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 . 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.