor site of peripheral and many solid tumors. These cells suppress the T cell response, thereby impairing the efficacy of CAR-expressing cell therapy. Although not theorized, MDSC modulator administration is believed to enhance the effects of the CAR-expressing cells described herein. In one embodiment, the subject has a solid tumor, such as the solid tumor described herein, such as glioblastoma. Exemplary MDSC modulators include, but are not limited to, MCS110 and BLZ945. MCS110 is a monoclonal antibody (mAb) against macrophage colony stimulating factor (M-CSF). For example, Clinical Trial Identifier No. See NCT00757757. BLZ945 is a small molecule inhibitor of colony stimulating factor 1 receptor (CSF1R). For example, Pyontech et al. Nat. Med. 19 (2013): 1264-72. The structure of BLZ945 is shown below.

In embodiments, CAR-expressing cells described herein are administered to a subject in combination with CD19 CART cells (eg, CTL019, eg, those described in WO 2012/079000, which is incorporated herein by reference). do. In embodiments, the subject has acute myeloid leukemia (AML), such as CD19-positive AML or CD19-negative AML. In embodiments, the subject has a CD19 + lymphoma, such as CD19 + non-Hodgkin's lymphoma (NHL), CD19 + FL or CD19 + DLBCL. In embodiments, the subject has relapsed or refractory CD19 + lymphoma. In embodiments, the lymphocyte depleting chemotherapeutic agent is administered (eg, infused) prior to, simultaneously with, or thereafter with the administration of CD19 CART cells. In one example, a lymphocyte depleting chemotherapeutic agent is administered to the subject prior to administration of CD19 CART cells. For example, lymphocyte depleting chemotherapeutic agents end 1 to 4 days (eg, 1 day, 2 days, 3 days or 4 days) before CD19 CART cell infusion. In embodiments, multiple doses of CD19 CAR T cells are administered, eg, as described herein. For example, a single dose contains about 5 × 10 ⁸ CD19 CART cells. In embodiments, the lymphocyte depleting chemotherapeutic agent is administered to the subject prior to, simultaneously with, or thereafter, the CAR-expressing cells described herein, eg, non-CD19 CAR-expressing cells (eg, infusion). In embodiments, CD19 CART is administered to the subject prior to, simultaneously with, or thereafter, administration of non-CD19 CAR-expressing cells, eg, non-CD19 CAR-expressing cells as described herein (eg, infusion).

In one embodiment, CAR-expressing cells described herein are referred to herein by CD19 CAR-expressing cells, eg, by reference, for the treatment of diseases associated with BCMA expression, such as cancer described herein. Administer to the subject in combination with, for example, CTL019 as described in the referenced International Publication No. 2012/079000 pamphlet. Without being bound by theory, administration of CD19 CAR-expressing cells in combination with CAR-expressing cells depletes regulatory B cells by regulating the immune response by targeting early cell lineage cancer cells, such as cancer stem cells. And / or by improving the tumor microenvironment, it is believed that the effects of the CAR-expressing cells described herein are improved. For example, CD19 CAR-expressing cells target early cell lineage markers, such as cancer stem cells and cancer cells expressing CD19-expressing cells, whereas the CAR-expressing cells described herein are late cell lineage markers, eg, Target cancer cells expressing BCMA. This pretreatment approach can improve the effects of CAR-expressing cells described herein. In such embodiments, the CD19 CAR-expressing cells are administered before, at the same time as, or after the administration (eg, infusion) of the CAR-expressing cells described herein.

In embodiments, the CAR-expressing cells described herein also express a CAR that targets CD19, such as the CD19 CAR. In one embodiment, cells expressing the CARs described herein and CD19 CARs are administered to a subject for the treatment of a cancer described herein, eg, AML. In one embodiment, the arrangement of one or both CAR molecules comprises a primary intracellular signaling domain and a costimulatory signaling domain. In another embodiment, the arrangement of one or both CAR molecules comprises a primary intracellular signaling domain and two or more, eg, two, three, four or five or more costimulatory signaling domains. include. In such embodiments, the CAR molecules and CD19 CARs described herein are the same or different primary intracellular signaling domains, the same or different costimulatory signaling domains, or the same or different numbers of costimulatory signaling. Can have a domain. Alternatively, the CARs and CD19 CARs described herein are arranged as split CARs, where one of the CAR molecules contains an antigen binding domain and a costimulatory domain (eg, 4-1BB) and the other CAR molecule. Includes an antigen-binding domain and a primary intracellular signaling domain (eg, CD3 zeta).

In one embodiment, the CAR-expressing cells described herein are the interleukin-15 (IL-15) polypeptide, the interleukin-15 receptor alpha (IL-15Ra) polypeptide or the IL-15 polypeptide and IL-. Both combinations of 15Ra polypeptides are administered to the subject in combination with, for example, hetIL-15 (Admune Therapeutics, LLC). hetIL-15 is a heterodimer non-covalent complex of IL-15 and IL-15Ra. HetIL-15 is, for example, US Pat. No. 8,124,084, US Patent Application Publication No. 2012/01775998, US Patent Application Publication No. 2009/0087229, which is incorporated herein by reference. , US Patent Application Publication No. 2012/0144131 and US Patent Application Publication No. 2011/0813111. In an embodiment, het-IL-15 is administered subcutaneously. In embodiments, the subject has a cancer, such as solid cancer, such as melanoma or colon cancer. In embodiments, the subject has a metastatic cancer.

In some embodiments, the subject can administer an agent that reduces or alleviates the side effects associated with the administration of CAR-expressing cells. Side effects associated with administration of CAR-expressing cells include, but are not limited to, CRS and hemophagocytic lymphohistiocytosis (HLH), also known as macrophage activation syndrome (MAS). Symptoms of CRS include high fever, nausea, transient hypotension, hypoxia and the like. CRS may include clinical constitutional signs and symptoms such as fever, fatigue, loss of appetite, myalgia, arthralgia, nausea, vomiting, headache. CRS may include clinical skin signs and symptoms such as rash. CRS may include clinical gastrointestinal signs and symptoms such as nausea, vomiting and diarrhea. CRS may include clinical respiratory signs and symptoms such as tachypnea and hypoxemia. CRS may include clinical cardiovascular signs and symptoms such as tachycardia, increased pulse pressure, hypotension, increased cardiac output (early) and decreased cardiac output (late). CRS may include symptoms such as signs of coagulation and increased d-dimer, hypofibrinogen blood with or without bleeding. CRS may include clinical renal signs and symptoms such as hypernitrogenemia. CRS may include clinical liver signs and symptoms such as hypertransaminaseemia and hyperbilirubinemia. CRS may include clinical neurological signs and symptoms such as headache, mental status changes, confusion, delirium, difficulty finding words or Frank's speech, hallucinations, tremor, dimetria, abnormal gait, and seizures.

Accordingly, the method described herein administers a subject the CAR-expressing cells described herein and further administers one or more agents that control the increased levels of soluble factors resulting from CAR-expressing cell treatment. Can include. In some embodiments, the soluble factor that can be increased in the subject is one or more of IFN-γ, TNFα, IL-2 and IL-6. In one embodiment, the increasing factor in the subject is one or more of IL-1, GM-CSF, IL-10, IL-8, IL-5 and fractalkine. Thus, the agent administered to treat these side effects can be an agent that neutralizes one or more of these soluble factors. In some embodiments, the agent that neutralizes one or more of these soluble forms is an antibody or antibody fragment. Examples of such agents include, but are not limited to, steroids (eg, corticosteroids), TNFα inhibitors and IL-6 inhibitors. Examples of TNFα inhibitors are anti-TNFα antibody molecules such as infliximab, adalimumab, ertolizumab pegol and golimumab. Another example of a TNFα inhibitor is a fusion protein such as etanercept. Small molecule TNFα inhibitors include, but are not limited to, xanthine derivatives (eg, pentoxifylline) and bupropion. Examples of IL-6 inhibitors are anti-IL- such as tocilizumab (toc), sarilumab, elcilimomab, CNTO 328, ALD518 / BMS-945429, CNTO 136, CPSI-2364, CDP6038, VX30, ARGX-109, FE301 and FM101. 6 antibody molecule. In some embodiments, the anti-IL-6 antibody molecule is tocilizumab. An example of an IL-1R-based inhibitor is Anakinra.

In one embodiment, the subject is administered, among other things, a corticosteroid such as, for example, methylprednisolone, hydrocortisone.

In one embodiment, the subject is administered a pressor agent such as, for example, norepinephrine, dopamine, phenylephrine, epinephrine, vasopressin or a combination thereof.

In one embodiment, the subject can be administered an antipyretic. In one embodiment, the subject can be administered an analgesic.

In some embodiments, agents that prevent the transport of BCMA CAR-expressing cells to the brain, such as natalizumab (TYSABRI®), can be administered to the subject. BCMA expression, such as splice variants thereof, has been detected in several parts of the brain, such as the cerebellum or medulla oblongata. Without being bound by a particular theory, preventing the transport of BCMA CAR-expressing cells to the brain is to prevent BCMA CAR-expressing cells from interacting with or acting on BCMA-expressing brain tissue. Is preferable.

