Bicistronic chimeric antigen receptors and their uses
View Patent ↗An embodiment of the invention provides bicistronic chimeric antigen receptor (CAR) amino acid constructs. Nucleic acids, recombinant expression vectors, host cells, populations of cells, and pharmaceutical compositions relating to the CAR constructs are disclosed. Methods of detecting the presence of cancer in a mammal and methods of treating or preventing cancer in a mammal are also disclosed. Methods of making the CAR constructs are disclosed.
1. A chimeric antigen receptor (CAR) amino acid construct comprising:
(a) two or more cleavable domains;
(b) a first CAR comprising
a first antigen binding domain,
a first transmembrane domain, and
a first intracellular T cell signaling domain; and
(c) a second CAR comprising
a second antigen binding domain,
a second transmembrane domain, and
a second intracellular T cell signaling domain;
wherein the first and second CARs are linked through the two or more cleavable domains, and
(i) wherein the first antigen binding domain comprises the heavy chain variable region CDR sequences of SEQ ID NOS: 4, 6, and 8and the light chain variable region CDR sequences of SEQ ID NOS: 12, 14 and 16, and
wherein when the first CAR is cleaved from the construct, the first antigen binding domain has antigenic specificity for CD22; or
(ii) wherein the first antigen binding domain comprises the light chain variable region CDR sequences of SEQ ID NOS: 24, 26, and 28, and the heavy chain variable region CDR sequences of SEQ ID NOS: 32, 34 and 36, and
wherein when the first CAR is cleaved from the construct, the first antigen binding domain has antigenic specificity for CD19.
2. The CAR construct according to claim 1 , wherein the first antigen binding domain comprises the heavy chain variable region CDR sequences of SEQ ID NOS: 4, 6, and 8, and the light chain variable region CDR sequences of SEQ ID NOS: 12, 14, and 16.
3. The CAR construct according to claim 1 , wherein the first antigen binding domain comprises the light chain variable region CDR sequences of SEQ ID NOS: 24, 26, and 28, and the heavy chain variable region CDR sequences of SEQ ID NOS: 32, 34, and 36.
4. The CAR construct according to claim 2 , wherein the second antigen binding domain comprises the light chain variable region CDR sequences of SEQ ID NOS: 24, 26, and 28, and the heavy chain variable region CDR sequences of SEQ ID NOS: 32, 34, and 36.
5. The CAR construct according to claim 4 , wherein the first or second transmembrane domain comprises a CD8 transmembrane domain and a CD8 hinge domain, and wherein the CD8 transmembrane domain comprises the amino acid sequence of IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 191 and the CD8 hinge domain comprises the amino acid sequence of TSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 18), SGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 86), or TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD of SEQ ID NO: 18.
6. The CAR construct according to claim 4 , wherein the first or second intracellular T cell signaling domain comprises a 4-1BB intracellular T cell signaling sequence, and wherein the 4-1BB intracellular T cell signaling sequence comprises the amino acid sequence of SEQ ID NO: 20.
7. The CAR construct according to claim 4 , wherein the first or second intracellular T cell signaling domain comprises a CD3 zeta (ζ) intracellular T cell signaling sequence, and wherein the CD3ζ intracellular T cell signaling sequence comprises the amino acid sequence of SEQ ID NO: 21.
8. The CAR construct according to claim 4 , wherein the two or more cleavable domains comprise a 2A cleavable domain and/or a furin cleavable domain.
9. The CAR construct according to claim 4 , wherein the CAR construct comprises exactly two CARs being the first and second CARs.
10. The CAR construct according to claim 1 , wherein the CAR comprises an amino acid sequence having 90% or greater sequence identity with, or comprising, any one of SEQ ID NOS: 49, 50, 51, or 52.
11. The CAR construct according to claim 1 , wherein there are exactly two cleavable domains.
12. A nucleic acid comprising a nucleotide sequence encoding the CAR amino acid construct according to claim 11 .
13. An isolated host cell comprising a recombinant expression vector comprising the nucleic acid according to claim 12 .
14. A pharmaceutical composition comprising a population of cells comprising at least one host cell of claim 13 , and a pharmaceutically acceptable carrier.
15. A method of treating cancer in a mammal, the method comprising administering to the mammal a population of cells comprising at least one host cell of claim 13 in an amount effective to treat cancer in the mammal.
16. The method of claim 15 , wherein the cancer is a hematological malignancy.
17. The CAR construct according to claim 1 , wherein the CAR comprises the amino acid sequence of SEQ ID NO: 49.
18. The CAR construct according to claim 1 , wherein the CAR comprises the amino acid sequence of SEQ ID NO: 50.
19. The CAR construct according to claim 1 , wherein the CAR comprises the amino acid sequence of SEQ ID NO: 51.
20. The CAR construct according to claim 1 , wherein the CAR comprises the amino acid sequence of SEQ ID NO: 52.