COMBINATION THERAPIES INCLUDING T CELL-ANTIGEN COUPLERS AND CHECKPOINT INHIBITORS FOR TREATING CANCER

CROSS-REFERENCE

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63/364,032, filed May 2, 2022, which is incorporated by reference in its entirety herein.

SUMMARY

[0002] Disclosed herein, in certain embodiments, are methods of treating a cancer expressing a target antigen in a subject in need thereof, the method comprising administering to the subject: (a) an engineered T cell comprising an expression vector encoding a T cell-antigen coupler (TAC) protein: (i) an antigen-binding domain that binds the target antigen; (ii) a ligand that binds a protein associated with a T cell receptor (TCR) complex on the engineered T cell; and (iii) a transmembrane domain and a cytosolic domain of a TCR co-receptor, wherein the antigen-binding domain, the ligand that binds a protein associated with TCR complex, and the transmembrane domain and cytosolic domain of a TCR co-receptor are fused directly to each other or joined by at least one linker; and (b) an immune checkpoint inhibitor. In some embodiments, the TAC protein is administered prior to administration of the immune checkpoint inhibitor. In some embodiments, the TAC protein is administered following administration of the immune checkpoint inhibitor. In some embodiments, the TAC protein and the immune checkpoint inhibitor are administered concurrently. In some embodiments, the target antigen is human epidermal grow th factor receptor 2 (HER2), B cell maturation antigen (BCMA), cluster of differentiation 19 (CD19), Claudin 18.2 (CLDN18.2), guanylate cyclase 2C (GUCY2C), or glypican 3 (GPC3). In some embodiments, the target antigen is HER2. In some embodiments, the antigen-binding domain is a designed ankyrin repeat protein (DARPin). In some embodiments, the antigen-binding domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 49. In some embodiments, the target antigen is BCMA. In some embodiments, the antigen-binding domain is a single-cham variable fragment (scFv). In some embodiments, the antigen-binding domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 50. In some embodiments, the target antigen is CD19. In some embodiments, the antigen-binding domain is an scFv. In some embodiments, the antigenbinding domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 51. In some embodiments, the target antigen is Claudin 18.2 (CLDN18.2). In some embodiments, the antigen-binding domain is a nanobody. In some embodiments, the antigenbinding domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 52 or 53. In some embodiments, the target antigen is GUCY2C. In some embodiments, the antigen-binding domain is an scFv. In some embodiments, the antigen-bmdmg domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 54- 117. In some embodiments, the antigen-binding domain is a nanobody. In some embodiments, the antigen-binding domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 118-136. In some embodiments, the target antigen is GPC3. In some embodiments, the antigen-binding domain is an scFv. In some embodiments, the antigenbinding domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 137. In some embodiments, the ligand that binds a protein associated with a TCR complex is an scFv derived from an antigen-binding domain selected from UCHT1, huUCHTl, OKT3, F6A, and L2K. In some embodiments, the ligand that binds a protein associated with a TCR complex is derived from a UCHT1 antigen-binding domain. In some embodiments, the ligand that binds a protein associated with a TCR complex comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 2. In some embodiments, the ligand that binds a protein associated with a TCR complex is derived from a UCHT1 antigen-binding domain and comprises a threonine (T) residue at amino acid position 177. In some embodiments, the ligand that binds a protein associated with a TCR complex comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 4. In some embodiments, the ligand that binds a protein associated with a TCR complex is derived from an huUCHTl antigen-binding domain. In some embodiments, the ligand that binds a protein associated with a TCR complex comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 6. In some embodiments, the ligand that binds a protein associated with a TCR complex is derived from an huUCHTl antigen-binding domain and comprises a threonine (T) residue at amino acid position 182. In some embodiments, the ligand that binds a protein associated with a TCR complex comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 8. In some embodiments, the ligand that binds a protein associated with a TCR complex is derived from an OKT3 antigenbinding domain. In some embodiments, the ligand that binds a protein associated with a TCR complex compnses an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 10. In some embodiments, the ligand that binds a protein associated with a TCR complex is derived from an F6A antigen-binding domain. In some embodiments, the ligand that binds a protein associated with a TCR complex comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 12. In some embodiments, the ligand that binds a protein associated with a TCR complex is derived from an L2K antigen-binding domain. In some embodiments, the ligand that binds a protein associated with a TCR complex comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 14. In some embodiments, the TCR co-receptor is CD4. In some embodiments, the TCR co-receptor is CD8, preferably CD8a. In some embodiments, the antigen-binding domain, the ligand that binds a protein associated with a TCR complex, and the transmembrane domain and cytosolic domain of a TCR co-receptor are directly fused. In some embodiments, the antigen-binding domain and the ligand that binds a protein associated with a TCR complex are directly fused and joined to the transmembrane domain and cytosolic domain of a TCR co-receptor by a linker. In some embodiments, the ligand that binds a protein associated with a TCR complex and the transmembrane domain and cytosolic domain of a TCR co-receptor are directly fused and joined to the antigen-binding domain by a linker. In some embodiments, the linker is a peptide linker, preferably a peptide linker comprising 5 to 30 amino acids, more preferably 5 amino acids, 10 amino acids, or 15 amino acids. In some embodiments, the peptide linker comprises a GiSi linker. In some embodiments, the TAC does not comprise a co-stimulatory domain. In some embodiments, the TAC does not comprise an activation domain. In some embodiments, the TAC comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to an amino acid sequence selected from any one of SEQ ID NOs: 138-207. In some embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor, a PD-L1 inhibitor, a PD-L2 inhibitor, a CTLA-4 inhibitor, a LAG3 inhibitor, a TIM-3 inhibitor, a BTLA inhibitor, a B7-H3 inhibitor, a B7-H4 inhibitor, or a KIR inhibitor. In some embodiments, the immune checkpoint inhibitor is a PD-1 or PD-L1 inhibitor. In some embodiments, the immune checkpoint inhibitor is nivolumab, pembrolizumab, cemiplimab, atezolizumab, avelumab, durvalumab. In some embodiments, the immune checkpoint inhibitor is a CTLA-4 inhibitor. In some embodiments, the immune checkpoint inhibitor is ipilimumab or tremelimumab. In some embodiments, the cancer is a solid or liquid cancer. In some embodiments, the cancer is a primary or metastatic cancer. In some embodiments, the cancer is unresectable. In some embodiments, the cancer is a salivary gland cancer, a lung cancer, a gastric cancer, a breast cancer, an ovarian cancer, a uterine cancer, a cervical cancer, a biliary tract cancer, a pancreatic cancer, a colorectal cancer, a bladder cancer, a prostate cancer, multiple myeloma, a glioblastoma, a gastroesophageal junction cancer, an esophageal cancer, a liver cancer, a thyroid cancer, a kidney cancer, a yolk sac tumor, a skin cancer, an endometrial cancer. In some embodiments, the cancer is an acute lymphoblastic leukemia, a chronic lymphocytic leukemia, a large B-cell lymphoma, or a diffuse large B-cell lymphoma. In some embodiments, the subject has received two or more prior lines of therapy. In some embodiments, the engineered T cells are autologous to the subject. In some embodiments, the engineered T cells are heterologous to the subject. In some embodiments, the engineered T cells are administered in a single dose. In some embodiments, the engineered T cells are administered at a dose of about 6* 10 ⁴ to about 6x 10 ^s cells/kg body weight. In some embodiments, the engineered T cells are administered at a dose of about IxlO ⁵ to about 8x l0 ⁶ cells/kg body weight. In some embodiments, the immune checkpoint inhibitor is administered at a dose of 200 mg. In some embodiments, the immune checkpoint inhibitor is administered 7 days after the engineered T cells are administered. In some embodiments, the immune checkpoint inhibitor is administered once every three weeks. [0003] Disclosed herein, in certain embodiments, are methods of treating systemic lupus erythematosus expressing a target antigen in a subject in need thereof, the method comprising administering to the subject: (a) an engineered T cell comprising an expression vector encoding a T cell-antigen coupler (TAC) protein: (i) an antigen-binding domain that binds the target antigen; (h) a ligand that binds a protein associated with a T cell receptor (TCR) complex on the engineered T cell; and (iii) a transmembrane domain and a cytosolic domain of a TCR coreceptor, wherein the antigen-binding domain, the ligand that binds a protein associated with TCR complex, and the transmembrane domain and cytosolic domain of a TCR co-receptor are fused directly to each other or joined by at least one linker; and (b) an immune checkpoint inhibitor. In some embodiments, the TAC protein is administered prior to administration of the immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is administered 7 days after the engineered T cells are administered. In some embodiments, the TAC protein is administered following administration of the immune checkpoint inhibitor. In some embodiments, the TAC protein and the immune checkpoint inhibitor are administered concurrently.

[0004] Disclosed herein, in certain embodiments, are uses of an engineered T cell comprising an expression vector encoding a T cell-antigen coupler (TAC) protein and an immune checkpoint inhibitor in combination for the manufacture of a medicament for treating a cancer expressing a target antigen in a subject in need thereof, comprising administering to the subject: (a) the engineered T cell comprising the expression vector encoding the T cell-antigen coupler (TAC) protein, the TAC protein comprising: (i) an antigen-binding domain that binds the target antigen; (ii) a ligand that binds a protein associated with a T cell receptor (TCR) complex on the engineered T cell; and (iii) a transmembrane domain and a cytosolic domain of a TCR coreceptor, wherein the antigen-binding domain, the ligand that binds a protein associated with a TCR complex, and the transmembrane domain and cytosolic domain of a TCR co-receptor are fused directly to each other or joined by at least one linker; and (b) an immune checkpoint inhibitor. In some embodiments, the TAC protein is administered prior to administration of the immune checkpoint inhibitor. In some embodiments, the TAC protein is administered following administration of the immune checkpoint inhibitor. In some embodiments, the TAC protein and the immune checkpoint inhibitor are administered concurrently In some embodiments, the target antigen is human epidermal growth factor receptor 2 (HER2), B cell maturation antigen (BCMA), cluster of differentiation 19 (CD19), Claudin 18.2 (CLDN18.2), guanylate cyclase 2C (GUCY2C), or glypican 3 (GPC3). In some embodiments, the target antigen is HER2. In some embodiments, the antigen-binding domain is a designed ankyrin repeat protein (DARPin). In some embodiments, the antigen-binding domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 49. In some embodiments, the target antigen is BCMA. In some embodiments, the antigen-binding domain is a single-chain variable fragment (scFv). In some embodiments, the antigen-binding domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 50. In some embodiments, the target antigen is CD19. In some embodiments, the antigen-binding domain is an scFv. In some embodiments, the antigenbinding domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 51. In some embodiments, the target antigen is Claudin 18.2 (CLDN18.2). In some embodiments, the antigen-binding domain is a nanobody. In some embodiments, the antigenbinding domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 52 or 53. In some embodiments, the target antigen is GUCY2C. In some embodiments, the antigen-binding domain is an scFv. In some embodiments, the antigen-binding domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 54- 117. In some embodiments, the antigen-binding domain is a nanobody. In some embodiments, the antigen-binding domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 118-136. In some embodiments, the target antigen is GPC3. In some embodiments, the antigen-binding domain is an scFv. In some embodiments, the antigenbinding domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 137. In some embodiments, the ligand that binds a protein associated with a TOR complex is an scFv derived from an antigen-binding domain selected from UCHT1, huUCHTl, OKT3, F6A, and L2K. In some embodiments, the ligand that binds a protein associated with a TCR complex is derived from a UCHT1 antigen-binding domain. In some embodiments, the ligand that binds a protein associated with a TCR complex comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 2. In some embodiments, the ligand that binds a protein associated with a TCR complex is derived from a UCHT1 antigen-binding domain and comprises a threonine (T) residue at amino acid position 177. In some embodiments, the ligand that binds a protein associated with a TCR complex comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 4. In some embodiments, the ligand that binds a protein associated with a TCR complex is derived from an huUCHTl antigen-binding domain. In some embodiments, the ligand that binds a protein associated with a TCR complex comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 6. In some embodiments, the ligand that binds a protein associated with a TCR complex is derived from an huUCHTl antigen-binding domain and comprises a threonine (T) residue at amino acid position 182. In some embodiments, the ligand that binds a protein associated with a TCR complex comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 8. In some embodiments, the ligand that binds a protein associated with a TCR complex is derived from an OKT3 antigen- binding domain. In some embodiments, the ligand that binds a protein associated with a TCR complex comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 10. In some embodiments, the ligand that binds a protein associated with a TCR complex is derived from an F6A antigen-binding domain. In some embodiments, the ligand that binds a protein associated with a TCR complex comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 12. In some embodiments, the ligand that binds a protein associated with a TCR complex is derived from an L2K antigen-binding domain. In some embodiments, the ligand that binds a protein associated with a TCR complex comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 14. In some embodiments, the TCR co-receptor is CD4. In some embodiments, the TCR co-receptor is CD8, preferably CD8a. In some embodiments, the antigen-binding domain, the ligand that binds a protein associated with a TCR complex, and the transmembrane domain and cytosolic domain of a TCR co-receptor are directly fused. In some embodiments, the antigen-binding domain and the ligand that binds a protein associated with a TCR complex are directly fused and joined to the transmembrane domain and cytosolic domain of a TCR co-receptor by a linker. In some embodiments, the ligand that binds a protein associated with a TCR complex and the transmembrane domain and cytosolic domain of a TCR co-receptor are directly fused and joined to the antigen-binding domain by a linker. In some embodiments, the linker is a peptide linker, preferably a peptide linker comprising 5 to 30 amino acids, more preferably 5 amino acids, 10 amino acids, or 15 ammo acids. In some embodiments, the peptide linker comprises a G4S3 linker. In some embodiments, the TAC does not comprise a co-stimulatory domain. In some embodiments, the TAC does not comprise an activation domain. In some embodiments, the TAC comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to an amino acid sequence selected from any one of SEQ ID NOs: 138-207. In some embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor, a PD-L1 inhibitor, a PD-L2 inhibitor, a CTLA-4 inhibitor, a LAG3 inhibitor, a TIM-3 inhibitor, a BTLA inhibitor, a B7-H3 inhibitor, a B7-H4 inhibitor, or a KIR inhibitor. In some embodiments, the immune checkpoint inhibitor is a PD-1 or PD-L1 inhibitor. In some embodiments, the immune checkpoint inhibitor is nivolumab, pembrolizumab, cemiplimab, atezolizumab, avelumab, durvalumab. In some embodiments, the immune checkpoint inhibitor is a CTLA-4 inhibitor. In some embodiments, the immune checkpoint inhibitor is ipilimumab or tremelimumab. In some embodiments, the cancer is a solid or liquid cancer. In some embodiments, the cancer is a primary or metastatic cancer. In some embodiments, the cancer is unresectable In some embodiments, the cancer is a salivary gland cancer, a lung cancer, a gastric cancer, a breast cancer, an ovarian cancer, a uterine cancer, a cervical cancer, a biliary tract cancer, a pancreatic cancer, a colorectal cancer, a bladder cancer, a prostate cancer, multiple myeloma, a glioblastoma, a gastroesophageal junction cancer, an esophageal cancer, a liver cancer, a thyroid cancer, a kidney cancer, a yolk sac tumor, a skin cancer, an endometrial cancer. In some embodiments, the cancer is an acute lymphoblastic leukemia, a chronic lymphocytic leukemia, a large B-cell lymphoma, or a diffuse large B-cell lymphoma. In some embodiments, the subject has received two or more prior lines of therapy. In some embodiments, the engineered T cells are autologous to the subject. In some embodiments, the engineered T cells are heterologous to the subject. In some embodiments, the engineered T cells are administered in a single dose. In some embodiments, the engineered T cells are administered at a dose of about 6x IO ⁴ to about 6x l0 ⁸ cells/kg body weight. In some embodiments, the engineered T cells are administered at a dose of about IxlO ⁵ to about 8x l0 ⁶ cells/kg body weight. In some embodiments, the immune checkpoint inhibitor is administered at a dose of 200 mg. In some embodiments, the immune checkpoint inhibitor is administered 7 days after the engineered T cells are administered. In some embodiments, the immune checkpoint inhibitor is administered once every three weeks. [0005] Disclosed herein, in certain embodiments, are engineered T cells comprising an expression vector encoding a T cell-antigen coupler (TAC) protein, the TAC protein comprising: (i) an antigen-binding domain that binds the target antigen; (ii) a ligand that binds a protein associated with a T cell receptor (TCR) complex on the engineered T cell; and (in) a transmembrane domain and a cytosolic domain of a TCR co-receptor; wherein the antigenbinding domain, the ligand that binds a protein associated with a TCR complex, and the transmembrane domain and cytosolic domain of a TCR co-receptor are fused directly to each other, or joined by at least one linker; for use in the treatment of cancer expressing a target antigen in a subject in need thereof, wherein the T cell-antigen coupler (TAC) protein is administered to the individual in combination with an immune checkpoint inhibitor. Disclosed herein, in certain embodiments, are engineered T cells comprising an expression vector encoding a T cell-antigen coupler (TAC) protein, the TAC protein comprising: (i) an antigenbinding domain that binds the target antigen; (ii) a ligand that binds a protein associated with a T cell receptor (TCR) complex on the engineered T cell; and (iii) a transmembrane domain and a cytosolic domain of a TCR co-receptor; wherein the antigen-binding domain, the ligand that binds a protein associated with a TCR complex, and the transmembrane domain and cytosolic domain of a TCR co-receptor are fused directly to each other, or joined by at least one linker, for use in the treatment of cancer expressing a tumor antigen in an individual in need thereof, wherein the engineered T cell comprising an expression vector encoding a TAC protein is administered simultaneously, separately or sequentially with an immune checkpoint inhibitor. In some embodiments, the target antigen is human epidermal growth factor receptor 2 (HER2), B cell maturation antigen (BCMA), cluster of differentiation 19 (CD19), Claudin 18.2 (CLDN18.2), guanylate cyclase 2C (GUCY2C), or glypican 3 (GPC3). In some embodiments, the target antigen is HER2. In some embodiments, the antigen-binding domain is a designed ankyrin repeat protein (DARPin). In some embodiments, the antigen-binding domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 49. In some embodiments, the target antigen is BCMA. In some embodiments, the antigen-binding domain is a single-chain variable fragment (scFv). In some embodiments, the antigen-binding domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 50. In some embodiments, the target antigen is CD 19. In some embodiments, the antigen-binding domain is an scFv. In some embodiments, the antigen-binding domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 51. In some embodiments, the target antigen is Claudin 18.2 (CLDN18.2). In some embodiments, the antigen-binding domain is a nanobody. In some embodiments, the antigen-binding domain comprises an ammo acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 52 or 53. In some embodiments, the target antigen is GUCY2C. In some embodiments, the antigen-binding domain is an scFv. In some embodiments, the antigen-binding domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 54-117. In some embodiments, the antigen-binding domain is a nanobody. In some embodiments, the antigen-binding domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 118-136. In some embodiments, the target antigen is GPC3. In some embodiments, the antigen-binding domain is an scFv. In some embodiments, the antigen-binding domain comprises an ammo acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 137. In some embodiments, the ligand that binds a protein associated with a TCR complex is an scFv derived from an antigen-binding domain selected from UCHT1, huUCHTl, OKT3, F6A, and L2K. In some embodiments, the ligand that binds a protein associated with a TCR complex is derived from a UCHT1 antigen-binding domain. In some embodiments, the ligand that binds a protein associated with a TCR complex comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 2. In some embodiments, the ligand that binds a protein associated with a TCR complex is derived from a UCHT1 antigenbinding domain and comprises a threonine (T) residue at amino acid position 177. In some embodiments, the ligand that binds a protein associated with a TCR complex comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 4. In some embodiments, the ligand that binds a protein associated with a TCR complex is derived from an huUCHTl antigen-binding domain. In some embodiments, the ligand that binds a protein associated with a TCR complex comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 6. In some embodiments, the ligand that binds a protein associated with a TCR complex is derived from an huUCHTl antigen-binding domain and comprises a threonine (T) residue at amino acid position 182. In some embodiments, the ligand that binds a protein associated with a TCR complex comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 8. In some embodiments, the ligand that binds a protein associated with a TCR complex is derived from an OKT3 antigen-binding domain. In some embodiments, the ligand that binds a protein associated with a TCR complex comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 10. In some embodiments, the ligand that binds a protein associated with a TCR complex is derived from an F6A antigen-binding domain. In some embodiments, the ligand that binds a protein associated with a TCR complex comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 12. In some embodiments, the ligand that binds a protein associated with a TCR complex is derived from an L2K antigen-binding domain. In some embodiments, the ligand that binds a protein associated with a TCR complex comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 14. In some embodiments, the TCR co-receptor is CD4. In some embodiments, the TCR co-receptor is CD8, preferably CD8a. In some embodiments, the antigen-binding domain, the ligand that binds a protein associated with a TCR complex, and the transmembrane domain and cytosolic domain of a TCR co-receptor are directly fused. In some embodiments, the antigen-binding domain and the ligand that binds a protein associated with a TCR complex are directly fused and joined to the transmembrane domain and cytosolic domain of a TCR co-receptor by a linker. In some embodiments, the ligand that binds a protein associated with a TCR complex and the transmembrane domain and cytosolic domain of a TCR co-receptor are directly fused and joined to the antigen-binding domain by a linker. In some embodiments, the linker is a peptide linker, preferably a peptide linker comprising 5 to 30 amino acids, more preferably 5 amino acids, 10 ammo acids, or 15 amino acids. In some embodiments, the peptide linker comprises a G4S3 linker. In some embodiments, the TAC does not comprise a co-stimulatory domain. In some embodiments, the TAC does not comprise an activation domain. In some embodiments, the TAC comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to an amino acid sequence selected from any one of SEQ ID NOs: 138-207. In some embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor, a PD-L1 inhibitor, a PD-L2 inhibitor, a CTLA-4 inhibitor, a LAG3 inhibitor, a TIM-3 inhibitor, a BTLA inhibitor, a B7-H3 inhibitor, a B7-H4 inhibitor, or a KIR inhibitor. In some embodiments, the immune checkpoint inhibitor is a PD-1 or PD-L1 inhibitor. In some embodiments, the immune checkpoint inhibitor is nivolumab, pembrolizumab, cemiplimab, atezohzumab, avelumab, durvalumab. In some embodiments, the immune checkpoint inhibitor is a CTLA-4 inhibitor. In some embodiments, the immune checkpoint inhibitor is ipilimumab or tremelimumab. In some embodiments, the cancer is a solid or liquid cancer. In some embodiments, the cancer is a primary or metastatic cancer. In some embodiments, the cancer is unresectable. In some embodiments, the cancer is a salivary gland cancer, a lung cancer, a gastric cancer, a breast cancer, an ovarian cancer, a uterine cancer, a cervical cancer, a biliary tract cancer, a pancreatic cancer, a colorectal cancer, a bladder cancer, a prostate cancer, multiple myeloma, a glioblastoma, a gastroesophageal junction cancer, an esophageal cancer, a liver cancer, a thyroid cancer, a kidney cancer, a yolk sac tumor, a skin cancer, an endometrial cancer. In some embodiments, the cancer is an acute lymphoblastic leukemia, a chronic lymphocytic leukemia, a large B-cell lymphoma, or a diffuse large B-cell lymphoma. In some embodiments, the subject has received two or more prior lines of therapy. In some embodiments, the engineered T cells are autologous to the subject. In some embodiments, the engineered T cells are heterologous to the subject. In some embodiments, the engineered T cells are administered in a single dose. In some embodiments, the engineered T cells are administered at a dose of about 6x 10 ⁴ to about 6x 10 ^s cells/kg body weight. In some embodiments, the engineered T cells are administered at a dose of about lx JO ⁵ to about 8x l0 ⁶ cells/kg body weight. In some embodiments, the immune checkpoint inhibitor is administered at a dose of 200 mg. In some embodiments, the immune checkpoint inhibitor is administered 7 days after the engineered T cells are administered. In some embodiments, the immune checkpoint inhibitor is administered once every three weeks.