In some embodiments, the subject can be administered an agent that enhances the activity of CAR-expressing cells. For example, in some embodiments, the agent can be an agent that inhibits an inhibitory molecule, for example, the agent is a checkpoint inhibitor. Inhibitor molecules, such as Programmed Cell Death 1 (PD1), may reduce the ability of CAR-expressing cells to initiate an immune effector response in some embodiments. Examples of inhibitory molecules are PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (eg 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. Inhibition of the inhibitory molecule by inhibition at the DNA, RNA or protein level can optimize CAR-expressing cell performance. In embodiments, inhibitory nucleic acids such as those described herein, such as inhibitory nucleic acids, such as dsRNAs, such as siRNA or shRNA, clustered evenly spaced short repetitive sequences (CRISPRs), transcription-activator-like effector nucleases. (TALEN) or zinc finger endonucleases (ZFNs) can be used to inhibit the expression of inhibitory molecules in CAR-expressing cells. In one embodiment, the inhibitor is shRNA. In one embodiment, the inhibitory molecule is inhibited within the CAR expressing cell. In these embodiments, the dsRNA molecule that inhibits the expression of the inhibitory molecule is ligated to a component of CAR, eg, a nucleic acid encoding the entire component. In embodiments, the CAR-expressing cells described herein are administered in combination with an inhibitor of the inhibitory molecule, eg, in combination with a checkpoint inhibitor, eg, in combination with an inhibitor of PD1 and / or PD-L1. .. In embodiments, the CAR-expressing cells described herein are administered in combination with an inhibitor of PD1. In embodiments, the CAR-expressing cells described herein are administered in combination with an inhibitor of PD-L1.

In one embodiment, a nucleic acid molecule encoding a dsRNA molecule that regulates or regulates, eg, inhibits, the expression of a molecule that inhibits T cell function, a dsRNA molecule that inhibits, for example, the expression of a molecule that regulates or regulates, eg, inhibits T cell function. Is operably linked to a promoter, such as an H1 or U6-driven promoter, such that is expressed in expression, eg, CAR-expressing cells. For example, Tiscornia G. et al. , "Development of Lentiviral Vectors Expressing siRNA," Chapter 3, in Gene Transfer: Delivery and Expression of DNA and RNA (eds. Friedmann). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2007; Brummelkamp TR, et al. (2002) Science 296: 550-553; Miyagishi M, et al. (2002) Nat. Biotechnol. 19: 497-500. In one embodiment, the nucleic acid molecule encoding a dsRNA molecule that regulates or regulates, eg, inhibits, the expression of a molecule that inhibits T cell function is the same vector comprising a component of CAR, eg, a nucleic acid molecule encoding all of the elements, eg. Present on the lentiviral vector. In such embodiments, the nucleic acid molecule encoding a dsRNA molecule that regulates or regulates, eg, inhibits, the expression of a molecule that inhibits T cell function is 5'or 3 in a nucleic acid that encodes a component of CAR, eg, all of the elements. 'The vector located in, for example, the lentiviral vector. A nucleic acid molecule encoding a dsRNA molecule that regulates or regulates, eg, inhibits, the expression of a molecule that inhibits T cell function can be transcribed in the same or different directions as the nucleic acid that encodes a component of CAR, eg, all of the elements. In one embodiment, the nucleic acid molecule encoding a dsRNA molecule that regulates or regulates, eg, inhibits, the expression of a molecule that inhibits T cell function is a vector other than a vector containing a component of CAR, eg, a nucleic acid molecule encoding all of the elements. Exists in. In one embodiment, a nucleic acid molecule encoding a dsRNA molecule that regulates or regulates T cell function, eg, inhibits expression of a molecule that inhibits it, is transiently expressed in CAR-expressing cells. In one embodiment, a nucleic acid molecule encoding a dsRNA molecule that regulates or regulates T cell function, eg, inhibits expression of a molecule that inhibits it, is stably integrated into the genome of CAR-expressing cells.

An example of a dsRNA molecule useful for regulating or regulating T cell function, eg, inhibiting the expression of a molecule that inhibits it, where the molecule that regulates or regulates, eg, inhibits T cell function is PD-1 below. To provide to.

In some embodiments, the inhibitor of the inhibitory signal can be, for example, an antibody or antibody fragment that binds to the inhibitory molecule. For example, the agent can be an antibody or antibody fragment that binds to PD1, PD-L1, PD-L2 or CTLA4 (eg, ipilimumab (also referred to as MDX-010 and MDX-101, commercially available as Yervoy®; Bristol). -Myers Squibb; tremelimumab (IgG2 monoclonal antibody available from Pfizer, formerly known as ticilimumab, CP-675,206)). In one embodiment, the agent is an antibody or antibody fragment that binds to TIM3. In one embodiment, the agent is an antibody or antibody fragment that binds to LAG3. In embodiments, an agent that enhances the activity of CAR-expressing cells, eg, an inhibitor of an inhibitory molecule, is an allogeneic CAR, eg, an allogeneic book. Administer in combination with the CARs described herein (eg, described in the allogeneic CARs herein).

PD1 is an inhibitory member of the CD28 family of receptors, including CD28, CTLA-4, ICOS and BTLA. PD-1 is expressed on activated B cells, T cells and bone marrow 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; Ratchman et. al. 2001 Nat Immunol 2: 261-8; Carter et al. 2002 Eur J Immunol 32: 634-43). PD-L1 is abundant in human cancer (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 Res10. : 5094). Immunosuppression can be reversed by inhibiting the local interaction between PD1 and PD-L1. Antibodies, antibody fragments and other inhibitors of PD1, PD-L1 and PD-L2 are available in the art and can be used in combination with the CAR of the present invention. For example, nivolumab (also referred to as BMS-936558 or MDX1106; Bristol-Myers Squibb) is a fully human IgG4 monoclonal antibody that specifically blocks PD1. Nivolumab (clone 5C4) and other human monoclonal antibodies that specifically bind to PD1 are disclosed in US Pat. No. 8,008,449 and WO 2006/121168. Pidilizumab (CT-011; Cure Tech) is a humanized IgG1k monoclonal antibody that binds to PD1. Pidilizumab and other humanized anti-PD1 monoclonal antibodies are disclosed in WO 2009/101611. Pembrolizumab (formerly known as rambrolizumab, also known as MK03475; Merck) is a humanized IgG4 monoclonal antibody that binds to PD1. Pembrolizumab and other humanized anti-PD1 antibodies are disclosed in US Pat. No. 8,354,509 and WO 2009/114335. MEDI4736 (Medimmune) is a human monoclonal antibody that binds to PDL1 and inhibits the interaction between the ligand and PD1. MDPL3280A (Genentech / Roche) is a human Fc-optimized IgG1 monoclonal antibody that binds to PD-L1. MDPL3280A and other human monoclonal antibodies to PD-L1 are disclosed in US Pat. No. 7,943,743 and US Patent Application Publication No. 20120039906. Other anti-PD-L1 binders include YW243.55. S70 (heavy and light chain variable regions are shown in SEQ ID NOs: 20 and 21 of International Publication No. 2010/077634) and MDX-1 105 (also known as BMS-936559 and, for example, International Publication No. 2007/005874). Includes the anti-PD-L1 binder disclosed in. AMP-224 (B7-DCIg; Amplimmune; disclosed, for example, in International Publication No. 2010/027827 and International Publication No. 2011/066342) is a PD-L2 Fc fusion that blocks the interaction of PD1 and B7-H1. It is a soluble receptor. Other anti-PD1 antibodies are described, among other things, in AMP 514, eg, US Pat. Includes the disclosed anti-PD1 antibody.

TIM3 (T cell immunoglobulin-3) also negatively regulates T cell function and plays an important role in T cell exhaustion, especially in IFN-g secreted CD4 + T helper 1 and CD8 + T cytotoxic 1 cells. Inhibition of the interaction of TIM3 and its ligands, such as galectin-9 (Gal9), phosphatidylserine (PS) and HMGB1, can enhance the immune response. Antibodies, antibody fragments and other inhibitors of TIM3 and its ligands are available in the art and can be used in combination with the CD19 or BCMA CAR described herein. For example, an antibody, antibody fragment, small molecule or peptide inhibitor that targets TIM3 binds to the IgV domain of TIM3 in order to inhibit its interaction with its ligand. Antibodies and peptides that inhibit TIM3 are disclosed in WO 2013/006490 and US Patent Application Publication No. 2010247521. Other anti-TIM3 antibodies are humanized versions of RMT3-23 (disclosed in Ngiow et al., 2011, Cancer Res, 71: 3540-3551) and clone 8B. Includes 2C12 (disclosed in Monney et al., 2002, Nature, 415: 536-541). Bispecific antibodies that inhibit TIM3 and PD-1 are disclosed in US Patent Application Publication No. 201301567774.

In other embodiments, agents that enhance the activity of CAR-expressing cells are CEACAM inhibitors (eg, CEACAM-1, CEACAM-3 and / or CEACAM-5 inhibitors). In some embodiments, the inhibitor of CEACAM is an anti-CEACAM antibody molecule. Exemplary anti-CEACAM-1 antibodies are described in International Publication No. 2010/125571, International Publication No. 2013/0823666, International Publication No. 2014/059251 and International Publication No. 2014/022332, eg. Monoclonal antibodies 34B1, 26H7 and 5F4; or recombinant forms thereof, for example as described in US Patent Application Publication No. 2004/0047858, US Patent No. 7,132,255 and International Publication No. 99/052552. Is. In other embodiments, the anti-CEACAM antibody is described, for example, by Zheng et al. PLoS One. 2010 Sep 2; 5 (9). Pii: e12529 (DOI: 10: 1371 / journal.pone. 0021146) to combine with CEACAM-5 or, for example, International Publication No. 2013/054331 and US Patent Application Publication No. 2014/0271618. Cross-reacts with CEACAM-1 and CEACAM-5 as described in the book.

