Prostate-specific membrane antigen cars and methods of use thereof

Abstract

The present disclosure provides modified immune cells (e.g., modified T cells) comprising a chimeric antigen receptor (CAR) having affinity for a prostate-specific membrane antigen (PSMA) (e.g., human PSMA). The present disclosure provides modified immune cells (e.g., modified T cells) comprising a CAR having affinity for PSMA and a dominant negative receptor and/or a switch receptor. The present disclosure provides modified immune cells (e.g., modified T cells) comprising a CAR having affinity for PSMA and a dominant negative receptor and/or a switch receptor, wherein the modified cell is capable of expressing and secreting a bispecific antibody.

1 . A modified immune cell or precursor cell thereof, comprising
 (a) a chimeric antigen receptor (CAR) capable of binding prostate specific membrane antigen (PSMA) comprising an antigen binding domain, a transmembrane domain, and an intracellular domain, wherein the antigen binding domain comprises:
 a heavy chain variable region (VH) that comprises the consensus sequence of SEQ ID NO:183; and a light chain variable region (VL) that comprises the consensus sequence of SEQ ID NO:184; and 
   (b) a dominant negative receptor and/or switch receptor.   
     
     
         2 . The modified cell of  claim 1 , wherein:
 (a) the VH comprises the sequence of SEQ ID NO:191; and/or   (b) the VL comprises the sequence of SEQ ID NO:192.   
     
     
         3 . (canceled) 
     
     
         4 . The modified cell of  claim 1 , wherein:
 (a) the antigen binding domain comprises an antibody or an antigen-binding fragment thereof; and/or   (b) the antigen binding domain comprises an antibody or an antigen-binding fragment thereof, and wherein the antigen-binding fragment is selected from the group consisting of a Fab, a single-chain variable fragment (scFv), or a single-domain antibody.   
     
     
         5 . (canceled) 
     
     
         6 . The modified cell of  claim 1 , wherein the transmembrane domain:
 (a) comprises a transmembrane region derived from CD8; and/or   (b) comprises a transmembrane region derived from CD8 comprising the amino acid sequence set forth in SEQ ID NO: 88; and/or   (c) further comprises a hinge region derived from CD8; and/or   (d) further comprises a hinge region derived from CD8 comprising the amino acid sequence set forth in SEQ ID NO: 86.   
     
     
         7 .- 9 . (canceled) 
     
     
         10 . The modified cell of  claim 6 , wherein the intracellular domain comprises:
 (a) a 4-1BB signaling domain and a CD3 zeta signaling domain; and/or   (b) an ICOS signaling domain and a CD3 zeta signaling domain; and/or   (c) a variant ICOS signaling domain and a CD3 zeta signaling domain; and/or   (d) a 4-1BB signaling domain comprising the amino acid sequence set forth in SEQ ID NO: 92; and/or   (e) an ICOS signaling domain and a CD3 zeta signaling domain, and wherein the ICOS signaling domain the amino acid sequence set forth in SEQ ID NO: 203; and/or   (f) a variant ICOS signaling domain and a CD3 zeta signaling domain, wherein the variant ICOS signaling domain comprises the amino acid sequence set forth in SEQ 1D NO: 95; and/or   (g) a CD3 zeta signaling domain comprising the amino acid sequence set forth in SEQ ID NOs: 97 or 100.   
     
     
         11 .- 16 . (canceled) 
     
     
         17 . The modified cell of  claim 1 , wherein the dominant negative receptor:
 (a) is a truncated variant of a wild-type protein associated with a negative signal; and/or   (b) a truncated variant of a wild-type protein associated with a negative signal comprising the amino acid sequence set forth in SEQ ID NO: 115.   
     
     
         18 . (canceled) 
     
