Chimeric antigen receptor and methods of use thereof

Abstract

The present disclosure provides a heterodimeric, conditionally active chimeric antigen receptor (CAR), and a nucleic acid comprising a nucleotide sequence encoding the CAR. The present disclosure provides cells genetically modified to produce the CAR. A CAR of the present disclosure can be used in various methods, which are also provided.

What is claimed is: 
     
         1 . A heterodimeric, conditionally active chimeric antigen receptor (CAR) comprising:
 a) a first polypeptide comprising:
 i) a first member of a specific binding pair; 
 ii) a first modulatory domain; 
 iii) a first member of a dimerization pair; and 
 iv) a transmembrane domain interposed between the first member of a specific binding pair and the first modulatory domain; and 
   b) a second polypeptide comprising:
 i) a transmembrane domain; 
 ii) a second modulatory domain; 
 iii) a second member of the dimerization pair; and 
 iv) an intracellular signaling domain; 
   or comprising:   a) a first polypeptide comprising:
 i) a first member of a specific binding pair; 
 ii) a modulatory domain; 
 iii) a first member of a dimerization pair; 
 iv) a transmembrane domain interposed between the first member of a specific binding pair and the modulatory domain; and 
   b) a second polypeptide comprising:
 i) a second member of the dimerization pair; and 
 ii) an intracellular signaling domain. 
   
     
     
         2 . The heterodimeric, conditionally active CAR of  claim 1 , wherein the first polypeptide comprises a hinge region interposed between the first member of the specific binding pair and the transmembrane domain. 
     
     
         3 . The heterodimeric, conditionally active CAR of  claim 1 , wherein the first member of the specific binding pair is an antibody or antibody fragment, a ligand, or a receptor. 
     
     
         4 . The heterodimeric, conditionally active CAR of  claim 2 , wherein the hinge region is an immunoglobulin IgG hinge region or a hinge derived from CD8. 
     
     
         5 . The heterodimeric, conditionally active CAR of  claim 1 , wherein the first and second modulatory domains are selected from 4-1BB (CD137), CD28, ICOS, BTLA, OX-40, CD27, CD30, GITR, HVEM, DAP10, DAP12, and CD28. 
     
     
         6 . The heterodimeric, conditionally active CAR of  claim 1 , wherein the intracellular signaling domain is selected from ZAP70 and CD3-zeta. 
     
     
         7 . The heterodimeric, conditionally active CAR of  claim 1 , wherein the intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM). 
     
     
         8 . The heterodimeric, conditionally active CAR of  claim 1 , wherein the first and second members of the dimerization pair form a homodimer in the presence of a small molecule dimerizer. 
     
     
         9 . The heterodimeric, conditionally active CAR of  claim 1 , wherein the first and second members of the dimerization pair form a heterodimer in the presence of a small molecule dimerizer. 
     
     
         10 . The heterodimeric, conditionally active CAR of  claim 1 , wherein the first and second members of the dimerization pair are selected from:
 a) FK506 binding protein (FKBP) and FKBP;   b) FKBP and calcineurin catalytic subunit A (CnA);   c) FKBP and cyclophilin;   d) FKBP and FKBP-rapamycin associated protein (FRB);   e) gyrase B (GyrB) and GyrB;   f) dihydrofolate reductase (DHFR) and DHFR;   g) DmrB and DmrB;   h) PYL and ABI;   i) Cry2 and CIP;   j) GAI and GID1.   
     
     
         11 . The heterodimeric, conditionally active CAR of  claim 1 , wherein:
 i) the first and second modulatory domains are derived from 4-1BB;   ii) the first and second members of the dimerization pair are FKBP and FRB; and   ii) the signaling domain comprises an ITAM.   
     
     
         12 . The heterodimeric, conditionally active CAR of  claim 1 , wherein the first member of the specific binding pair is a single-chain Fv. 
     
     
         13 . The heterodimeric, conditionally active CAR of  claim 1 , wherein the first member of the specific binding pair binds an epitope present on a cell, on a solid surface, or a lipid bilayer. 
     
     
         14 . The heterodimeric, conditionally active CAR of  claim 13 , wherein the cell is a cancer cell. 
     