Without wishing to be bound by theory, carcinoembryonic antigen cell adhesion molecules (CEACAM) such as CEACAM-1 and CEACAM-5 are thought to mediate at least partial inhibition of the antitumor immune response (eg, for example. Markel et al. J Immunol. 2002 Mar 15; 168 (6): 2803-10; Markel et al. J Immunol. 2006 Nov 1; 177 (9): 6062-71; Markel et al. Immunology. 2009 Feb; (2): 186-200; Markel et al. Cancer Immunol Immunother. 2010 Feb; 59 (2): 215-30; Ortenberg et al. Mol Cancer Ther. 2012 Jun; 11 (6): 1300-10; Stern et. See al. J Immunol. 2005 Jun 1; 174 (11): 6692-701; Zheng et al. PLoS One. 2010 Sep 2; 5 (9) .pii: e12529). For example, CEACAM-1 has been described as a heteroaffinity ligand for TIM-3 and has a role in TIM-3 mediated T cell tolerance and exhaustion (eg, WO 2014/022332; Huang, et al. et al. (2014) Nature doi: 10.1038 / nature13848). In embodiments, co-blocking of CEACAM-1 and TIM-3 has been shown to enhance an antitumor immune response in a xenograft colorectal cancer model (eg, WO 2014/022332; Huang, et al. et al. (2014), see above). In other embodiments, co-blocking of CEACAM-1 and PD-1 reduces T cell tolerance, eg, as described in WO 2014/059251. Therefore, CEACAM inhibitors can be used with other immunomodulators described herein (eg, anti-PD-1 and / or anti-TIM-3 inhibitors) to use cancers such as melanoma, lung cancer (eg, anti-PD-1 and / or anti-TIM-3 inhibitors). It can enhance the immune response against NSCLC), bladder cancer, colon cancer, ovarian cancer and other cancers described herein.

LAG-3 (lymphocyte activation gene-3 or CD223) is a cell surface molecule expressed on activated T cells and B cells that has been shown to play a role in CD8 + T cell exhaustion. Antibodies, antibody fragments and other inhibitors of LAG3 and its ligands are available in the art and can be used in combination with the CD19 or BCMA CAR described herein. For example, BMS-986016 (Bristol-Myers Squib) is a monoclonal antibody that targets LAG3. IMP701 (Immutep) is an antagonist LAG-3 antibody and IMP731 (Immutep and GSK plcMithKline) is a depleting LAG-3 antibody. Other LAG-3 inhibitors are recombinant fusion proteins of the soluble portion of LAG3 and Ig of MHC class II molecules, including IMP321 (Immutep), which activates antigen presenting cells (APCs). Other antibodies are disclosed, for example, in International Publication No. 2010/019570.

In one embodiment, the agent that enhances the activity of CAR-expressing cells can be, for example, a fusion protein comprising a first domain and a second domain, the first domain being an inhibitory molecule or fragment thereof, the first. Domain 2 is a polypeptide associated with a positive signal, eg, a polypeptide comprising an intracellular signaling domain described herein. In one embodiment, the polypeptide that binds to a positive signal is the costimulatory domain of CD28, CD27, ICOS, eg CD28, CD27 and / or the intracellular signaling domain of ICOS and / or eg CD3 as described herein. It may include the primary signaling domain of the zeta. In some embodiments, the fusion protein is expressed by the same cells that express CAR. In another embodiment, the fusion protein is expressed by cells that do not express anti-BCMA CAR, such as T cells or NK cells.

In some embodiments, the agent that enhances the activity of CAR-expressing cells described herein is miR-17-92.

In some embodiments, the agents that enhance the activity of CAR described herein are cytokines. Cytokines have important functions related to T cell proliferation, differentiation, survival and homeostasis. Cytokines that can be administered to subjects receiving CAR-expressing cells as described herein include IL-2, IL-4, IL-7, IL-9, IL-15, IL-18 and IL-21 or combinations thereof. include. In a preferred embodiment, the cytokine to be administered is IL-7, IL-15 or IL-21 or a combination thereof. Cytokines can be administered once or more than once a day, for example twice a day, three times a day or four times a day. Cytokines can be administered for more than 1 day, eg, cytokines are administered for 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks or 4 weeks. For example, cytokines are administered once daily for 7 days.

In embodiments, cytokines are administered in combination with CAR-expressing T cells. Cytokines can be administered simultaneously or with CAR-expressing T cells, eg, on the same day. Cytokines can be formulated in the same pharmaceutical composition as CAR-expressing T cells, or in a different pharmaceutical composition. Alternatively, cytokines may be administered shortly after administration of CAR-expressing T cells, eg, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days after administration of CAR-expressing T cells. In embodiments in which cytokines are administered in a dosing regimen of more than one day, the first day of the cytokine dosing regimen can be the same day as the administration of CAR-expressing T cells, or the first day of the cytokine dosing regimen is CAR expression. It can be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days after administration of T cells. In some embodiments, CAR-expressing T cells are administered to the subject on day 1 and cytokines are administered once daily for the next 7 days on day 2. In a preferred embodiment, the cytokine administered in combination with CAR-expressing T cells is IL-7, IL-15 or IL-21.

In other embodiments, cytokines are applied for a period of time after administration of CAR-expressing cells, eg, at least 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 4 months after administration of CAR-expressing cells. Administer after 5, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or 1 year or more. In some embodiments, cytokines are administered after evaluation of the subject's response to CAR-expressing cells. For example, subjects are administered CAR-expressing cells according to the doses and regimens described herein. Subject's response to CAR-expressing cell therapy, including administration of CAR-expressing cells using any of the methods described herein, including tumor growth inhibition, circulating tumor cell reduction or tumor regression, 2 weeks, 3 weeks, It will be evaluated after 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or 1 year or more. Cytokines can be administered to subjects who do not respond adequately to CAR-expressing cell therapy. Administration of cytokines to subjects whose response to CAR-expressing cell therapy is suboptimal improves CAR-expressing cell efficacy or anticancer activity. In a preferred embodiment, the cytokine administered after administration of CAR-expressing cells is IL-7.

In some embodiments, the BCMA CAR T cells described herein can be used in combination with low immunopotentiating doses of mTOR inhibitors. The methods described herein use low immunopotentiating doses of mTOR inhibitors, such as allosteric mTOR inhibitors containing lapalogs such as RAD001. Administration of a low immunopotentiating dose of an mTOR inhibitor (eg, a dose that itself is insufficient to completely suppress the immune system but sufficient to improve immune function) is an immune effector cell in the subject, eg, The performance of T cells or CAR-expressing cells can be optimized. Methods, doses, treatment regimens and suitable pharmaceutical compositions for measuring mTOR inhibition are described in US Patent Application Publication No. 2015/01240036, which is incorporated herein by reference. For a method of combining BCMA CAR T cells with an mTOR inhibitor, see, for example, paragraphs [0826]-[0861] of US Patent Application Publication No. 20160046724, which is incorporated herein by reference in its entirety.

Methods and Biomarkers for Assessing CAR Efficacy or Sample Suitability In another embodiment, the invention relates to CAR-expressing cell therapy (eg, BCMA CAR therapy) in a subject (eg, a subject with cancer, eg, blood cancer). It relates to a method of assessing or monitoring efficacy or suitability of a sample (eg, apheresis sample) for CAR treatment (eg, BCMA CAR treatment). The method comprises obtaining a value of efficacy for CAR therapy or sample aptitude, wherein said value is an indicator of efficacy or aptitude for CAR-expressing cell therapy.

In embodiments, efficacy values or sample suitability for CAR therapy include one, two, three, four, five, six or more (all) measures:
(I) Resting _TEFF cells, resting T _REG cells, younger T cells (eg, younger CD4 or CD8 cells or gamma / delta T cells) in a sample (eg, an afferesis sample or a produced CAR-expressing cell product sample). ) Or early memory T cells or a combination thereof at a level or activity of one, two, three or more (eg, all);
(Ii) Activated _TEFF cells, activated T _REG cells, older T cells (eg, older CD4 or CD8 cells) or late memory in a sample (eg, an afferesis sample or a produced CAR-expressing cell product sample). Levels or activity of one, two, three or more (eg, all) of T cells or combinations thereof;
(Iii) Immune cell depletion markers in samples (eg, apheresis samples or produced CAR-expressing cell product samples), eg, one, two or more immune checkpoint inhibitors (eg, PD-1, etc.). Level or activity of PD-L1, TIM-3 and / or LAG-3). In some embodiments, the immune cell has a depleted phenotype, eg, co-expresses at least two depletion markers, eg, PD-1 and TIM-3. In other embodiments, immune cells have a depleted phenotype, eg, co-express at least two depletion markers, eg, PD-1 and LAG-3;
(Iv) Levels or activity of CD27 and / or CD45RO- (eg, CD27 + CD45RO-) immune effector cells in a sample (eg, an apheresis sample or a produced CAR-expressing cell product sample), eg, in a CD4 + or CD8 + T cell population;
(V) One or two selected from CCL20, IL-17a and / or IL-6, PD-1, PD-L1, LAG-3, TIM-3, CD57, CD27, CD122, CD62L, KLRG1. Levels or activity of 3, 4, 5, 10, 20 or more biomarkers;
(Vi) Cytokine level or activity in a CAR-expressing cell product sample, such as a BCMA-expressing cell product sample (eg, quality of the cytokine repertoire); or (vi) Transduction efficiency of CAR-expressing cells in a produced CAR-expressing cell product sample. ..

In some embodiments of any of the methods disclosed herein, CAR-expressing cell therapy comprises multiple (eg, population) CAR-expressing immune effector cells, eg, multiple (eg, population) T cells or NK. Includes cells or combinations thereof. In one embodiment, the CAR expression cell therapy is BCMACAR therapy.