BRIEF DESCRIPTION OF THE DRAWINGS

[0006] FIGs. 1A-1B depict an in vivo experiment assessing the effects of HER2-TAC T cells on HER ⁺ xenograft tumors in mice. FIG. 1A diagrams the design of the experiment. FIG. IB is a graph depicting tumor volumes measured over the course of the experiment. FIG. 1C depicts measurement of CD69 (vertical axis) and PD-1 (horizonal axis) in cells collected from xenografted mice sacrificed 30 minutes after engraftment of HER2-TAC T cells. FIG. ID depicts measurement of CD69 (vertical axis) and PD-1 (horizonal axis) in cells collected from xenografted mice sacrificed 7 days after engraftment of HER2-TAC T cells.

[0007] FIGs. 2A-2C depict an in vitro experiment measuring the effects of HER2-TAC T cells on HER2 ⁺ tumor spheroids engineered to express green fluorescent protein (GFP). FIG. 2A depicts fluorescence images of spheroids treated with HER2-TAC T cells or controls at indicated effector target (E:T) ratios. FIG. 2B depicts percent viability of tumor cells in indicated treatment groups after 4 days of treatment, as measured by normalized GFP fluorescence. FIG. 2C depicts images of tumor spheroids treated with HER2-TAC T cells (top panels) or control (bottom panels) that were fixed and stained for (from left-to-right) hematoxylin and eosin (HE), CD3, granzyme B, PD-L1, HER2, and cleaved caspase 3.

[0008] FIGs. 3A-3C depict an experiment assessing the effects of anti-PD-1 treatment on TAC T cells activated against PD-L1 overexpressing tumor cells. FIG. 3A depicts flow cytometry measurement of surface expression of PD-L1 in BT-474 cells engineered to overexpress PD-L1. FIG. 3B depicts flow cytometry measurement of surface expression of PD-L1 in N87 cells engineered to overexpress PD-L1. FIG. 3C is a graph showing proliferation of HER2-TAC T cells as assessed by CTV dye staining after exposure to indicated HER2 ⁺ cell lines with and without pembrolizumab co-treatment.

[0009] FIGs. 4A-4B depict an in vitro experiment assessing the effects of HER2-TAC T cell treatment, administered with or without pembrolizumab, on HER2-expressing N87 ^{WT/nuc GFP} or N87 ^PD ' ^L1 ' ^{IIlsh/nuc GFP} cancer cells. FIG. 4A depicts cell images taken at the experimental endpoint (i.e., day 6, hour 144), with live tumor cells shown in green and death dye DRAQ7 shown in red. FIG. 4B is a graph showing the percentage of live cells quantified at each timepoint of the experiment (i.e., every 8 hours, up until hour 144).

[0010] FIG. 5 depicts the design of a clinical trial assessing a HER2-TAC T cell + pembrolizumab combination therapy for treating cancer patients.

DETAILED DESCRIPTION

[0011] Disclosed herein, in certain embodiments, are methods of treating cancer using a combination therapy comprising T cells expressing an antigen-specific T cell-antigen coupler (TAC) and an immune checkpoint inhibitor.

[0012] Cancer is a major health challenge, with over 150,000 cases of cancer expected to be diagnosed in Canada alone. While patients with early -stage disease are sometimes treated effectively by conventional therapies (surgery, radiation, chemotherapy), few' options are available to patients with advanced disease, and those options are typically palliative in nature. [0013] One option for treating cancers is treatment with engineered T cells. Engineered T cells can be genetically modified to yield: (i) forced expression of T cell receptor (TCR); or (ii) a chimeric antigen receptor (CAR) specific for antigen targets on the tumor. To date, the chimeric antigen receptors used for engineering T cells consist of: (i) a targeting domain, usually a singlechain fragment variable (scFv); (ii) a transmembrane domain; and (iii) a cytosolic domain that contains signaling elements from the T cell receptor and associated proteins. Such chimeric antigen receptors have also been referred to as “T-body” or “Chimeric Immune Receptor” (CIR), but currently, most researchers use the term “CAR”. One advantage of the CAR approach is that it allows any patient’s immune cells to be targeted against any desirable target in a major histocompatibility complex (MHC) independent manner. This is appealing as MHC presentation is often defective in tumor cells.

[0014] While CAR-engineered T cells have shown considerable promise in clinical application, they rely on a synthetic method for replacing the native activation signal that is provided by the T cell receptor (TCR). Furthermore, since the CAR signaling domains are disconnected from their natural regulatory partners by the very nature of the CAR structure, there is an inherent risk that CARs may lead to a low-level of constitutive activation, which could result in off-target toxicities. Given these limitations, it is preferable to re-direct T cells to attack tumors via their natural TCR. To this end, a class of recombinant proteins termed “Bispecific T-cell Engagers” (BiTEs) has been created. These proteins employ bispecific antibody fragments to crosslink T- cell TCR receptors with target antigens. This leads to efficient T-cell activation, triggering cytotoxicity. Similarly, bi-specific antibodies have been generated that accomplish this goal and some scientists have simply linked anti-CD3 antibodies to tumor-specific antibodies employing chemical linkage. While these bi-specific proteins have demonstrated some activity in vitro, GMP production, short biological half-lives, and limited bioavailability represent significant challenges to the successful use of these molecules in cancer treatment. Additionally, these molecules also fail to properly recapitulate natural TCR signaling because they do not engage the TCR co-receptors (CD8 and CD4).

[0015] An alternate chimeric receptor, termed a T cell Antigen Coupler (TAC) receptor, has been developed which employs a distinct biology to direct the T cell to attack tumors. While the CAR is a fully synthetic receptor that stitches together components of T cell receptor (TCR) signaling complex, the TAC receptor re-directs the TCR towards tumor targets and recapitulates the native TCR signaling structure. For example, in some embodiments, the TACs disclosed herein activate natural Major Histocompatibility complex (MHC) signaling through the T-cell receptor (TCR), while retaining MHC -unrestricted targeting. Further, the TACs disclosed herein recruit the T-Cell Receptor (TCR) in combination with co-receptor stimulation. Moreover, in some embodiments, TACs disclosed herein show enhanced activity and safety.

Certain Terminology

[0016] The term “antigen-binding domain,” refers to any substance or molecule that binds, directly or indirectly, to a target (e.g, Claudin 18.2). Antigen-binding domains include antibodies or fragments thereof, peptides, peptidomimetics, proteins, glycoproteins, proteoglycans, carbohydrates, lipids, nucleic acids, or small molecules that bind to a target. [0017] As used herein, unless otherwise indicated, the term “antibody” is understood to mean an intact antibody (e.g., an intact monoclonal antibody), or a fragment thereof, such as a Fc fragment of an antibody (e.g, an Fc fragment of a monoclonal antibody), or an antigen-binding fragment of an antibody (e.g., an antigen-binding fragment of a monoclonal antibody), including an intact antibody, antigen-binding fragment, or Fc fragment that has been modified, engineered, or chemically conjugated. In general, antibodies are multimeric proteins that contain four polypeptide chains. Two of the polypeptide chains are called immunoglobulin heavy chains (H chains), and two of the polypeptide chains are called immunoglobulin light chains (L chains). The immunoglobulin heavy and light chains are connected by an interchain disulfide bond. The immunoglobulin heavy chains are connected by interchain disulfide bonds. A light chain consists of one variable region (VL) and one constant region (CL). The heavy chain consists of one variable region (VH) and at least three constant regions (CHI, CH2 and CH3). The variable regions determine the binding specificity of the antibody. Each variable region contains three hypervariable regions known as complementarity determining regions (CDRs) flanked by four relatively conserved regions known as framework regions (FRs). The extent of the FRs and CDRs has been defined (Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91- 3242; and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917). The three CDRs, referred to as CDR1, CDR2, and CDR3, contribute to the antibody binding specificity. Naturally occurring antibodies have been used as starting material for engineered antibodies, such as chimeric antibodies and humanized antibodies. Examples of antibody-based antigen-binding fragments include Fab, Fab’, (Fab’)2, Fv, single chain antibodies (e.g., scFv), minibodies, and diabodies. Examples of antibodies that have been modified or engineered include chimeric antibodies, humanized antibodies, and multispecific antibodies (e.g, bispecific antibodies). An example of a chemically conjugated antibody is an antibody conjugated to a toxin moiety.

[0018] The term “polynucleotide” and/or “nucleic acid sequence” and/or “nucleic acid” as used herein refers to a sequence of nucleoside or nucleotide monomers consisting of bases, sugars and intersugar (backbone) linkages. The term also includes modified or substituted sequences comprising non-naturally occurnng monomers or portions thereof. The nucleic acid sequences of the present application may be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA) and may include naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil The sequences may also contain modified bases. Examples of such modified bases include aza and deaza adenine, guanine, cytosine, thymidine and uracil; and xanthine and hypoxanthine. The nucleic acids of the present disclosure may be isolated from biological organisms, formed by laboratory methods of genetic recombination or obtained by chemical synthesis or other known protocols for creating nucleic acids.

[0019] The term “recombinant nucleic acid” or “engineered nucleic acid” as used herein refers to a nucleic acid or polynucleotide that is not found in a biological organism. For example, recombinant nucleic acids may be formed by laboratory methods of genetic recombination (such as molecular cloning) to create sequences that would not otherwise be found in nature. Recombinant nucleic acids may also be created by chemical synthesis or other known protocols for creating nucleic acids.

[0020] The terms “peptide,” “polypeptide,” and “protein” as used herein mean a chain of amino acids. The term protein as used herein further means a large molecule comprising one or more chains of amino acids and, in some embodiments, is a fragment or domain of a protein or a full- length protein. Furthermore, as used herein, the term protein either refers to a linear chain of amino acids or to a chain of amino acids that has been processed and folded into a functional protein. The protein structure is divided into four distinct levels: (1) primary structure - referring to the sequence of amino acids in the polypeptide chain, (2) secondary structure - referring to the regular local sub-structures on the polypeptide backbone chain, such as a-helix and -sheets, (3) tertiary structure - referring to the three-dimensional structure if monomeric and multimeric protein molecules, and (4) quaternary structure - referring to the three-dimensional structure comprising the aggregation of two or more individual polypeptide chains that operate as a single functional unit. The use of peptide or polypeptide herein does not mean that the chain of amino acids is not also a protein (i.e., a chain of amino acids having a secondary , tertiary or quaternary structure).

[0021] The term “vector” as used herein refers to a polynucleotide that is used to deliver a nucleic acid to the inside of a cell. In some embodiments, a vector is an expression vector comprising expression control sequences (for example, a promoter) operatively linked to a nucleic acid to be expressed in a cell. Vectors known in the art include, but are not limited to, plasmids, phages, cosmids and viruses.

[0022] The term “tumor antigen” or “tumor associated antigen” as used herein refers to a substance produced by tumor cells, which is recognized by the immune system, and which elicits an antigen-specific immune response in a host (e.g., which is presented by MHC complexes). In some embodiments, a tumor antigen is on the surface of a tumor cell.

[0023] As used herein, the term “transmembrane and cytosolic domain” refers to a polypeptide that comprises a transmembrane domain and a cytosolic domain of a protein associated with the T cell receptor (TCR) complex. In some embodiments, such transmembrane and cytosolic domain may include, but is not limited to, protein domains that (a) associate with the lipid raft and/or (b) bind Lek.

[0024] A “TCR co-receptor” as used herein, refers to a molecule that assists the T cell receptor (TCR) in communicating with an antigen-presenting cell. Examples of TCR co-receptors include, but are not limited to, CD4, LAG3, and CD8.

[0025] A “TCR co-stimulator” or “co-stimulatory domain” as used herein, refers to a molecule that enhances the response of a T cell to an antigen and may be considered a signal that leads to the activation of the TCR. Examples of TCR co-stimulators include, but are not limited to, ICOS, CD27, CD28, 4-1BB (CD 137), 0X40 (CD134), CD30, CD40, lymphocyte fiction- associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds CD83.

[0026] The terms “recipient,” “individual,” “subject,” “host,” and “patient,” are used interchangeably herein and in some embodiments, refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans. “Mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and laboratory, zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys etc. In some embodiments, the mammal is human. None of these terms require the supervision of medical personnel.

[0027] As used herein, the terms “treatment,” “treating,” and the like, in some embodiments, refer to administering an agent, or carrying out a procedure, for the purposes of obtaining an effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of affecting a partial or complete cure for a disease and/or symptoms of the disease. “Treatment,” as used herein, may include treatment of a disease or disorder (e.g. , cancer) in a mammal, particularly in a human, and includes: (a) preventing the disease or a symptom of a disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it (e.g, including diseases that may be associated with or caused by a primary disease; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease. Treating may refer to any indicia of success in the treatment or amelioration or prevention of a cancer, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms; or making the disease condition more tolerable to the patient; slowing in the rate of degeneration or decline; or making the final point of degeneration less debilitating. The treatment or amelioration of symptoms is based on one or more objective or subjective parameters; including the results of an examination by a physician. Accordingly, the term "treating" includes the administration of the compounds or agents of the present invention to prevent, delay, alleviate, arrest or inhibit development of the symptoms or conditions associated with diseases (e.g., cancer). The term "therapeutic effect" refers to the reduction, elimination, or prevention of the disease, symptoms of the disease, or side effects of the disease in the subject. [0028] As used herein, singular forms “a”, “and,” and “the” include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to “an antibody” includes a plurality of antibodies and reference to “an antibody” in some embodiments includes multiple antibodies, and so forth.

[0029] As used herein, all numerical values or numerical ranges include whole integers within or encompassing such ranges and fractions of the values or the integers within or encompassing ranges unless the context clearly indicates otherwise. Thus, for example, reference to a range of 90-100%, includes 91%, 92%, 93%, 94%, 95%, 95%, 96%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc., and so forth. In another example, reference to a range of 1-5,000-fold includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, fold, etc., as well as 1.1, 1.2, 1.3, 1.4, 1.5, fold, etc., 2.1, 2.2, 2.3, 2.4, 2.5, fold, etc., and so forth.

[0030] “About” a number, as used herein, refers to range including the number and ranging from 10% below that number to 10% above that number. “About” a range refers to 10% below the lower limit of the range, spanning to 10% above the upper limit of the range.

[0031] “Percent (%) identity” refers to the extent to which two sequences (nucleotide or amino acid) have the same residue at the same positions in an alignment. For example, “an amino acid sequence is X% identical to SEQ ID NO: Y” refers to % identity of the amino acid sequence to SEQ ID NO: Y and is elaborated as X% of residues in the amino acid sequence are identical to the residues of sequence disclosed in SEQ ID NO: Y. Generally, computer programs are employed for such calculations. Exemplary programs that compare and align pairs of sequences, include ALIGN (Myers and Miller, 1988), FASTA (Pearson and Lipman, 1988; Pearson, 1990) and gapped BLAST (Altschul et al., 1997), BLASTP, BLASTN, or GCG (Devereux et al., 1984).

[0032] As used herein, the term “selective binding” refers to the higher affinity with which a molecule (e.g., protein such as an antigen-binding domain of TAC) binds its target molecule (e.g, target antigen such as HER2) over other molecules. Unless indicated otherwise, the terms “selective binding” and “specific binding” are used interchangeably herein.

Methods of Treatment and Use

[0033] Disclosed herein, in certain embodiments, are methods of treating a cancer expressing a target antigen in a subject in need thereof, comprising administering to the individual an engineered TAC T cell that targets said antigen in combination with an immune checkpoint inhibitor, for example a checkpoint inhibitor disclosed herein. In some embodiments, a target antigen-binding domain of a TAC polypeptide disclosed herein specifically binds to a target antigen on a tumor cell.

[0034] Further disclosed herein, in certain embodiments, are methods of treating systemic lupus erythematosus expressing a target antigen in a subject in need thereof, comprising administering to the individual an engineered TAC T cell that targets said antigen in combination with an immune checkpoint inhibitor, for example a checkpoint inhibitor disclosed herein.

[0035] In some embodiments, the TAC protein is administered prior to administration of the immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is administered 7 days after the engineered T cells are administered. In some embodiments, the immune checkpoint inhibitor is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more than 14 days after the engineered T cells are administered. In some embodiments, the immune checkpoint inhibitor is administered 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, or more than 8 weeks after the engineered T cells are administered.

[0036] In some embodiments, the TAC protein is administered following administration of the immune checkpoint inhibitor.

[0037] In some embodiments, the TAC protein and the immune checkpoint inhibitor are administered concurrently.

[0038] In some embodiments, the methods further comprise administering a lymphodepl eting therapy, or are administered to a subject who has received a lymphodepleting therapy. Examples of lymphodepl eting therapies include nonmyeloablative lymphodepleting chemotherapy, myeloablative lymphodepleting chemotherapy, fludarabine, cyclophosphamide, corticosteroids, alemtuzumab, total body irradiation (TBI), and any combination thereof. [0039] Cancers that may be treated with engineered T cells disclosed herein in combination with an immune checkpoint inhibitor include any form of neoplastic disease. In some embodiments, cancers that are treated include, but are not limited to, a salivary gland cancer, a lung cancer, a gastric cancer, a breast cancer, an ovarian cancer, a uterine cancer (e.g, an endometrial cancer), a cervical cancer, a biliary tract cancer, a pancreatic cancer, a colorectal cancer, a bladder cancer (e.g., a urothelial cancer), a prostate cancer, multiple myeloma, a glioblastoma, a gastroesophageal junction cancer, an esophageal cancer, a liver cancer, a thyroid cancer, a kidney cancer, a yolk sac tumor, a skin cancer, or a sarcoma. In some embodiments, the cancer is breast, lung, pancreatic, colorectal, gastric, endometrial, or ovarian cancer. In some embodiments, the cancer is rectosigmoid cancer, gastro-esophageal cancer, or gastric adenocarcinoma.

[0040] In some embodiments, the cancer is a liver cancer (for example, HCC), gastric carcinoma, ovarian carcinoma (for example, ovarian clear cell carcinoma), melanoma, colorectal carcinoma, thyroid cancer, squamous cell carcinoma of the lung, hepatoblastoma, nephroblastoma, or yolk sac tumor. In some embodiments, cancers that are treated include, but are not limited to, a pancreatic cancer (e.g., pancreatic adenocarcinoma, a gastric cancer (e.g, gastric adenocarcinoma), a gastroesophageal cancer (e.g., gastroesophageal junction (GEJ) adenocarcinoma), an esophageal cancer, an ovarian cancer, or a lung cancer (e.g., non-small cell lung cancer). In some embodiments, the cancer is a primary colorectal cancer, a primary gastric cancer, a primary gastroesophageal junction cancer, a primary esophageal cancer, or a primary pancreatic cancer. In some embodiments, the cancer is a metastatic colorectal cancer, a metastatic gastric cancer, a metastatic gastroesophageal junction cancer, a metastatic esophageal cancer, or a metastatic pancreatic cancer. In some embodiments, cancers that are treated include, but are not limited to, multiple myeloma, B cell lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), or Non-Hodgkin’s Lymphoma.

In some embodiments, the cancer to be treated is non-Hodgkin’s lymphoma, such as a B-cell lymphoma. In some embodiments, the non-Hodgkin’s lymphoma is a B-cell lymphoma, such as a large B-cell lymphoma (LBCL), a diffuse large B-cell lymphoma (DLBCL), primary mediastinal B-cell lymphoma, high grade B-cell lymphoma, follicular lymphoma, small lymphocytic lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia, chronic lymphocytic leukemia (CLL), or primary central nervous system lymphoma. In some embodiments, the cancer to be treated is multiple myeloma. In some embodiments, the cancer to be treated is acute lymphoblastic leukemia (ALL). In some embodiments, the ALL is relapsed/refractory adult and pediatric ALL.