     
         19 . The modified cell of  claim 1 , wherein the switch receptor:
 (a) comprises a first domain, wherein the first domain is derived from a first polypeptide that is associated with a negative signal; and a second domain, wherein the second domain is derived from a second polypeptide that is associated with a positive signal; and/or   (b) comprises a first domain, wherein the first domain is derived from a first polypeptide that is associated with a negative signal, wherein the first domain comprises at least a portion of the extracellular domain of the first polypeptide that is associated with a negative signal; and a second domain, wherein the second domain is derived from a second polypeptide that is associated with a positive signal, and wherein the second domain comprises at least a portion of the intracellular domain of the second polypeptide that is associated with a positive signal; and/or   (c) further comprises a switch receptor transmembrane domain, and optionally wherein the transmembrane domain comprises the transmembrane domain of the first polypeptide that is associated with a negative signal; or the transmembrane domain of the second polypeptide that is associated with a positive signal; and/or   (d) comprises a first domain, wherein the first domain is derived from a first polypeptide that is associated with a negative signal; and a second domain, wherein the second domain is derived from a second polypeptide that is associated with a positive signal, wherein the first polypeptide that is associated with a negative signal is selected from the group consisting of CTLA4, PD-1, BTLA, TIM-3, and a TGFβR, and/or   (e) comprises a first domain, wherein the first domain is derived from a first polypeptide that is associated with a negative signal; and a second domain, wherein the second domain is derived from a. second polypeptide that is associated with a positive signal, wherein the second polypeptide that is associated with a positive signal is selected from the group consisting of CD28, ICOS, 4-1BB, and a IL-12R; and/or   (f) comprises a first domain comprising at least a portion of the extracellular domain of PD1; a switch receptor transmembrane domain comprising at least a portion of the transmembrane domain of CD28; and a second domain comprising at least a portion of the intracellular domain of CD28; and/or   (g) comprises the amino acid sequence set forth in SEQ ID NO: 117; and/or   (h) comprises a first domain comprising at least a portion of the extracellular domain of PD1; a switch receptor transmembrane domain comprising at least a portion of the transmembrane domain of PD1; and a second domain comprising at least a portion of the intracellular domain of CD28, and optionally wherein the first domain comprises at least a portion of the extracellular domain of PD1 comprises an alanine (A) to leucine (L) substitution at amino acid position 132; and/or   (i) comprises the amino acid sequence set forth in SEQ ID NO: 119; and/or   (j) comprises the amino acid sequence set forth in SEQ ID NO: 121; and/or   (k) the switch receptor comprises a first domain comprising at least a portion of the extracellular domain of PD1 comprising an alanine (A) to leucine (L) substitution at amino acid position 132; and a second domain comprising at least a portion of the intracellular domain of CD28; and/or   (l) comprises a first domain comprising at least a portion of the extracellular domain of PD1 comprising an alanine (A) to leucine (L) substitution at amino acid position 132; and a second domain comprising at least a portion of the intracellular domain of 4-1BB; and/or   (m) comprises the amino acid sequence set forth in SEQ ID NO: 215; and/or   (n) comprises: a first domain comprising at least a portion of the extracellular domain of TIM-3; and a second domain comprising at least a portion of the intracellular domain of CD28; and/or   (o) comprises the amino acid sequence set forth in SEQ ID NO: 127; and/or   (p) comprises a first domain comprising at least a portion of the extracellular domain of a TGFβR; and a second domain comprising at least a portion of the intracellular domain of ILI2Rα1; and/or   (q) comprises the amino acid sequence set forth in SEQ ID NO: 123; and/or   (r) comprises a first domain comprising at least a portion of the extracellular domain of a TGFβR; and a second domain comprising at least a portion of the intracellular domain of IL12Rβ1; and/or   (s) comprises the amino acid sequence set forth in SEQ ID NO: 125.   
     
     
         20 .- 40 . (canceled) 
     
     
         41 . A modified immune cell or precursor cell thereof, comprising:
 (a) a chimeric antigen receptor (CAR) having affinity for a prostate specific membrane antigen (PSMA) on a target cell, wherein the CAR comprises a PSMA binding domain comprising a heavy chain variable region (VH) that comprises the sequence of SEQ ID NO:191; and a light chain variable region (VL) that comprises the sequence of SEQ ID NO:192; and a dominant negative receptor comprising the amino acid sequence set forth in SEQ ID NO: 115; or   (b) a chimeric antigen receptor (CAR) having affinity for a prostate specific membrane antigen (PSMA) on a target cell, wherein the CAR comprises a PSMA binding domain comprising a heavy chain variable region (VH) that comprises the sequence of SEQ ID NO: 191; and a light chain variable region (VL) that comprises the sequence of SEQ ID NO: 192; and a switch receptor comprising the amino acid sequence set forth in SEQ ID NO: 213 or 215; or   (c) a chimeric antigen receptor (CAR) having affinity for a prostate specific membrane antigen (PSMA) on a target cell, wherein the CAR comprises a PSMA binding domain comprising a heavy chain variable region (VH) that comprises the sequence of SEQ ID NO: 191; and a light chain variable region (VL) that comprises the sequence of SEQ ID NO: 192; and a switch receptor comprising the amino acid sequence set forth in SEQ ID NOs: 117 or 119; or   (d) a chimeric antigen receptor (CAR) having affinity for a prostate specific membrane antigen (PSMA) on a target cell, wherein the CAR comprises a PSMA binding domain comprising a heavy chain variable region (VH) that comprises the sequence of SEQ ID NO: 191; and a light chain variable region (VL) that comprises the sequence of SEQ ID NO: 192; and a switch receptor comprising the amino acid sequence set forth in SEQ ID NO: 121; or   (e) a chimeric antigen receptor (CAR) having affinity for a prostate specific membrane antigen (PSMA) on a target cell, wherein the CAR comprises a PSMA binding domain comprising a heavy chain variable region (VH) that comprises the sequence of SEQ ID NO: 191; and a light chain variable region (VL) that comprises the sequence of SEQ ID NO: 192; and a switch receptor comprising the amino acid sequence set forth in SEQ ID NO: 127; or   (f) a chimeric antigen receptor (CAR) having affinity for a prostate specific membrane antigen (PSMA) on a target cell, wherein the CAR comprises a PSMA binding domain comprising a heavy chain variable region (VH) that comprises the sequence of SEQ ID NO: 191; and a light chain variable region (VL) that comprises the sequence of SEQ ID NO: 192; and a switch receptor comprising the amino acid sequence set forth in SEQ ID NO: 123; or   (g) a chimeric antigen receptor (CAR) having affinity for a prostate specific membrane antigen (PSMA) on a target cell, wherein the CAR comprises a PSMA binding domain comprising a heavy chain variable region (VH) that comprises the sequence of SEQ ID NO: 191; and a light chain variable region (VL) that comprises the sequence of SEQ ID NO: 192; and a switch receptor comprising the amino acid sequence set forth in SEQ ID NO: 125.   
     