In some embodiments of any of the methods disclosed herein, one or more measures (i)-(vii) are obtained from apheresis samples obtained from the subject. Apheresis samples can be evaluated prior to infusion or reinjection.

In some embodiments of any of the methods disclosed herein, one or more measures (i)-(vii) are from a produced CAR-expressing cell product sample, such as a BCMACAR-expressing cell product sample. can get. The CAR-expressing cell products produced can be evaluated prior to infusion or reinjection.

In some embodiments of any of the methods disclosed herein, a subject is evaluated before, during, or after receiving CAR-expressing cell therapy.

In some embodiments of any of the methods disclosed herein, one or more measures (i)-(vii) include one or more profiles of gene expression, flow cytometry or protein expression. evaluate.

In some embodiments of any of the methods disclosed herein, the method targets respondents, non-responders, relapsers based on one or more measures (i)-(vii). Or further includes recognizing as a non-recurrence person.

In some embodiments of any of the methods disclosed herein, responders (eg, complete responders) are one of GZMK, PPF1BP2 or naive T cells, 2 as compared to non-responders. One or more (all) of higher levels or activities are or are recognized as having it.

In some embodiments of any of the methods disclosed herein, non-responders are IL22, IL-2RA, IL-21, IRF8, IL8, CCL17, CCL22, effectors as compared to responders. Having or having a higher level or activity of one, two, three, four, five, six, seven or more (eg, all) of T cells or regulatory T cells. Be recognized.

In one embodiment, the relapser is one or more of the following genes: MIR199A1, MIR1203, uc021ovp, ITM2C and HLA-DQB1 (eg, 2, 3, 4, or all) as compared to the non-relapser. One or more of the following genes: PPIAL4D, TTTY10, TXLNG2P, MIR465-1, KDM5D, USP9Y, PRKY, RPS4Y2, RPS4Y1, NCRNA00185, SULT1E1 and EIF1AY compared to increased expression levels and / or non-relapsed individuals. For example, 2, 3, 4, 5, 6, 7, 7, 8, 9, 10, 11 or all) have or are recognized to have reduced expression levels. I am a patient.

In some embodiments of any of the methods disclosed herein, complete responders are greater, eg, statistically significant, compared to a reference value, eg, the percentage of non-responders of CD8 + T cells. Has or is recognized as having a large percentage of CD8 + T cells.

In some embodiments of any of the methods disclosed herein, the complete responder is greater than the reference value, eg, in the CD8 + population, as compared to the CD27 + CD45RO-immune effector cell count of the non-responder. Has or is recognized to have a percentage of CD27 + CD45RO-immune effector cells.

In some embodiments of any of the methods disclosed herein, a complete or partial responder is larger, eg, compared to a reference value, eg, the percentage of non-responders of CD4 + T cells. It has or is recognized to have a statistically significant percentage of CD4 + T cells.

In some embodiments of any of the methods disclosed herein, complete responders are referred to as reference values such as resting _TEFF cells, resting T _REG cells, younger T cells (eg, more) of non-responders. A larger percentage of resting _TEFF cells, resting T _REG cells, younger T cells (eg, younger CD4 or CD8 cells or gamma / delta T cells) compared to the number of young CD4 or CD8 cells) or initial memory T cells. ) Or early memory T cells or combinations thereof have, or are recognized as having, two, three or more (eg, all).

In some embodiments of any of the methods disclosed herein, the non-responder is a reference value, eg, the responder's activated _TEFF cells, activated T _REG cells, older T cells (eg, eg). Older CD4 or CD8 cells) or larger percentages of activated T _EFF cells, activated T _REG cells, older T cells (eg, older CD4 or CD8 cells) or late memory T cell counts. Memory T cells have or are recognized to have one, two, three or more (eg, all) of T cells or combinations thereof.

In some embodiments of any of the methods disclosed herein, non-responders are associated with a larger percentage of immune cell depletion markers, eg, one, two or more immune checkpoint inhibitors. Have or are recognized as having (eg, PD-1, PD-L1, TIM-3 and / or LAG-3). In some embodiments, the non-responder is a larger percentage of PD-1, PD-L1 or LAG-compared to the percentage of immune effector cells expressing PD-1 or LAG-3 from the responder. Immune effector cells expressing 3 (eg, CD4 + T cells and / or CD8 + T cells) (eg, CAR-expressing CD4 + cells and / or CD8 + T cells) are possessed or recognized as having it.

In some embodiments, non-responders co-express a larger percentage of immune cells with a depleted phenotype, such as at least two depletion markers, such as PD-1, PD-L1 and / or TIM-. It has or is recognized as having immune cells that co-express 3. In other embodiments, the non-responder is an immune cell that co-expresses a larger percentage of immune cells with a depleted phenotype, eg, at least two depletion markers, eg PD-1 and LAG-3. Or is recognized as having it.

In some embodiments of any of the methods disclosed herein, a non-responder is a CAR-expressing cell population (eg, a complete responder) as compared to a responder (eg, a complete responder) to CAR-expressing cell therapy. BCMACAR + cell population) has or is recognized to have a larger percentage of PD-1 / PD-L1 + / LAG-3 + cells.

In some embodiments of any of the methods disclosed herein, partial responders have a higher percentage of PD-1 / PD- in the CAR-expressing cell population (eg, BCMACAR + cell population) than the respondents. Has or is recognized as having L1 + / LAG-3 + cells.

In some embodiments of any of the methods disclosed herein, the non-responder is depleted of the PD1 / PD-L1 + CAR + phenotype and co-existence of LAG3 in a CAR-expressing cell population (eg, BCMACAR + cell population). Has or is recognized as having expression.

In some embodiments of any of the methods disclosed herein, the non-responder is in a CAR-expressing cell population (eg, BCMACAR + cell population) as compared to a responder (eg, a complete responder). , Has or is recognized as having a larger percentage of PD-1 / PD-L1 + / TIM-3 + cells.

In some embodiments of any of the methods disclosed herein, partial responders have a higher percentage of PD-1 / PD- in the CAR-expressing cell population (eg, BCMACAR + cell population) than the respondents. Has or is recognized as having L1 + / TIM-3 + cells.

In some embodiments of any of the methods disclosed herein, the presence of CD8 + CD27 + CD45RO-T cells in an apheresis sample is a positive predictor of a subject's response to CAR-expressing cell therapy (eg, BCMACAR therapy). Is.

In some embodiments of any of the methods disclosed herein, a high percentage of PD1 + CAR + and LAG3 + or TIM3 + T cells in an apheresis sample prognoses the subject's response to CAR-expressing cell therapy (eg, BCMACAR therapy). It is a predictor of failure.

In some embodiments of any of the methods disclosed herein, a respondent (eg, a complete or partial responder) is a number of one, two, three, or more of the following profiles. Have (or all):
(I) Have a larger number of CD27 + immune effector cells compared to the reference value, eg, the number of non-responder CD27 + immune effector cells;
(Ii) (i) has a larger number of CD8 + T cells compared to a reference value, eg, the number of non-responder CD8 + T cells;
(Iii) A smaller number, eg PD-1, PD-L1, LAG-3, TIM-3, compared to a reference value, eg, the number of non-responder cells expressing one or more checkpoint inhibitors. Or have one or more checkpoint inhibitors selected from KLRG-1 or a combination thereof; or (iv) reference values such as dormant T _EFF cells, dormant T _REG cells, naive CD4 cells, unstimulated memory cells. Or a larger number of 1, 2, 3, 4, or more (all) dormant _TEFF cells, dormant T _REG cells, compared to the number of non-responders of early memory T cells. It has naive CD4 cells, unstimulated memory cells or early memory T cells, or a combination thereof.

In some embodiments of any of the methods disclosed herein, the cytokine level or activity of (vi) is the cytokine CCL20 / MIP3a, IL17A, IL6, GM-CSF, IFNγ, IL10, IL13, IL2, Selected from one, two, three, four, five, six, seven, eight or more (or all) of IL21, IL4, IL5, IL9, TNFα or combinations thereof. .. Cytokines can be selected from one, two, three, four or more (all) of IL-17a, CCL20, IL2, IL6 or TNFa. In some embodiments, increased levels or activity of cytokines are selected from one or both of IL-17a and CCL20, indicating increased responsiveness or decreased recurrence.

In some embodiments of any of the methods disclosed herein, a transduction efficiency of 15% or greater in (vii) indicates an increase in responsiveness or a decrease in recurrence.

In some embodiments of any of the methods disclosed herein, a transduction efficiency of less than 15% in (vii) indicates a decrease in responsiveness or an increase in recurrence.

In embodiments, responders, non-responders, relapsers or non-relapsers recognized by the methods herein can be further evaluated according to clinical criteria. For example, a complete responder is recognized as having or having a disease (eg, cancer) and exhibits a complete response to the treatment, eg, complete remission. Complete responses are described, for example, in NCCN Guidelines® or Cheson et al, J Clin Oncol 17: 1244 (1999) and Cheson et al. , "Revised Response Criteria for Malignant Lymphoma", J Clin Oncol 25: 579-586 (2007), both of which are incorporated herein by reference in their entirety. Partial responders are identified as having or having a disease (eg, cancer) and exhibiting a partial response to treatment, eg, partial remission. Partial responses can be identified using, for example, NCCN Guidelines® or the Cheson criteria as described herein. Non-responders are identified as having or having a disease (eg, cancer) and do not respond to treatment, eg, the patient has a stable or progressive disease. Non-responders can be identified using, for example, NCCN Guidelines® or the Cheson criteria as described herein.