T cell-antigen coupler (TAC)

[0041] In certain embodiments, the methods disclosed herein employ a T cell-antigen coupler (TAC) polypeptide. In some embodiments, the TAC polypeptide includes a target antigenbinding domain, which binds to a target antigen of interest (e.g., an antigen expressed on a cancer cell); a ligand that binds a protein associated with a T cell receptor (TCR) (e.g, a CD3 protein, e.g., CD3E); and a transmembrane and cytosolic domain of a TCR co-receptor (e.g, CD4 or CD8). The target antigen-binding domain can allow the T cell to specifically bind to a cell expressing the target antigen (e.g., a cancer cell). The ligand that binds a protein associated with a TCR can localize the TAC to the TCR on a T cell. The transmembrane and cytosolic domain of a TCR co-receptor can allow T cell activation through endogenous TCR signaling by inducing Lck-mediated phosphorylation of immunotyrosine activation motifs (ITAMs) on TCRs. In some embodiments, the TAC polypeptide does not include a co-stimulatory domain. In some embodiments, the TAC polypeptide does not include a co-activation domain.

Ligand that binds a TCR complex

[0042] In certain embodiments, the TAC comprises an antigen-binding domain that binds a protein associated with the TCR complex. A “TCR complex protein antigen-binding domain,” also referred to as a “TCR complex antigen-binding domain,” “antigen-binding domain that binds the TCR complex,” or “antigen-binding domain that binds a protein associated with the TCR complex,” refers to any substance or molecule that binds, directly or indirectly, toa protein associated with a TCR complex. In some embodiments, the antigen-binding domain that binds a protein associated with a TCR complex selectively binds to a protein of the TCR. In some embodiments, the antigen-binding domain that binds a protein associated with a TCR complex comprises a substance that specifically binds to a protein of the TCR.

[0043] In some embodiments, the TCR complex protein antigen-binding domain is selected from antibodies or fragments thereof, for example, single chain antibodies (e.g, single-chain fragment variable antibodies (scFvs)), single domain antibodies (e.g., nanobodies (VHH), shark heavy-chain-only antibodies (VNAR)), diabodies, minibodies, Fab fragments, Fab' fragments, F(ab’)2 fragments, or Fv fragments that bind to a protein of the TCR. In some embodiments, the TCR complex protein antigen-binding domain is selected from ankyrin repeat proteins (DARPins), affibodies, adnectins, affilins, phylomers: fynomers, affimers, peptide aptamers, lectins, knottins, centyrins, anticalins, peptides, peptidomimetics, proteins, glycoproteins, or proteoglycans that bind to a protein of the TCR, or naturally occurring ligands for a protein of the TCR. In some embodiments, the TCR complex protein antigen-binding domain is a nonprotein compound that binds to a protein of the TCR, including but not limited to carbohydrates, lipids, nucleic acids, or small molecules. In some embodiments, the TCR complex protein antigen-binding domain is a designed ankyrin repeat (DARPin) targeted to a protein of the TCR. In some embodiments, the TCR complex protein antigen-binding domain is a single-chain variable fragment (ScFv) targeted to a protein of the TCR. In some embodiments, the TCR complex protein antigen-binding domain is a nanobody targeted to a protein of the TCR.

[0044] Proteins associated with the TCR include, but are not limited, to the TCR alpha (a) chain, TCR beta (P) chain, TCR gamma (y) chain, TCR delta (5) chain, CD3y chain, CD33 chain and CD3e chains. In some embodiments, an antigen-binding domain that binds a protein associated with the TCR complex is an antibody to the TCR alpha (a) chain, TCR beta ( ) chain, TCR gamma (y) chain, TCR delta (3) chain, CD3y chain, CD33 chain and/or CD3E chain. In some embodiments, the protein associated with a TCR complex is CD3. In some embodiments, the protein associated with a TCR complex is CD3E. In some embodiments, the antigen-binding domain that binds CD3 is an antibody, for example, a single chain antibody, for example a single-chain variable fragment (scFv). Examples of CD3 antibodies, include, but are not limited to, UCHT1, OKT3, F6A, L2K, muromonab, otelixizumab, teplizumab, visilizumab, CD3-12, MEM-57, 4D10A6, CD3D, or TR66.

[0045] In some embodiments, the antigen-binding domain that binds the TCR complex is UCHT1, or a variant thereof. In some embodiments, the UCHT1 antigen-binding domain is encoded by SEQ ID NO: 1. In some embodiments, the UCHT1 antigen-binding domain comprises SEQ ID NO: 2. In some embodiments, the UCHT1 antigen-binding domain is mutated. In some embodiments, the UCHT1 antigen-binding domain comprises a Y to T mutation at a position corresponding to amino acid 182 of SEQ ID NO: 32 (Y182T). In some embodiments, the UCHT1 (Y182T) antigen-binding domain is encoded by SEQ ID NO: 3. In some embodiments, the UCHT1 (Y182T) antigen-binding domain comprises SEQ ID NO: 4. In some embodiments, the antigen-binding domain that binds the TCR complex is a humanized UCHT1 (huUCHTl). In some embodiments, the huUCHTl antigen-binding domain is encoded by SEQ ID NO: 5. In some embodiments, the huUCHTl antigen-binding domain comprises SEQ ID NO: 6. In some embodiments, the huUCHTl has a Y to T mutation at a position corresponding to amino acid 177 of SEQ ID NO: 6 (Y177T). In some embodiments, the huUCHTl (Y177T) antigen-binding domain is encoded by SEQ ID NO: 7. In some embodiments, the huUCHTl antigen-binding domain comprises SEQ ID NO: 8.

[0046] In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 70% sequence identity with the nucleotide sequence of SEQ ID NO: 1 (UCHT1). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 75% sequence identity with the nucleotide sequence of SEQ ID NO: 1 (UCHT1). In some embodiments, the polynucleotide encoding the antigen-bmdmg domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 80% sequence identity' with the nucleotide sequence of SEQ ID NO: 1 (UCHT1). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 85% sequence identity' with the nucleotide sequence of SEQ ID NO: 1 (UCEIT1). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 90% sequence identity' with the nucleotide sequence of SEQ ID NO: 1 (UCHT1). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 95% sequence identity' with the nucleotide sequence of SEQ ID NO: 1 (UCHT1). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 96% sequence identity' with the nucleotide sequence of SEQ ID NO: 1 (UCHT1 ). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 97% sequence identity' with the nucleotide sequence of SEQ ID NO: 1 (UCHT1). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 98% sequence identity' with the nucleotide sequence of SEQ ID NO: 1 (UCHT1). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex compnses a nucleotide sequence having at least 99% sequence identity' with the nucleotide sequence of SEQ ID NO: 1 (UCHT1). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises the nucleotide sequence of SEQ ID NO: 1 (UCHT1).

[0047] In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 2 (UCHT1). In some embodiments, the antigenbinding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 75% sequence identity with the amino acid sequence of SEQ ID NO: 2 (UCHT1). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex compnses an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 2 (UCHT1). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 2 (UCHT1). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 2 (UCHT1). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 2 (UCHT1). In some embodiments, the antigenbinding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 96% sequence identity' with the amino acid sequence of SEQ ID NO: 2 (UCHT1). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 97% sequence identity with the amino acid sequence of SEQ ID NO: 2 (UCHT1). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 2 (UCHT1). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 2 (UCHT1). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises the amino acid sequence of SEQ ID NO: 2 (UCHT1). In some embodiments, the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 2 (UCHT1) (z.e., the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence comprising a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, each having 100% identity to the corresponding CDR in the amino acid sequence of SEQ ID NO: 2 (UCHT1). In some embodiments, the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 2 (UCHT1), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 80% sequence identity with the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 2 (UCHT1). In some embodiments, the CDR sequences of the antigenbinding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 2 (UCHT1), and the non- CDR (e.g., framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 85% sequence identity with the non-CDR (e.g., framew ork) sequences of the amino acid sequence of SEQ ID NO: 2 (UCHT1). In some embodiments, the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 2 (UCHT1), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 90% sequence identity with the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 2 (UCHT1). In some embodiments, the CDR sequences of the antigenbinding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 2 (UCHT1), and the non- CDR (e .g., framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 95% sequence identity with the non-CDR (e.g., framew ork) sequences of the amino acid sequence of SEQ ID NO: 2 (UCHT1). In some embodiments, the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 2 (UCHT1), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 96% sequence identity with the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 2 (UCHT1). In some embodiments, the CDR sequences of the antigenbinding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 2 (UCHT1), and the non- CDR (e.g., framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 97% sequence identity with the non-CDR (e.g, framework) sequences of the amino acid sequence of SEQ ID NO: 2 (UCHT1). In some embodiments, the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 2 (UCHT1), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 98% sequence identity with the non-CDR (e.g, framework) sequences of the amino acid sequence of SEQ ID NO: 2 (UCHT1). In some embodiments, the CDR sequences of the antigenbinding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 2 (UCHT1), and the non- CDR (e.g., framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 99% sequence identity with the non-CDR (e.g, framework) sequences of the amino acid sequence of SEQ ID NO: 2 (UCHT1).

[0048] In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 70% sequence identity with the nucleotide sequence of SEQ ID NO: 3 (UCHT1 (Y182T)). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 75% sequence identity with the nucleotide sequence of SEQ ID NO: 3 (UCHT1 (Y182T)). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 80% sequence identity' with the nucleotide sequence of SEQ ID NO: 3 (UCHT1 (Y182T)). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 85% sequence identity' with the nucleotide sequence of SEQ ID NO: 3 (UCHT1 (Y182T)). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 90% sequence identity' with the nucleotide sequence of SEQ ID NO: 3 (UCHT1 (Y182T)). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 95% sequence identity' with the nucleotide sequence of SEQ ID NO: 3 (UCHT1 (Y182T)). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 96% sequence identity with the nucleotide sequence of SEQ ID NO: 3 (UCHT1 (Y182T)). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 97% sequence identity' with the nucleotide sequence of SEQ ID NO: 3 (UCHT1 (Y182T)). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 98% sequence identity' with the nucleotide sequence of SEQ ID NO: 3 (UCHT1 (Y182T)). In some embodiments, the polynucleotide encoding the antigen-bmdmg domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 99% sequence identity' with the nucleotide sequence of SEQ ID NO: 3 (UCHT1 (Y182T)). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises the nucleotide sequence of SEQ ID NO: 3 (UCHT1 (Y182T)).

[0049] In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 75% sequence identity with the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)). In some embodiments, the antigenbinding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 90% sequence identity' with the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 95% sequence identity' with the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 96% sequence identity with the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)). In some embodiments, the antigen- binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 97% sequence identity with the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)). In some embodiments, the antigen- binding domain that binds the protein associated with the TCR complex comprises the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)). In some embodiments, the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)) (i.e., the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence comprising a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, each having 100% identity to the corresponding CDR in the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)). In some embodiments, the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 80% sequence identity with the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)). In some embodiments, the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 85% sequence identity with the non-CDR (e.g, framework) sequences of the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)). In some embodiments, the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 90% sequence identity with the non-CDR (e.g. , framework) sequences of the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)). In some embodiments, the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)), and the non- CDR (e.g., framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 95% sequence identity with the non-CDR (e.g, framework) sequences of the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)). In some embodiments, the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)), and the non-CDR (e.g, framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 96% sequence identity with the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)). In some embodiments, the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 97% sequence identity with the non-CDR (e.g. , framework) sequences of the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)). In some embodiments, the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)), and the non- CDR (e.g., framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 98% sequence identity with the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)). In some embodiments, the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 99% sequence identity with the non-CDR (e.g, framework) sequences of the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)).

[0050] In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 70% sequence identity with the nucleotide sequence of SEQ ID NO: 5 (huUCHTl). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 75% sequence identity with the nucleotide sequence of SEQ ID NO: 5 (huUCHTl). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 80% sequence identity' with the nucleotide sequence of SEQ ID NO: 5 (huUCHTl). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 85% sequence identity' with the nucleotide sequence of SEQ ID NO: 5 (huUCHTl). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 90% sequence identity' with the nucleotide sequence of SEQ ID NO: 5 (huUCHTl). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 95% sequence identity' with the nucleotide sequence of SEQ ID NO: 5 (huUCHTl). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 96% sequence identity' with the nucleotide sequence of SEQ ID NO: 5 (huUCHTl). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 97% sequence identity' with the nucleotide sequence of SEQ ID NO: 5 (huUCHTl). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 98% sequence identity' with the nucleotide sequence of SEQ ID NO: 5 (huUCHTl). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 99% sequence identity' with the nucleotide sequence of SEQ ID NO: 5 (huUCHTl). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises the nucleotide sequence of SEQ ID NO: 5 (huUCHTl).

[0051] In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 70% sequence identity with the ammo acid sequence of SEQ ID NO: 6 (huUCHTl). In some embodiments, the antigenbinding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 75% sequence identity' with the amino acid sequence of SEQ ID NO: 6 (huUCHTl). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 6 (huUCHTl). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 6 (huUCHTl). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 6 (huUCHTl). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 95% sequence identity' with the amino acid sequence of SEQ ID NO: 6 (huUCHTl). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 96% sequence identity with the amino acid sequence of SEQ ID NO: 6 (huUCHTl). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 97% sequence identity with the amino acid sequence of SEQ ID NO: 6 (huUCHTl). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 98% sequence identity' with the amino acid sequence of SEQ ID NO: 6 (huUCHTl). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 99% sequence identity with the ammo acid sequence of SEQ ID NO: 6 (huUCHTl). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises the amino acid sequence of SEQ ID NO: 6 (huUCHTl). In some embodiments, the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 6 (huUCHTl) (i.e., the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence comprising a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, each having 100% identity to the corresponding CDR in the amino acid sequence of SEQ ID NO: 6 (huUCHTl). In some embodiments, the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 6 (huUCHTl), and the non-CDR (e.g, framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 80% sequence identity with the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 6 (huUCHTl). In some embodiments, the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 6 (huUCHTl), and the non-CDR (e.g, framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 85% sequence identity with the non-CDR (e.g. , framework) sequences of the amino acid sequence of SEQ ID NO: 6 (huUCHTl). In some embodiments, the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 6 (huUCHTl), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 90% sequence identity with the non-CDR (e.g, framework) sequences of the amino acid sequence of SEQ ID NO: 6 (huUCHTl). In some embodiments, the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 6 (huUCHTl), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 95% sequence identity with the non-CDR (e.g. , framework) sequences of the amino acid sequence of SEQ ID NO: 6 (huUCHTl). In some embodiments, the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 6 (huUCHTl), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 96% sequence identity with the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 6 (huUCHTl). In some embodiments, the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 6 (huUCHTl), and the non-CDR (e.g, framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 97% sequence identity with the non-CDR (e.g. , framework) sequences of the amino acid sequence of SEQ ID NO: 6 (huUCHTl). In some embodiments, the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 6 (huUCHTl), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 98% sequence identity with the non-CDR (e g., framework) sequences of the amino acid sequence of SEQ ID NO: 6 (huUCHTl). In some embodiments, the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 6 (huUCHTl), and the non-CDR (e.g, framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 99% sequence identity with the non-CDR (e.g. , framework) sequences of the amino acid sequence of SEQ ID NO: 6 (huUCHTl).

[0052] In some embodiments, the polynucleotide encoding the antigen-bindmg domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 70% sequence identity with the nucleotide sequence of SEQ ID NO: 7 (huUCHTl (Y177T)). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 75% sequence identity with the nucleotide sequence of SEQ ID NO: 7 (huUCHTl (Y177T)). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence of SEQ ID NO: 7 (huUCHTl (Y177T)). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 85% sequence identity with the nucleotide sequence of SEQ ID NO: 7 (huUCHTl (Y177T)). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 90% sequence identity with the nucleotide sequence of SEQ ID NO: 7 (huUCHTl (Y177T)). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 95% sequence identity with the nucleotide sequence of SEQ ID NO: 7 (huUCHTl (Y177T)). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 96% sequence identity with the nucleotide sequence of SEQ ID NO: 7 (huUCHTl (Y177T)). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex compnses a nucleotide sequence having at least 97% sequence identity with the nucleotide sequence of SEQ ID NO: 7 (huUCHTl (Y177T)). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 98% sequence identity with the nucleotide sequence of SEQ ID NO: 7 (huUCHTl (Y177T)). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 99% sequence identity with the nucleotide sequence of SEQ ID NO: 7 (huUCHTl (Y177T)). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises the nucleotide sequence of SEQ ID NO: 7 (huUCHTl (Y177T)).

[0053] In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 75% sequence identity with the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 96% sequence identity with the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 97% sequence identity with the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex compnses an amino acid sequence having at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)). In some embodiments, the antigen-binding domain that binds the protein associated wi th the TCR complex comprises an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)). In some embodiments, the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)) (i.e., the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence comprising a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, each having 100% identity to the corresponding CDR in the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)). In some embodiments, the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)), and the non-CDR (e.g, framework) sequences of the antigenbinding domain that binds the protein associated with the TCR complex have at least 80% sequence identity' with the non-CDR (e.g, framework) sequences of the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)). In some embodiments, the CDR sequences of the antigenbinding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 85% sequence identity with the non-CDR (e.g, framework) sequences of the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)). In some embodiments, the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)), and the non-CDR (e.g, framew ork) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 90% sequence identity with the non-CDR (e.g, framework) sequences of the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)). In some embodiments, the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)), and the non-CDR (e.g, framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 95% sequence identity with the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)). In some embodiments, the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)), and the non-CDR (e.g, framework) sequences of the antigenbinding domain that binds the protein associated with the TCR complex have at least 96% sequence identity with the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)). In some embodiments, the CDR sequences of the antigenbinding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 97% sequence identity with the non-CDR (e.g, framework) sequences of the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)). In some embodiments, the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)), and the non-CDR (e.g., framew ork) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 98% sequence identity with the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)). In some embodiments, the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 99% sequence identity with the non-CDR (e.g, framework) sequences of the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)).

[0054] In some embodiments, the antigen-binding domain that binds to the protein associated with the TCR complex is OKT3. In some embodiments, the murine OKT3 antigen-binding domain is encoded by SEQ ID NO: 9. In some embodiments, the OKT3 antigen-binding domain comprises SEQ ID NO: 10.

[0055] In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 70% sequence identity with the nucleotide sequence of SEQ ID NO: 9 (OKT3). In some embodiments, the polynucleotide encoding the antigen-bmdmg domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 80% sequence identity' with the nucleotide sequence of SEQ ID NO: 9 (OKT3). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 90% sequence identity with the nucleotide sequence of SEQ ID NO: 9 (OKT3). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 95% sequence identity' with the nucleotide sequence of SEQ ID NO: 9 (OKT3). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 99% sequence identity' with the nucleotide sequence of SEQ ID NO: 9 (OKT3). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises the nucleotide sequence of SEQ ID NO: 9 (OKT3). [0056] In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 10 (OKT3). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 10 (OKT3). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 10 (OKT3). In some embodiments, the antigenbinding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 95% sequence identity' with the amino acid sequence of SEQ ID NO: 10 (OKT3). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 99% sequence identity' with the amino acid sequence of SEQ ID NO: 10 (OKT3). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises the amino acid sequence of SEQ ID NO: 10 (OKT3).

[0057] In some embodiments, the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 10 (OKT3) (i.e., the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence comprising a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, each having 100% identity to the corresponding CDR in the amino acid sequence of SEQ ID NO: 10 (OKT3). [0058] In some embodiments, the antigen-binding domain that binds to the protein associated with the TCR complex is F6A. In some embodiments, the murine F6A antigen-binding domain is encoded by SEQ ID NO: 11. In some embodiments, the F6A antigen-binding domain comprises SEQ ID NO: 12.

[0059] In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 70% sequence identity with the nucleotide sequence of SEQ ID NO: 11 (F6A). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence of SEQ ID NO: 11 (F6A). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 90% sequence identity' with the nucleotide sequence of SEQ ID NO: 11 (F6A). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 95% sequence identity' with the nucleotide sequence of SEQ ID NO: 11 (F6A). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 99% sequence identity' with the nucleotide sequence of SEQ ID NO: 11 (F6A). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises the nucleotide sequence of SEQ ID NO: 11 (F6A). [0060] In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 12 (F6A). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 12 (F6A). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 12 (F6A). In some embodiments, the antigenbinding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 95% sequence identity' with the amino acid sequence of SEQ ID NO: 12 (F6A). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 12 (F6A). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises the amino acid sequence of SEQ ID NO: 12 (F6A).

[0061] In some embodiments, the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 12 (F6A) (i.e., the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence comprising a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, each having 100% identity to the corresponding CDR in the amino acid sequence of SEQ ID NO: 12 (F6A).

[0062] In some embodiments, the antigen-binding domain that binds to the protein associated with the TCR complex is L2K. In some embodiments, the murine L2K antigen-binding domain is encoded by SEQ ID NO: 13. In some embodiments, the L2K antigen-binding domain comprises SEQ ID NO: 14.

[0063] In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 70% sequence identity with the nucleotide sequence of SEQ ID NO: 13 (L2K). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 80% sequence identity' with the nucleotide sequence of SEQ ID NO: 13 (L2K). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 90% sequence identity' with the nucleotide sequence of SEQ ID NO: 13 (L2K). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 95% sequence identity' with the nucleotide sequence of SEQ ID NO: 13 (L2K). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 99% sequence identity' with the nucleotide sequence of SEQ ID NO: 13 (L2K). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises the nucleotide sequence of SEQ ID NO: 13 (L2K). [0064] In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 14 (L2K). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 14 (L2K). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 14 (L2K). In some embodiments, the antigenbinding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 14 (L2K). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex compnses an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 14 (L2K). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises the amino acid sequence of SEQ ID NO: 14 (L2K). In some embodiments, the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 14 (L2K) (i.e., the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence comprising a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, each having 100% identity to the corresponding CDR in the amino acid sequence of SEQ ID NO: 14 (L2K).

[0065] Amino acid and nucleotide sequences of exemplary antigen-binding domains that bind a protein associated with the TCR complex are provided in Table 1.