     
         15 . A mammalian cell genetically modified to produce the heterodimeric, conditionally active CAR of  claim 1 . 
     
     
         16 . The cell of  claim 15 , wherein the cell is a stem cell, a progenitor cell, or a cell derived from a stem cell or a progenitor cell. 
     
     
         17 . The cell of  claim 15 , wherein the cell is a T lymphocyte or an NK cell. 
     
     
         18 . A nucleic acid comprising nucleotide sequences encoding the heterodimeric, conditionally active CAR of  claim 1 . 
     
     
         19 . The nucleic acid of  claim 18 , wherein the nucleotide sequences are operably linked to a T lymphocyte-specific promoter or an NK cell-specific promoter. 
     
     
         20 . The nucleic acid of  claim 18 , wherein the nucleic acid is in vitro transcribed RNA. 
     
     
         21 . A recombinant expression vector comprising the nucleic acid of  claim 18 . 
     
     
         22 . A method of activating a T lymphocyte, the method comprising contacting the T lymphocyte with a dimerizing agent and a second member of a specific binding pair, wherein the T lymphocyte is genetically modified to produce a heterodimeric, conditionally active CAR of  claim 1 , and wherein, in the presence of the dimerizing agent and the second member of a specific binding pair, the heterodimeric, conditionally active CAR dimerizes and activates the T lymphocyte, thereby producing an activated T lymphocyte. 
     
     
         23 . The method of  claim 22 , wherein the second member of a specific binding pair is an antigen. 
     
     
         24 . The method of  claim 22 , wherein said contacting occurs in vivo. 
     
     
         25 . The method of  claim 22 , wherein the activated T lymphocyte mediates killing of a target cell. 
     
     
         26 . The method of  claim 22 , wherein the activated T lymphocyte produces IL-2 and/or IFN-γ. 
     
     
         27 . The method of  claim 25 , wherein the target cell is a cancer cell. 
     
     
         28 . The method of  claim 22 , wherein the first member of the specific binding pair of the heterodimeric, conditionally active CAR is an antibody specific for an epitope on a cancer cell. 
     
     
         29 . A method of making the cell of  claim 15 , the method comprising genetically modifying a mammalian cell with an expression vector comprising nucleotide sequences encoding the heterodimeric, conditionally active CAR of  claim 1 , or genetically modifying a mammalian cell with an RNA comprising nucleotide sequences encoding the heterodimeric, conditionally active CAR of  claim 1 . 
     
     
         30 . The method of  claim 29 , wherein said genetic modification is carried out ex vivo. 
     
     
         31 . The method of  claim 29 , wherein the cell is a T lymphocyte, a stem cell, an NK cell, a progenitor cell, a cell derived from a stem cell, or a cell derived from a progenitor cell. 
     
     
         32 . A method of treating a cancer in an individual, the method comprising:
 i) genetically modifying T lymphocytes obtained from the individual with an expression vector comprising nucleotide sequences encoding the heterodimeric, conditionally active CAR of  claim 1 , wherein the antigen-binding domain of the heterodimeric, conditionally active CAR is specific for an epitope on a cancer cell in the individual, and wherein said genetic modification is carried out ex vivo;   ii) introducing the genetically modified T lymphocytes into the individual; and   iii) administering to the individual an effective amount of a dimerizing agent, wherein the dimerizing agent induces dimerization of the heterodimeric, conditionally active CAR, wherein said dimerization provides for activation of the genetically modified T lymphocytes and killing of the cancer cell, thereby treating the cancer.   
     
     
         33 . The method of  claim 32 , wherein the dimerizing agent is a rapalog. 
     
     
         34 . A method of modulating the activity of a host cell, the method comprising contacting the host cell with a dimerizing agent and a second member of a specific binding pair, wherein the T lymphocyte is genetically modified to produce a heterodimeric, conditionally active CAR of  claim 1 , and wherein, in the presence of the dimerizing agent and the second member of a specific binding pair, the heterodimeric, conditionally active CAR dimerizes and modulates at least one activity of the host cell. 
     
     
         35 . The method of  claim 34 , wherein the activity is proliferation, cell survival, apoptosis, gene expression, or immune activation. 
     
     
         36 . The method of  claim 34 , wherein the second member of a specific binding pair is an antigen.