Alternatively or in combination with the methods disclosed herein, in response to said values, perform one, two, three, four or more of the following:
Administering CAR-expressing cell therapy, for example, to responders or non-relapsers;
Administering modified doses of CAR-expressing cell therapy;
Changing the schedule or time course of CAR-expressing cell therapy;
Administration of additional agents in combination with CAR-expressing cell therapy, such as checkpoint inhibitors, such as the checkpoint inhibitors described herein, to, for example, non-responders or partial responders;
Prior to treatment with CAR-expressing cell therapy, administration of therapy to increase the number of younger T cells in the subject to non-responders or partial responders;
Subjects recognized for modifying the manufacturing process of CAR-expressing cell therapy, eg, enriching younger T cells or transducing efficiency prior to introducing the nucleic acid encoding CAR, eg, non-responders or partial responders. To enhance about;
Alternative therapy is administered, for example, to non-responders or partial responders or relapsers; or if the subject is or is recognized as a non-responder or relapser, the _TREG cell population and / or _TREG . Gene signatures are reduced, for example, by one or more doses of CD25 depletion, cyclophosphamide, anti-GITR antibodies or combinations thereof.

In certain embodiments, the subject is pretreated with an anti-GITR antibody. In certain embodiments, the subject is treated with anti-GITR antibody prior to infusion or reinjection.

Biopolymer Delivery Method In one embodiment, one or more CAR-expressing cells disclosed herein can be administered or delivered to a subject by a biopolymer scaffold, such as a biopolymer implant. The biopolymer can be administered or delivered to the subject by a biopolymer scaffold, such as a biopolymer implant. Biopolymer scaffolds include biocompatible (eg, substantially non-inducing inflammatory or immune responses) and / or biodegradable polymers that may be naturally occurring or are synthetic.

Examples of suitable biopolymers are agar, agarose, alginic acid, alginic acid / calcium phosphate cement (CPC), β-galactosidase (β-GAL), (1,2,3,4,6-pentaacetyl a-D-galactose). , Cellulose, chitin, chitosan, collagen, elastin, gelatin, hyaluronic acid collagen, hydroxyapatite, poly (3-hydroxybutyrate-co-3-hydroxy-hexanoate) (PHBHHx), poly (lactide), poly (caprolactone) ( PCL), poly (lactide-co-glycolide) (PLG), polyethylene oxide (PEO), poly (lactic acid-co-glycolic acid) (PLGA), polypropylene oxide (PPO), polyvinyl alcohol) (PVA), silk, soybeans. Contains, but is not limited to, a mixture of proteins and soybean protein isolates alone or with any other polymer composition in any concentration and ratio. Biopolymers are enhanced or modified with adhesion-promoting molecules such as collagen mimetic peptides that bind to collagen receptors in lymphocytes and / or stimulatory molecules that enhance the delivery, proliferation or function of the delivering cells, such as anticancer activity. obtain. The biopolymer scaffold can be an injectable eg gel or semi-solid or solid composition.

In one embodiment, the CAR-expressing cells described herein are seeded on a biopolymer scaffold prior to delivery to a subject. In embodiments, the biopolymer scaffold is, for example, one or more additional therapeutic agents described herein that have been incorporated or conjugated to the biopolymer of the scaffold (eg, another CAR-expressing cell, antibody). Or small molecule) or agents that enhance the activity of CAR-expressing cells. In embodiments, the biopolymer scaffold is injected, for example, intratumorally, or surgically implanted proximally to the tumor sufficiently to mediate the tumor or antitumor effect. Further examples of biopolymer compositions and methods for delivery thereof are described in Stephan et al. , Nature Biotechnology, 2015, 33: 97-101; and International Publication No. 2014/110591.

Pharmaceutical Compositions and Treatments The pharmaceutical compositions of the invention, as described herein, dilute CAR-expressing cells, eg, multiple CAR-expressing cells, into one or more pharmaceutically or physiologically acceptable carriers. Included in combination with agents or additives. Such compositions are buffers such as neutral buffered saline, phosphate buffered saline; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants. Agents; chelating agents such as EDTA or glutathione; adjuvants (eg, aluminum hydroxide); and preservatives may be included. The compositions of the invention are formulated for intravenous administration in one embodiment.

The pharmaceutical composition of the present invention may be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the patient's condition and the type and severity of the patient's disease, but the appropriate dose may be determined by clinical trials.

In some embodiments, the pharmaceutical composition is, for example, endotoxin, mycoplasma, replicable lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag, residual anti-CD3 / anti-CD28 coated beads, mouse antibody, stored human. There are virtually no contaminants selected from the group consisting of serum, bovine serum albumin, bovine serum, culture medium elements, vector packaging cells or plasmid components, bacteria and fungi, eg, not present at detectable levels. In some embodiments, the bacterium is Alcaligenes faecalis, Candida albicans, Escherichia coli, Haemophilus ninesenis seri Mengingitides), Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumone (Streptococcus pneumonia), Streptococcus pneumonia, Streptococcus pneumonia ..

When an "immunologically effective amount", "anti-tumor effective amount", "tumor inhibitory effective amount" or "therapeutic amount" is indicated, the exact amount of the composition of the invention to be administered is age, body weight, and the like. It can be determined by the physician in consideration of individual differences in tumor size, degree of infection or metastasis and patient (subject) condition. Pharmaceutical compositions comprising the immune effector cells described herein ⁽ eg, T cells, NK cells) are used in ^104-109 cells / kg body weight, in some examples ^{105-106 cells} / kg body weight. It can generally be stated that doses in the range (including all integer values within these ranges) can be administered. The T cell composition may also be administered multiple times at this dose. Cells can be administered using infusion techniques commonly known in immunotherapy (see, eg, Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988).

In one embodiment, activated T cells are administered to a subject, followed by blood sampling (or apheresis), which activates the T cells from which according to the invention, and these activated and proliferated T cells. It may be desirable to reinject into the patient. This process can be performed multiple times every few weeks. In one embodiment, T cells can be activated from 10 cc to 400 cc of collected blood. In one embodiment, T cells are activated from 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc or 100 cc of collected blood.

Administration of the subject composition can be performed by any convenient method including aerosol inhalation, injection, ingestion, infusion, indwelling or transplantation. The compositions described herein can be administered to a patient transarterically, subcutaneously, intradermally, intratumorally, intranodesally, intramedullarily, intramuscularly, or by intravenous (iv) injection. It can be administered intraperitoneally. In one embodiment, the CAR-expressing cell (eg, T cell or NK cell) composition of the invention is administered to a patient by intradermal or subcutaneous injection. In one embodiment, the T cell composition of the present invention is used in i. v. Administer by injection. The composition of CAR-expressing cells (eg, T cells or NK cells) can be injected directly into the tumor, lymph node or site of infection.

In certain exemplary embodiments, the subject collects leukocytes and enriches or depletes them with Exvivo to select and / or isolate cells of interest, such as immune effector cells (eg, T cells or NK cells). Can be removed. Isolators of these immune effector cells (eg, T cells or NK cells) are grown by methods known in the art to introduce one or more CAR constructs of the invention thereby the CAR expressing cells of the invention. (Eg, CAR T cells or CAR-expressing NK cells) can be treated to be created. Subjects in need of treatment may then be subjected to standard treatment with high dose chemotherapeutic agents followed by peripheral blood stem cell transplantation. In one embodiment, the subject receives an infusion of grown CAR-expressing cells of the invention (eg, CAR T cells or NK cells) after or at the same time as transplantation. In a further embodiment, the proliferating cells are administered before or after surgery.

In embodiments, lymphatic depletion is performed, for example, prior to administration of one or more cells expressing the CARs described herein (eg, BCMA-bound CARs described herein). .. In embodiments, lymphatic depletion comprises administering one or more of melphalan, cytoxane, cyclophosphamide and fludarabine.

The dose of the above treatment to be administered to the patient depends on the exact nature of the condition to be treated and the recipient of the treatment. Scaling for human administration can be performed according to the practices recognized in the art. The dose of CAMPATH is generally in the range of 1 to about 100 mg for adult patients, and is usually administered daily over a period of 1 to 30 days. The preferred daily dose is 1-10 mg / day, but in some cases high doses up to 40 mg / day may be used (described in US Pat. No. 6,120,766).

In some embodiments, CAR is introduced into an immune effector cell (eg, T cell or NK cell) using, for example, in vitro transcription, and the subject (eg, human) is the CAR immune effector cell of the invention (eg, human). For example, an initial dose of T cells or NK cells) followed by one or more doses of the CAR immune effector cells of the invention (eg, T cells or NK cells), where one or more subsequent doses. Administration is 15 days, for example, 14 days, 13 days, 12 days, 11 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days or 2 days after the previous administration. Do it within. In some embodiments, more than one dose of the CAR immune effector cells of the invention (eg, T cells or NK cells) is given to a subject (eg, human) per week, eg, the CAR immune effector of the invention. Cells (eg, T cells or NK cells) are administered twice, three or four times per week. In some embodiments, the subject (eg, a human subject) receives more than one dose of CAR immune effector cells (eg, T cells or NK cells) per week (eg, twice per week, 3). One or four doses) (also referred to herein as a cycle) followed by no CAR immune effector cells (eg, T cells or NK cells) for 1 week followed by CAR immune effector cells (eg, eg, NK cells). One or more additional doses of T cells (eg, T cells or NK cells) (eg, more than one dose of CAR immune effector cells (eg, T cells or NK cells) per week) are administered to the subject. In another embodiment, the subject (eg, a human subject) undergoes more than one cycle of CAR immune effector cells (eg, T cells or NK cells), with a duration of 10 days, 9 days, between each cycle. Shorter than 8th, 7th, 6th, 5th, 4th or 3rd. In some embodiments, CAR immune effector cells (eg, T cells or NK cells) are administered every other day three times per week. In some embodiments, the CAR immune effector cells of the invention (eg, T cells or NK cells) are at least 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks or more. Is administered.