Table 1: Table of Sequences ¹ Light chain, nucleotides 1-324; Linker, nucleotides 325-387; Heavy chain, nucleotides 388- 750

² Light chain, amino acids 1 -108; Linker, amino acids 109-128; Heavy chain, amino acids 129- 250

Configurations, Linkers, and Connectors

[0066] In some embodiments, a nucleic acid disclosed herein is in an order of (1) a polynucleotide encoding a target antigen-binding domain; (2) a polynucleotide encoding a UCHT1 (e.g., huUCHTl); (3) a polynucleotide encoding a CD4 transmembrane domain and a cytosolic domain. In some embodiments, a nucleic acid disclosed herein is in an order of (1) a target antigen-binding domain; (2) a polynucleotide encoding a UCHT1 (e.g., huUCHTl); (3) a polynucleotide encoding a CD4 transmembrane domain and a cytosolic domain, wherein the order is 5’ end to 3’ end. In some embodiments, a nucleic acid disclosed herein is in an order of (1) a target antigen-binding domain; (2) a polynucleotide encoding a UCHT1 (e.g., huUCHTl); (3) a polynucleotide encoding a CD4 transmembrane domain and a cytosolic domain, wherein the order is 3’ end to 5’ end.

[0067] In some embodiments, a nucleic acid described herein is in an order of (1) a polynucleotide encoding a UCHT1 (e.g., huUCHTl); (2) a target antigen-binding domain; (3) a polynucleotide encoding a CD4 transmembrane domain and a cytosolic domain. In some embodiments, a nucleic acid described herein is in an order of (1) a polynucleotide encoding a UCHT1 (e.g., huUCHTl); (2) a target antigen-binding domain; (3) a polynucleotide encoding a CD4 transmembrane domain and a cytosolic domain, wherein the order is 5’ end to 3’ end. In some embodiments, a nucleic acid described herein is in an order of (1) a polynucleotide encoding a UCHT1 (e.g., huUCHTl); (2) a target antigen-binding domain; (3) a polynucleotide encoding a CD4 transmembrane domain and a cytosolic domain, wherein the order is 3’ end to 5’ end.

[0068] In some embodiments, the target antigen-binding domain, the UCHT1 (e.g., huUCHTl), and CD4 transmembrane domain and a cytosolic domain polypeptides are directly fused. For example, the target antigen-binding domain and the CD4 transmembrane domain and a cytosolic domain polypeptide are both fused to the UCHT1 (e.g, huUCHTl). In some embodiments, the target antigen-binding domain, the UCHT1 (e.g., huUCHTl), and CD4 transmembrane domain and a cytosolic domain polypeptides are joined by at least one linker. In some embodiments, the first polypeptide and the second polypeptide are directly fused, and joined to the third polypeptide by a linker. In some embodiments, the second polypeptide and the third polypeptide are directly fused, and joined to the first polypeptide by a linker. [0069] In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker comprises 1 to 40 amino acids. In some embodiments, the peptide linker comprises 1 to 30 amino acids. In some embodiments, the peptide linker comprises 1 to 15 amino acids. In some embodiments, the peptide linker comprises 1 to 10 amino acids. In some embodiments, the peptide linker comprises 1 to 6 amino acids. In some embodiments, the peptide linker comprises 30 to 40 amino acids. In some embodiments, the peptide linker comprises 32 to 36 amino acids. In some embodiments, the peptide linker comprises 5 to 30 amino acids. In some embodiments, the peptide linker comprises 5 amino acids. In some embodiments, the peptide linker comprises 10 ammo acids. In some embodiments, the peptide linker comprises 15 ammo acids. In some embodiments, the peptide linker comprises 20 amino acids. In some embodiments, the peptide linker comprises 25 amino acids. In some embodiments, the peptide linker comprises 30 amino acids.

[0070] In some embodiments, the at least one linker comprises an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 18 ((G4S)4-based linker), SEQ ID NO: 20 (G4S-based linker), SEQ ID NO: 26 (CD4 based linker), SEQ ID NO: 28 (short helix connector), SEQ ID NO: 30 (long helix connector), SEQ ID NO: 32 (large domain connector), SEQ ID NO: 41 (flexible connector), SEQ ID NO: 45 (G4S flexible linker), or SEQ ID NO: 46 (G4S3 flexible linker). In some embodiments, the at least one linker comprises an amino acid sequence having at least 85% identity with the amino acid sequence of SEQ ID NO: 18 ((G4S)4-based linker), SEQ ID NO: 20 (G4S-based linker), SEQ ID NO: 26 (CD4 based linker), SEQ ID NO: 22 (short helix connector), SEQ ID NO: 24 (long helix connector), SEQ ID NO: 26 (large domain connector), SEQ ID NO: 209 (flexible connector), SEQ ID NO: 211 (G4S flexible linker), or SEQ ID NO: 213 (G4S3 flexible linker). In some embodiments, the at least one linker comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 18 ((G4S)4-based linker), SEQ ID NO: 20 (G4S-based linker), SEQ ID NO: 26 (CD4 based linker), SEQ ID NO: 28 (short helix connector), SEQ ID NO: 30 (long helix connector), SEQ ID NO: 32 (large domain connector), SEQ ID NO: 209 (flexible connector), SEQ ID NO: 211 (G4S flexible linker), or SEQ ID NO: 213 (G4S3 flexible linker). In some embodiments, the at least one linker comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 18 ((G4S)4-based linker), SEQ ID NO: 20 (G4S-based linker), SEQ ID NO: 26 (CD4 based linker), SEQ ID NO: 28 (short helix connector), SEQ ID NO: 30 (long helix connector), SEQ ID NO: 32 (large domain connector), SEQ ID NO: 209 (flexible connector), SEQ ID NO: 211 (G4S flexible linker), or SEQ ID NO: 213 (G4S3 flexible linker). In some embodiments, the at least one linker comprises an amino acid sequence having at least 96% identity with the amino acid sequence of SEQ ID NO: 18 ((G4S)4-based linker), SEQ ID NO: 20 (G4S-based linker), SEQ ID NO: 26 (CD4 based linker), SEQ ID NO: 28 (short helix connector), SEQ ID NO: 30 (long helix connector), SEQ ID NO: 32 (large domain connector), SEQ ID NO: 209 (flexible connector), SEQ ID NO: 211 (G4S flexible linker), or SEQ ID NO: 213 (G4S3 flexible linker). In some embodiments, the at least one linker comprises an amino acid sequence having at least 97% identity with the amino acid sequence of SEQ ID NO: 18 ((G4S)4-based linker), SEQ ID NO: 20 (G4S-based linker), SEQ ID NO: 26 (CD4 based linker), SEQ ID NO: 28 (short helix connector), SEQ ID NO: 30 (long helix connector), SEQ ID NO: 32 (large domain connector), SEQ ID NO: 209 (flexible connector), SEQ ID NO: 211 (G4S flexible linker), or SEQ ID NO: 213 (G4S3 flexible linker). In some embodiments, the at least one linker comprises an amino acid sequence having at least 98% identity with the amino acid sequence of SEQ ID NO: 18 ((G4S)4-based linker), SEQ ID NO: 20 (G4S-based linker), SEQ ID NO: 26 (CD4 based linker), SEQ ID NO: 28 (short helix connector), SEQ ID NO: 30 (long helix connector), SEQ ID NO: 32 (large domain connector), SEQ ID NO: 209 (flexible connector), SEQ ID NO: 211 (G4S flexible linker), or SEQ ID NO: 213 (G4S3 flexible linker). In some embodiments, the at least one linker comprises an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 18 ((G4S)4-based linker), SEQ ID NO: 20 (G4S-based linker), SEQ ID NO: 26 (CD4 based linker), SEQ ID NO: 28 (short helix connector), SEQ ID NO: 30 (long helix connector), SEQ ID NO: 32 (large domain connector), SEQ ID NO: 209 (flexible connector), SEQ ID NO: 211 (G4S flexible linker), or SEQ ID NO: 213 (G4S3 flexible linker). In some embodiments, the at least one linker comprises the amino acid sequence of SEQ ID NO: 18 ((G4S)4-based linker), SEQ ID NO: 20 (G4S-based linker), SEQ ID NO: 26 (CD4 based linker), SEQ ID NO: 28 (short helix connector), SEQ ID NO: 30 (long helix connector), SEQ ID NO: 32 (large domain connector), SEQ ID NO: 209 (flexible connector), SEQ ID NO: 211 (G4S flexible linker), or SEQ ID NO: 213 (G4S3 flexible linker).

[0071] In some embodiments, the peptide linker that joins the target antigen-binding domain to the antigen-binding domain that binds a TCR complex (e.g., UCHT1) is known as the connector to distinguish this protein domain from other linkers in the TAC. The connector may be of any size. In some embodiments, the connector between the antigen-binding domain that binds a TCR complex and the target antigen-binding domain is a short helix comprising SEQ ID NO: 28. In some embodiments, the connector between the antigen-binding domain that binds a TCR complex and the target antigen-binding domain is a short helix encoded by SEQ ID NO: 27. In some embodiments, the connector between the antigen-binding domain that binds a TCR complex and the target antigen-binding domain is a long helix comprising SEQ ID NO: 29. In some embodiments, the connector between the antigen-binding domain that binds a TCR complex and the target antigen-binding domain is a long helix encoded by SEQ ID NO: 28. In some embodiments, the connector between the antigen-binding domain that binds a TCR complex and the target antigen-binding domain is a large domain comprising SEQ ID NO: 32. In some embodiments, the connector between the antigen-binding domain that binds a TCR complex and the target antigen-bindmg domain is a large domain encoded by SEQ ID NO: 30. [0072] In some embodiments, a nucleic acid or TAC disclosed herein comprises a leader sequence. In some embodiments, the leader sequence is encoded by a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence of SEQ ID NO: 33 (muIgG leader), SEQ ID NO: 35 (huIgG leader), or SEQ ID NO: 37 (huCDSa leader). In some embodiments, the leader sequence is encoded by a nucleotide sequence having at least 85% sequence identity with the nucleotide sequence of SEQ ID NO: 33 (muIgG leader), SEQ ID NO: 35 (huIgG leader), or SEQ ID NO: 37 (huCD8a leader). In some embodiments, the leader sequence is encoded by a nucleotide sequence having at least 90% sequence identity with the nucleotide sequence of SEQ ID NO: 33 (muIgG leader), SEQ ID NO: 35 (huIgG leader), or SEQ ID NO: 37 (huCD8a leader). In some embodiments, the leader sequence is encoded by a nucleotide sequence having at least 95% sequence identity with the nucleotide sequence of SEQ ID NO: 33 (muIgG leader), SEQ ID NO: 35 (huIgG leader), or SEQ ID NO: 37 (huCD8a leader). In some embodiments, the leader sequence is encoded by a nucleotide sequence having at least 96% sequence identity with the nucleotide sequence of SEQ ID NO: 33 (muIgG leader), SEQ ID NO: 35 (huIgG leader), or SEQ ID NO: 37 (huCD8a leader). In some embodiments, the leader sequence is encoded by a nucleotide sequence having at least 97% sequence identity with the nucleotide sequence of SEQ ID NO: 33 (muIgG leader), SEQ ID NO: 35 (huIgG leader), or SEQ ID NO: 37 (huCD8a leader). In some embodiments, the leader sequence is encoded by a nucleotide sequence having at least 98% sequence identity with the nucleotide sequence of SEQ ID NO: 33 (muIgG leader), SEQ ID NO: 35 (huIgG leader), or SEQ ID NO: 37 (huCD8a leader). In some embodiments, the leader sequence is encoded by a nucleotide sequence having at least 99% sequence identity with the nucleotide sequence of SEQ ID NO: 33 (mulgG leader), SEQ ID NO: 35 (huIgG leader), or SEQ ID NO: 37 (huCD8a leader). In some embodiments, the leader sequence comprises the nucleotide sequence of SEQ ID NO: 33 (muIgG leader), SEQ ID NO: 35 (huIgG leader), or SEQ ID NO: 37 (huCD8a leader).

[0073] In some embodiments, a nucleic acid or TAC disclosed herein comprises a leader sequence. In some embodiments, the leader sequence comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 34 (muIgG leader), SEQ ID NO: 36 (huIgG leader), or SEQ ID NO: 38 (huCD8a leader). In some embodiments, the leader sequence comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 34 (muIgG leader), SEQ ID NO: 36 (huIgG leader), or SEQ ID NO: 38 (huCD8a leader). In some embodiments, the leader sequence comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 34 (muIgG leader), SEQ ID NO: 36 (huIgG leader), or SEQ ID NO: 38 (huCD8a leader). In some embodiments, the leader sequence comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 34 (muIgG leader), SEQ ID NO: 36 (huIgG leader), or SEQ ID NO: 38 (huCD8a leader). In some embodiments, the leader sequence comprises an amino acid sequence having at least 96% sequence identity with the amino acid sequence of SEQ ID NO: 34 (muIgG leader), SEQ ID NO: 36 (huIgG leader), or SEQ ID NO: 38 (huCD8a leader). In some embodiments, the leader sequence comprises an amino acid sequence having at least 97% sequence identity with the amino acid sequence of SEQ ID NO: 34 (muIgG leader), SEQ ID NO: 36 (huIgG leader), or SEQ ID NO: 38 (huCD8a leader). In some embodiments, the leader sequence comprises an amino acid sequence having at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 34 (muIgG leader), SEQ ID NO: 36 (huIgG leader), or SEQ ID NO: 38 (huCD8a leader). In some embodiments, the leader sequence comprises an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 34 (muIgG leader), SEQ ID NO: 36 (huIgG leader), or SEQ ID NO: 38 (huCD8a leader). In some embodiments, the leader sequence comprises the amino acid sequence of SEQ ID NO: 34 (muIgG leader), SEQ ID NO: 36 (huIgG leader), or SEQ ID NO: 38 (huCD8a leader).

[0074] Amino acid and nucleotide sequences of exemplary linkers, connectors, tags, and leader sequences are provided in Table 2.

Table 2: Table of Sequences

Transmembrane domain and Cytosolic domain

[0075] In some embodiments, the T cell receptor signaling domain polypeptide comprises a TCR co-receptor domain. In some embodiments, the TCR signaling domain polypeptide comprises a transmembrane domain and/or a cytosolic domain of a TCR co-receptor. In some embodiments, the TCR co-receptor is CD4, CD8, LAG3, or a chimeric variation thereof. [0076] In some embodiments, the TCR co-receptor is CD4. In some embodiments, the TAC comprises a transmembrane domain and a cytosolic domain of a CD4 co-receptor. In some embodiments, the polynucleotide encoding the cytosolic and transmembrane domain comprises a nucleotide sequence having at least 70% sequence identity with the nucleotide sequence of SEQ ID NO: 41 (CD4 transmembrane and cytosolic domain). In some embodiments, the polynucleotide encoding the cytosolic and transmembrane domain comprises a nucleotide sequence having at least 75% sequence identity with the nucleotide sequence of SEQ ID NO: 41 (CD4 transmembrane and cytosolic domain). In some embodiments, the polynucleotide encoding the cytosolic and transmembrane domain comprises a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence of SEQ ID NO: 41 (CD4 transmembrane and cytosolic domain). In some embodiments, the polynucleotide encoding the cytosolic and transmembrane domain comprises a nucleotide sequence having at least 85% sequence identity with the nucleotide sequence of SEQ ID NO: 41 (CD4 transmembrane and cytosolic domain). In some embodiments, the polynucleotide encoding the cytosolic and transmembrane domain comprises a nucleotide sequence having at least 90% sequence identity with the nucleotide sequence of SEQ ID NO: 41 (CD4 transmembrane and cytosolic domain). In some embodiments, the polynucleotide encoding the cytosolic and transmembrane domain comprises a nucleotide sequence having at least 95% sequence identity with the nucleotide sequence of SEQ ID NO: 41 (CD4 transmembrane and cytosolic domain). In some embodiments, the polynucleotide encoding the cytosolic and transmembrane domain comprises a nucleotide sequence having at least 96% sequence identity with the nucleotide sequence of SEQ ID NO: 41 (CD4 transmembrane and cytosolic domain). In some embodiments, the polynucleotide encoding the cytosolic and transmembrane domain comprises a nucleotide sequence having at least 97% sequence identity with the nucleotide sequence of SEQ ID NO: 41 (CD4 transmembrane and cytosolic domain). In some embodiments, the polynucleotide encoding the cytosolic and transmembrane domain comprises a nucleotide sequence having at least 98% sequence identity with the nucleotide sequence of SEQ ID NO: 41 (CD4 transmembrane and cytosolic domain). In some embodiments, the polynucleotide encoding the cytosolic and transmembrane domain comprises a nucleotide sequence having at least 99% sequence identity' with the nucleotide sequence of SEQ ID NO: 41 (CD4 transmembrane and cytosolic domain). In some embodiments, the polynucleotide encoding the cytosolic and transmembrane domain comprises the nucleotide sequence of SEQ ID NO: 41 (CD4 transmembrane and cytosolic domain). In some embodiments, the cytosolic and transmembrane domain comprise an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 42 (CD4 transmembrane and cytosolic domain). In some embodiments, the cytosolic and transmembrane domain comprise an amino acid sequence having at least 75% sequence identity with the amino acid sequence of SEQ ID NO: 42 (CD4 transmembrane and cytosolic domain). In some embodiments, the cytosolic and transmembrane domain comprise an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 42 (CD4 transmembrane and cytosolic domain). In some embodiments, the cytosolic and transmembrane domain comprise an amino acid sequence having at least 85% sequence identity with the ammo acid sequence of SEQ ID NO: 42 (CD4 transmembrane and cytosolic domain). In some embodiments, the cytosolic and transmembrane domain comprise an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 42 (CD4 transmembrane and cytosolic domain). In some embodiments, the cytosolic and transmembrane domain comprise an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 42 (CD4 transmembrane and cytosolic domain). In some embodiments, the cytosolic and transmembrane domain comprise an amino acid sequence having at least 96% sequence identity with the amino acid sequence of SEQ ID NO: 42 (CD4 transmembrane and cytosolic domain). In some embodiments, the cytosolic and transmembrane domain comprise an amino acid sequence having at least 97% sequence identity with the amino acid sequence of SEQ ID NO: 42 (CD4 transmembrane and cytosolic domain). In some embodiments, the cytosolic and transmembrane domain comprise an amino acid sequence having at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 42 (CD4 transmembrane and cytosolic domain). In some embodiments, the cytosolic and transmembrane domain comprise an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 42 (CD4 transmembrane and cytosolic domain). In some embodiments, the cytosolic and transmembrane domain comprise the amino acid sequence of SEQ ID NO: 42 (CD4 transmembrane and cytosolic domain).

[0077] In some embodiments, the TCR co-receptor is CD8. In some embodiments, the TCR coreceptor is CD8a. In some embodiments, the polynucleotide encoding the cytosolic and transmembrane domain comprises a nucleotide sequence having at least 70% sequence identity with the nucleotide sequence of SEQ ID NO: 43 (CD8 transmembrane and cytosolic domain. In some embodiments, the polynucleotide encoding the cytosolic and transmembrane domain comprises a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence of SEQ ID NO: 43 (CD8 transmembrane and cytosolic domain). In some embodiments, the polynucleotide encoding the cytosolic and transmembrane domain comprises a nucleotide sequence having at least 90% sequence identity with the nucleotide sequence of SEQ ID NO: 43 (CD8 transmembrane and cytosolic domain). In some embodiments, the polynucleotide encoding the cytosolic and transmembrane domain comprises a nucleotide sequence having at least 95% sequence identity with the nucleotide sequence of SEQ ID NO: 43 (CD8 transmembrane and cytosolic domain In some embodiments, the polynucleotide encoding the cytosolic and transmembrane domain comprises a nucleotide sequence having at least 99% sequence identity with the nucleotide sequence of SEQ ID NO: 43 (CD8 transmembrane and cytosolic domain). In some embodiments, the polynucleotide encoding the cytosolic and transmembrane domain comprises the nucleotide sequence of SEQ ID NO: 43 (CD8 transmembrane and cytosolic domain). In some embodiments, the cytosolic and transmembrane domain comprise an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 44 (CD8 transmembrane and cytosolic domain). In some embodiments, the cytosolic and transmembrane domain comprise an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 44 (CD8 transmembrane and cytosolic domain). In some embodiments, the cytosolic and transmembrane domain comprise an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 44 (CD8 transmembrane and cytosolic domain). In some embodiments, the cytosolic and transmembrane domain comprise an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 44 (CDS transmembrane and cytosolic domain). In some embodiments, the cytosolic and transmembrane domain comprise an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 44 (CD8 transmembrane and cytosolic domain). In some embodiments, the cytosolic and transmembrane domain comprise the amino acid sequence of SEQ ID NO: 44 (CD8 transmembrane and cytosolic domain).

[0078] In some embodiments, the TCR signaling domain polypeptide comprises a chimera of sequences or domains from co-receptors. In some embodiments, the TCR signaling domain polypeptide comprises a chimera of CD8a and CD8P, wherein the CD8a arginine rich region is replaced with the CD8P arginine rich region (CD8a+R(P) chimera). In some embodiments, the polynucleotide encoding the cytosolic and transmembrane domain composes a nucleotide sequence having at least 70% sequence identity with the nucleotide sequence of SEQ ID NO: 45 (CD8a+R(P) chimera). In some embodiments, the polynucleotide encoding the cytosolic and transmembrane domain comprises a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence of SEQ ID NO: 45 (CD8a+R(P) chimera). In some embodiments, the polynucleotide encoding the cytosolic and transmembrane domain comprises a nucleotide sequence having at least 90% sequence identity with the nucleotide sequence of SEQ ID NO: 45 (CD8a+R(P) chimera). In some embodiments, the polynucleotide encoding the cytosolic and transmembrane domain comprises a nucleotide sequence having at least 95% sequence identity with the nucleotide sequence of SEQ ID NO: 45 (CD8a+R(P) chimera). In some embodiments, the polynucleotide encoding the cytosolic and transmembrane domain comprises a nucleotide sequence having at least 99% sequence identity with the nucleotide sequence of SEQ ID NO: 45 (CD8a+R(P) chimera). In some embodiments, the polynucleotide encoding the cytosolic and transmembrane domain comprises the nucleotide sequence of SEQ ID NO: 45 (CD8a+R(P) chimera). In some embodiments, the cytosolic and transmembrane domain comprise an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 46 (CD8a+R(P) chimera). In some embodiments, the cytosolic and transmembrane domain comprise an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 46 (CD8a+R(P) chimera). In some embodiments, the cytosolic and transmembrane domain comprise an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 46 (CD8a+R(P) chimera). In some embodiments, the cytosolic and transmembrane domain comprise an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 46 (CD8a+R(P) chimera). In some embodiments, the cytosolic and transmembrane domain comprise an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 46 (CD8a+R(P) chimera). In some embodiments, the cytosolic and transmembrane domain comprise the amino acid sequence of SEQ ID NO: 46 (CD8a+R(P) chimera).

