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 - 30 . (canceled)
31 . 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 a T cell.
32 . The recombinant nucleic acid molecule of claim 31 , wherein the single subunit is CD3 epsilon.
33 . The recombinant nucleic acid molecule of claim 31 , wherein the single subunit is CD3 gamma.
34 . The recombinant nucleic acid molecule of claim 31 , wherein the single subunit comprises an amino acid sequence that is at least 89% identical to SEQ ID NO: 57.
35 . The recombinant nucleic acid molecule of claim 31 , wherein the extracellular domain of CD3 gamma has an amino acid sequence identical to amino acids 23-116 of SEQ ID NO: 57.
36 . The recombinant nucleic acid molecule of claim 31 , 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.
37 . The recombinant nucleic acid molecule of claim 31 , 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.
38 . The recombinant nucleic acid molecule of claim 31 , wherein the single subunit comprises an amino acid sequence identical to amino acids 23-182 of SEQ ID NO: 57.
39 . The recombinant nucleic acid molecule of claim 31 , wherein the single subunit comprises an amino acid sequence that is at least 89% identical to SEQ ID NO: 56.
40 . The recombinant nucleic acid molecule of claim 31 , wherein the extracellular domain of CD3 epsilon has an amino acid sequence identical to amino acids 23-126 of SEQ ID NO: 56.
41 . The recombinant nucleic acid molecule of claim 31 , 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.
42 . The recombinant nucleic acid molecule of claim 31 , 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.
43 . The recombinant nucleic acid molecule of claim 31 , wherein the single subunit comprises an amino acid sequence identical to amino acids 23-207 of SEQ ID NO: 56.
44 . The recombinant nucleic acid molecule of claim 31 , wherein the single subunit consists of an amino acid sequence identical to amino acids 23-207 of SEQ ID NO: 56.
45 . The recombinant nucleic acid molecule of claim 31 , wherein the TFP comprises the murine, human or humanized single domain antibody.
46 . The recombinant nucleic acid molecule of claim 45 , wherein the murine, human or humanized single domain antibody is a V HH domain.
47 . The recombinant nucleic acid molecule of claim 31 , wherein the TFP comprises the murine, human or humanized scFv.
48 . The recombinant nucleic acid molecule of claim 31 , wherein the linker sequence connects the antigen binding domain to the extracellular domain.
49 . The recombinant nucleic acid molecule of claim 48 , 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.
50 . The recombinant nucleic acid molecule of claim 31 , wherein the TFP lacks a costimulatory domain.
51 . The recombinant nucleic acid molecule of claim 31 , wherein the TFP lacks a heterologous stimulatory domain.
52 . A pharmaceutical composition comprising the recombinant nucleic acid molecule of claim 31 or a cell comprising the recombinant nucleic acid molecule of claim 31 , and a pharmaceutically acceptable carrier.