In one embodiment, BCMA CAR-expressing cells (eg, BCMA CART or BCMA CAR-expressing NK cells) are produced using a lentiviral viral vector such as lentivirus. CAR-expressing cells produced by this method (eg, CART or CAR-expressing NK cells) have stable CAR expression.

In one embodiment, CAR-expressing cells, such as CART, are produced using a gamma retroviral vector, eg, a viral vector such as the gamma retroviral vector described herein. CART produced using these vectors may have stable CAR expression.

In one embodiment, CAR-expressing cells (eg, CART or CAR-expressing NK cells) are transduced 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days. After 14 and 15 days, the CAR vector is transiently expressed. Transient expression of CAR can be achieved by RNA CAR vector delivery. In one embodiment, CAR RNA is transduced into cells (eg, T cells or NK cells) by electroportation.

Occurs in patients treated with transiently expressed CAR-expressing cells (eg, CART or CAR-expressing NK cells), particularly with mouse scFv-carrying CAR-expressing cells (eg, CART or CAR-expressing NK cells). A potential problem may be anaphylacticity after multiple treatments.

Although not bound by this theory, such anaphylactic responses are believed to be due to patients who develop a humoral anti-CAR response, i.e., anti-CAR antibodies with anti-IgE isotypes. The antibody of the patient producing the cells is considered to undergo a class switch from the IgG isotype (which does not produce anaphylaxis) to the IgE isotype when there is a 10-14 day interruption of exposure to the antigen.

Infusion of CAR-expressing cells (eg, CART cells or CAR-expressing NK cells) if the patient is at high risk of producing an anti-CAR antibody response during transient CAR treatment (eg, completed by RNA transduction). The interruption should not continue for more than 10-14 days.

The present invention will be described in more detail with reference to the following experimental examples. These examples are provided for illustration purposes only and are not intended to be limiting unless otherwise noted. Accordingly, the invention should by no means be construed as being limited to the following embodiments, but rather to include any and all variants that become apparent as a result of the teachings provided herein. be.

Without further description, one of ordinary skill in the art will be able to manufacture and utilize the compounds of the invention and practice the methods of the present invention using the previous description and the following explanatory examples. The following working examples specifically illustrate various aspects of the invention and should not be construed as limiting this disclosure.

Example 1: In vitro characterization of human BCMA CAR A set of fully human single chain variable fragments (scFv) was cloned into a lentivirus CAR expression vector with a CD3 zeta chain and a 4-1BB stimulating molecule: R1B6, R1F2, R1G5, PI61, B61-10, B61-02, Hy03 and Hy52. Constructs are first screened using an automated cell reporter assay, followed by expression in primary T cells and effector T cell responses to BCMA expression (“BCMA +” or “BCMA positive”) targets (“BCMA CART” or “BCMA CART” or “BCMA CART” or “BCMA positive”. Optimal clones were selected based on the quantity and quality of BCMA CAR T cells). Responses of effector T cells include, but are not limited to, cell expansion, proliferation, doubling, cytokine production and target cell killing or cytolytic activity (degranulation).

BCMA CAR Lentivirus Generation All of the above scFvs encoding lentivirus introduction vectors were used to produce genomic material packaged in VSVg pseudotyped lentivirus particles. Lentivirus-introduced vector DNA encoding CAR was mixed with three packaging components VSVg, gag / pol and rev in combination with lipofectamine reagents to transfect Lenti-X 293T cells (Clontech) 12-18 thereof. The medium was changed after hours. After 30 hours of medium replacement, the medium was collected, filtered and stored at −80 ° C.

BCMA CAR JNL and JNL screening reporter assay using automated system In the reporter assay, the lentivirus encoding BCMA CAR was introduced in two different cell densities (40,000 cells (1xH293) or 80,000 cells (2xH293)) in HEK293 cells. ), Generated in a 96-well plate by an automated small-scale method, where the virus-containing supernatant was harvested 48 hours after transfection and transduced into the Jurkat T cell reporter cell line. Used fresh without freezing. The Jurkat NFAT luciferase (JNL) reporter cell line is based on the acute T-cell leukemia strain Jurkat. This strain was modified to express luciferase under the control of the nuclear factor (NFAT) response element of activated T cells. For transduction by BCMA CAR, 50 μl of fresh 45 μm filtered virus-containing supernatant was transduced into a 96-well plate of 10,000 JNL cells / well. Plates were cultured for 5 days prior to co-culturing with target cells.

To assess the functional ability of BCMA CARs to activate JNL cells, they were co-cultured with target cancer cells at different effector to target cell ratios (E: T ratios) and they were quantified for luciferase expression. Read the activation of. The scFv-based CARs R1B6, R1F2, R1G5, PI61, B61-10, B61-02, Hy03 and Hy52 were evaluated. CD19 JNL CAR cells were used as target-specific controls and only medium containing no target cells served as a negative control.

The above-mentioned 5-day transduced JNL CAR cells were co-cultured with BCMA-positive multiple myeloma (MM) cell line KMS11 or acute lymphocytic leukemia cell line NALM6 and functioned as a BCMA-negative control. The remaining JNL CAR T cells were evaluated for BCMA CAR expression by flow cytometry. Co-cultures set the effector to target (E: T) ratio to 4: 1, 2: 1, 1: 1 and 0.5: 1 on a 384-well plate, incubated for 24 hours, and then activated. Expression of luciferase by JNL CAR T cells was quantified by the Bright-Glo ™ luciferase assay system (Promega, Madison, WI). The amount of light (emission) emitted from each well was a direct reading of JNL activation by each CAR. JNL cells were considered activated when the luminescence level was more than twice that of UTD cells. The BCMA + KMS11 cell line resulted in activation of JNL cells expressing R1B6, R1F2, R1G5, PI61, B61-10, B61-02, Hy03 and Hy52 (FIGS. 1A and 1C). None of the BCMA CARs showed activation by the BCMA negative line NALM6 (FIGS. 1E and 1F). None of the CAR transduced JNLs tested was activated in media containing no target cells alone (FIGS. 1G and 1H). FACS analysis showed that BCMA-CAR expression in transduced JNL was detected to varying degrees. CAR% is generally positively correlated with JNL activation by KMS11 cells in the most active JNL CART (FIGS. 1B and 1D).

BCMA CAR T Cell Generation The following eight CARs were selected for analysis of CAR expression, stability and efficacy in primary T cells: R1B6, R1F2, R1G5, PI61, B61-10, B61-02, Hy03 and Hy52. BCMA CAR T cells were generated starting from blood from a healthy apheretic donor from which T cells (CD4 + and CD8 + lymphocytes) were obtained by negative selection of CD3 + T cells. These cells are T cell medium (RPMI1640, 10% heat-inactivated bovine fetal serum (FCS), 2 mM L-glutamine, 1x penicillin / streptomycin, 100 μM non-essential amino acids, 1 mM sodium pyruvate, 10 mM Hepes and 55 μM 2-mercapto. It was activated by the addition of CD3 / CD28 beads (Dynabeads® Human T-Expander CD3 / CD28, Thermo Fisher Scientific) in a ratio of 1: 3 (T cells vs. beads) in ethanol). T cells were cultured at 37 ° C., 5% CO ₂ in 0.5 × 10 ⁶ T cells in 1 mL of medium per well of a 24-well plate. Twenty-four hours later, while the T cells were blasting, the T cells were transduced with BCMA CAR virus at a multiplicity of infection (MOI) of 5. T cells begin to divide in an exponential growth pattern, which is monitored by measuring the number of cells per mL, T cells diluted with fresh medium every 2 days, beads removed and further on day 9. Collected for analysis. T cell aliquots were stained and CAR expression was measured by flow cytometry on days 5 and 9 with FACS Fortessa (BD). All BCMA CAR T cells were produced under research grade (ie, non-clinical grade) production conditions.

BCMA-CAR surface expression and its stability were assessed by measuring CAR% and MFI (mean fluorescence intensity) on days 5 and 9 using flow cytometric analysis of rBCMA_Fc-AF647 stained cells. (Fig. 2 and Table 17). Expression of BCMA CAR in the final product on day 9 varies from construct to construct and ranges from 18% to 42.4%, with MFI ranging from 672 to 5238. Constructs from PALLAS-derived clones R1F2, R1B6 and R1G5 and hybridoma clone Hy03 show a loss of approximately 30% to 50% CAR from day 5 to day 9, while PI61, B61-10 and -02 and Hy52. Is relatively stable with respect to the percentage of CAR expression, but all CAR constructs show a decrease in MFI from day 5 to day 9, which is probably the T cell's resting phase on day 9. Because it was smaller in size. Cell numbers in CAR T cell cultures have no detectable negative effect of BCMA CAR-carrying human scFv on the ability of cells to proliferate normally when compared to non-transduced T cells (“UTD”). showed that.

Evaluation of BCMA CAR Redirected T Cell Functionality To assess the functional capacity of BCMA CAR-T cells, co-culture with BCMA positive and negative cancer strains was set up. CAR-T cells were thawed, counted, and co-cultured with target cells to read their killing ability and cytokine secretion. BCMA CAR clones R1B6, R1F2, R1G5, B61-02, B61-10, PI61, Hy03 and Hy52 were tested. Non-transduced T cells (UTDs) were used as non-targeted T cell controls.

