Heterodimeric inactivatable chimeric antigen receptors
The invention relates to heterodimeric inactivatable chimeric antigen receptors (CARs) and their use for treatment.
1 . A heterodimeric inactivatable chimeric antigen receptor (CAR) comprising:
a) a first polypeptide chain comprising:
i) an extracellular region;
ii) a first transmembrane (TM) region;
iii) a first co-stimulatory endodomain (ED); and
iv) a first member of a dimerization pair; and
b) a second polypeptide chain comprising:
i) optionally, an extracellular region which does not comprise a target-binding region;
ii) a second TM region;
iii) optionally, a second co-stimulatory ED;
iv) a second member of the dimerization pair; and
v) an intracellular signaling ED,
wherein the first and second member of the dimerization pair form a heterodimer, wherein the heterodimer formed by the first and second member of the dimerization pair is disrupted in the presence of an inhibitory molecule resulting in inhibition of CAR-mediated signaling, and wherein (1) the first member of the dimerization pair comprises a sequence selected from SEQ ID NO: 5, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 24 comprising residues 35-50 of SEQ ID NO: 22 in place of residues 51-91 of SEQ ID NO: 24, and SEQ ID NO: 30, and the second member of the dimerization pair comprises a sequence IAXXLXXIGXXF (SEQ ID NO: 179), wherein X is any natural amino acid and L is optionally substituted with A, or (2) the first member of the dimerization pair comprises a sequence IAXXLXXIGXXF (SEQ ID NO: 179), wherein X is any natural amino acid and L is optionally substituted with A, and the second member of the dimerization pair comprises a sequence selected from SEQ ID NO: 5, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 24 comprising residues 35-50 of SEQ ID NO: 22 in place of residues 51-91 of SEQ ID NO: 24, and SEQ ID NO: 30, and
the inhibitory molecule is venetoclax, A-1331852, or A-1155463.
2 . The CAR of claim 1 , wherein the second polypeptide chain comprises an extracellular region which does not comprise a target-binding region.
3 . The CAR of claim 1 , wherein the first polypeptide chain does not comprise an intracellular signaling ED.
4 . The CAR of claim 1 , comprising:
a) a first polypeptide chain consisting essentially of in the direction from the N terminus to the C terminus:
i) an extracellular target-binding region;
ii) a first linker region;
iii) a first transmembrane (TM) region;
iv) a first co-stimulatory endodomain (ED); and
v) a first member of a dimerization pair; and
b) a second polypeptide chain consisting essentially of in the direction from the N terminus to the C terminus:
i) an extracellular region which does not comprise a target-binding region;
ii) a second linker region;
iii) a second TM region;
iv) a second co-stimulatory ED;
v) a second member of the dimerization pair; and
vi) an intracellular signaling ED,
wherein the first polypeptide chain does not comprise an intracellular signaling ED.
5 . The CAR of claim 1 , wherein the extracellular region of the first polypeptide chain comprises an antigen-binding polypeptide, a receptor, or a natural ligand for a target cell antigen or receptor.
6 . The CAR of claim 5 , wherein the antigen recognized by the antigen-binding polypeptide is CD19, prostate-specific membrane antigen (PSMA), or mesothelin.
7 . The CAR of claim 5 , wherein the natural ligand for a target cell antigen or receptor is an NKG2D ectodomain.
8 . The CAR of claim 1 , wherein the extracellular region of the first polypeptide chain comprises a T-cell receptor (TCR)-based recognition domain.
9 . The CAR of claim 1 , wherein
a) the first and/or second transmembrane (TM) region is derived from CD8, CD8α, CD4, CD3-zeta, CD3-epsilon, CD28, CD45, CD4, CD5, CD7, CD9, CD16, CD22, CD33, CD37, CD40, CD64, CD80, CD86, CD134 (OX-40), CD137, CD154, DAP10, or DAP12;
b) the first and/or second co-stimulatory ED is derived from 4-1BB (CD137), CD28, ICOS, CD134 (OX-40), BTLA, CD27, CD30, GITR, CD226, or HVEM;
c) the intracellular signaling ED of the second polypeptide chain is derived from DAP10, DAP12, Fc epsilon receptor I gamma chain (FCER1G), FcR beta CD3-delta, CD3-epsilon, CD3-gamma, CD3-zeta, CD226, CD66d, CD79A, or CD79B; and/or
d) the first and/or second polypeptide chain further comprises one or more polypeptide sequences, wherein said one or more polypeptide sequences are selected from one or more additional co-stimulatory EDs, signal sequences, separation sequences, epitope tags, and polypeptides that produce a detectable signal.