[0079] In some embodiments, the TCR signaling domain polypeptide comprises a chimera of CD8a and CD8P, where the CD8a CXCP domain, which contains an Lek binding motif, is appended to the C-terminus of the CD8P cytosolic domain (CD8P+Lck chimera). In some embodiments, the polynucleotide encoding the cytosolic and transmembrane domain comprises a nucleotide sequence having at least 70% sequence identity with the nucleotide sequence of SEQ ID NO: 47 (CD8p+Lck chimera). In some embodiments, the polynucleotide encoding the cytosolic and transmembrane domain comprises a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence of SEQ ID NO: 47 (CD8P+Lck chimera). In some embodiments, the polynucleotide encoding the cytosolic and transmembrane domain comprises a nucleotide sequence having at least 90% sequence identity with the nucleotide sequence of SEQ ID NO: 47 (CD8p+Lck chimera). In some embodiments, the polynucleotide encoding the cytosolic and transmembrane domain comprises a nucleotide sequence having at least 95% sequence identity with the nucleotide sequence of SEQ ID NO: 47 (CD8p+Lck chimera). In some embodiments, the polynucleotide encoding the cytosolic and transmembrane domain comprises a nucleotide sequence having at least 99% sequence identity with the nucleotide sequence of SEQ ID NO: 47 (CD8p+Lck chimera). In some embodiments, the polynucleotide encoding the cytosolic and transmembrane domain comprises the nucleotide sequence of SEQ ID NO: 47 (CD8p+Lck chimera). In some embodiments, the cytosolic and transmembrane domain comprise an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 48 (CD8p+Lck chimera). In some embodiments, the cytosolic and transmembrane domain comprise an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 48 (CD8P+Lck chimera). In some embodiments, the cytosolic and transmembrane domain comprise an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 48 (CD8p+Lck chimera). In some embodiments, the cytosolic and transmembrane domain comprise an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 48 (CD8P+Lck chimera). In some embodiments, the cytosolic and transmembrane domain comprise an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 48 (CD8p+Lck chimera). In some embodiments, the cytosolic and transmembrane domain comprise the amino acid sequence of SEQ ID NO: 48 (CD8P+Lck chimera).

[0080] In some embodiments, the TCR signaling domain polypeptide includes both a cytosolic domain and a transmembrane domain of a TCR co-receptor protein. In some embodiments, the cytosolic domain and transmembrane domain are from the same co-receptor or from different co-receptors.

[0081] Amino acid and nucleotide sequences of exemplary transmembrane and cytosolic domains are provided in Table 3.

Table 3: Table of Sequences

¹ Extracellular linker, nucleotides 1-66; Transmembrane domain, nucleotides 67-132; Cytosolic domain, nucleotides 133-254

² Extracellular linker, amino acids 1-22; Transmembrane domain, amino acids 23-44; Cytosolic domain, amino acids 45-84

Target Antigen-Binding Domain

[0082] The target antigen-binding domain refers to any substance or molecule that binds, directly or indirectly, to a target antigen. Target-specific antigen-binding domains include, but are not limited to, antibodies and fragments thereof, for example single chain antibodies such as single-chain antibodies (scFvs), single domain antibodies (e.g., nanobodies), peptides, peptidomimetics, proteins, glycoproteins, or proteoglycans that bind to the target cell and/or antigen. In some embodiments, the target-specific ligands include, but are not limited to, designed ankyrin repeat proteins (DARPins), lectins, knottins, centryrins, anticalins, or naturally occurring ligands for the tumor antigen, such as growth factors, enzyme substrates, receptors or binding proteins. In some embodiments, target specific ligands include non-protein compounds that bind to target cells and/or antigens, including but not limited to carbohydrates, lipids, nucleic acids, or small molecules. In some embodiments, a target-specific ligand is a designed ankyrin repeat protein (DARPin) targeted to a specific cell and/or antigen. In some embodiments, a target-specific ligand is a single-chain variable fragment (scFv) targeted to a specific cell and/or antigen, antigen. In some embodiments, a target-specific ligand is a nanobody (VHH) targeted to a specific cell and/or antigen.

[0083] In some embodiments, the target antigen is expressed on a cancer/tumor cell. In some embodiments, the target antigen is expressed on a metastatic cancer cell. In some embodiments, the target antigen is human epidermal growth factor receptor 2 (HER2), B cell maturation antigen (BCMA), cluster of differentiation 19 (CD19), Claudin-18.2 (CLDN18.2), guanylate cyclase 2C (GUCY2C), or glypican 3 (GPC3).

[0084] In some embodiments, the target antigen is HER2. In some embodiments, the HER2- binding domain comprises an antigen-binding domain of an antibody selected from Trastuzumab, Pertuzumab, Lapatinib, Neratinib, Ado-trastuzmab Emtansine, Gancotamab, Margetuximab, Timigutuzumab, and Ertumaxomab. In some embodiments, the HER2-binding domain is a DARPin that specifically binds a HER2 antigen. In some embodiments, the DARPin targeted to HER2 comprises SEQ ID NO: 49.

[0085] In some embodiments, the HER2-binding domain comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 49. In some embodiments, the HER2- bmding domain comprises an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 49. In some embodiments, the HER2-binding domain comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 49. In some embodiments, the HER2-binding domain comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 49. In some embodiments, the HER2 -binding domain comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 49. In some embodiments, the HER2-binding domain comprises an amino acid sequence having at least 95% sequence identity' with SEQ ID NO: 49. In some embodiments, the HER2-binding domain comprises an amino acid sequence of SEQ ID NO: 49.

[0086] In some embodiments, the target antigen is BCMA. In some embodiments, the BCMA- binding domain comprises an antigen-binding domain of an antibody selected from Belantamab mafodotin, and GSK2857916. In some embodiments, the target-binding domain is an scFv that specifically binds BCMA. In some embodiments, the scFv that binds BCMA comprises SEQ ID NO: 50.

[0087] In some embodiments, the BCMA-binding domain comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 50. In some embodiments, the BCMA- binding domain comprises an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 50. In some embodiments, the BCMA-binding domain comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 50. In some embodiments, the BCMA-binding domain comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 50. In some embodiments, the BCMA-binding domain comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 50. In some embodiments, the BCMA-binding domain comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 50. In some embodiments, the BCMA-binding domain comprises an amino acid sequence of SEQ ID NO: 50.

[0088] In some embodiments, the target antigen is CD19. In some embodiments, the CD19- binding domain is an scFv that selectively binds a CD 19 antigen. In some embodiments, the scFv targeted to CD 19 comprises SEQ ID NO: 51. [0089] In some embodiments, the CD19-binding domain comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 51. In some embodiments, the CD19- binding domain comprises an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 51. In some embodiments, the CD19-binding domain comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 51. In some embodiments, the CD19-binding domain comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 51. In some embodiments, the CD19-binding domain comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 51. In some embodiments, the CD19-bindmg domain comprises an ammo acid sequence having at least 95% sequence identity' with SEQ ID NO: 51. In some embodiments, the CD19-binding domain comprises an amino acid sequence of SEQ ID NO: 51.

[0090] In some embodiments, the target antigen is Claudin 18.2. In some embodiments, the Claudin 18.2-binding domain is a nanobody that selectively binds a Claudin 18.2 antigen. In some embodiments, the scFv targeted to Claudin 18.2 comprises SEQ ID NO: 52 or 53.

[0091] In some embodiments, the Claudin 18.2-binding domain comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 52. In some embodiments, the Claudin 18.2-binding domain comprises an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 52. In some embodiments, the Claudin 18.2-binding domain comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 52. In some embodiments, the Claudin 18.2-binding domain comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 52. In some embodiments, the Claudin 18.2-binding domain comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 52. In some embodiments, the Claudin 18.2-binding domain comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 52. In some embodiments, the Claudin 18.2-binding domain comprises an amino acid sequence of SEQ ID NO: 52.

[0092] In some embodiments, the Claudin 18.2-binding domain comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 53. In some embodiments, the Claudin 18.2-binding domain comprises an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 53. In some embodiments, the Claudin 18.2-binding domain comprises an ammo acid sequence having at least 80% sequence identity with SEQ ID NO: 53. In some embodiments, the Claudin 18.2-binding domain comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 53. In some embodiments, the Claudin 18.2-binding domain comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 53. In some embodiments, the Claudin 18.2-binding domain comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 53. In some embodiments, the Claudin 18.2-binding domain comprises an amino acid sequence of SEQ ID NO: 53.

[0093] In some embodiments, the target antigen is GUCY2C. In some embodiments, the GUCY2C -binding domain is an scFv that selectively binds a GUCY2C antigen. In some embodiments, the scFv targeted to GUCY2C comprises any one of SEQ ID NOs: 54-117. In some embodiments, the GUCY2C -binding domain is a nanobody that selectively binds a GUCY2C antigen. In some embodiments, the scFv targeted to GUCY2C comprises any one of SEQ ID NOs: 118-136.

[0094] In some embodiments, the GUCY2C-binding domain comprises an amino acid sequence having at least 70% sequence identity with any one of SEQ ID NOs: 54-117. In some embodiments, the GUCY2C-binding domain comprises an amino acid sequence having at least 75% sequence identity with any one of SEQ ID NOs: 54-117. In some embodiments, the GUCY2C -binding domain comprises an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 54-117. In some embodiments, the GUCY2C-binding domain comprises an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 54-117. In some embodiments, the GUCY2C -binding domain comprises an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NOs: 54- 117. In some embodiments, the GUCY2C-binding domain comprises an amino acid sequence having at least 95% sequence identity with any one of SEQ ID NOs: 54-117. In some embodiments, the GUCY2C-binding domain comprises an amino acid sequence of SEQ ID NO: 54-117.

[0095] In some embodiments, the Claudin 18.2-binding domain comprises an amino acid sequence having at least 70% sequence identity with any one of SEQ ID NOs: 118-136. In some embodiments, the Claudin 18.2-binding domain comprises an amino acid sequence having at least 75% sequence identity with any one of SEQ ID NOs: 118-136. In some embodiments, the Claudin 18.2-binding domain comprises an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 118-136. In some embodiments, the Claudin 18.2-binding domain comprises an ammo acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 118-136. In some embodiments, the Claudin 18.2-binding domain comprises an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NOs: 118-136. In some embodiments, the Claudin 18.2-binding domain comprises an amino acid sequence having at least 95% sequence identity with any one of SEQ ID NOs: 118-136. In some embodiments, the Claudin 18.2-binding domain comprises an amino acid sequence of SEQ ID NO: 118-136.

[0096] In some embodiments, the target antigen is GPC3. In some embodiments, the GPC3- binding domain is a nanobody that selectively binds a GPC3 antigen. In some embodiments, the scFv targeted to GPC3 comprises SEQ ID NO: 137.

[0097] In some embodiments, the GPC3-binding domain comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 137. In some embodiments, the GPC3- binding domain comprises an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 137. In some embodiments, the GPC3-binding domain comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 137. In some embodiments, the GPC3-binding domain comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 137. In some embodiments, the GPC3-binding domain comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 137. In some embodiments, the GPC3-binding domain comprises an amino acid sequence having at least 95% sequence identity' with SEQ ID NO: 137. In some embodiments, the GPC3-binding domain comprises an amino acid sequence of SEQ ID NO: 137.

[0098] Amino acid sequences of exemplary antigen-binding domains that bind a target antigen are provided in Table 4.

Table 4: Table of Sequences

Specific TACs

[0099] In some embodiments, the TAC disclosed herein is a HER2-TAC. In some embodiments, the HER2-TAC comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 138. In some embodiments, the HER2-TAC comprises an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 138. In some embodiments, the HER2- TAC comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 138. In some embodiments, the HER2-TAC comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 138. In some embodiments, the HER2-TAC an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 138. In some embodiments, the HER2-TAC comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 138. In some embodiments, the HER2-TAC comprises an amino acid sequence of SEQ ID NO: 138.

[0100] In some embodiments, the HER2-TAC comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 139. In some embodiments, the HER2-TAC compnses an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 139. In some embodiments, the HER2-TAC comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 139. In some embodiments, the HER2-TAC comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 139. In some embodiments, the HER2-TAC an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 139. In some embodiments, the HER2-TAC comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 139. In some embodiments, the HER2-TAC comprises an amino acid sequence of SEQ ID NO: 139.

[0101] In some embodiments, the HER2-TAC comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 140. In some embodiments, the HER2-TAC comprises an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 140. In some embodiments, the HER2-TAC comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 140. In some embodiments, the HER2-TAC comprises an ammo acid sequence having at least 85% sequence identity with SEQ ID NO: 140. In some embodiments, the HER2-TAC an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 140. In some embodiments, the HER2-TAC comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 140. In some embodiments, the HER2-TAC comprises an amino acid sequence of SEQ ID NO: 140.

[0102] In some embodiments, the TAC disclosed herein is a BCMA-TAC. In some embodiments, the BCMA-TAC comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 141. In some embodiments, the BCMA-TAC comprises an amino acid sequence having at least 75% sequence identity' with SEQ ID NO: 141. In some embodiments, the BCMA-TAC comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 141. In some embodiments, the BCMA-TAC comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 141. In some embodiments, the BCMA-TAC an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 141. In some embodiments, the BCMA-TAC comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 141. In some embodiments, the BCMA-TAC comprises an amino acid sequence of SEQ ID NO: 141.

[0103] In some embodiments, the BCMA-TAC comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 142. In some embodiments, the BCMA-TAC comprises an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 142. In some embodiments, the BCMA-TAC comprises an ammo acid sequence having at least 80% sequence identity' with SEQ ID NO: 142. In some embodiments, the BCMA-TAC comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 142. In some embodiments, the BCMA-TAC an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 142. In some embodiments, the BCMA-TAC comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 142. In some embodiments, the BCMA-TAC comprises an amino acid sequence of SEQ ID NO: 142.

[0104] In some embodiments, the BCMA-TAC comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 143. In some embodiments, the BCMA-TAC comprises an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 143. In some embodiments, the BCMA-TAC comprises an amino acid sequence having at least 80% sequence identity' with SEQ ID NO: 143. In some embodiments, the BCMA-TAC comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 143. In some embodiments, the BCMA-TAC an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 143. In some embodiments, the BCMA-TAC comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 143. In some embodiments, the BCMA-TAC comprises an amino acid sequence of SEQ ID NO: 143.

[0105] In some embodiments, the BCMA-TAC comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 144. In some embodiments, the BCMA-TAC comprises an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 144. In some embodiments, the BCMA-TAC comprises an amino acid sequence having at least 80% sequence identity' with SEQ ID NO: 144. In some embodiments, the BCMA-TAC comprises an ammo acid sequence having at least 85% sequence identity with SEQ ID NO: 144. In some embodiments, the BCMA-TAC an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 144. In some embodiments, the BCMA-TAC comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 144. In some embodiments, the BCMA-TAC comprises an amino acid sequence of SEQ ID NO: 144.

[0106] In some embodiments, the TAC disclosed herein is a CD19-TAC. In some embodiments, the CD19-TAC comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 145. In some embodiments, the CD19-TAC comprises an ammo acid sequence having at least 75% sequence identity with SEQ ID NO: 145. In some embodiments, the CD19- TAC comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 145. In some embodiments, the CD19-TAC comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 145. In some embodiments, the CD19-TAC an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 145. In some embodiments, the CD19-TAC comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 145. In some embodiments, the CD19-TAC comprises an amino acid sequence of SEQ ID NO: 145.

[0107] In some embodiments, the TAC disclosed herein is a Claudin 18.2-TAC. In some embodiments, the Claudin 18.2-TAC comprises an amino acid sequence having at least 70% sequence identity' with SEQ ID NO: 146. In some embodiments, the Claudin 18.2-TAC comprises an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 146. In some embodiments, the Claudin 18.2-TAC comprises an ammo acid sequence having at least 80% sequence identity with SEQ ID NO: 146. In some embodiments, the Claudin 18.2-TAC comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 146. In some embodiments, the Claudin 18.2-TAC an amino acid sequence having at least 90% sequence identity' with SEQ ID NO: 146. In some embodiments, the Claudin 18.2-TAC comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 146. In some embodiments, the Claudin 18.2-TAC comprises an amino acid sequence of SEQ ID NO: 146.

[0108] In some embodiments, the Claudin 18.2-TAC comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 147. In some embodiments, the Claudin 18.2- TAC comprises an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 147. In some embodiments, the Claudin 18.2-TAC comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 147. In some embodiments, the Claudin 18.2-TAC comprises an ammo acid sequence having at least 85% sequence identity with SEQ ID NO: 147. In some embodiments, the Claudin 18.2-TAC an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 147. In some embodiments, the Claudin 18.2- TAC comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 147. In some embodiments, the Claudin 18.2-TAC comprises an amino acid sequence of SEQ ID NO: 147.

[0109] In some embodiments, the Claudin 18.2-TAC comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 148. In some embodiments, the Claudin 18.2- TAC comprises an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 148. In some embodiments, the Claudin 18.2-TAC comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 148. In some embodiments, the Claudin 18.2-TAC comprises an ammo acid sequence having at least 85% sequence identity with SEQ ID NO: 148. In some embodiments, the Claudin 18.2-TAC an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 148. In some embodiments, the Claudin 18.2- TAC comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 148. In some embodiments, the Claudin 18.2-TAC comprises an amino acid sequence of SEQ ID NO: 148.

[0110] In some embodiments, the Claudin 18.2-TAC comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 149. In some embodiments, the Claudin 18.2- TAC comprises an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 149. In some embodiments, the Claudin 18.2-TAC comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 149. In some embodiments, the Claudin 18.2-TAC comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 149. In some embodiments, the Claudin 18.2-TAC an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 149. In some embodiments, the Claudin 18.2- TAC comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 149. In some embodiments, the Claudin 18.2-TAC comprises an amino acid sequence of SEQ ID NO: 149.

[OHl] In some embodiments, the TAC disclosed herein is a GUCY2C-TAC. In some embodiments, the GUCY2C-TAC comprises an amino acid sequence having at least 70% sequence identity with any one of SEQ ID NOs: 150-206. In some embodiments, the GUCY2C- TAC comprises an amino acid sequence having at least 75% sequence identity with any one of SEQ ID NOs: 150-206. In some embodiments, the GUCY2C-TAC comprises an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 150-206. In some embodiments, the GUCY2C-TAC comprises an amino acid sequence having at least 85% sequence identity' with any one of SEQ ID NOs: 150-206. In some embodiments, the GUCY2C- TAC an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NOs: 150-206. In some embodiments, the GUCY2C-TAC comprises an amino acid sequence having at least 95% sequence identity with any one of SEQ ID NOs: 150-206. In some embodiments, the GUCY2C-TAC comprises an amino acid sequence of any one of SEQ ID NOs: 150-206.

[0112] In some embodiments, the TAC disclosed herein is a GPC3-TAC. In some embodiments, the GPC3-TAC comprises an amino acid sequence having at least 70% sequence identity' with SEQ ID NO: 207. In some embodiments, the GPC3-TAC comprises an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 207. In some embodiments, the GPC3- TAC comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 207. In some embodiments, the GPC3-TAC comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 207. In some embodiments, the GPC3-TAC an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 207. In some embodiments, the GPC3-TAC comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 207. In some embodiments, the GPC3-TAC comprises an amino acid sequence of SEQ ID NO: 207.

[0113] Amino acid sequences of exemplary TACs are provided in Table 5.

Table 5: Table of Sequences

Immune Checkpoint Inhibitors

[0114] In certain embodiments, the TACs disclosed herein are expressed in T cells (e.g., a(3 T cells, y5 T cells). In some embodiments, TAC-expressing T cells disclosed herein are administered to subjects in need thereof as part of a combination therapy. In some embodiments, the combination therapy comprises co-administration of an immune-checkpoint inhibitor.

Immune checkpoint inhibitors can be used to support sustained activation of engineered T cells, for example, by blocking negative feedback loops that suppress effector T cell activation.

[0115] One example of a negative feedback loop suppressing sustained T cell activation is the PD-1 immune checkpoint. During activation, T cells increase expression of programmed death protein 1 (PD-1 or PD1), which can bind to its ligands PD-1 ligand 1 (PD-L1) or PD-1 ligand 2 (PD-L2) expressed on normal cells. While this inhibitory negative feedback loop is a natural pathway to regulate and control T cell activation, prolonged binding to PD-L1 can result in functionally exhausted T cells. As a maladaptive function, cancer cells often upregulate PD-L1 expression as a mechanism of immune evasion inhibiting and ultimately causing the accumulation of functionally exhausted T cells. Inhibition of such immune checkpoints can therefore be used to protect T cells from inhibition and exhaustion induced by factors such as PD-1/PD-L1 binding. Other immune checkpoint proteins include CTLA-4, LAG3, BTLA, B7- H3, B7-H4, TIM-3, and KIR.