     
         42 .- 47 . (canceled) 
     
     
         48 . The modified cell of  claim 1 , wherein the modified cell is a modified T cell. 
     
     
         49 . The modified T cell of  claim 48 , wherein the modified T cell is an autologous cell. 
     
     
         50 . The modified T cell of  claim 48 , wherein the modified T cell is an allogeneic cell. 
     
     
         51 . The modified cell of  claim 1 , wherein the modified cell is a cytotoxic T lymphocyte (CTL). 
     
     
         52 . The modified cell of  claim 1 , wherein the modified cell is derived from a human cell. 
     
     
         53 . An isolated nucleic acid, comprising:
 (a) a first nucleic acid sequence encoding a chimeric antigen receptor (CAR) capable of binding prostate specific membrane antigen (PSMA) comprising an antigen binding domain, a transmembrane domain, and an intracellular domain, wherein the antigen binding domain comprises: a heavy chain variable region (VH) that comprises the consensus sequence of SEQ ID NO:183; and a light chain variable region (VL) that comprises the consensus sequence of SEQ ID NO:184; and   (b) a second nucleic acid sequence encoding a dominant negative receptor and/or a switch receptor.   
     
     
         54 . The isolated nucleic acid of  claim 53 , wherein:
 (a) the VH comprises the sequence of SEQ ID NO:191; and/or   (b) the VL comprises the sequence of SEQ ID NO:192; and/or   (c) the first nucleic acid sequence comprises the nucleic acid sequence set forth in any one of SEQ ID NOs: 246, 248, 250, 252, 254, or 256; and/or   (d) the second nucleic acid sequence comprises the nucleic acid sequence set forth in any one of SEQ ID NOs: 116, 118, 120, 122, 124, 126 128, 214, or 216; and/or   (e) the first nucleic acid sequence and the second nucleic acid sequence are separated by a linker; and/or   (f) the first nucleic acid sequence and the second nucleic acid sequence are separated by a linker, wherein the linker comprises a nucleic acid sequence encoding an internal ribosome entry site (IRES); and/or   (g) the first nucleic acid sequence and the second nucleic acid sequence are separated by a linker, wherein the linker comprises a nucleic acid sequence encoding a self-cleaving peptide; and/or   (h) the first nucleic acid sequence and the second nucleic acid sequence are separated by a linker, wherein the linker comprises a nucleic acid sequence encoding a self-cleaving 2A peptide; and/or   (i) the first nucleic acid sequence and the second nucleic acid sequence are separated by a linker, wherein the linker comprises a nucleic acid sequence encoding a self-cleaving 2A peptide, wherein the 2A peptide is selected from the group consisting of porcine teschovirus-1 2 A (P2A), Thoseaasigna virus 2A (T2A), equine rhinitis A virus 2A (E2A), and foot-and-mouth disease virus 2A (F2A); and/or   (j) the first nucleic acid sequence and the second nucleic acid sequence are separated by a linker, wherein the linker comprises a nucleic acid sequence encoding a self-cleaving 2A peptide, wherein the 2A peptide is T2A; and/or   (k) the first nucleic acid sequence and the second nucleic acid sequence are separated by a linker, wherein the linker comprises a nucleic acid sequence encoding a self-cleaving 2A peptide, wherein the 2A peptide is F2A; and/or   (l) the isolated nucleic acid comprises from 5′ to 3′ the first nucleic acid sequence, the linker, and the second nucleic acid sequence; and/or   (m) the isolated nucleic acid comprises from 5′ to 3′ the second nucleic acid sequence, the linker, and the first nucleic acid sequence.   
     
