Engineered T cell receptors fused to antibody-derived binding domains

Abstract

The present disclosure provides improved T cell receptors, polynucleotides, polypeptides, vectors, cells, and methods of using them. In particular, the present invention relates to T cell receptor-based constructs engineered to include one or more additional binding domains, and methods of using the same. In certain embodiments, the one or more binding domains are fused to one or both TCR variable domains. In certain embodiments, the one or more additional binding domains are linked to the TCR using one or more polypeptide linkers.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 63 / 221,819, filed July 14, 2021, which is incorporated by reference herein in its entirety.

[0002] Description of sequence listing The sequence listing associated with this application is provided in sequence listing XML format in lieu of a paper copy and is incorporated herein by reference. The name of the XML file containing the sequence listing is 137080-03620_SL.xml. The text file is 198,833 bytes in size, was created on July 14, 2022, and is being submitted electronically via the Patent Center simultaneously with the filing of this application.

[0003] The present invention relates to engineered T cell receptors (TCRs). In particular, the present invention relates to TCR-based constructs and complexes engineered to include one or more additional antigen binding domains, and methods of using the same. In certain embodiments, the one or more antigen binding domains are linked to the TCR alpha, TCR beta, TCR gamma, and / or TCR delta variable domains. In certain embodiments, the one or more additional antigen binding domains are linked to the TCR variable domains via one or more polypeptide linkers. [Background technology]

[0004] Description of related fields Adoptive T cell therapy can be engineered to target either cell surface antigens (via chimeric antigen receptors; CARs) or intracellular antigens (via engineered T cell receptors; TCRs). CAR T cell activation and antitumor activity is achieved by linking a targeting moiety to a compound intracellular signaling region that contains one or more costimulatory signaling domains fused to a CD3-zeta signaling domain. Conversely, engineered TCR T cells are activated through natural intracellular signaling events orchestrated by the CD3 complex and other proximal signaling molecules, resulting in increased susceptibility to CAR T cells.

[0005] Although TCR T cell response sensitivity to CAR T cells is desirable, TCR T cells are limited by other characteristics. For example, target recognition is controlled by MHC restriction, so TCRs are typically deployed against HLA haplotypes present in less than 40% of the general population. This represents an upper limit for patient eligibility / recruitment before standard cuts resulting from target expression and other exclusions and restrictions. MHC restriction also creates ample opportunity for target cells (e.g., tumors) to evolve escape pathways via genetic mutations or suppression of antigen processing and presentation mechanisms.

[0006] Thus, there remains a need for improved TCR-based constructs and therapies for treating disease. Summary of the Invention [Means for solving the problem]

[0007] The present disclosure relates generally, in part, to engineered T cell receptors, fusion proteins, polynucleotides, compositions, medicaments and uses thereof.

[0008] In one aspect, an engineered T cell receptor (TCR) is provided, the engineered TCR receptor comprising one or more antigen binding domain(s) linked to one or both TCR variable domains.

[0009] In another aspect, an engineered T cell receptor (TCR) is provided that comprises: (a) a TCR alpha polypeptide comprising a TCR alpha variable domain; (b) a TCR beta polypeptide comprising a TCR beta variable domain; and (c) one or more antigen binding domains linked to the TCR alpha variable domain and / or the TCR beta variable domain.

[0010] In another aspect, an engineered T cell receptor (TCR) is provided that comprises: (a) a TCR gamma polypeptide comprising a TCR gamma variable domain; (b) a TCR delta polypeptide comprising a TCR delta variable domain; and (c) one or more antigen binding domains linked to the TCR gamma variable domain and / or the TCR delta variable domain.

[0011] In another aspect, a fusion polypeptide is provided that includes: (a) a TCR β polypeptide comprising a TCR β variable domain; (b) a polypeptide cleavage signal; and (c) a TCR α polypeptide comprising one or more antigen binding domains, a polypeptide linker, and a TCR α variable domain.

[0012] In another aspect, a fusion polypeptide is provided comprising: (a) a TCR β polypeptide comprising one or more antigen binding domains, a polypeptide linker, and a TCR β variable domain; (b) a polypeptide cleavage signal; and (c) a TCR α polypeptide comprising a TCR α variable domain.

[0013] In another aspect, a fusion polypeptide is provided comprising: (a) a TCR β polypeptide comprising one or more antigen binding domains, a polypeptide linker, and a TCR β variable domain; (b) a polypeptide cleavage signal; and (c) a TCR α polypeptide comprising one or more antigen binding domains, a polypeptide linker, and a TCR α variable domain.

[0014] In another aspect, a fusion polypeptide is provided comprising: (a) a TCRγ polypeptide comprising a TCRγ variable domain; (b) a polypeptide cleavage signal; and (c) a TCRδ polypeptide comprising one or more antigen binding domains, a polypeptide linker, and a TCRδ variable domain.