[0116] In some embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor, a PD-L1 inhibitor, a PD-L2 inhibitor, a CTLA-4 inhibitor, a LAG3 inhibitor, a TIM-3 inhibitor, a BTLA inhibitor, a B7-H3 inhibitor, a B7-H4 inhibitor, or a KIR inhibitor. In some embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor. In some embodiments, the immune checkpoint inhibitor is a PD-L1 inhibitor. In some embodiments, the immune checkpoint inhibitor is a PD- L2 inhibitor. In some embodiments, the immune checkpoint inhibitor is a CTLA-4 inhibitor. In some embodiments, the immune checkpoint inhibitor is a LAG3 inhibitor. In some embodiments, the immune checkpoint inhibitor is a TIM-3 inhibitor. In some embodiments, the immune checkpoint inhibitor is a BTLA inhibitor. In some embodiments, the immune checkpoint inhibitor is aB7-H3 inhibitor. In some embodiments, the immune checkpoint inhibitor is a B7-H4 inhibitor. In some embodiments, the immune checkpoint inhibitor is a KRI inhibitor.

[0117] In some embodiments, the immune checkpoint inhibitor is an antibody or antigenbinding fragment derived therefrom. In some embodiments, the immune checkpoint inhibitor is a small molecule.

[0118] In some embodiments, the immune checkpoint inhibitor is nivolumab, pembrolizumab, cemiplimab, atezolizumab, avelumab, durvalumab, ipilimumab, tremelimumab, or relatlimab. In some embodiments, the immune checkpoint inhibitor is nivolumab. In some embodiments, the immune checkpoint inhibitor is pembrolizumab. In some embodiments, the immune checkpoint inhibitor is cemiplimab. In some embodiments, the immune checkpoint inhibitor is atezolizumab. In some embodiments, the immune checkpoint inhibitor is avelumab. In some embodiments, the immune checkpoint inhibitor is durvalumab. In some embodiments, the immune checkpoint inhibitor is ipilimumab. In some embodiments, the immune checkpoint inhibitor is tremelimumab. In some embodiments, the immune checkpoint inhibitor is relatlimab.

Polypeptides and Vector Constructs

[0119] In some embodiments, the TACs disclosed herein are delivered to a T cell via a vector. In some embodiments, the vectors further comprise a promoter. In some embodiments, the promoter is functional in a mammalian cell. Promoters, regions of DNA that initiate transcription of a particular nucleic acid sequence, are well known in the art. A “promoter functional in a mammalian cell” refers to a promoter that drives expression of the associated nucleic acid sequence in a mammalian cell. A promoter that drives expression of a nucleic acid sequence is referred to as being “operably connected” to the nucleic acid sequence.

[0120] A variety of delivery vectors and expression vehicles are employed to introduce nucleic acids described herein into a cell.

[0121] In some embodiments, the TAC coding sequence is operably connected to the promoter. [0122] In some embodiments, the vector is designed for expression in mammalian cells such as T cells. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a retroviral vector.

[0123] In some embodiments, vectors that are useful comprise vectors derived from lentiviruses, Murine Stem Cell Viruses (MSCV), pox viruses, oncoretroviruses, adenoviruses, and adeno- associated viruses. Other delivery vectors that are useful comprise vectors derived from herpes simplex viruses, transposons, vaccinia viruses, human papilloma virus, Simian immunodeficiency viruses, HTLV, human foamy virus and variants thereof. Further vectors that are useful comprise vectors derived from spumaviruses, mammalian type B retroviruses, mammalian type C retroviruses, avian type C retroviruses, mammalian type D retroviruses and HTLV/BLV type retroviruses. One example of a lentiviral vector useful in the disclosed compositions and methods is the pCCL4 vector.

Expression in T cells

[0124] In some embodiments, the T cell is transduced or transfected with a nucleic acid sequence encoding a HER2 T cell antigen coupler, for example a vector comprising a nucleic acid sequence encoding a HER2. In some embodiments, the T cell is an isolated T cell.

[0125] T cells are obtained from a number of sources, including, but not limited to blood (for example, peripheral blood mononuclear cells), bone marrow, thymus tissue, lymph node tissue, cord blood, thymus tissue, tissue from an infection site, spleen tissue, or tumors.

[0126] In some embodiments, the T cells are autologous T cells. In some embodiments, the T cells are allogenic. In some embodiments, the T cells are a(3 (alpha beta) T cells (z.e., T cell with a TCR composed of one a (alpha) chain and (beta) chain. In some embodiments, the T cells are gamma delta (y5) T cells (i.e. , T cells with a TCR composed of one y (gamma) chain and one 5 (delta) chain.

[0127] In some embodiments, the T cells are obtained from a cell line of T cells. In some embodiments, the T cells are obtained from donors (allogeneic T cells). In some embodiments, the T cells are obtained by differentiation of embryonic or adult stem cells or from induced pluripotent stem cells. In some embodiments, regardless of the source of T cells, the T cells have been modified so that they lack expression of an endogenous TCR and/or permanently or transiently lack expression of MHC/HLA molecules (universal donor T cells). In some embodiments, the T cells are autologous with respect to the subject. In some embodiments, the cells are allogeneic, syngeneic, or xenogeneic with respect to the subject.

[0128] In some embodiments, once obtained, the T cells are optionally enriched in vitro. In some embodiments, a population of cells is enriched by positive or negative selection. Further, the T cells are optionally frozen or cryopreserved and then thawed at a later date.

[0129] In some embodiments, T cells are activated and/or expanded before or after introducing the TAC to the T cells. In some embodiments, the T cells are expanded by contact with a surface having attached thereto an agent that stimulates a CD3/TCR complex associated signal and a ligand that stimulates a co-stimulator molecule on the surface of the T cells. In some embodiments, the T cells are expanded by contact with one or more soluble agents that stimulate CD3/TCR complex signaling and co-stimulator molecule signaling.

[0130] In some embodiments, the T cells are transduced or transfected with nucleic acid sequences. The transduced or transfected T cells express proteins coded for by the transfected or transduced nucleic acid sequences. A nucleic acid may be introduced into a cell by physical, chemical, or biological means. Physical means include, but are not limited to, microinjection, electroporation, particle bombardment, lipofection and calcium phosphate precipitation.

Biological means include the use of DNA and RNA vectors.

[0131] Viral vectors, including retroviral vectors, are used to introduce and express a nucleic acid into a T cell. Viral vectors include vectors derived from lentivirus, Murine Stem Cell Viruses (MSCV), pox viruses, herpes simplex virus I, adenovirus and adeno-associated viruses. The vector optionally includes a promoter that drives expression of the transduced nucleic acid molecule in a T cell (e.g, a CMV promoter, eFla promoter, or MSCV promoter).

[0132] Any suitable assay is used to confirm the presence and/or expression of the transduced nucleic acid sequence and/or the polypeptide encoded by the nucleic acid in the T cell. Assays include, but are not limited to, Southern and Northern blotting, RT-PCR and PCR, ELISA, Western blotting, and flow cytometry.

[0133] A T cell expressing a TAC has increased T cell activation in the presence of an antigen compared to a T cell not expressing a TAC and/or as compared to a T cell expressing a traditional CAR. Increased T cell activation is ascertained by numerous methods, including but not limited to, increased tumor cell line killing, increased cytokine production, increased cytolysis, increased degranulation and/or increased expression of activation markers such as CD107a, IFNy, IL2 or TNFa. In some embodiments, increases are measured in an individual cell or in a population of cells.

[0134] The terms “increased” or “increasing” as used herein refer to at least a 1%, 2%, 5%, 10%, 25%, 50%, 100% or 200% increase in a T cell or population of T cells expressing a TAC compared to a T cell or population of T cells not expressing a TAC and/or as compared to a T cell or population of T cells expressing a traditional CAR.

Pharmaceutical Compositions

[0135] In some embodiments, the T cell expressing the HER2 TAC is administered to an individual in need in a pharmaceutical compositions comprising a HER2 TAC T cell (e.g., an a[l T cell, a y5 T cell) disclosed herein (transduced with and/or expressing a TAC), and a pharmaceutically acceptable carrier.

[0136] Pharmaceutically acceptable carriers include, but are not limited to, buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants {e.g., aluminum hydroxide); or preservatives. In some embodiments, the engineered T cells (e.g., a|3 T cells, y<5 T cells) are formulated for intravenous administration.

[0137] Pharmaceutical compositions are administered in a manner appropriate to the disease to be treated (or prevented). The quantity and frequency of administration is determined by such factors as the condition of the patient, and the type and severity of the patient’s disease, although appropriate dosages are determined by clinical trials. When “an immunologically effective amount,” “an anti-tumor effective amount,” “a tumor-inhibiting effective amount,” or “therapeutic amount” is indicated, the precise amount of the compositions of the present invention to be administered is determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject).

[0138] In some embodiments, the modified T cells (e.g., 0.(3 T cells, y6 T cells) and/or pharmaceutical compositions described herein are administered at a dosage of 10 ¹ to 10 ¹ ’ cells/kg body weight, 10 ⁴ to 10 ⁹ cells/kg body weight, optionally 10 ⁵ to 10 ⁸ cells cells/kg body weight, 10 ⁶ to IO ⁷ cells/kg body weight or 10 ⁵ to 10 ⁶ cells/kg body weight, including all integer values within those ranges. In some embodiments, the modified T cells and/or pharmaceutical compositions described herein are administered at a dosage of greater than 10 ¹ cells/kg body weight. In some embodiments, the modified T cells and/or pharmaceutical compositions described herein are administered at a dosage of less than 10 ¹⁵ cells/kg body weight. In some embodiments, the modified T cells are administered at a dosage of about 6* 10 ⁴ to about 6* 10 ⁸ cells/kg body weight. In some embodiments, the modified T cells are administered at a dosage of about I x lO ⁵ to about 8xio ⁶ cells/kg body weight. In some embodiments, the modified T cells are administered at a dosage of about 6xl0 ⁴ to 8xl0 ⁴ cells/kg body weight. In some embodiments, the modified T cells are administered at a dosage of about I x lO ⁵ to 3*10 ⁵ cells/kg body weight. In some embodiments, the modified T cells are administered at a dosage of about 6x l0 ⁵ to 8x l0 ⁵ cells/kg body weight. In some embodiments, the modified T cells are administered at a dosage of about 1 xio ⁶ to 3xl0 ⁶ cells/kg body weight. In some embodiments, the modified T cells are administered at a dosage of about 6xl0 ⁶ to 8x l0 ⁶ cells/kg body weight. [0139] In some embodiments, the modified T cells (e.g., a0 T cells, y5 T cells) and/or pharmaceutical compositions described herein are administered at a dosage of 0.5 x 10 ⁴ cells, I x lO ⁴ cells, 2xl0 ⁴ cells, 4 l0 ⁴ cells, 0.5x l0 ⁵ cells, 2xl0 ⁵ cells, 4xl0 ⁵ cells, 0.5xl0 ⁶ cells, 2xl0 ⁶ cells, 4xl0 ⁵ cells, 5xl0 ⁶ cells, 1.2xl0 ⁷ cells, 2xl0 ⁷ cells, 5xl0 ⁷ cells, 2x l0 ⁸ cells, 5xl0 ⁸ cells, or 2x l0 ⁹ cells. In some embodiments, the modified T cells and/or pharmaceutical compositions described herein are administered at a dosage in a range of 0.005-2000x 10 ⁶ cells, 0.01-2000x 10 ⁶ cells, 0.025-2000xl0 ⁶ cells, 0.05-2000xl0 ⁶ cells, 0.1-2000x l0 ⁶ cells, 0.25-2000xl0 ⁶ cells, 0.5- 2000xl0 ⁶ cells, 0.5-2xl0 ⁴ cells, 0.5-2xl0 ⁵ cells, 0.5-2xl0 ⁶ cells, 0.5-2xl0 ⁷ cells, 0.5-2x l0 ⁸ cells, or 0.5-2xl0 ⁹ cells, including all integer values within those ranges.

[0140] Also disclosed herein are pharmaceutical compositions comprising engineered/modified and unmodified T cells (e.g., ot(3 T cells, y6 T cells), or comprising different populations of engineered/modified T cells with or without unmodified T cells. One of ordinary skill in the art would understand that a therapeutic quantity of engineered/modified T cells need not be homogenous in nature. In some embodiments, activated engineered/modified T cells further activate unmodified T cells (e.g., a bystander T cell) within the same pharmaceutical composition/cell population (referred hereinto as the “bystander effect”). In some embodiments, engineered/modified T cells activate unmodified T cells only when activated in response to binding of the antigen to the TAC expressed by the engineered/modified T cell.

[0141] In some embodiments, T cell (e.g., an a.f> T cell, a yd T cell) compositions are administered multiple times at these dosages. In some embodiments, the dosage is administered a single time or multiple times, for example daily, weekly, biweekly, or monthly, hourly, or is administered upon recurrence, relapse or progression of the cancer being treated. The cells, in some embodiments, are administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988).

[0142] In some embodiments, the modified T cells (e.g., a|3 T cells, y5 T cells) and/or pharmaceutical compositions described herein are administered in combination with an immune checkpoint inhibitor (e.g., nivolumab, pembrolizumab, cemiplimab, atezolizumab, avelumab, and durvalumab). In some embodiments, the immune checkpoint inhibitor is pembrolizumab.

[0143] In some embodiments, pembrolizumab is administered at a dose of about 0.1-1 mg/kg, 1- 5 kg/mg, 1-10 kg/mg, 1-15 kg/mg, 5-10 kg/mg, 5-15 kg/mg, or 10-15 kg/mg. In some embodiments, pembrolizumab is administered at a dose of about 1 kg/mg. In some embodiments, pembrolizumab is administered at a dose of about 2 kg/mg. In some embodiments, pembrolizumab is administered at a dose of about 3 kg/mg. In some embodiments, pembrolizumab is administered at a dose of about 4 kg/mg. In some embodiments, pembrolizumab is administered at a dose of about 5 kg/mg. In some embodiments, pembrolizumab is administered at a dose of about 6 kg/mg. In some embodiments, pembrolizumab is administered at a dose of about 7 kg/mg. In some embodiments, pembrolizumab is administered at a dose of about 8 kg/mg. In some embodiments, pembrolizumab is administered at a dose of about 9 kg/mg. In some embodiments, pembrolizumab is administered at a dose of about 10 kg/mg. In some embodiments, pembrolizumab is administered at a dose of about 11 kg/mg. In some embodiments, pembrolizumab is administered at a dose of about 12 kg/mg. In some embodiments, pembrolizumab is administered at a dose of about 13 kg/mg. In some embodiments, pembrolizumab is administered at a dose of about 14 kg/mg. In some embodiments, pembrolizumab is administered at a dose of about 15 kg/mg.

[0144] In some embodiments, pembrolizumab is administered as a single dose. In some embodiments, the pembrolizumab is administered over multiple doses. In some embodiments, multiple equivalent dose amounts of pembrolizumab are administered. In some embodiments, multiple different dose amounts of pembrolizumab are administered.

[0145] In some embodiments, the dosage interval is about 1-2 days, 2-3 days, 3-5 days, 4-5 days, 5-6 days, 6-7 days, 1-2 weeks, 2-3 weeks, 3-4 weeks, or 4-5 weeks for subsequent doses to the first dose. In some embodiments, the dosage interval is approximately 4-5 days for subsequent doses to the first dose.

[0146] In some embodiments, the first dose is administered at a dose of about 1-10 kg/mg. In some embodiments, the first dose is administered at a dose of about 10 kg/mg. In some embodiments, the first dose is administered at a dose of about 9 kg/mg. In some embodiments, the first dose is administered at a dose of about 8 kg/mg. In some embodiments, the first dose is administered at a dose of about 7 kg/mg. In some embodiments, the first dose is administered at a dose of about 6 kg/mg. In some embodiments, the first dose is administered at a dose of about 5 kg/mg. In some embodiments, the first dose is administered at a dose of about 4 kg/mg. In some embodiments, the first dose is administered at a dose of about 3 kg/mg. In some embodiments, the first dose is administered at a dose of about 2 kg/mg. In some embodiments, the first dose is administered at a dose of about 1 kg/mg.

[0147] In some embodiments, the subsequent dose is administered at a dose of about 1-10 kg/mg. In some embodiments, the subsequent dose is administered at a dose of about 10 kg/mg. In some embodiments, the subsequent dose is administered at a dose of about 9 kg/mg. In some embodiments, the subsequent dose is administered at a dose of about 8 kg/mg. In some embodiments, the subsequent dose is administered at a dose of about 7 kg/mg. In some embodiments, the subsequent dose is administered at a dose of about 6 kg/mg. In some embodiments, the subsequent dose is administered at a dose of about 5 kg/mg. In some embodiments, the subsequent dose is administered at a dose of about 4 kg/mg. In some embodiments, the subsequent dose is administered at a dose of about 3 kg/mg. In some embodiments, the subsequent dose is administered at a dose of about 2 kg/mg. In some embodiments, the subsequent dose is administered at a dose of about 1 kg/mg. In some embodiments, a first 10 mg/kg dose of pembrolizumab is followed by subsequent dose of 5 mg/kg of pembrolizumab every 4-5 days over a full course of treatment.

[0148] The pharmaceutical composition is substantially free of, e.g, there are no detectable levels of a contaminant, e.g., selected from the group consisting of endotoxin, mycoplasma, replication competent lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag, residual anti- CD3/anti-CD28 coated beads, mouse antibodies, pooled human serum, bovine serum albumin, bovine serum, culture media components, vector packaging cell or plasmid components, a bacterium a fungus, mycoplasma, IL-2, and IL-7.

[0149] In some embodiments, engineered T-cells disclose herein are administered to a subject and blood is subsequently redrawn (or apheresis performed), T-cells therefrom are activated and reinfused into the patient with engineered T cells. This process, in some embodiments, is carried out multiple times every few weeks. T-cells are activated from blood draws of from 10 cc to 400 cc. T-cells are activated from blood draws of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc. [0150] The modified/engineered T cells and/or pharmaceutical compositions are administered by methods including, but not limited to, aerosol inhalation, injection, infusion, ingestion, transfusion, implantation or transplantation. The modified T cells and/or pharmaceutical compositions are administered to a subject transarterially, subcutaneously, intradermally, intratumorally, intranodally, intrameduliary, intramuscularly, by intravenous (i.v.) injection, by intravenous (i.v.) infusion, or intraperitoneally. The modified/engineered T cells and/or pharmaceutical compositions thereof are administered to a patient by intradermal or subcutaneous injection. The modified/engineered T cells and/or pharmaceutical compositions thereof are administered by i.v. injection. The modified/engineered T cells and/or pharmaceutical compositions thereof are injected directly into a tumor, lymph node, or site of infection.

[0151] The modified/engineered T cells T cells and/or pharmaceutical compositions are administered in a volume of about 5 mL, 10 mL, 15 mL, 20 mL, 25 mL, 30 mL, 35 mL, 40 mL, 45 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 110 mL, 120 mL, 130 mL, 140 mL, 150 mL, 200 mL, 300 mL, 400 mL, or 500 mL.

[0152] The modified/engineered T cells T cells and/or pharmaceutical compositions are administered in a volume of at greater than at most about 5 mL, 10 mL, 15 mL, 20 mL, 25 mL, 30 mL, 35 mL, 40 mL, 45 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 110 mL, 120 mL, 130 mL, 140 mL, 150 mL, 200 mL, 300 mL, 400 mL, or 500 mL.

[0153] The modified/engineered T cells T cells and/or pharmaceutical compositions are administered in a volume of at least about 5 mL, 10 mL, 15 mL, 20 mL, 25 mL, 30 mL, 35 mL, 40 mL, 45 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 110 mL, 120 mL, 130 mL, 140 mL, 150 mL, 200 mL, 300 mL, 400 mL, or 500 mL.

[0154] A pharmaceutical composition is prepared by per se known methods for the preparation of pharmaceutically acceptable compositions that are administered to subjects, such that an effective quantity of the T cells is combined in a mixture with a pharmaceutically acceptable carrier. Suitable carriers are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, 20th ed., Mack Publishing Company, Easton, Pa., USA, 2000). On this basis, the compositions include, albeit not exclusively, solutions of the substances in association with one or more pharmaceutically acceptable carriers or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids.

[0155] Suitable pharmaceutically acceptable carriers include essentially chemically inert and nontoxic compositions that do not interfere with the effectiveness of the biological activity of the pharmaceutical composition. Examples of suitable pharmaceutical carriers include, but are not limited to, water, saline solutions, glycerol solutions, N-(l(2,3-dioleyloxy)propyl)N,N,N- trimethylammonium chloride (DOTMA), diolesylphosphotidyl-ethanolamine (DOPE), and liposomes. In some embodiments, such compositions contain a therapeutically effective amount of the compound, together with a suitable amount of carrier so as to provide the form for direct administration to the patient.

[0156] Pharmaceutical compositions include, without limitation, lyophilized powders or aqueous or non-aqueous sterile injectable solutions or suspensions, which may further contain antioxidants, buffers, bacteriostats and solutes that render the compositions substantially compatible with the tissues or the blood of an intended recipient. Other components that may be present in such compositions include water, surfactants (such as Tween), alcohols, polyols, glycerin and vegetable oils, for example. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, tablets, or concentrated solutions or suspensions.

[0157] A pharmaceutical composition disclosed herein is formulated into a variety of forms and administered by a number of different means. A pharmaceutical formulation is administered orally, rectally, or parenterally, in formulations containing conventionally acceptable carriers, adjuvants, and vehicles as desired. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, or intrastemal injection and infusion techniques. Administration includes injection or infusion, including intra-arterial, intracardiac, intracerebroventricular, intradermal, intraduodenal, intramedullary , intramuscular, intraosseous, intraperitoneal, intrathecal, intravascular, intravenous, intravitreal, epidural and subcutaneous), inhalational, transdermal, transmucosal, sublingual, buccal and topical (including epicutaneous, dermal, enema, eye drops, ear drops, intranasal, vaginal) administration. In some exemplary embodiments, a route of administration is via an injection such as an intramuscular, intravenous, subcutaneous, or intraperitoneal injection.

