Compositions and methods for TCR reprogramming using fusion proteins

1. A pharmaceutical composition comprising

(I) 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; and

(II) a pharmaceutically acceptable carrier;

wherein the TCR subunit and the antigen binding domain are operatively linked;

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 a full length extracellular domain of the single subunit;

wherein the TFP functionally interacts with an endogenous TCR when expressed in a T cell; and

wherein the T cell exhibits increased cytotoxicity to a human cell expressing an antigen that specifically interacts with the antigen binding domain compared to a T cell not containing the TFP.

2. The pharmaceutical composition of claim 1 , wherein the antigen binding domain is connected to the TCR extracellular domain by a linker.

3. The pharmaceutical composition of claim 2 , wherein the linker comprises (G 4 S), wherein G is glycine, S is serine, and n is an integer from 1 to 4.

4. The pharmaceutical composition of claim 1 , wherein the antigen binding domain comprises an anti-CD19 binding domain or an anti-BCMA binding domain.

5. The pharmaceutical composition of claim 1 , wherein the antigen binding domain comprises

(i) a light chain (LC) CDR1, LC CDR2 and LC CDR3 sequence of SEQ ID NO: 25, SEQ ID NO: 27 and SEQ ID NO: 29, respectively;

(ii) a heavy chain (HC) CDR1, HC CDR2 and HC CDR3 sequence of SEQ ID NO: 31, SEQ ID NO: 33 and SEQ ID NO: 35, respectively; or

(iii) a combination thereof.

6. The pharmaceutical composition of claim 1 , wherein the pharmaceutical composition is substantially free of serum.

7. The pharmaceutical composition of claim 1 , wherein the TFP comprises the murine, human or humanized scFv.

8. The pharmaceutical composition of claim 1 , wherein the TCR subunit lacks a costimulatory domain.

9. The pharmaceutical composition of claim 1 , wherein the TFP comprises the murine, human or humanized single domain antibody.

10. The pharmaceutical composition of claim 1 , wherein in the presence of a human cell expressing an antigen that specifically interacts with the antigen binding domain the T cell has greater than or more efficient cytotoxic activity than a T cell comprising a nucleic acid encoding a chimeric antigen receptor (CAR) comprising (a) the antigen binding domain operatively linked to (b) a CD28 extracellular domain (c) a CD28 transmembrane domain (d) a CD28 intracellular domain and (e) a CD3 zeta intracellular domain.

11. The pharmaceutical composition of claim 4 , wherein the antigen binding domain comprises the anti-CD19 binding domain and the TFP functionally interacts with an endogenous TCR complex, at least one endogenous TCR polypeptide, or a combination thereof when expressed in the T cell.

12. The pharmaceutical composition of claim 4 , wherein the antigen binding domain comprises the anti-BCMA binding domain and the TFP functionally interacts with an endogenous TCR complex, at least one endogenous TCR polypeptide, or a combination thereof when expressed in the T cell.

13. The pharmaceutical composition of claim 9 , wherein the murine, human or humanized single domain antibody is a V H domain.

14. The pharmaceutical composition of claim 1 , wherein production of a pro-inflammatory cytokine by the T cell is lower compared to production of the pro-inflammatory cytokine by a T cell comprising a nucleic acid encoding a CAR comprising the antigen binding domain operatively linked to a CD28 extracellular domain, a CD28 transmembrane domain, a CD28 intracellular domain, and a CD3 zeta intracellular domain in the presence of a human cell expressing an antigen that specifically interacts with the antigen binding domain.

15. The pharmaceutical composition of claim 1 , wherein the T cell is a CD8+ human T cell, a CD4+ human T cell, or a combination thereof.

16. The pharmaceutical composition 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.

17. The pharmaceutical composition of claim 1 , wherein the single subunit comprises SEQ ID NO: 56.

18. The pharmaceutical composition of claim 1 , wherein in the presence of a human cell expressing an antigen that specifically interacts with the antigen binding domain production of IL-2 by the T cell is increased compared to a T cell not containing the TFP.

19. The pharmaceutical composition of claim 1 , wherein the single subunit comprises SEQ ID NO: 57.

20. The pharmaceutical composition of claim 14 , wherein the pro-inflammatory cytokine is TNFα.

21. The pharmaceutical composition of claim 14 , wherein the pro-inflammatory cytokine is GM-CSF.

22. The pharmaceutical composition of claim 14 , wherein the pro-inflammatory cytokine is IL-2.

23. The pharmaceutical composition of claim 1 , wherein the TCR subunit lacks a heterologous stimulatory domain.

24. The pharmaceutical composition of claim 1 , wherein the single subunit is CD3 epsilon.

25. The pharmaceutical composition of claim 1 , wherein the single subunit is CD3 gamma.

26. The pharmaceutical composition of claim 1 , wherein the intracellular domain comprises a full length intracellular domain of the single subunit.

27. The pharmaceutical composition of claim 1 , wherein the intracellular domain comprises at least one ITAM.