[0015] In another aspect, a fusion polypeptide is provided comprising: (a) a TCRγ polypeptide comprising one or more antigen binding domains, a polypeptide linker, and a TCRγ variable domain; (b) a polypeptide cleavage signal; and (c) a TCRδ polypeptide comprising a TCRδ variable domain.

[0016] In another aspect, a fusion polypeptide is provided comprising: (a) a TCRγ polypeptide comprising one or more antigen binding domains, a polypeptide linker, and a TCRγ variable domain; (b) a polypeptide cleavage signal; and (c) a TCRδ polypeptide comprising one or more antigen binding domains, a polypeptide linker, and a TCRδ variable domain.

[0017] In various embodiments, the TCR alpha polypeptide comprises a TCR alpha constant domain and the TCR beta polypeptide comprises a TCR beta constant domain.

[0018] In various embodiments, the TCR gamma polypeptide comprises a TCR gamma constant domain and the TCR delta polypeptide comprises a TCR delta constant domain.

[0019] In various embodiments, the one or more antigen binding domains comprise a first antigen binding domain linked to a TCR alpha or TCR gamma variable domain. In some embodiments, the one or more antigen binding domains comprise a first antigen binding domain linked to a TCR beta or TCR delta variable domain. In some embodiments, the one or more antigen binding domains comprise (i) a first antigen binding domain linked to a TCR alpha or TCR gamma variable domain, and (ii) a first antigen binding domain linked to a TCR beta or TCR delta variable domain. In some embodiments, the first antigen binding domain is linked to the N-terminus of the variable domain. In some embodiments, the first antigen binding domains are the same or different and / or bind to the same or different target antigens.

[0020] In various embodiments, the one or more antigen binding domains comprise a second antigen binding domain linked to a first antigen binding domain. In various embodiments, the one or more antigen binding domains comprise a second antigen binding domain linked to a first antigen binding domain linked to a TCR alpha or TCR gamma variable domain. In some embodiments, the one or more antigen binding domains comprise a second antigen binding domain linked to a first antigen binding domain linked to a TCR beta or TCR delta variable domain. In some embodiments, the one or more antigen binding domains comprise (i) a second antigen binding domain linked to a first antigen binding domain linked to a TCR alpha or TCR gamma variable domain, and (ii) a second antigen binding domain linked to a first antigen binding domain linked to a TCR beta or TCR delta variable domain.

[0021] In various embodiments, the second antigen binding domain is linked to the N-terminus of the first antigen binding domain.In some embodiments, the second antigen binding domain is the same or different, and / or binds to the same or different target antigen.In some embodiments, the first and second antigen binding domains are the same or different, and / or bind to the same or different target antigen.

[0022] In various embodiments, the one or more antigen binding domains are selected from the group consisting of folate receptor alpha (FRα), α v β 6Integrins, ADGRE2, BACE2, B cell maturation antigen (BCMA), B7-H3 (CD276), B7-H4, B7-H6, CA19.9, carbonic anhydrase IX (CAIX), CCR1, CD7, CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD133, CD138, CD171, CD244, carcinoembryonic antigen (CEA), C-type lectin-like molecule-1 (CLL-1), CD2 subset 1 (CS-1), CLDN6, cMET, chondroitin sulfate proteoglycan 4 (CSPG4), CLDN18.2, cutaneous T-cell lymphoma-associated antigen 1 (CTAGE1), DLL3, epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), EGFR806, epithelial glycoprotein 2 (EGP2), epithelial glycoprotein 40 (EGP40), EPHB2, ERBB4, epithelial cell adhesion molecule (EPCAM), ephrin type A receptor 2 (EPHA2), fibroblast activation protein (FAP), Fc receptor-like 5 (FCRL5), fetal acetylcholinesterase receptor (AchR), FLT3, FN, FN-EDB, FRBeta, ganglioside G2 (GD2), ganglioside G3 (GD3), glypican-3 (GPC3), EGFR family including ErbB2 (HER2), HER2p95, EGFRv3, IL-10Rα, IL-13Rα2, kappa, cancer / testis antigen 2 (LAGE-1A), K-Ras, K-Ras G12C, K-Ras G12D, K-Ras G12V, lambda, Lewis-Y (LeY), L1 cell adhesion molecule (L1-CAM), LILRB2, LY6G6GD, melanoma antigen recognized by T cells 1 (MelanA or MART1), mesothelin (MSLN), MMP10, MUC1, MUC16, MHC class I chain-related protein A (MICA), MHC class I chain-related protein B (MICB), neural cell adhesion molecule (NCAM), prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), The present invention binds to a target antigen selected from the group consisting of synovial sarcoma, X-breakpoint 2 (SSX2), survivin, tumor-associated glycoprotein 72 (TAG72), transmembrane activator and CAML interactor (TACI), tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), TIM3, trophoblast glycoprotein (TPBG), UL16-binding protein (ULBP) 1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, and vascular endothelial growth factor receptor 2 (VEGFR2).