     
         55 .- 66 . (canceled) 
     
     
         67 . An isolated nucleic acid, comprising:
 (a) a first nucleic acid sequence encoding a chimeric antigen receptor (CAR) capable of binding prostate specific membrane antigen (PSMA) comprising an antigen binding domain, a transmembrane domain, and an intracellular domain, wherein the antigen binding domain comprises: a heavy chain variable region (VH) that comprises the sequence of SEQ ID NO:191; and a light chain variable region (VL) that comprises the sequence of SEQ ID NO:192; and a second nucleic acid sequence encoding a dominant negative receptor and/or switch receptor comprising the nucleic acid sequence set forth in any one of SEQ ID NOs: 116 118, 120, 122, 124, 126, 128, 214, or 216; or   (b) a first nucleic acid sequence encoding a chimeric antigen receptor (CAR) capable of binding prostate specific membrane antigen (PSMA) comprising an antigen binding domain, a transmembrane domain, and an intracellular domain, wherein the antigen binding domain comprises: a heavy chain variable region (VH) that comprises the sequence of SEQ ID NO:191; and a light chain variable region (VL) that comprises the sequence of SEQ ID NO:192; and a second nucleic acid sequence encoding a dominant negative receptor and/or switch receptor comprising the nucleic acid sequence set forth in SEQ ID NO: 116.   
     
     
         68 . (canceled) 
     
     
         69 . The isolated nucleic acid of  claim 67 , wherein:
 (a) the first nucleic acid sequence and the second nucleic acid sequence is separated by a linker comprising a nucleic acid sequence encoding T2A; or   (b) the first nucleic acid sequence and the second nucleic acid sequence is separated by a linker comprising a nucleic acid sequence encoding F2A.   
     
     
         70 . (canceled) 
     
     
         71 . An expression construct comprising the isolated nucleic acid of  claim 53 . 
     
     
         72 . The expression construct of  claim 71 , wherein:
 (a) the expression construct is a viral vector selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector; and/or   (b) the expression construct is a lentiviral vector; and/or   (c) the expression construct is a lentiviral vector, and wherein the lentiviral vector further comprises an EF-1 a promoter; and/or   (d) the expression construct is a lentiviral vector, and wherein the lentiviral vector further comprises a rev response element (RRE); and/or   (e) the expression construct is a lentiviral vector, and wherein the lentiviral vector further comprises a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE); and/or   (f) the expression construct is a lentiviral vector, and wherein the lentiviral vector further comprises a cPPT sequence; and/or   (g) the expression construct is a lentiviral vector, and wherein the lentiviral vector further comprises an EF-1 a promoter, a rev response element (RRE), a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), and a cPPT sequence; and/or   (h) the expression construct is a lentiviral vector, and wherein the lentiviral vector is a self-inactivating lentiviral vector.   
     
     
         73 .- 80 . (canceled) 
     
     
         81 . A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition comprising the modified immune cell of  claim 1 . 
     
     
         82 . The method of  claim 81 , further comprising:
 (a) administering to the subject a lymphodepleting chemotherapy, and/or   (b) administering to the subject a lymphodepleting chemotherapy, wherein the lymphodepleting chemotherapy comprises administering to the subject a therapeutically effective amount of cyclophosphamide and/or fludarabine.   
     
     
         83 . (canceled) 
     
     
         84 . The method of  claim 81 , wherein the method is directed to treating prostate cancer in a subject in need thereof, the method comprising:
 administering to the subject a lymphodepleting chemotherapy comprising administering to the subject a therapeutically effective amount of cyclophosphamide; and   administering to the subject a modified T cell comprising:
 a chimeric antigen receptor (CAR) having affinity for a prostate specific membrane antigen (PSMA) on a target cell, wherein the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular domain, wherein the antigen binding domain comprises: a heavy chain variable region (VH) that comprises the sequence of SEQ ID NO:191; and a light chain variable region (VL) that comprises the sequence of SEQ ID NO:192; and 
 a dominant negative receptor comprising an amino acid sequence set forth in SEQ ID NO: 115. 
   
     
     
         85 . The method of  claim 81 , wherein the method is directed to treating metastatic castrate resistant prostate cancer in a subject in need thereof, the method comprising:
 administering to the subject a lymphodepleting chemotherapy comprising administering to the subject a therapeutically effective amount of cyclophosphamide; and   administering to the subject a modified T cell comprising:
 a chimeric antigen receptor (CAR) having affinity for a prostate specific membrane antigen (PSMA) on a target cell, wherein the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular domain, wherein the antigen binding domain comprises: a heavy chain variable region (VH) that comprises the sequence of SEQ ID NO:191; and a light chain variable region (VL) that comprises the sequence of SEQ ID NO:192; and 
 a dominant negative receptor comprising an amino acid sequence set forth in SEQ NO: 115.