Chimeric antigen receptor and methods of use thereof
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.
1. An isolated nucleic acid comprising a nucleotide sequence encoding a first polypeptide and a second polypeptide of a heterodimeric chimeric antigen receptor (CAR), wherein the first and the second polypeptides are two separate polypeptides, such that when present in a eukaryotic cell membrane, the CAR dimerizes in the presence of a small molecule dimerizer and an antigen to which the first polypeptide of the CAR binds to produce titratable activity, wherein:
a) the first polypeptide comprises, in order from N-terminus to C-terminus:
i) an antigen binding domain comprising an antigen-binding single-chain Fv (scFv);
ii) a transmembrane domain; and
iii) a first member of a dimerization pair; and
b) the second polypeptide comprises, in order from N-terminus to C-terminus:
i) a transmembrane domain;
ii) a second member of the dimerization pair; and
iii) an intracellular signaling domain comprising an immunoreceptor tyrosine-based activation motif (ITAM), wherein the intracellular signaling domain provides signal transduction activity,
wherein the second polypeptide does not comprise an antigen-binding domain,
wherein the first polypeptide, the second polypeptide or both the first and second polypeptides comprise a costimulatory polypeptide interposed between the transmembrane domain and the member of the dimerization pair, and wherein the costimulatory polypeptide is selected from the group consisting of: a 4-1BB polypeptide, a CD28 polypeptide and an OX-40 polypeptide.
2. The nucleic acid of claim 1 , wherein the first polypeptide comprises a hinge region interposed between the scFv and the transmembrane domain.
3. The nucleic acid of claim 2 , wherein the hinge region is an immunoglobulin IgG hinge region or a hinge derived from CD8.
4. The nucleic acid of claim 1 , wherein the intracellular signaling domain comprising the ITAM is selected from the group consisting of CD3-zeta and ZAP70.
5. The nucleic acid of claim 1 , wherein the first and second members of the dimerization pair form a heterodimer in the presence of the small molecule dimerizer.
6. The nucleic acid of claim 1 , wherein the first and second members of the dimerization pair form a homodimer in the presence of the small molecule dimerizer.
7. The nucleic acid of claim 5 , wherein the first and second members of the dimerization pair are selected from the group consisting of:
a) FK506 binding protein (FKBP) and FKBP-rapamycin associated protein (FRB);
b) a Gibberellic Acid Insensitive (GAI) protein and a gibberellin receptor (GID1) protein;
c) FKBP and calcineurin catalytic subunit A (CnA);
d) an abscisic acid receptor (PYL) protein and an abscissic acid insensitive (ABI) protein;
e) a cryptochrome 2 (Cry2) protein and a transcription factor bHLH63 (CIB1) protein; and
f) FKBP and cyclophilin.
8. The nucleic acid of claim 6 , wherein the first and second members of the dimerization pair are selected from the group consisting of:
a) FK506 binding protein (FKBP) and FKBP;
b) gyrase B (GyrB) and GyrB;
c) dihydrofolate reductase (DHFR) and DHFR; and
d) DmrB and DmrB.
9. The nucleic acid of claim 1 , wherein the first member of the dimerization pair is an FK506 binding protein (FKBP), and wherein the second member of the dimerization pair is a FKBP-rapamycin associated protein (FRB).
10. The nucleic acid of claim 1 , wherein the first member of the dimerization pair is GID1, and wherein the second member of the dimerization pair is GAI.
11. The nucleic acid of claim 1 , wherein the scFv binds an epitope present on a cancer cell.
12. The nucleic acid of claim 11 , wherein the cancer cell is a breast cancer cell, a B cell lymphoma, a Hodgkin lymphoma cell, an ovarian cancer cell, a prostate cancer cell, a mesothelioma, a lung cancer cell, a non-Hodgkin B-cell lymphoma cell, an ovarian cancer cell, a prostate cancer cell, a mesothelioma cell, a melanoma cell, a chronic lymphocytic leukemia cell, an acute lymphocytic leukemia cell, a neuroblastoma cell, a glioma, a glioblastoma, a medulloblastoma, or a colorectal cancer cell.
13. The nucleic acid of claim 1 , wherein the nucleotide sequence is operably linked to a T lymphocyte-specific promoter.
14. The nucleic acid of claim 1 , wherein the nucleotide sequence is operably linked to an NK cell-specific promoter.
15. The nucleic acid of claim 1 , wherein the 4-1BB polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:24.
16. The nucleic acid of claim 1 , wherein the OX-40 polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:65.
17. The nucleic acid of claim 1 , wherein the first polypeptide comprises a first costimulatory polypeptide comprising an amino acid sequence and the second polypeptide comprises a second costimulatory polypeptide comprising an amino acid sequence that has at least 95% amino acid identity to the amino acid sequence of the first costimulatory polypeptide in the first polypeptide.
18. The nucleic acid of claim 1 , wherein the intracellular signaling domain comprising the ITAM comprises an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence set forth in one of SEQ ID NOs:98-119.
19. The nucleic acid of claim 9 , wherein the FKBP comprises an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:12, and wherein the FRB comprises an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:14.
20. The nucleic acid of claim 10 , wherein the GID1 polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence set forth in one of SEQ ID NOs:95-97, and wherein the GAI polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:94.
21. The nucleic acid of claim 1 , wherein:
a) the scFv binds an epitope present on a cancer cell;
b) the first member of the dimerization pair is an FK506binding protein (FKBP);
c) the second member of the dimerization pair is a FKBP-rapamycin associated protein (FRB);
d) the first polypeptide comprises a first costimulatory polypeptide comprising a 4-1BB polypeptide;
e) the second polypeptide comprises a second costimulatory polypeptide comprising a 4-1BB polypeptide; and
f) the intracellular signaling domain is a CD3-zeta polypeptide.
22. The nucleic acid of claim 21 , wherein the first polypeptide comprises a hinge region derived from CD8 interposed between the scFv and the transmembrane domain.
23. The nucleic acid of claim 1 , wherein:
a) the scFv binds an epitope present on a cancer cell;
b) the first member of the dimerization pair is an FK506 binding protein (FKBP);
c) the second member of the dimerization pair is a FKBP-rapamycin associated protein (FRB);
d) the first polypeptide comprises a first costimulatory polypeptide comprising an OX-40 polypeptide;
e) the second polypeptide comprises a second costimulatory polypeptide comprising an OX-40 polypeptide; and
f) the intracellular signaling domain is a CD3-zeta polypeptide.
24. The nucleic acid of claim 23 , wherein the first polypeptide comprises a hinge region derived from CD8 interposed between the scFv and the transmembrane domain.
25. A recombinant expression vector comprising the nucleic acid of claim 1 .
26. A T lymphocyte comprising the recombinant expression vector of claim 25 .