[0023] In various embodiments, the one or more antigen binding domains are selected from the group consisting of alpha-fetoprotein (AFP), ASCL2, B melanoma antigen (BAGE) family members, regulator of imprinted sites Brother (BORIS), cancer-testis antigen, cancer-testis antigen 83 (CT-83), carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), cytomegalovirus (CMV) antigen, cytotoxic T cell (CTL) recognition antigen on melanoma (CAMEL), Epstein-Barr virus (EBV) antigen, and / or EBV-specific antigen (EGFR). EBV) antigen, EPHB2, G antigen 1 (GAGE-1), GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, GAGE-8, glycoprotein 100 (GP100), Hepatitis B virus (HBV) antigen, Hepatitis C virus (HCV) nonstructural protein 3 (NS3), human papillomavirus (HPV)-E6, HPV-E7, human telomerase reverse transcriptase (hTERT), IGF2BP3 / A3, IGF2BP1, K-Ras, K-Ras G12C, K-Ras G12D, K-Ras G12V, latent membrane protein 2 (LMP2), LY6G6D, melanoma antigen family A,1 (MAGE-A1), MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, melanoma antigen recognized by T cells (MART-1), mesothelin (MSLN), mucin 1 (MUC1), mucin 16 (MUC16), New York esophageal squamous cell carcinoma-1 (NYESO-1), P53,P antigen (PAGE) family member, PAP, PIK3CA, PIK3CA The present invention binds to a target polypeptide derived from a protein selected from the group consisting of H1047R, placenta specific 1 (PLAC1), preferentially expressed antigen in melanoma (PRAME), prostate specific antigen PSA, survivin, synovial sarcoma X1 (SSX1), synovial sarcoma X2 (SSX2), synovial sarcoma X3 (SSX3), synovial sarcoma X4 (SSX4), synovial sarcoma X5 (SSX5), synovial sarcoma X8 (SSX8), thyroglobulin, TP53 R175H, tyrosinase, tyrosinase-related protein (TRP) 1, TRP2, UBD, Wilms tumor protein (WT-1), Wnt10A, X antigen family member 1 (XAGE1), and X antigen family member 2 (XAGE2).

[0024] In some embodiments, the one or more antigen binding domains bind to CD33, CLL1, CD19, CD20, CD22, CD79A, CD79B, or BCMA. In some embodiments, the one or more antigen binding domains bind to CD19, CD20, CD22, CD33, CD79A, CD79B, B7H3, Muc16, Her2, EGFR, FN-EDB, CLDN18.2, DLL3, FLT3, CLL1, CD123, or BCMA. In some embodiments, the one or more antigen binding domains comprise an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 1-32.

[0025] In various embodiments, the one or more antigen binding domains are selected from the group consisting of camel Ig, llama Ig, alpaca Ig, Ig NAR, Fab' fragments, F(ab') 2 Fragments, bispecific Fab dimers (Fab2), trispecific Fab trimers (Fab3), Fv, single chain Fv proteins ("scFv"), bis-scFv, (scFv) 2 In some embodiments, the one or more antigen binding domains comprise one or more single chain variable fragments (scFv). In some embodiments, the one or more antigen binding domains comprise one or more single domain antibodies (sdAbs). In some embodiments, the sdAb is a camelid VHH, a nanobody, or a heavy chain only antibody (HcAb). In some embodiments, the sdAb is a camelid VHH. In some embodiments, the antibody or antigen binding fragment thereof is human or humanized.

[0026] In various embodiments, the one or more antigen binding domains comprise a ligand.

[0027] In various embodiments, the one or more antigen binding domains are linked to the TCR variable domain by one or more polypeptide linkers. In some embodiments, the one or more polypeptide linkers comprise a linker about 2 to about 25 amino acids in length. In some embodiments, the one or more polypeptide linkers comprise a linker about 4 to about 15 amino acids in length. In some embodiments, the one or more polypeptide linkers comprise a linker about 4 to about 10 amino acids in length. In some embodiments, the one or more polypeptide linkers comprise a linker about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15 amino acids in length. In some embodiments, the one or more polypeptide linkers comprise a linker about 9 or about 10 amino acids in length.