10 . The CAR of claim 1 , wherein
i) the first member of the dimerization pair comprises the sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 19;
ii) the second member of the dimerization pair comprises the sequence of SEQ ID NO: 5, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 30;
iii) the extracellular region of the first polypeptide chain comprises the sequence of SEQ ID NO: 6, or SEQ ID NO: 49;
iv) the intracellular signaling ED of the second polypeptide chain comprises the sequence of SEQ ID NO: 7;
v) the first and/or second linker region comprises the sequence of SEQ ID NO: 9;
vi) the first and/or second TM region comprises the sequence of SEQ ID NO: 10; and/or
vii) the first and/or second co-stimulatory ED comprises the sequence or SEQ ID NO: 11.
11 . The CAR of claim 1 , wherein:
a) the first polypeptide chain comprises the sequence of SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, or SEQ ID NO: 112; and/or
b) the second polypeptide chain comprises the sequence of SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, or SEQ ID NO: 117.
12 . The CAR of claim 1 comprising:
a) the first polypeptide chain comprising the sequence of SEQ ID NO: 109 and the second polypeptide chain comprising the sequence of SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, or SEQ ID NO: 117;
b) the first polypeptide chain comprising the sequence of SEQ ID NO: 110 and the second polypeptide chain comprising the sequence of SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, or SEQ ID NO: 117;
c) the first polypeptide chain comprising the sequence of SEQ ID NO: 111 and the second polypeptide chain comprising the sequence of SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, or SEQ ID NO: 117; or
d) the first polypeptide chain comprising the sequence of SEQ ID NO: 112 and the second polypeptide chain comprising the sequence of SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, or SEQ ID NO: 117.
13 . A nucleic acid molecule comprising a nucleotide sequence encoding the first polypeptide chain of the heterodimeric inactivatable CAR of claim 1 , the second polypeptide chain of the heterodimeric inactivatable CAR of claim 1 , or the first and second polypeptide chains of the heterodimeric inactivatable CAR of claim 1 .
14 . A recombinant vector comprising the nucleic acid molecule of claim 13 .
15 . An isolated, genetically modified host cell comprising the heterodimeric inactivatable CAR of claim 1 .
16 . The host cell of claim 15 , which is selected from a cytotoxic cell, a T cell, a stem cell, a progenitor cell, a cell derived from a stem cell, and a cell derived from a progenitor cell.
17 . A pharmaceutical composition comprising the host cell of claim 15 and a pharmaceutically acceptable carrier and/or excipient.
18 . A method for producing a host cell comprising the heterodimeric inactivatable CAR of claim 1 , said method comprising genetically modifying said cell with (i) a nucleic acid molecule comprising a nucleotide sequence encoding the first polypeptide chain of the heterodimeric inactivatable CAR of claim 1 and a nucleic acid molecule comprising a nucleotide sequence encoding the second polypeptide chain of the heterodimeric inactivatable CAR of claim 1 or (ii) a nucleic acid molecule comprising nucleotide sequence(s) encoding the first and second polypeptide chains of heterodimeric inactivatable CAR of claim 1 .
19 . A method for stimulating elimination of a prostate-specific membrane antigen (PSMA)-expressing cell or CD19-expressing cell, said method comprising contacting said cell with a T cells or natural killer (NK) cells comprising the heterodimeric inactivatable CAR of claim 1 , wherein the extracellular region of the first polypeptide chain of said CAR binds to PSMA or CD19.
20 . A method for treating a cancer in a subject having the cancer, said method comprising administering to the subject a therapeutically effective amount of T cells or natural killer (NK) cells comprising the heterodimeric inactivatable CAR of claim 1 , wherein the cancer is prostate cancer, leukemia or lymphoma.
21 . The method of claim 20 , said method further comprising inhibiting the activity of the CAR by administering to the subject an effective amount of an inhibitory molecule, wherein the inhibitory molecule is venetoclax, A-1331852, or A-1155463.
22 . A method for inhibiting the activity of the heterodimeric inactivatable CAR in the host cell of claim 15 , comprising contacting the host cell with an inhibitory molecule, wherein the inhibitory molecule is venetoclax, A-1331852, or A-1155463.
23 . The CAR of claim 1 , wherein the extracellular region of the second polypeptide chain comprises a DAP10 ectodomain.
24 . The CAR of claim 1 , wherein the second member of the dimerization pair in (1) or the first member of the dimerization pair in (2) comprises Apolipoprotein E (ApoE) which comprises the sequence IAXXLXXIGXXF (SEQ ID NO: 179), wherein X is any natural amino acid and L is optionally substituted with A.
25 . The CAR of claim 1 , wherein the second member of the dimerization pair in (1) or the first member of the dimerization pair in (2) comprises the amino acid sequence SEQ ID NO: 1, 2, 3, 4, or 19.