Cart cell killing was performed by co-culturing CART cells with KMS11-Luc and NALM6-Luc target cells at different E: T ratios for 20 hours. The CAR T cell population was normalized to an equivalent percentage of CAR positive cells prior to plating. Cytokine IFNγ is a supernatant from a 20-hour co-culture of target cells and CAR-T cells at an effector-to-target ratio of 2.5: 1 using Meso Scale Discovery (MSD; Gaithersburg, MD). Measured and the results for each cytokine were calculated at pg / ml using known criteria. All assays were repeated from a single source of donor cells. Death data show that all BCMA CAR clones effectively kill KMS11 cancer cells (FIG. 3A). Control target cells NALM6 were not killed by any of these BCMA-specific CARs (FIG. 3B). The ability of these CARs to produce IFN-γ when cultured with KMS11 was also tested (FIG. 3C). BCMA CAR R1F2, R1G5 and PI61 produced the most IFN-γ. The levels of cytokines produced by BCMA CART after exposure to control NALM6 cells were low (FIG. 3C), indicating no non-specific activation by BCMA CAR.

CONCLUSIONS: New BCMA-bound scFvs were tested in the context of CAR T cells. Eight CARs were assayed with the JNL reporter assay and primary T cells: R1B6, R1F2, R1G5, B61-02, B61-10, PI61, Hy03 and Hy52. All eight CAR-T cells undergo target-specific killing. Indicated. T cells expressing R1F2, R1G5 or PI61 produced the highest amount of IFN-γ in the presence of target cells. Overall, introduction of BCMA CAR into primary T cells induced anti-BCMA CAR reactivity, but not off-target function.

Equivalents The disclosures of each and all patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety. Although the present invention is disclosed with reference to specific embodiments, other embodiments and variations of the invention may be devised by those skilled in the art without departing from the true spirit and scope of the invention. Is clear. The claims below are intended to be construed to include all such embodiments and even variants.

Claims (43)