[0028] In various embodiments, the one or more polypeptide linkers are selected from the group consisting of GG, GS, SG, SS, GSS, SSG, GSG, SGS, SGG, GGS, GGGS (SEQ ID NO:53), (GGGGS) 1~5 The polypeptide linker comprises a linker selected from the group consisting of a polypeptide (SEQ ID NOs: 35-39), a linker from a marsupial γμ TCR (e.g., LEKT; SEQ ID NO: 33), and any combination thereof. In some embodiments, the one or more polypeptide linkers comprise a linker from a marsupial γμ TCR comprising the amino acid sequence set forth in SEQ ID NO: 33. In some embodiments, the one or more polypeptide linkers comprise a GGGGS (SEQ ID NO: 35) linker (G4S). In some embodiments, the one or more polypeptide linkers comprise a marsupial γμ TCR linker and a G4S linker set forth in SEQ ID NO: 34. In some embodiments, the one or more polypeptide linkers comprise two GGGGS linkers (2×G4S) (SEQ ID NO: 36). In some embodiments, the one or more polypeptide linkers comprise three GGGGS linkers (3×G4S) (SEQ ID NO: 37). In certain embodiments, the one or more polypeptide linkers comprise the amino acid sequence set forth in any one of SEQ ID NOs: 33-53.

[0029] In various embodiments, the first and second antigen-binding domains are separated by a second polypeptide linker. In some embodiments, the second polypeptide linker is about 2 to about 25 amino acids in length. In some embodiments, the second polypeptide linker is about 4 to about 15 amino acids in length.

[0030] In various embodiments, the second polypeptide linker is selected from the group consisting of GG, GS, SG, SS, GSS, SSG, GSG, SGS, SGG, GGS, GGGS (SEQ ID NO: 53), (GGGGS) 1~5 In a specific embodiment, the second polypeptide linker comprises an amino acid sequence set forth in any one of SEQ ID NOs: 33-53.

[0031] In various embodiments, the TCR variable domain binds to a target polypeptide presented by an MHC complex.

[0032] In various embodiments, the TCR variable domain is selected from the group consisting of alpha-fetoprotein (AFP), ASCL2, B melanoma antigen (BAGE) family members, regulator of imprinted sites Brother (BORIS), cancer-testis antigen, cancer-testis antigen 83 (CT-83), carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), cytomegalovirus (CMV) antigen, cytotoxic T cell (CTL) recognition antigen on melanoma (CAMEL), Epstein-Barr virus (EBV) antigen, and / or cytotoxic T cell (CTL) recognition antigen on melanoma (CAMEL). ) antigen, EPHB2, G antigen 1 (GAGE-1), GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, GAGE-8, glycoprotein 100 (GP100), hepatitis B virus (HBV) antigen, hepatitis C virus (HCV) nonstructural protein 3 (NS3), human papillomavirus (HPV)-E6, HPV-E7, human telomerase reverse transcriptase (hTERT), IGF2BP3 / A3, IGF2BP1, K-Ras, K-Ras G12C, K-Ras G12D, K-Ras G12V, latent membrane protein 2 (LMP2), LY6G6D, melanoma antigen family A,1 (MAGE-A1), MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, melanoma antigen recognized by T cells (MART-1), mesothelin (MSLN), mucin 1 (MUC1), mucin 16 (MUC16), New York esophageal squamous cell carcinoma-1 (NYESO-1), P53,P antigen (PAGE) family member, PAP, PIK3CA, PIK3CA The present invention binds to a target polypeptide derived from a protein selected from the group consisting of H1047R, placenta specific 1 (PLAC1), preferentially expressed antigen in melanoma (PRAME), prostate specific antigen PSA, survivin, synovial sarcoma X1 (SSX1), synovial sarcoma X2 (SSX2), synovial sarcoma X3 (SSX3), synovial sarcoma X4 (SSX4), synovial sarcoma X5 (SSX5), synovial sarcoma X8 (SSX8), thyroglobulin, TP53 R175H, tyrosinase, tyrosinase-related protein (TRP) 1, TRP2, UBD, Wilms tumor protein (WT-1), Wnt10A, X antigen family member 1 (XAGE1), and X antigen family member 2 (XAGE2).In some embodiments, the TCR variable domain binds to a target polypeptide derived from MAGE-A4, PRAME, K-Ras, TP53R175H, PSA, or IGF2BP3. In some embodiments, the TCR variable domain binds to a target polypeptide derived from MAGE-A4.

[0033] In various embodiments, the TCR alpha constant domain comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 82 or 88, and / or the TCR beta constant domain comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 80, 81, 86, or 87.

[0034] In various embodiments, the TCR gamma constant domain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 83 or 84, and / or the TCR delta constant domain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 85.

