Compositions and methods for TCR reprogramming using fusion proteins
Provided herein are T-cell receptor (TCR) fusion proteins (TFPs), T-cells engineered to express one or more TFPs, and methods of use thereof for the treatment of diseases, including cancer.
1. A T cell from a human subject, wherein the T cell comprises a recombinant nucleic acid molecule encoding a T cell receptor (TCR) fusion protein (TFP) comprising:
(a) a TCR subunit comprising (i) an extracellular domain, (ii) a transmembrane domain, and (iii) a TCR intracellular domain comprising a stimulatory domain from an intracellular signaling domain; and
(b) a murine, human or humanized scFv or single domain antibody comprising an antigen binding domain;
wherein the TCR subunit and the antigen binding domain are operatively linked by a linker sequence;
wherein the extracellular domain, the transmembrane domain and the intracellular signaling domain are derived from a single subunit, wherein the single subunit is CD3 epsilon or wherein the single subunit is CD3 gamma;
wherein the extracellular domain comprises at least a portion of an extracellular domain of the single subunit;
wherein the single subunit comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 56 or SEQ ID NO: 57; and
wherein the TFP functionally interacts with an endogenous TCR subunit when expressed in the T cell.
2. The T cell of claim 1 , wherein the single subunit is CD3 epsilon.
3. The T cell of claim 1 , wherein the single subunit is CD3 gamma.
4. The T cell of claim 1 , wherein the single subunit comprises an amino acid sequence that is at least 89% identical to SEQ ID NO: 57.
5. The T cell of claim 1 , wherein the extracellular domain of CD3 gamma has an amino acid sequence identical to amino acids 23-116 of SEQ ID NO: 57.
6. The T cell of claim 1 , wherein the single subunit comprises a transmembrane domain that has an amino acid sequence identical to amino acids 117-137 of SEQ ID NO: 57.
7. The T cell of claim 1 , wherein the single subunit comprises an intracellular domain that has an amino acid sequence identical to amino acids 138-182 of SEQ ID NO: 57.
8. The T cell of claim 1 , wherein the single subunit comprises an amino acid sequence identical to amino acids 23-182 of SEQ ID NO: 57.
9. The T cell of claim 1 , wherein the single subunit comprises an amino acid sequence that is at least 89% identical to SEQ ID NO: 56.
10. The T cell of claim 1 , wherein the extracellular domain of CD3 epsilon has an amino acid sequence identical to amino acids 23-126 of SEQ ID NO: 56.
11. The T cell of claim 1 , wherein the single subunit comprises a transmembrane domain that has an amino acid sequence identical to amino acids 127-152 of SEQ ID NO: 56.
12. The T cell of claim 1 , wherein the single subunit comprises an intracellular domain that has an amino acid sequence identical to amino acids 153-207 of SEQ ID NO: 56.
13. The T cell of claim 1 , wherein the single subunit comprises an amino acid sequence identical to amino acids 23-207 of SEQ ID NO: 56.
14. The T cell of claim 1 , wherein the single subunit consists of an amino acid sequence identical to amino acids 23-207 of SEQ ID NO: 56.
15. The T cell of claim 1 , wherein the TFP comprises the murine, human or humanized single domain antibody.
16. The T cell of claim 15 , wherein the murine, human or humanized single domain antibody is a VHH domain.
17. The T cell of claim 1 , wherein the TFP comprises the murine, human or humanized scFv.
18. The T cell of claim 1 , wherein the linker sequence connects the antigen binding domain to the extracellular domain.
19. The T cell of claim 18 , wherein the linker sequence comprises (G 4 S) n wherein G is glycine, S is serine, and n is an integer from 1 to 4.
20. The T cell of claim 1 , wherein the TFP lacks a costimulatory domain.
21. The T cell of claim 1 , wherein the TFP lacks a heterologous stimulatory domain.
22. The T cell of claim 1 , wherein the T cell is formulated as a pharmaceutical composition.
23. The T cell of claim 1 , wherein the T cell is a CD8+ human T cell, a CD4+ human T cell, or a combination thereof.
24. The T cell of claim 1 , wherein production of IFNγ by the T cell is increased compared to a T cell not containing the TFP in the presence of a human cell expressing an antigen that specifically interacts with the antigen binding domain.
25. A pharmaceutical composition comprising the T cell of claim 1 and a pharmaceutically acceptable carrier.
26. A population of T cells comprising at least 2×10{circumflex over ( )}5, at least 1×10{circumflex over ( )}6, or at least 5×10{circumflex over ( )}6 T cells according to the T cell of claim 1 .
27. The population of T cells of claim 26 , wherein the population of T cells comprises a population of CD8+ T cells, a population of CD4+ T cells, or a combination thereof.