[0158] Liquid formulations include an oral formulation, an intravenous formulation, an intranasal formulation, an ocular formulation, an otic formulation, an aerosol, and the like. In certain embodiments, a combination of various formulations is administered. In certain embodiments a composition is formulated for an extended-release profile.

EXAMPLES [0159] The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion. The present examples, along with the methods described herein are presently representative of preferred embodiments, are exemplary , and are not intended as limitations on the scope of the invention. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.

Example 1: Analysis of immune checkpoint protein expression in TAC-Expressing T cells [0160] T cells were engineered to express aHER2-TAC (SEQ ID NO: 36) and injected at a dosage of 6xl0 ⁶ TAC+ T cells into NSG mice bearing HER2-positive NCI-N87 subcutaneous xenografts once tumors reached a size of approximately 100 mm ³ . Non-transduced T cells were used as negative control. 30 minutes following T cell engraftment, half of mice in treatment and control groups were sacrificed and tissues and blood samples were collected, at which point TAG T cells were not detected in the tumor. Tumor volumes were monitored in remaining mice for 7 days, after which, mice were sacrificed, and tissues were collected and further processed to isolate HER2-TAC T cells for subsequent flow cytometry analysis. A summary of the experiment is depicted in FIG. 1A.

[0161] After 7 days, nearly all mice treated with HER2-TAC T cells showed tumor regression, whereas no tumor regression was observed in mice treated with non-transduced T cells. (FIG. IB). Furthermore, HER2-TAC T cells showed no CD69 or PD-1 upregulation when first administered into animals, indicating that these T cells have not yet encountered any HER2 antigen (FIG. 1C). In contrast, TAC T cells recovered from tumors 7 days post-engraftment showed both upregulation of CD69 and PD-1 , indicating activation (FIG. ID). These observations suggest that TAC T cells become activated by HER2-expressing tumor cells and, consequently, express PD-1, which can lead to T cell exhaustion. Therefore, blocking PD1 signaling may protect T cell from PD-L1 mediated inhibition and ultimately exhaustion and increase the potency of TAC-T cells.

[0162] In a separate experiment, tumor spheroids were created with HER2 ⁺ , GFP-expressing N87 cancer cells grown in the presence of human dermal fibroblasts. Spheroids were treated with T cells engineered to express a HER2-TAC (SEQ ID NO: 36) at effector: target (E:T) ratios of 2: 1, 10: 1, and 20: 1 and observed over 4 days. Non-transduced T cells (NTD) were used at the same ratios as negative controls. Spheroids were also cultured in media only (negative control) or 0.5 pM Staurosporin (STS) (positive control). Spheroids were imaged daily by fluorescence microscopy.

[0163] Over the course of the experiment, spheroids treated with TAC T cells showed a marked reduction in size, particularly at E:T ratios of 10: 1 and 20: 1 (FIG. 2A), whereas there was no discernable reduction of spheroid size in NTD control groups. Furthermore, spheroid cells treated with TAC T cells had reduced percent viability at all E:T ratios based on reduction of GFP fluorescence, as did SPS-treated spheroids. By contrast, NTD control groups did not show any reduced viability (FIG. 2B). Spheroids treated with control or TAC-expressing T cells at an E:T ratio of 2: 1 were fixed with 4% paraformaldehyde after four days of treatment and analyzed via immunohistochemistry. Paraffin sections were stained for hematoxylin and eosin (HE), CD3 (a T cell marker), granzyme B (an activated T cell marker), PD-L1, HER2, and cleaved caspase- 3 (a marker of apoptosis). HER2 expression was confirmed in all tissues. T cell infiltration (CD3) was only observed in HER2-TAC T treated cells. TAC T cell infiltration was accompanied with a significant upregulation of PD-L1 (PD-L1) as well as the secretion of Granzyme B and activation of Caspase 3 (cleaved caspase 3) (FIG. 2C). This data demonstrates that HER2-TAC T cells are capable of infiltrating spheroid tissues and indicates T cell induced tumor cell death via the caspase pathway. Concomitantly, a significant upregulation of PD-L1 in tumor tissue was observed. Since PD-L1 can interact with PD-1 expressed on activated T cells and inhibit T cell function, blocking PD-1 may counteract PD-1 -induced T cell inhibition and ultimately exhaustion and, consequently, increase the potency of TAC T cells.

Example 2: In vitro analysis of an anti-PD-1 antibody/TAC T cell combination therapy [0164] HER2-expressing cancer cell lines BT-474 (breast cancer) and N87 (gastric carcinoma) were engineered to overexpress PD-L1. This led to a significant upregulation in PD-L1 in both BT-474 (BT-474 ^PD ‘ ^L1/H ' ^gh ) (FIG. 3A) and N87 (N87 ^PD - ^L1/Higl1 ) (FIG. 3B) cells relative to the nonengineered parental cell lines.

[0165] The impact of PD-L1 overexpression on HER2-TAC T cells was investigated in a T cell proliferation assay (FIG. 3C). To this end, HER2-TAC T cells or non-transduced control T cells (NTD) were incubated with Cell Trace Violet (CTV) and co-cultured with BT-474, and BT- _anc | N87 ^PD ' ^L1/Hlgh cells. These co-cultures were carried out in the presence and absence of the PD-1 blocking agent, pembrolizumab. After 4 days, T cells were harvested and analyzed for CTV intensity by flow cytometry, which is indicative of the number of cell divisions based on dilution of the CTV stain. [0166] Data shown represents the average of 4 independent experiments. All data was normalized to the division index in non-engineered BT-474 or N87 cells (re., normalized to the wild-type co-culture), respectively. NTD cells were used as a negative control and showed no meaningful proliferation. Non-engineered BT-474 and N87 showed no significant differences in proliferation with and without the addition of pembrolizumab. However. HER2-TAC T cells cocultured with N87 ^PD ' ^L1/Hlgh or BT-474 ^PD ' ^{L l l llgh} showed a significant reduction in their respective proliferation index. This T cell impairment was rescued by addition of pembrolizumab, leading to a significant increase in the division index.

[0167] The data demonstrate that PD-L1 expressed by tumor cells directly impairs the activity of TAC T cells. Blocking PD1 signaling via checkpoint blockade, such as pembrolizumab, can restore TAC T cell activity, thereby increasing the potency of TAC T cells in the presence of tumor cells with high PD-L1 levels.

[0168] In a separate experiment, HER2-expressing N87 ^{WT/nuc GFP} or N87 ^PD ' ^{L14Ilgh/nuc GFP} cancer cells were co-cultured with HER2-TAC T cells, administered with or without anti-PD-1 antibody pembrolizumab, and assessed for viability (FIG. 4A-4B).

[0169] Cells were co-cultured for 6 days, and images were collected every 8 hours. Endpoint images (taken on day 6) are shown for an experiment E:T ratio of 1 : 10 (HER2-TAC T cells:Tumor cells) in FIG 4A. Both tumor target and T cells are shown in white against a black background. When target N87 ^WT and N87 ^PD444Ilgh tumor cells were co-cultured with nontransduced (NTD) T cells, tumor cells were seen uniformly spread across the w ell with no signs of significant tumor cell death. For target N87 ^WT tumor cells co-cultured with HER2-TAC T cells, dense cell clusters were observed and, in contrast to the NTD controls, large areas of the cell culture well were devoid of tumor cells, demonstrating target cell killing in both (“Untreated”) or treated with pembrolizumab (“+Pembro”). For target N87 ^PD ' ^L1 ' ^Hlgh tumor cells co-cultured with HER2-TAC T cells, little clearing was seen in the untreated well (“Untreated”), while in comparison, the pembrolizumab-treated well (“+Pembro”) displayed significantly cell clustering and large areas where no tumor cells could be detected, demonstrating extensive N87 ^PD ' ^L1 ' ^Hlgh tumor cell death in the pembrolizumab-treated well.

[0170] Quantification of the percentage of live cancer cells in each well at each timepoint for each condition reveals a significant decrease in live N87 ^PD ' ^L1 ' ^Hlgh target cells when treated with TAC T cells combined pembrolizumab (FIG. 4B). Data was statistically analyzed using a two- way ANOVA and Tukey post-hoc analysis on the AUC of each time-course plot. [0171] The experiment (FIG. 4A-4B) was repeated three times in triplicate. In all cases, pembrolizumab reproducibly enhanced the potency of TAC T cells in co-culture with N87 ^PD ' ^L1 ‘ ^Hlgl1 target cells, although statistical significance was not reached in one well.

[0172] The cell imaging results (FIG. 4A) and quantification of percentage of live cancer cells (FIG. 4B) demonstrate that HER2-TAC T cell treatment of PD-L1 expressing tumor cells, administered in combination with pembrolizumab, had a positive impact on reducing the number of live PD-L1 high tumor cells over the course of the experiment, thereby indicating that PD1 signaling blockade can increase the potency of TAC T cells in the presence of tumor cells with high PD-L1 levels.

Example 3: Clinical Trial of HER2 TAC

[0173] Study Rationale

[0174] Despite recent therapeutic developments for patients with advanced, metastatic, unresectable HER2 positive (HER2 ⁺ ) solid tumors, a significant unmet medical need still exists. The TAC technology is a novel approach to modify a patient’s own T cells, herein referred to as TAC T cells, and to use them in the treatment of patients with solid tumors. TAC T cells are produced through genetic engineering, incorporating TAC receptors into a patient’s own T cells. This redirects these enhanced T cells to target specific cancer antigens; and upon tumor recognition, activate them by co-opting the natural signaling pathways of the T cell receptor (TCR).

[0175] TAC T cells use HER2 antigens, present on the surface of cancer cells, to recognize and eradicate tumor cells. This approach has resulted in promising outcomes in mouse models, demonstrating TAC T cells accumulate within solid tumors and lead to robust anti-tumor efficacy, while yielding a favorable safety profile. Importantly, TAC T cells can persist for extended periods of time in mice and protect the host from tumor regrowth. Consequently, it is hypothesized TAC T cell monotherapy will be safe and effective in treating patients with HER2 ⁺ solid tumors and can provide a significant therapeutic benefit in an area of high unmet medical need.

[0176] The dose finding portion of this trial (Phase 1) will evaluate increasing dose levels of TAC T cells used as a monotherapy, and in combination with pembrolizumab, to identify recommended Phase 2 doses (RP2Ds) in patients with solid tumors who are HER2+ (with immunohistochemistry [IHC] expression levels of 3+, 2+, and 1+) and have progressed after 2 lines of systemic therapy. The combination arm will specifically investigate the safety and clinical activity of adding an anti-PDl checkpoint inhibitor to TAC T cell monotherapy. It is hypothesized that the addition of pembrolizumab may enhance TAC T cell activity and antitumor potency, while preventing TAC T cell exhaustion, through attenuation of inhibitory pathways which occur once PD-1 receptors on T cells bind to PD-L1 and PD-L2 ligands expressed on cancer cells and other immune cells.

[0177] In Phase 2, the following dose expansion groups will further evaluate the safety', efficacy, and pharmacokinetics (PK) of TAC T cell monotherapy and TAC T cells + pembrolizumab combination therapy at the RP2Ds: breast, lung, pancreatic, colorectal, gastric, endometrial, ovarian, and all others.

[0178] In summary, HER2 amplification is known to occur in breast, gastric, salivary, vaginal, endometrial, bladder, colorectal, and cervical cancers, activating numerous oncogenic signaling axes (e.g. , PI3K/AKT and Ras/Raf/ERK) resulting in improved malignant cell survival, proliferation, migration, and resistance to immunotherapy. Novel therapies are desperately needed to more effective treat these patient populations.

[0179] Pre-clinical Rationale for Combination with anti-PDl Therapy:

[0180] Programmed cell death protein 1 (PD-1) is a well-known immune checkpoint and surface receptor on T cells which promotes self-tolerance and guards against autoimmunity. Since numerous tumors frequently express high levels of PD-L1 to escape immune surveillance, anti- PD-1 strategies, such as, pembrolizumab treatment (i.e., anti-PD-1 monoclonal antibody) have successfully led to the reinvigoration of exhausted T cells and T cell-mediated anti-tumor responses.

[0181] Tumors that are associated with improved clinical responses to checkpoint blockade are those with known high PD-L1 expression, however responses can also be observed in tumors without PD-L1 expression (Garon et al. N Engl J Med. 2015; 372(21) and Topalian et al. N Engl J Med. 2012; 366(26)). PD-L1 expression is believed to be induced in these tumor cells, following the influx of T cell infiltrate and after reinvigoration via checkpoint blockade, through the secretion of T cell cytokines, IFN-y and TNF-a (Spranger et al. Sci Transl Med. 2013;

5(200); Tumeh et al. Nature. 2014; 515(7528); and Herbst et al. Nature. 2014; 515(7528)). [0182] Preclinical studies using TAC T cells in an isogenic tumor cell model indicated TAC T cell activity was reduced when ectopic PD-L1 expression was high relative to a parental line expressing low PD-L1 levels. In addition, upregulation of PD-1 receptors has been observed on TAC T cells following antigen-specific activation. These observations suggest the importance of combining TAC T cell therapy with PD-1 blockade to potentially enhance the anti-tumor activity of administered TAC T cells. Attenuating the eventual exhaustion of TAC T cells may also facilitate the recruitment of endogenous polyclonal immune cells to overcome tumor heterogeneity and prevent antigen escape (Grosser et al. Cancer Cell. 2019;36(5)).

[0183] Similar results were reported with CAR T cells in preclinical mouse models (Adusumilli et al. Cancer Discov . 2021; 11(11)), i.e., tumor cells upregulated PD-L1 in response to CAR T- cell-secreted cytokines, and CAR T cells also upregulated PD-1. To overcome this tumor- mediated adaptive resistance, Adusumilli et al. added pembrolizumab following CAR T-cell administration. Results indicated exhausted CAR T cells were rescued by pembrolizumab treatment, yielding enhanced efficacy and longer persistence of the CAR T cells.

[0184] In summary, pre-clinical studies performed to date using TAC T cells indicate the addition of pembrolizumab has the potential of synergistic efficacy effects.

[0185] Clinical Rationale for Combination with anti-PDl Therapy:

[0186] The safety and clinical activity of adding pembrolizumab to HER2 -targeted therapies has recently been demonstrated with trastuzumab in metastatic oesophagogastric cancer (O’Donnell et al. Nat Rev Clin Oncol. 2019; 16(3)), and margetuximab in gastro-oesophageal adenocarcinoma (Catenacci et al. Lancet Oncol. 2020; 21(8)). Pembrolizumab has also been safety added to CD19-directed CAR T cell therapy in B cell lymphomas (Chong et al. Blood. 2022; 139(7)) and to mesothelm-targeted CAR T cell therapy in metastatic lung and breast cancers, and malignant pleural mesothelioma (MPM) (Adusumilli et al. 2021).

[0187] Through examination of the Phase 1 clinical trial using the mesothelin-targeted CAR T cell + pembrolizumab treatment combination (Adusumilli et al. 2021), it is likely TAC T cells can also be used in combination with pembrolizumab to yield safe and more effective outcomes in patients with solid tumors. Specifically, in the mesothelin study, 27 total patients were infused with CAR T cells: 25 had malignant pleural mesothelioma (MPM), and 1 each had metastatic lung and metastatic breast cancers. Eight monotherapy dose escalation cohorts consisted of 3 patients each (unless otherwise noted): 1.3*10 ⁵ , 2.3x 10’, 3.1 x l0 ⁶ , 4.3xl0 ⁶ , 5.6x l0 ⁶ , 6.1 xl0 ⁷ (6 patients), 7.3xl0 ⁷ , and 8.6x l0 ⁷ cells/kg. All patients except the first 3 received a single dose of cyclophosphamide (CYC) preconditioning (1500 mg/m ² ). From these 24 CYC + CAR T cell- treated patients, 23 had MPM, 18 of whom were treated with pembrolizumab 200 mg Q3W for a minimum 3 doses, with > 3 months of follow-up after the 3rd dose. The other 5 MPM patients did not receive pembrolizumab treatment and remained in the CAR T cell monotherapy group (i.e., CYC + CAR T cell), with 1 breast cancer patient also not receiving pembrolizumab treatment. [0188] The most frequently occurring treatment-emergent adverse events (TEAEs) over the 1st month following CAR T cell infusion for all 27-treated patients can be found below (/.<?., before pembrolizumab treatment):

Table 6: TEAEs in > 15% of Patients Over First Month (Monotherapy; N=27)

[0189] The most frequently occurring TEAEs in > 1 combination-treated patient up to 6 months following CAR T cell infusion were as follows:

Table 7: TEAEs Over First 6 Months (CAR T cell + Pembrolizumab Treated Patients;

N=18)

TEAEs are listed in descending order in the proportion of patients with Grade 1 TEAEs. TEAE, treatment-emergent adverse event.

[0190] Overall, the addition of 3 doses of pembrolizumab Q3W appeared to decrease, not increase, the incidence and severity of TEAEs observed at 6 months versus 1 month following CAR T cell infusion. The combination arm included 18 of the 23 MPM patients. Notably, 7 of these patients (39%) had received > 2 lines of therapy before CAR T-cell infusion and 3 of the 7 had received > 3 prior lines. The median time to initiation of pembrolizumab after CAR T-cell administration was 6 weeks (range 4-17 weeks), and median overall survival times were 23.9 vs 6.1 months for the combination (N=18) vs monotherapy (CAR T cell + CYC; N=5) groups, respectively.

[0191] In summary, no DLTs or deaths were observed in the monotherapy or combination groups, and the addition of pembrolizumab to CAR T cell treatment appeared to decrease the incidence and severity of TEAEs over 6 months versus those observed at 1 month. Clinical activity was also markedly enhanced by the addition of pembrolizumab to CAR T cell monotherapy; however, this result should be interpreted with caution due to the small sample sizes.

[0192] Another Phase 1/2 trial using a CAR T cell treatment targeting CD19 and CD22 followed by limited duration pembrolizumab treatment in patients with relapsed or refractory diffuse large B-cell lymphoma (DLBCL) (Osborne et al. J Clin Oncol. 2020; 38(15)) yielded similar results. Three CAR T cell dose levels were investigated: 50, 150, and 450xl0 ⁶ total cells. Patients received CAR T cells alone, or with 3 doses of pembrolizumab 200 mg Q3 W starting on Day 14 (regimen A), or with a single dose of pembrolizumab 200 mg on Day -1 (regimen B). As of Jan 21, 2020, dose escalation from 50 to 450x106 cells was completed with pembrolizumab regimen A and B without any DLTs. Across all dose levels, there were no severe CRS events with primary infusion and 5% severe neurotoxicity (1/19) which resolved. There were no cases of severe CRS and no neurotoxicity of any grade at > 50xl0 ⁶ cells. Overall, it was concluded CAR T cells dose levels > 50xl0 ⁶ used in combination with pembrolizumab induced complete responses (CRs) without severe CRS or neurotoxicities of any grade.

[0193] The combination of CAR T cells + pembrolizumab have also been safely used to treat 3 patients with neuroblastoma, with no DLTs being observed (Heczey 2017). In this case, PD-1 inhibition was not found to enhance CAR T cell expansion or persistence, and anti-tumor responses were modest. It was speculated low PD-1 expression levels on CD4 (< 20.6%) and CD8 (< 9.04%) CAR T cells and the presence of additional immune suppressive mechanisms may have led to these results (Heczey et al. Mol Ther. 2017; 25(9)). [0194] Lastly, the clinical activity & safety of ICT01, an anti-BTN3A monoclonal antibody (mAb) which activates y982 T cells is also being investigated as a monotherapy and in combination with pembrolizumab in patients with advanced solid tumors (i.e., EVICTION trial). To date, the mAb/yST cell + pembrolizumab treatment combination arm has been well-tolerated during dose escalation (SITC 36th annual meeting December 2021).

[0195] TAC T cells Monotherapy Dose Rationale:

[0196] Dose levels used in preclinical pharmacology studies in mice are determined empirically and typically ranged from 2.5* 10 ⁷ to 6xl0 ⁸ TAC T cells per kg (c/kg), assuming a mouse body weight of 20 g for various TAC T cell products. The Maximum Feasible Dose (MFD) was ty pically 3-fold higher (e.g., 6x l0 ⁸ c/kg). Human equivalent dose levels are calculated based on a direct c/kg conversion and, separately, on body surface area. These calculations indicate the proposed clinical monotherapy starting dose (i.e., Dose Level 1) is approximately 2-3 logs lower than dose levels used in mice.

[0197] However, similar to other forms of cellular immune therapy, dose levels investigated in mice do not accurately predict dose levels in humans for a number of reasons (CAR T cell Products guidance). Therefore, additional information is considered to estimate the starting dose in humans, such as, dose levels of axicabtagene ciloleucel and tisagenlecleucel CAR T cell therapies, which were both safe and biologically active in trials in humans.

[0198] Pembrolizumab Dose Rationale for Combination Therapy:

[0199] Preclinical PK data suggest peak expansion of TAC T cells occurs on or after Day 7 within the tumor, following TAC T cell administration on Day 1. Hence, pembrolizumab may have the greatest effect when given 7 days after TAC T infusion.

[0200] Primary Study Objectives:

[0201] The primary objectives of the study are:

• To evaluate the safety and tolerability of TAC T cell monotherapy and when used in combination with pembrolizumab in patients with HER2+ solid tumors; and

• To determine the RP2Ds for TAC T cell monotherapy and when used in combination therapy with pembrolizumab; with endpoints being:

• Incidence of dose limiting toxicities (DLTs) over the first 28 days.

• Frequency, severity, and duration of treatment emergent adverse events (TEAEs) and laboratory abnormalities.