[0035] In various embodiments, the TCR alpha or TCR gamma polypeptide comprises a TCR alpha or TCR gamma variable domain comprising (i) an amino acid sequence set forth in any one of SEQ ID NOs: 105-111, or (ii) an amino acid sequence set forth in any one of SEQ ID NOs: 62, 64, 66, 68, 70, 72, 74, 76, and 78.

[0036] In various embodiments, the TCR β or TCR δ polypeptide comprises a TCR β or TCR δ variable domain comprising (i) the amino acid sequence set forth in SEQ ID NO: 103 or 104, or (ii) any one of SEQ ID NOs: 63, 65, 67, 69, 71, 73, 75, 77, and 79.

[0037] In various embodiments, the polypeptide cleavage signal of the fusion polypeptide is a viral autocleavage peptide or a ribosomal skipping sequence. In some embodiments, the polypeptide cleavage signal is a viral 2A peptide. In some embodiments, the polypeptide cleavage signal is an aphthovirus 2A peptide, a potyvirus 2A peptide, or a cardiovirus 2A peptide. In some embodiments, the polypeptide cleavage signal is a viral 2A peptide and is selected from the group consisting of a foot and mouth disease virus (FMDV) 2A peptide, an equine rhinitis A virus (ERAV) 2A peptide, a zosea signavirus (TaV) 2A peptide, a porcine teschovirus-1 (PTV-1) 2A peptide, a tylovirus 2A peptide, and an encephalomyocarditis virus 2A peptide. In some embodiments, the polypeptide cleavage signal comprises a furin recognition site upstream of the autocleavage peptide, optionally, the furin recognition site comprises an amino acid sequence set forth in SEQ ID NO: 112. In some embodiments, the polypeptide cleavage signal comprises an amino acid sequence set forth in any one of SEQ ID NOs: 113-137.

[0038] In various embodiments, the TCR β or TCR δ polypeptide of the fusion polypeptide is N-terminal to the TCR α or TCR γ polypeptide.

[0039] In various embodiments, the TCR alpha or TCR gamma polypeptide of the fusion polypeptide is N-terminal to a TCR beta or TCR delta polypeptide.

[0040] In various embodiments, the TCR alpha and TCR beta polypeptides each comprise an N-terminal signal sequence. In various embodiments, the TCR gamma and TCR delta polypeptides each comprise an N-terminal signal sequence. In some embodiments, the signal sequences are the same or different. In some embodiments, the signal sequence is an IgK or TCR alpha signal sequence. In some embodiments, the signal sequence is a CD8 alpha signal sequence.

[0041] In various embodiments, the fusion polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in any one of SEQ ID NOs:91-97, 100, and 102.

[0042] In another aspect, there is provided a polynucleotide encoding an engineered TCR or fusion polypeptide contemplated herein.

[0043] In another aspect, a vector is provided that includes one or more polynucleotides contemplated herein. In some embodiments, the vector is an expression vector, a retroviral vector, or a lentiviral vector.

[0044] In another aspect, a cell is provided that comprises an engineered TCR, fusion polypeptide, polynucleotide, or vector contemplated herein. In some embodiments, the cell is a hematopoietic cell. In some embodiments, the cell is a T cell, an αβ-T cell, or a γδ-T cell. In some embodiments, the cell is a CD3 + , CD4 + and / or CD8 + The cell is a cell. In some embodiments, the cell is an immune effector cell. In some embodiments, the cell is a cytotoxic T lymphocyte (CTL), a tumor infiltrating lymphocyte (TIL), or a helper T cell. In some embodiments, the cell is a T cell, a natural killer (NK) cell, or a natural killer T (NKT) cell. In some embodiments, the source of the cell is a peripheral blood mononuclear cell, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, or a tumor. In some embodiments, the cell is an isolated non-natural cell. In some embodiments, the cell is obtained from a subject. In some embodiments, the cell is a human cell.

[0045] In another aspect, there is provided a composition comprising an engineered TCR, fusion polypeptide, polynucleotide, vector, or cell contemplated herein.

[0046] In another aspect, there is provided a pharmaceutical composition comprising an engineered TCR, fusion polypeptide, polynucleotide, vector, or cell as contemplated herein.

[0047] In another aspect, there is provided a method of treating a subject in need thereof comprising administering to the subject an effective amount of a cell, composition, or pharmaceutical composition contemplated herein.

[0048] In another aspect, there is provided a method for treating, preventing, or alleviating at least one symptom of cancer, an infectious disease, an autoimmune disease, an inflammatory disease, and an immune disorder, or a condition associated therewith, comprising administering to a subject an effective amount of a cell, composition, or pharmaceutical composition contemplated herein.