An isolated nucleicus molecule encoding a chimeric antigen receptor (CAR), said CAR comprising an anti-BCMA binding domain (eg, human anti-BCMA binding domain), transmembrane domain and intracellular signaling domain. BCMA binding domains include heavy chain complementarity determining regions 1 (HC CDR1), heavy chain complementarity determining regions 2 (HC CDR2) and heavy chain complementarity determining regions 3 (HC CDR3), as well as heavy chain variable regions (VH). HC CDR1 comprising a light chain variable region (VL) comprising a light chain complementarity determining region 1 (LC CDR1), a light chain complementarity determining region 2 (LC CDR2) and a light chain complementarity determining region 3 (LC CDR3). , HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR3
SEQ ID NOs : 86, 130, 88, 95, 131 and 132, respectively
An isolated nucleic acid molecule comprising the amino acid sequence of. An isolated CAR comprising an anti-BCMA binding domain (eg, human anti BCMA binding domain), transmembrane domain and intracellular signaling domain, wherein the anti BCMA binding domain is a heavy chain complementarity determining region 1 (HC CDR1). , Heavy Chain Complementarity Determine Regions 2 (HC CDR1), Heavy Chain Variable Regions (VH) Containing Heavy Chain Complementarity Determine Regions 2 (HC CDR2) and Heavy Chain Complementarity Determine Regions 3 (HC CDR3), Light Chain Complementarity Determine Regions 1 (LC CDR1), Light Chain Complementarity. The HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR3s include determining regions 2 (LC CDR2s) and light chain complementarity determining regions 3 (LC CDR3s).
SEQ ID NOs : 86, 130, 88, 95, 131 and 132, respectively
An isolated CAR comprising the amino acid sequence of. The HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR3 are
SEQ ID NOs: 86, 87, 88, 95, 96 and 97 , respectively
The isolated nucleic acid molecule or CAR according to claim 1 or 2 , which comprises the amino acid sequence of. (I) The VH comprises 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 with it ; and / or.
(Ii) The VL comprises 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 with it.
The isolated nucleic acid molecule or CAR according to any one of claims 1 to 3 . (I) Containing the nucleic acid sequence encoding the VH, the nucleic acid sequence is the nucleic acid sequence of SEQ ID NO: 260, 94 or 113 or a nucleic acid having at least about 85%, 90%, 95% or 99% sequence identity thereof. Contains sequences ; and / or
(Ii) Containing the nucleic acid sequence encoding the VL, the nucleic acid sequence has the nucleic acid sequence of SEQ ID NO: 261, 103, 119 or 125 or at least about 85%, 90%, 95% or 99% sequence identity thereof. Contains nucleic acid sequences
The isolated nucleic acid molecule according to any one of claims 1 to 4 . The VH and VL are
(I) SEQ ID NOs: 93 and 102;
(Ii) SEQ ID NOs: 112 and 118, respectively; or (iii) SEQ ID NOs: 112 and 124, respectively.
The isolated nucleic acid molecule or CAR according to any one of claims 1 to 5 , which comprises the amino acid sequence of. 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 thereof. The isolated nucleic acid molecule or CAR according to any one of claims 1 to 6 , which comprises. The anti-BCMA binding domain comprises scFv, the nucleic acid molecule comprises a nucleic acid sequence encoding said scFv, said nucleic acid sequence is the nucleic acid sequence of SEQ ID NO: 253, 106, 121 or 127 or at least about 85% thereof. The isolated nucleic acid molecule according to any one of claims 1 to 7 , which comprises a nucleic acid sequence having 90%, 95% or 99% sequence identity. One of claims 1-8 , wherein the CAR comprises 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 thereof. The isolated nucleic acid molecule or CAR according to. One of claims 1-9 , comprising the nucleic acid sequence of SEQ ID NO: 259, 258, 108, 123 or 129 or a nucleic acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereof. The isolated nucleic acid molecule according to. The isolated nucleic acid molecule or CAR according to any one of claims 1 to 10 , wherein the VH and VL are connected by a linker, and optionally, the linker comprises the amino acid sequence of SEQ ID NO: 63 or 104. (I) The transmembrane domain is an alpha chain, beta chain or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, Does it contain a transmembrane domain of a protein selected from CD86, CD134, CD137 or CD154;
(Ii) The transmembrane domain comprises 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 thereof; or (iii) the nucleic acid molecule. Containing a nucleic acid sequence encoding the transmembrane domain, said nucleic acid sequence comprises 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 with it. The isolated nucleic acid molecule or CAR according to any one of claims 1 to 11 . The anti-BCMA binding domain is connected to the transmembrane domain by a hinge region and, optionally,
(I) Does the hinge region contain 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 with it; or (ii) said. The nucleic acid molecule comprises a nucleic acid sequence encoding the hinge region, wherein the nucleic acid sequence has at least about 85%, 90%, 95% or 99% sequence identity with the nucleic acid sequence of SEQ ID NO: 13, 14 or 15. The isolated nucleic acid molecule or CAR according to any one of claims 1 to 12 , which comprises a nucleic acid sequence having. The intracellular signaling domain comprises a primary signaling domain and, optionally,
(I) The primary signaling domain is CD3 zeta, TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (ICOS), FcεRI, DAP10, DAP12 or Does it contain a functional signaling domain derived from CD66d;
(Ii) The primary signaling domain comprises 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 with it; or (iii) said. The nucleic acid molecule comprises the nucleic acid sequence encoding the primary signaling domain, wherein the nucleic acid sequence is the nucleic acid sequence of SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 256 or at least about 85%, 90%, 95% or 99 thereof. The isolated nucleic acid molecule or CAR according to any one of claims 1 to 13 , which comprises a nucleic acid sequence having% sequence identity. The intracellular signaling domain comprises a co-stimulation signaling domain and, optionally,
(I) The costimulatory signal transduction domain includes MHC class I molecule, TNF receptor protein, immunoglobulin-like protein, cytokine receptor, integulin, signaling lymphoid activation molecule (SLAM protein), and activated NK cell receptor. , BTLA, Toll ligand receptor, OX40, CD2, 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 , ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, NKG2C, TNFR2. / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), 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, CD28-OX40, CD28-4-1BB Or does it contain a functional signaling domain derived from a ligand that specifically binds to CD83;
(Ii) The costimulatory signaling domain comprises 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 with it; or (iii) the nucleic acid. The molecule comprises a nucleic acid sequence encoding the costimulatory signaling domain, wherein the nucleic acid sequence is the nucleic acid sequence of SEQ ID NO: 18 or SEQ ID NO: 255 or at least about 85%, 90%, 95% or 99% sequence identical thereto. The isolated nucleic acid molecule or CAR according to any one of claims 1 to 14 , comprising a nucleic acid sequence having sex. The intracellular signaling domain comprises a functional signaling domain derived from 4-1BB and a functional signaling domain derived from the CD3 zeta, and optionally the intracellular signaling domain is the amino acid of SEQ ID NO: 7. The sequence (or an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence identity with it) and the amino acid sequence of SEQ ID NO: 9 or 10 (or at least about 85%, 90%, 95% or 99 with it). Amino acid sequence having% sequence identity), and optionally, the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 7 and the amino acid sequence of SEQ ID NO: 9 or 10. The isolated nucleic acid molecule or CAR according to item 1. The isolated nucleic acid molecule or CAR according to any one of claims 1 to 16 , further comprising a leader sequence comprising the amino acid sequence of SEQ ID NO: 1. The CAR has the following characteristics:
(I) When the CAR is expressed in a cell (eg, a T cell), it activates NFAT signaling in the cell in the presence of BCMA expressing cells;
(Ii) Induces cytotoxicity of BCMA - expressing cells when expressed in cells (eg, T cells); and (iii) said CARs are expressed in cells (eg, T cells). If one or more (eg, one, two or all) of inducing the expression of a cytokine (eg, IFN - γ) in said cell in the presence of BCMA expressing cells. 17. The isolated nucleic acid molecule or CAR according to any one of 17. An isolated polypeptide molecule encoded by the nucleic acid molecule according to any one of claims 1 or 3-18 . Heavy chain variable regions (VHs) and light chain complementarity determining, including heavy chain complementarity determining regions 1 (HC CDR1), heavy chain complementarity determining regions 2 (HC CDR2) and heavy chain complementarity determining regions 3 (HC CDR3). An anti-BCMA binding domain comprising a light chain variable region (VL) comprising a region 1 (LC CDR1), a light chain complementarity determining region 2 (LC CDR2) and a light chain complementarity determining region 3 (LC CDR3). HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR3 are
SEQ ID NOs : 86, 130, 88, 95, 131 and 132, respectively
An anti-BCMA binding domain comprising the amino acid sequence of. The HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR3 are
(I) SEQ ID NOs: 86, 87, 88, 95, 96 and 97;
( Ii ) SEQ ID NOs: 86, 109, 88, 95, 114 and 115; or
( Iii ) SEQ ID NOs: 86, 109, 88, 95, 114 and 97 , respectively.
20. The anti-BCMA binding domain according to claim 20 , comprising the amino acid sequence of. The VH and VL are
(I) SEQ ID NOs: 93 and 102;
( Ii ) SEQ ID NOs: 112 and 118; or , respectively.
( Iii ) SEQ ID NOs: 112 and 124, respectively .
The anti-BCMA binding domain of claim 20 or 21 , comprising the amino acid sequence of. The nucleic acid molecule according to any one of claims 1 or 3 to 18 , the nucleic acid molecule encoding the CAR according to any one of claims 2, 4, 6, 7 , 9, and 11-18 , claim . A vector containing the nucleic acid molecule encoding the polypeptide molecule according to Item 19, or the nucleic acid molecule encoding the anti-BCMA binding domain according to any one of claims 20 to 22, and optionally a DNA vector. , An RNA vector, a plasmid, a lentivirus vector, an adenovirus vector or a retroviral vector, further optionally comprising an EF-1 promoter comprising the nucleic acid sequence of SEQ ID NO: 11 . The nucleic acid molecule according to any one of claims 1 or 3 to 18 , the CAR according to any one of claims 2, 4, 6, 7 , 9, or 11 to 18 , the CAR according to claim 19 . A cell (eg, T cell or NK cell) comprising a polypeptide molecule , the anti-BCMA binding domain according to any one of claims 20-22, or the vector according to claim 23 . A method for producing cells, which comprises transducing cells (eg, T cells or NK cells) with the vector according to claim 23 . A method of producing RNA-manipulated cells comprising introducing in vitro transcribed RNA or synthetic RNA into a cell (eg, T cell or NK cell), wherein the RNA is claimed 1 or 3-18 . The nucleic acid molecule according to any one, the nucleic acid molecule encoding the CAR according to any one of claims 2, 4, 6, 7 , 9, or 11-18, or any of claims 20 to 22. A method comprising a nucleic acid molecule encoding the anti-BCMA binding domain according to one . Use of an effective amount of a cell according to claim 24 for the manufacture of a pharmaceutical for anti-tumor immunity in a subject. 24. Use of an effective amount of cells according to claim 24 for the manufacture of a pharmaceutical agent for treating a subject having a disease associated with BCMA expression. 28. The use according to claim 27 or 28 , wherein the cell is an autologous T cell or an allogeneic T cell. The disease associated with BCMA expression
(I) A precancerous condition selected from one or more of cancer or malignant tumor or myelodysplastic syndrome, myelodysplastic syndrome or preleukemia, or (ii) a non-cancer-related indication associated with the expression of BCMA. 28 or 29 . The use according to any one of claims 28 to 30 , wherein the disease is a blood cancer or a solid cancer. The 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), pre-B cell lymphocytic leukemia, blast plasma cell-like dendritic cell neoplasm, Berkit lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small cells Type or large cell follicular lymphoma, malignant lymphoproliferative disorder, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodystrophy and myelopathy syndrome, non-hodgkin lymphoma, plasmablast lymphoma, Plasma cell-like dendritic cell neoplasms, Waldenstrem macroglobulinemia, prostate cancer (eg, castration-resistant or treatment-resistant prostate cancer or metastatic prostate cancer), pancreatic cancer, lung cancer, plasma cell proliferative disorders (For example, asymptomatic myeloma (smoldering multiple myeloma or painless myeloma), unclear monoclonal hypergamma globulinemia (MGUS), Waldenstrem macroglobulinemia, plasmacytoma (for example) For example, plasma cell overgrowth, solitary myeloma, solitary plasmacytoma, extramedullary plasmacytoma and multiple plasmacytoma), systemic amyloid light chain amyloidosis or POEMS syndrome (Crow-Fukase syndrome, Takatsuki disease). And PEP syndrome))) or a combination thereof, according to any one of claims 28-31 . The use according to any one of claims 28-32 , wherein the disease is multiple myeloma. The pharmaceutical is formulated for administration in combination with a second therapeutic agent, and optionally the second therapeutic agent is.
(I) PD-1 inhibitor selected from the group consisting of PDR001, nivolumab, pembrolizumab , MEDI0680, REGN2810, TSR-042, PF-06801591 and AMP-224, which are PD-1 inhibitors. ;
(Ii) A PD-L1 inhibitor selected from the group consisting of FAZ053, atezolizumab, avelumab, durvalumab and BMS-936559, which is a PD-L1 inhibitor;
(Iii) A LAG-3 inhibitor, optionally selected from the group consisting of LAG525, BMS-986016, TSR-033, MK-4280 and REGN3767;
(Iv) TIM-3 inhibitor, optionally selected from the group consisting of MBG453, TSR-022 and LY3321367;
(V) CTLA-4 inhibitor, optionally ipilimumab or tremelimumab, CTLA-4 inhibitor;
(Vi) Interleukin-15 (IL-15) polypeptide, interleukin-15 receptor alpha (IL-15Ra) polypeptide or a combination of both IL-15 and IL-15Ra polypeptides, such as hetIL-15. ;
(Vii) Interleukin-12 (IL-12) polypeptide; or (viii) mTOR inhibitor, optionally selected from RAD001 or mTOR inhibitor which is rapamycin, any of claims 27-33 . Use as described in item 1. From N-terminus to C-terminus:
(I) Reader sequence of SEQ ID NO: 1;
(Ii) HC CDR1 of SEQ ID NO: 86;
(Iii) HC CDR2 of SEQ ID NO: 87;
(Iv) HC CDR3 of SEQ ID NO: 88;
(V) LC CDR1 of SEQ ID NO: 95;
(Vi) LC CDR2 of SEQ ID NO: 96;
(Vii) LC CDR3 of SEQ ID NO: 97;
(Viii) CD8 transmembrane domain and hinge of SEQ ID NO: 202;
(Ix) Functional signaling domain of 4-1BB of SEQ ID NO: 7; and
(X) Functional signaling domain of the CD3 zeta of SEQ ID NO: 10.
Chimeric antigen receptor (CAR) that binds to BCMA. 35. The CAR of claim 35, 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. The CAR according to claim 35 or 36, comprising the amino acid sequence of SEQ ID NO: 257 or SEQ ID NO: 107. An isolated nucleic acid molecule encoding the CAR molecule according to any one of claims 35 to 37. A vector containing a nucleic acid molecule encoding a chimeric antigen receptor (CAR) that binds to BCMA, where CAR is from the N-terminus to the C-terminus:
(I) Reader sequence of SEQ ID NO: 1;
(Ii) HC CDR1 of SEQ ID NO: 86;
(Iii) HC CDR2 of SEQ ID NO: 87;
(Iv) HC CDR3 of SEQ ID NO: 88;
(V) LC CDR1 of SEQ ID NO: 95;
(Vi) LC CDR2 of SEQ ID NO: 96;
(Vii) LC CDR3 of SEQ ID NO: 97;
(Viii) CD8 transmembrane domain and hinge of SEQ ID NO: 202;
(Ix) Functional signaling domain of 4-1BB of SEQ ID NO: 7; and
(X) Functional signaling domain of the CD3 zeta of SEQ ID NO: 10.
Including the vector. A cell containing a chimeric antigen receptor (CAR) that binds to BCMA (eg, T cells or NK cells), where the CAR is from N-terminus to C-terminus:
(I) Reader sequence of SEQ ID NO: 1;
(Ii) HC CDR1 of SEQ ID NO: 86;
(Iii) HC CDR2 of SEQ ID NO: 87;
(Iv) HC CDR3 of SEQ ID NO: 88;
(V) LC CDR1 of SEQ ID NO: 95;
(Vi) LC CDR2 of SEQ ID NO: 96;
(Vii) LC CDR3 of SEQ ID NO: 97;
(Viii) CD8 transmembrane domain and hinge of SEQ ID NO: 202;
(Ix) Functional signaling domain of 4-1BB of SEQ ID NO: 7; and
(X) Functional signaling domain of the CD3 zeta of SEQ ID NO: 10.
A cell (eg, T cell or NK cell) comprising. A cell containing the CAR molecule according to any one of claims 35 to 37, the nucleic acid molecule according to claim 38, or the vector according to claim 39 (for example, T cells or NK cells). Use of the CAR according to any one of claims 35-38 or the cells according to claim 40 or 41 for the manufacture of a pharmaceutical agent for treating a disease associated with the expression of BCMA. 42. The use according to claim 42, wherein the disease associated with the expression of BCMA is multiple myeloma.