[0202] Secondary Study Objectives: [0203] The secondary objectives of the study are:

• To characterize PK profiles of TAC T cell monotherapy and when used in combination with pembrolizumab, with endpoints being: o Cmax, Tmax, and AUC; and o Duration of persistence

• To evaluate the efficacy of TAC T cell monotherapy and when used in combination with pembrolizumab, with endpoints being Investigator Assessment of the following Response Evaluation Criteria in Solid Tumors (RECIST), Version 1.1 : o Overall Response Rate (ORR); o Duration of Response (DOR); o Disease Control Rate (DCR); o Overall Survival (OS); and o Progression-free survival (PFS); and

• To evaluate the immunogenicity of TAC T cell monotherapy and when used in combination with pembrolizumab, and to assess potential impacts on PK exposure and biological activity, with the endpoint being immunogenicity of TAC T cell and pembrolizumab.

[0204] Exploratory Study Objectives:

[0205] The exploratory objective of the study is to explore biomarkers that may predict pharmacologic activity or response to TAC T cell monotherapy and when used in combination with pembrolizumab, with endpoints being:

• Characterize TAC T cell engraftment;

• Correlate T cell activation markers with clinical outcomes;

• Correlate changes in cytokine levels with cytokine release syndrome (CRS) and neurotoxicity;

• Compare pre- and post-treatment tumor biopsies for efficacy biomarkers.

[0206] Study Design:

[0207] This is a first-in-human, open-label. Phase 1/2 multicenter trial. In Phase 1, the safety and tolerability of increasing dose levels of TAC T cell monotherapy are investigated first (Dose Levels -1, 1, 2, 3, 4; beginning with Dose Level 1). The dose levels of TAC T cells are summarized in Table 8. A summary of the study design is depicted in FIG. 5.

Table 8: Summary of Experimental TAC T Cell Monotherapy Dose Levels

[0208] Once a monotherapy dose level has met the DLT criteria for dose escalation or de- escalation (as indicated by the keyboard decision table), and the Data Safety Monitoring committee (DSMC) has reviewed all current safety and pharmacokinetic (PK) data and has approved dose escalation to the next highest monotherapy dose level, combination therapy can begin in parallel using the previously investigated lowest DSMC-reviewed TAC T cell dose level + pembrolizumab (200 mg every 3 weeks [Q3W]). Once additional monotherapy dose levels have been reviewed and approved by the DSMC, dose escalating combination cohorts may be initiated using the investigated TAC T cell dose levels:

Table 9: Summary of Combination Therapy Dose Levels a. Unless the specific disease indication recommends a different dosage.

[0209] HER2 ⁺ tumor types allowed in Phase 1 include, but are not limited to, salivary gland, breast, stomach, ovary, uterus, cervix, lung, biliary tract, pancreas, colorectum, bladder, and prostate. HER2 expression levels of 1+, 2+, and 3+ by IHC are allowed in Phase 1.

[0210] In Phase 2, the following dose expansion groups further evaluate the safety, efficacy, and PK of TAC T cell monotherapy and TAC T cells + pembrolizumab combination therapy at the RP2Ds in 7 prespecified tumor types: breast, lung, pancreatic, colorectal, gastric, endometrial, ovarian, and all others. The investigations of these proposed indications are subject to change based on preliminary data obtained in Phase 1, i.e., some may be deleted and others added.

[0211] Duration: [0212] Patients in the monotherapy and combination therapy dose cohorts continue participation in the trial for up to 2 years, or until progressive disease (PD), or unacceptable toxicity is reported, whichever occurs first.

[0213] Methodology:

[0214] Upon enrollment, patients undergo leukapheresis to obtain T cells for the manufacture of TAC T cells. Patients may receive bridging anticancer therapy, after leukapheresis and before lymphodepleting chemotherapy, if deemed necessary by the Investigator. Bridging therapies must be discontinued at least 14 days prior to initiation of lymphodepletion, patients must continue to meet eligibility criteria pertaining to adequate organ function (except hematologic parameters), active infections, pregnancy, measurable disease confirmed by imaging and medication washout before initiation of lymphodepletion. If TAC T cells cannot be manufactured from the first leukapheresis product, additional leukapheresis may be allowed after consultation with the Sponsor.

[0215] Upon the successful manufacture of TAC T cells, patients enter the treatment phase. It includes lymphodepleting chemotherapy, followed by a single dose of TAC T cells administered intravenously (IV) approximately 48 hours (± 24 hours) after completion of lymphodepleting chemotherapy, unless clinical or logistical circumstances require modification of this timing. Any dose adjustments to the lymphodepleting chemotherapy must be discussed in advance with the Medical Monitor. A post-treatment tumor biopsy (in patients with accessible disease) is obtained approximately 8 days following the TAC T cell dose.

[0216] Follow-up Periods:

[0217] Patients treated with TAC T cell monotherapy, and TAC T cell + pembrolizumab combination therapy, continue to be followed for up to 2 years, or until PD, or unacceptable toxicity is reported, whichever occurs first. After trial completion, patients are enrolled in a separate long-term follow-up (LTFU) protocol recording survival, long-term toxicity, and viral vector safety for up to 15 years.

[0218] Assessments:

[0219] Monotherapy Assessments

[0220] Safety, PK, PD, and biomarker values are closely assessed over the first month; after which, assessments occur during visits every 3 months for up to 2 years, or until PD, or unacceptable toxicity is reported, whichever occurs first. Radiographic disease assessments are performed pre-treatment and approximately at 1, 3, 6, 9, 12, 18 and 24 months following the TAC T cells dose. [0221] Combination Therapy Assessments

[0222] Safety, PK, PD, and biomarker values are closely assessed over the first 2 months; after which, assessments will occur during visits every 3 weeks (i.e., at pembrolizumab dosing visits) for up to 2 years, or until PD, or unacceptable toxicity is reported, whichever occurs first. Radiographic disease assessments (following RECIST 1.1 criteria) are performed pre-treatment, at 1 month, then every 8 weeks (Q8W) up to 24 months following the TAC T cells dose administration.

[0223] General Inclusion Criteria

[0224] Patients must meet all the following criteria to participate in this trial:

• Signed, written informed consent obtained prior to any study procedures.

• Age > 18 years at the time of informed consent.

• For the Phase 1 component of the trial: either a fresh tumor sample or the most recent archival tumor sample available with corresponding pathology report for histological disease diagnosis to confirm HER2 -protein expression on tumor cell surface by central analysis. The most recent archival tissue available is an acceptable alternative regardless of when it was obtained during prior treatment or lines of therapy. Tumor samples are screened centrally for HER2 status by both fluorescent in-situ hybridization (FISH) and immunohistochemistry (IHC) assays. If a patient’s fresh or archival tumor sample can be obtained for central confirmation and is HER2 ⁺ on local laboratory results by one of the following 3 methods: IHC, FISH, orNGS, screening and enrollment may proceed, and the central confirmation can be completed after the patient has enrolled in the trial

• For the Phase 2 component of the trial: A recent tumor sample obtained at the time of or since last relapse and after last line of therapy including neoadjuvant therapy must be available with corresponding pathology report for histological disease diagnosis to confirm HER2-protein expression on tumor cell surface by central analysis. Tumor samples are screened centrally for HER2 status by both FISH and IHC assays.

• Histologically confirmed advanced, metastatic, unresectable solid tumors with metastatic disease after at least two prior lines of any therapy. For breast cancer patients, both prior lines of therapy must have included HER2-targeted agents.

• Patients with solid tumors with genetic alterations and mutations (such as BRAF, BRCA, EGFR mutations, and ALK translocation) where approved targeted therapies are available to their specific cancers must have been previously treated with such approved therapies, or refused such approved targeted therapy for their cancers prior to enrollment, or in the opinion of the investigator would be unlikely to tolerate or derive clinically meaningful benefit from standard of care therapies.

• Measurable disease on imaging per RECIST Criteria Version 1.1 at time of enrollment

• Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1 at screening.

• Life expectancy of at least 12 weeks.

• Adequate organ and bone marrow reserve function, as defined in the table below, prior to leukapheresis.

• Recovery to < Grade 1 or baseline of any toxicities due to previous therapy, except alopecia, anemia, thrombocytopenia, and neutropenia. o If patient received major surgery, they must have recovered adequately from the toxicity and/or complications from the intervention prior to starting therapy. o Toxicity that has not recovered to < Grade 1 is allowed if it meets the inclusion requirements for laboratory parameters.

• Adequate vascular access for leukapheresis as per institutional guidelines.

• For women physiologically capable of becoming pregnant, agreement to use highly effective methods of contraception starting 28 days prior to study treatment and for 1 year after the last HER2-TAC T cell dose. For men who have partners physiologically capable of becoming pregnant, agreement to use an effective barrier contraceptive method during study treatment and for 1 year after the last HER2-TAC T cell dose.

Table 10: Adequate Organ Function Laboratory Values

[0225] Exclusion Criteria:

[0226] Patients who meet any of the following criteria are excluded from participation in this trial:

• Intolerant to any component of the TAC T cells product.

• Prior treatment with any of the following: o Adoptive cell transfer of any kind, including Chimeric Antigen Receptor (CAR) T cells o Gene therapy Investigational medicinal product within 5 half-lives or 28 days prior to leukapheresis, whichever is shorter.

• Live vaccine within 28 days prior to initiation of trial therapy

• Monoclonal antibody 28 days prior to initiation of trial therapy.

• Radiation within 28 days prior to lymphodepletion. Radiation to a single lesion, if additional non-irradiated lesions are present, is allowed up to 14 days prior to lymphodepletion.

• Chemotherapy or targeted small molecule therapy within 14 days prior to initiation of trial therapy. (1 week for erlotinib, gefitinib, afatimb, or crizotimb).

• Requirement for any other form of antmeoplastic therapy while on study except for the TAC T cell monotherapy treatment arms, where patients are allowed to start on antineoplastic therapy should they progress during the 24 months study duration.

• Colony stimulating factors, including granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), erythropoietin, and other hematopoietic cytokines, within 14 days prior to leukapheresis

• Immunosuppressive medication within 14 days and corticosteroid treatment < 72 hours prior to leukapheresis, except for physiological replacement doses (< 12 mg/m ² /24h) of hydrocortisone or equivalent and topical or inhaled steroids.

• History or presence of clinically relevant central nervous system (CNS) pathology such as epilepsy, seizure, paresis, aphasia, stroke, severe brain injury, dementia, Parkinson’s disease, cerebellar disease, organic brain syndrome, or psychosis. (Brain metastasis - nonprogressive or previously treated and currently stable - are permitted.)

• Active inflammatory neurological disorders (e.g., Guillain-Barre Syndrome, amyotrophic lateral sclerosis, multiple sclerosis).

• Active autoimmune disease (e.g, lupus, rheumatoid arthritis, Sjogren’s syndrome) requiring systemic disease modifying agents in the past 2 years. Replacement therapy (e.g. , thyroxine, insulin, or physiologic corticosteroid replacement therapy for adrenal or pituitary insufficiency, etc.) is not considered a form of systemic treatment. • Active hepatitis B or C (HCV RNA positive) infection or any history of or active human immunodeficiency virus (HIV) infection.

• Uncontrolled, acute, or life-threatening bacterial, viral, or fungal infection. Patients with ongoing use of prophylactic antibiotics, antifungals, or antivirals are eligible if no evidence of active infection.

• Class III or IV heart failure (as defined by the New York Heart Association - NYHA), cardiac angioplasty or stenting, myocardial infarction, unstable angina, or other clinically significant cardiac disease within 6 months prior to screening.

• Cardiac arrhy thmia not controlled by medical management.

• Clinically significant thrombotic events within 6 months prior to leukapheresis and/or inability to stop anti-coagulation for at least 4 weeks around time of HER2-TAC T cell infusion without compromising patient’s health.

• Known additional malignancy that is progressing or requires active treatment, exceptions include basal cell carcinoma of the skin, squamous cell carcinoma of the skin, or in situ cervical cancer that has undergone potentially curative therapy.

• Pregnant or nursing (lactating). Females physiologically capable of becoming pregnant must have a negative serum beta human chorionic gonadotropin (P-hCG) pregnancy test result at screening and within 48 hours prior to the first dose of lymphodepleting chemotherapy.

• As determined by the Investigator, any uncontrolled medical, psychological, familial, sociological, or geographical condition(s) that do(es) not permit compliance with the protocol.

• Risk factors for bowel obstruction or bowel perforation.

• Symptomatic ascites or pleural effusion.

• History of pneumonitis or interstitial lung disease.

• Previous severe hypersensitivity reaction to treatment with another monoclonal antibody.

[0227] Dose

[0228] The dose finding part of this trial utilizes the keyboard design to determine the safety and tolerability of various TAC T cell monotherapy dose levels and used in combination with pembrolizumab. The first 3 trial-treated patients are administered TAC T cells at Dose Level 1 (1 to 3* 10 ⁵ cells/kg). Within the first dose cohort, treatment of the 2 ^nd patient must occur > 28 days after the first patient’s infusion. Treatment of the 3 ^rd patient must occur > 14 days after the 2 ^nd patient’s infusion. Assuming no DLTs are observed, subsequent patients within this dose cohort may be treated without staggering. At each higher monotherapy dose level, the interval between treatment of the 1 ^st and 2 ^nd patient must also be staggered by at least 28 days. After staggering has been completed, no more than one patient may be treated with TAC T cells in any dose cohort per day. As indicated in the trial design, once all the data of a completed monotherapy dose level has been reviewed by the SMC, and dose escalation has been approved, dosing can begin in the combination arm using the previously tested and reviewed TAC T cell dose level(s).

[0229] Dose-Limiting Toxicity

[0230] TAC T Cell Dose-Limiting Toxicity’

[0231] For TAC-T cells one time administration will be evaluated and graded according to the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) Criteria Version 5.0, CRS as per Lee 20143 and neurotoxicity as per American Society for Transplantation and Cellular Therapy (ASTCT) Consensus Grading.

[0232] A DLT is defined as:

• Any Grade 4 or any Grade 5 event determined by the Investigator to be related to investigational product with the exception of Grade 4 laboratory abnormalities (such as electrolyte abnormalities) that may be at least possibly related to the investigational product but are rapidly reversible or correctable without substantial safety concerns.

• Grade > 3 TAC T-cell associated acute infusion reactions persisting for > 24 hours

• Grade > 3 TAC T-cell associated CRS or neurotoxicity persisting for > 72 hours

• Grade > 3 cardiovascular, or pulmonary toxicity persisting for > 72 hours

• Grade > 3 immune-related toxicities (colitis, nephritis, hepatitis, myocarditis, hypophysitis, salivary gland toxicity, etc.)

• Grade > 3 organ toxicities or non-hematological toxicities that do not improve to baseline within 7 days

• Grade >4 thrombocytopenia, or clinically consequential Grade 3 neutropenia or thrombocytopenia for more than 42 days after TAC T cell treatment neutropenia or

[0233] Combination Therapy Dose-Limiting Toxicity [0234] DLT is defined as toxicity that is possibly, probably, or definitely related to studytherapy and may result in a change in the given dose. DLTs include

• All DLT criteria listed for the monotherapy TAC T cell group apply to the Combination group;

• Grade 4 non-hematologic toxicity (not laboratory);

• Grade 4 hematologic toxicity lasting > 7 days (except for neutropenia and thrombocytopenia);

• Most non-hematologic AEs Grade > 3 in severity;

• Any Grade 3 or Grade 4 non-hematologic laboratory- value that requires clinically significant medical intervention, leads to hospitalization, persists for >1 week, or results in a drug-induced liver injury-;

• Grade 3 or Grade 4 febrile neutropenia;

• A prolonged delay beyond 14 days in initiating Cycle 3 due to treatment-related toxicity;

• Any- treatment-related toxicity that causes the participant to discontinue treatment during the DLT observation period, and Grade 5 toxicity.

[0235] Monotherapy DLT Period

[0236] DLTs are assessed from the time of the TAC T cell dose until 28 days following the TAC T cell dose, and up to 42 days should clinically consequential severe neutropenia or thrombocytopenia occur.

[0237] Combination Therapy DLT Period

[0238] DLTs are assessed during the first 7 weeks of treatment (i.e., after 2 cycles of pembrolizumab starting on Day 7) during dose escalation and for the first 4 weeks (i.e., after 1 cycle of pembrolizumab starting on Day 7) during dose expansion.

[0239] Dosage Form.

[0240] The HER2-TAC T cell product is a suspension of genetically modified autologous T cells expressing HER2-TAC (SEQ ID NO: 36) for infusion (i.e., HER2-targeted TAC T cells) containing 10% dimethyl sulfoxide (DMSO) in approximately 20 mL.

[0241] Administration:

[0242] TAC T cells are administered as a single IV infusion approximately 48 hours after completion of lymphodepleting chemotherapy. Lymphodepleting chemotherapy consists of 3 consecutive days of fludarabine (Flu) IV (30 mg/m ² ) and cyclophosphamide (Cy) IV (300 mg/m ² ) with or without Mesna IV. Central venous access is recommended for the infusion of TAC T cells. The patient’s identity must match the patient identifiers on the HER2-TAC T cell product bag.

[0243] For Pembrolizumab:

[0244] Pembrolizumab 200 mg I.V. is administered every' 3 weeks starting on Day 7 from administration of the TAC-T cells. Dose modifications will follow the guidance described in the approved current USPI.

[0245] Efficacy Assessments:

[0246] Disease assessments should be performed by using CT (or MRI if indicated) and PET scans at baseline (screening and/or after bridging anticancer therapy), confirmation of CR and PD. Scans will be reviewed and assessed locally by the PI and radiology using RECIST Criteria vl.l.

[0247] Safety Assessments:

[0248] Adverse events (AEs) are assessed throughout the study according to CTCAE Criteria Version 5,4 Lee 20143 for CRS and ASTCT5 Consensus Criteria. Adverse events, serious adverse events (SAEs), and laboratory abnormalities (type, frequency, and severity) are collected.

[0249] Disease progression, e.g., exacerbation of clinical signs or symptoms or worsening of a patient’s underlying primary malignant disease, is not recorded as an AE, unless it is considered to be a treatment-related adverse event (TRAE) by the Investigator.

[0250] Potential TAC T cell related toxicities include infusion reactions, CRS, neurotoxicity, macrophage activation syndrome, and tumor lysis syndrome (TLS); the list of these risks may be updated during the study based on observed safety signals.

[0251] Replication-competent lentivirus (RCL) and if positive, follow-on viral vector sequence testing is performed at specified timepoints during the trial using polymerase chain reaction (PCR)-based assays.

[0252] Potential pembrolizumab related toxicities include fatigue, musculoskeletal pain, rash, diarrhea, pyrexia, cough, decreased appetite, pruritus, dyspnea, constipation, pain, abdominal pain, nausea, and hypothyroidism. Infusion related reactions and Immune-Mediated Adverse Reactions which may be severe or fatal, can occur in any organ system or tissue for which monitoring for early identification and management is required per current package insert.

[0253] Other Assessments:

[0254] PK Assessments: [0255] Assessment of TAC T cell expansion and persistence in blood (and if available also on cerebrospinal fluid [CSF] and bone marrow [BM] samples) is determined by qPCR to detect the HER2-TAC transgene.

[0256] Biomarker Assessments:

[0257] Biomarker assessments are performed to evaluate HER2+ tumor status, and immune system characteristics that may be associated with TAC T cell toxicity, efficacy, and resistance mechanisms to TAC T cell treatment.

[0258] Statistical Methods:

[0259] The keyboard dose escalation design governs the number of patients to be enrolled in Phase 1. It is estimated up to 60 patients may be enrolled following the design, i.e., ~6 patients for each of the 5 dose levels across 2 arms.

[0260] In Phase 2, up to 23 patients are enrolled in the breast cancer monotherapy expansion cohort, with a Simon 2-stage design assessing futility. If < 7 patients have a response among the first 14 patients in the cohort, the study is stopped for a lack of efficacy. If > 7 patients have a response, then another 9 patients are enrolled, for a total of 23 breast cancer monotherapy patients. If < 15 of these patients have a response, then treatment with TAC T cells, as a monotherapy, is not considered as having adequate efficacy to continue investigation in HER2 ⁺ breast cancer patients. If > 15 patients have a response, then treatment with TAC T cells, as a monotherapy, is considered efficacious and continued clinical trial investigations will be warranted.

[0261] Treatment with TAC T cells is evaluated both as a monotherapy, and in combination with pembrolizumab, in patients with 7 different HER2 ⁺ cancers. This “basket” of tumor types will include lung, pancreatic, colorectal, gastric, endometrial, ovarian, and all others. Each treatment arm enrolls up to 35 total basket cancer patients, with a target of at least 5 patients per tumor type. Rapidly assessing potential efficacy signals across a wide range of cancer indications, using the minimum number of patients, is the goal of this “basket” strategy.

[0262] Since the objective of the basket approach is exploratory in nature, i.e., to generate hypotheses for future studies, formal sample size requirements have not been formulated. All data from all available patients (i.e., dose escalation and dose expansion phases) are used when evaluating the potential efficacy of TAC T cells. Efficacy and safety of TAC T cells, both as a monotherapy and in combination with pembrolizumab, are explored for each type of cancer individually, and for all types of cancer combined. [0263] Patients who are in DLT evaluation cohorts are considered evaluable if they complete the defined DLT observation period.

[0264] Dose Escalation:

[0265] Statistical analyses of the primary, secondary, and exploratory endpoints are descriptive for the trial. Summaries are provided by dose level, overall, and by treatment arm. The incidence of DLTs, the incidence and severity of TEAEs, and laboratory abnormalities are described and summarized.

[0266] Dose Expansion:

[0267] The primary efficacy analysis is based on all patients who have measurable disease at the last disease assessment, prior to initiation of study treatments (i.e., TAC T cell monotherapy or TAC T cell + pembrolizumab combination therapy) and who receive study products at the RP2Ds

[0268] Estimates of ORR and other response proportions (e.g., DCR) are presented together with 95% confidence intervals calculated using the Clopper-Pearson method. Kaplan-Meier estimations are used for the analysis of time-to-event endpoints, including duration of response (DOR), overall survival (OS), and progression-free survival (PFS).

[0269] The cellular kinetics of TAC T cells are determined from individual concentration time profiles of circulating TAC T cells and characterized in peripheral blood and summarized by treatment arm.

SEQUENCE LISTING