[0049] In another aspect, a method for treating solid cancer is provided, comprising administering to a subject an effective amount of a cell, composition, or pharmaceutical composition as contemplated herein.In various embodiments, the solid cancer is selected from the group consisting of lung cancer, squamous cell carcinoma, colorectal cancer, pancreatic cancer, breast cancer, thyroid cancer, bladder cancer, cervical cancer, esophageal cancer, ovarian cancer, gastric cancer, endometrial cancer, brain cancer, or sarcoma.In some embodiments, the solid cancer is non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck squamous cell carcinoma, colorectal cancer, pancreatic cancer, breast cancer, thyroid cancer, bladder cancer, cervical cancer, esophageal cancer, ovarian cancer, gastric cancer, endometrial cancer, glioma, glioblastoma, oligodendroglioma, sarcoma, or osteosarcoma.

[0050] In another aspect, a method for treating hematological malignancies comprises administering to a subject an effective amount of a cell, composition, or pharmaceutical composition as contemplated herein. In various embodiments, the malignant hematological disease is leukemia, lymphoma, or multiple myeloma. In some embodiments, the hematological malignancies are selected from the group consisting of non-Hodgkin's lymphoma, acute myeloid leukemia (AML), and acute lymphoblastic leukemia (ALL). [Brief description of the drawings]

[0051] [Figure 1]Figure 1A shows an exemplary MAGE TCR, CD33 DARIC, and engineered TCR (VHH-TCR) construct design. Figure 1B shows an exemplary engineered TCR with a VHH linked to the TCR. [Figure 2A] FIG. 2A shows VHH expression on immune effector cells. [Figure 2B] FIG. 2B shows engineered TCR / receptor cytokine responses against A549.CD33 cells. [Figure 2C] FIG. 2C shows engineered TCR / receptor cytotoxicity against A549.CD33 cells. [Figure 3A] FIG. 3A shows engineered TCR / receptor expression on immune effector cells. [Figure 3B] FIG. 3B shows engineered TCR / receptor cytokine responses against A549.A2.MAGEA4 cells. [Figure 3C] FIG. 3C shows engineered TCR / receptor cytotoxicity against A549.A2.MAGEA4 cells. [Figure 4A] FIG. 4A shows engineered TCR cytokine responses to MAGEA4 peptides. [Figure 4B] Figures 4B and 4C show engineered TCR cytokine responses to cells electroporated with various amounts of CD33 mRNA. [Figure 4C] Same as above. [Figure 5A] 5A-5C show engineered TCR and DARIC cytotoxicity against HL-60, Kasumi1, and OCI-AML3 cells. [Figure 5B] Same as above. [Figure 5C] Same as above. [Figure 6] FIG. 6 shows an exemplary engineered TCR construct. [Figure 7A] FIG. 7A shows VHH expression on immune effector cells. [Figure 7B] FIG. 7B shows engineered TCR / receptor cytokine responses against A549.CD33 cells. [Figure 7C] FIG. 7C shows engineered TCR / receptor cytotoxicity against A549.CD33 cells. [Figure 8A] FIG. 8A shows VHH expression on immune effector cells. [Figure 8B] FIG. 8B shows engineered TCR / receptor cytokine responses against A549.MAGEA4.A2 cells. [Figure 8C] FIG. 8C shows engineered TCR / receptor cytotoxicity against A549.MAGEA4.A2 cells. [Figure 9] FIG. 9 shows an exemplary engineered TCR construct. [Figure 10A] FIG. 10A shows VHH expression on immune effector cells. [Figure 10B] FIG. 10B shows engineered TCR / receptor cytokine responses against A549.CD33 cells. [Figure 10C] FIG. 10C shows engineered TCR / receptor cytotoxicity against A549.CD33 cells. [Figure 11A] FIG. 11A shows VHH expression on immune effector cells. [Figure 11B] FIG. 11B shows engineered TCR cytokine responses against A549.MAGEA4.A2 cells. [Figure 11C] FIG. 11C shows engineered TCR / receptor cytotoxicity against A549.MAGEA4.A2 cells. [Figure 12A] FIG. 12A shows exemplary MAGE TCR, CD33 DARIC, CLL1 DARIC, CLL1-CD33 DARIC, and engineered TCR (CLL1-CD33 VHH TCR) construct designs. [Figure 12B] FIG. 12B shows an exemplary engineered TCR with two VHHs linked to the TCR. [Figure 13A] FIG. 13A shows CD33-based receptor expression on immune effector cells. [Figure 13B]FIG. 13B shows engineered TCR / receptor cytokine responses against A549.CD33 cells. [Figure 14A] FIG. 14A shows CLL1-based receptor expression on immune effector cells. [Figure 14B] FIG. 14B shows engineered TCR / receptor cytokine responses against A549.CLL1 cells. [Figure 15A] FIG. 15A shows TCR expression on immune effector cells. [Figure 15B] FIG. 15B shows engineered TCR / receptor cytokine responses against A549.MAGEA4 cells. [Figure 16] FIG. 16 shows TCR and CAR expression on immune effector cells. [Figure 17] FIG. 17 shows engineered TCR and CAR cytokine responses against A375.NLR (MAGEA4+;BCMA-) cells. [Figure 18A] FIG. 18A shows engineered TCR and CAR IFNg cytokine responses against Toledo cells. [Figure 18B] FIG. 18B shows engineered TCR and CAR IL-2 cytokine responses against Toledo cells. [Figure 19] Figure 19 shows antigen-independent IFNg cytokine responses using engineered TCR and CAR T cells alone. [Figure 20A] FIG. 20A shows exemplary MAGEA4 TCR, scFv CAR, and engineered TCR (scFv TCR) construct designs. [Figure 20B] FIG. 20B shows an exemplary engineered TCR with an scFv linked to the TCR. [Figure 21A] FIG. 21A shows BCMA-based receptor expression on immune effector cells. [Figure 21B] FIG. 21B shows engineered TCR / receptor IFNg cytokine responses against HT1080.BCMA, RPMI-8226, and Toledo cells. [Figure 21C] FIG. 21C shows engineered TCR / receptor IL-2 cytokine responses on HT1080.BCMA, RPMI-8226, and Toledo cells. [Figure 21D] FIG. 21D shows engineered TCR / receptor TNFα cytokine responses on HT1080.BCMA, RPMI-8226, and Toledo cells. [Figure 21E] Figure 21E shows engineered TCR / receptor cytotoxicity against HT1080.BCMA cells. [Figure 22A] FIG. 22A shows TCR expression on immune effector cells. [Figure 22B] FIG. 22B shows engineered TCR / receptor IFNg, IL2, and TNFα cytokine responses to A375 cells. [Diagram 23] FIG. 23 shows HL-60.FP (CD33+ MAGEA4-) tumor growth in the NGS systemic tumor model treated with UTD T cells, CD33 DARIC T cells, MAGEA4 TCR T cells, or VHH-TCR T cells. [Figure 24] FIG. 24 shows NCI-H2023 (CD33− MAGEA4+) tumor growth in the NGS subcutaneous tumor model treated with UTD T cells, CD33 DARIC T cells, MAGEA4 TCR T cells, or VHH-TCR T cells. [Figure 25A] FIG. 25A shows TCR / ATOMIC expression on immune effector cells. [Figure 25B] FIG. 25B shows engineered TCR / TCR IFNg cytokine responses to RPMI-8226 cells. [Figure 25C] FIG. 25C shows engineered TCR / Atomic IFNg cytokine responses to K562.CD19 cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0052] A brief explanation of sequence numbers

[0053] SEQ ID NOs: 1 to 32 show the amino acid sequences of representative target antigen-binding domains.

[0054] SEQ ID NOs: 33 to 53 describe the amino acid sequences of representative polypeptide linkers.

[0055] SEQ ID NOs:54 to 79 provide the amino acid sequences of representative TCR components (eg, TCR variable regions).

[0056] SEQ ID NOs: 80 to 88 show the amino acid sequences of representative TCR constant domains.

[0057] SEQ ID NO:89 sets forth the amino acid sequence of a representative MAGEA4-targeting TCR.

[0058] SEQ ID NOs:90, 98, and 99 set forth the amino acid sequences of representative DARIC.

[0059] SEQ ID NOs: 91-97, 100, and 102 set forth the amino acid sequences of representative engineered TCR constructs / atoms ICs.

[0060] SEQ ID NO: 101 sets forth the amino acid sequence of a representative anti-BCMA CAR.

[0061] SEQ ID NOs:103-111 set forth the amino acid sequences of representative TRA or TRB polypeptides.

[0062] SEQ ID NO:112 sets forth the amino acid sequence of a representative furin cleavage site.

[0063] SEQ ID NOs:113-137 set forth the amino acid sequences of representative polypeptide cleavage signals (eg, self-cleaving peptides).

[0064] In the above sequences, X refers to any amino acid, if present, or to the absence of an amino acid. [Mode for carrying out the invention]

[0065] A. Overview The present disclosure relates generally, in part, to TCR-based constructs engineered to include one or more additional binding domains (e.g., antigen binding domains), and methods of using the same. Without wishing to be bound by any particular theory, the inventors have unexpectedly discovered that TCRs engineered to include both a TCR binding domain (e.g., a TCR variable domain) and one or more additional antigen binding domains are surprisingly effective at cell killing and can target cells expressing either a TCR antigen, a non-TCR antigen, or both.

[0066] The multi-chain structure of the TCR poses significant structural obstacles to grafting secondary binders into the TCR structure, and success has been achieved primarily by co-expressing scFv-CD3 chain fusions or by replacing the TCR variable regions with antibody-based binders. Overall, the complexity and MHC-restricted nature of the TCR structure have hindered the development of broadly applicable technologies that achieve high levels of sensitivity and / or multiplexing. At the very least, there are few potential solutions to these significant challenges that do not consume a large portion of the available vector (e.g., lentiviral) payload space.

[0067] Thus, an efficient and effective engineered / hybridized TCR structure is disclosed herein that allows simultaneous TCR targeting and secondary binding agent targeting.Specifically, an antigen-binding domain (e.g., VHH or scFv) is linked to a TCR component, such as TCRα, TCRβ, TCRγ, and / or TCRδ variable domain / chain, in a manner that preserves TCR function.In certain embodiments, the engineered TCR includes a linker between the antigen-binding domain and the TCR component, so that the function of each targeting molecule (i.e., TCR component and secondary antigen-binding domain) is preserved.Thus, the present invention allows simultaneous targeting of intracellular and extracellular antigens.

[0068] In various embodiments, the engineered / hybridized TCR comprises one or more additional antigen binding domains. In some embodiments, the engineered / hybridized TCR comprises two or more additional antigen binding domains. In some embodiments, the two or more additional antigen binding domains target the same antigen or different antigens.

[0069] In various embodiments, the one or more antigen binding domains are selected from the group consisting of camel Ig, llama Ig, alpaca Ig, Ig NAR, Fab' fragments, F(ab') 2 Fragments, bispecific Fab dimers (Fab2), trispecific Fab trimers (Fab3), Fv, single chain Fv proteins ("scFv"), bis-scFv, (scFv) 2 , minibodies, diabodies, triabodies, tetrabodies, disulfide stabilized Fv proteins ("dsFv"), and single domain antibodies (sdAbs, camelid VHHs, nanobodies). In certain embodiments, the one or more antigen binding domains comprise one or more single chain variable fragments (scFvs) or single domain antibodies (sdAbs, e.g., camelid VHHs).

[0070] In various embodiments, the linker is a polypeptide linker of about 2 to about 25 amino acids in length. In some embodiments, the linker is selected from the group consisting of GG, GS, SG, SS, GSS, SSG, GSG, SGS, SGG, GGS, GGGS (SEQ ID NO: 53), (GGGGS) 1~5 The linker is selected from the group consisting of a polypeptide (SEQ ID NOs: 35-39), a linker derived from a marsupial γμ TCR (e.g., LEKT; SEQ ID NO: 33), and any combination thereof. In certain embodiments, the linker comprises an amino acid sequence set forth in any one of SEQ ID NOs: 33-53.

[0071] In various embodiments, an engineered TCR comprises one or more TCR components that comprise one or more TCR variable domains that bind to a target polypeptide presented by an MHC complex.

[0072] In various embodiments, the TCR component of the engineered TCR comprises a TCR constant region. In some embodiments, the TCR constant region is selected from a TCR alpha, TCR beta, TCR gamma, or TCR delta constant region. In some embodiments, the TCR constant domain comprises an amino acid sequence at least 90% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 80-88.

[0073] In some embodiments, a non-functional TCR can be used when antibody-based targeting alone is sufficient.

[0074] Recombinant (i.e., engineered) DNA, peptide and oligonucleotide synthesis, immunoassays, tissue culture, transformation (e.g., electroporation, lipofection), enzymatic reactions, purification and related techniques and procedures may be generally performed as described in various general and more specific references in microbiology, molecular biology, biochemistry, molecular genetics, cell biology, virology and immunology, which are cited and discussed throughout this specification. For example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2008), Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Glover, DNA Cloning: A Practical Approach, vol. I & II (IRL Press, Oxford Univ. Press USA, 1985), Current Protocols in Immunology (Edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M.Shevach,Warren Strober 2001 John Wiley & Sons,NY,NY)、Real-Time PCR:Current Technology and Applications,Edited by Julie Logan,Kirstin Edwards and Nick Saunders,2009,Caister Academic Press,Norfolk,UK、Anand,Techniques for the Analysis of Complex Genomes,(Academic Press,New York,1992)、Guthrie and Fink,Guide to Yeast Genetics and Molecular Biology(Academic Press,New York,1991)、Oligonucleotide Synthesis(N.Gait,Ed.,1984)、Nucleic Acid The Hybridization(B.Hames & S.Higgins,Eds.,1985)、Transcription and Translation(B.Hames & S.Higgins,Eds.,1984)、Animal Cell Culture(R.Freshney,Ed.,1986)、Perbal,A Practical Guide to Molecular Cloning(1984)、Next-Generation Genome Sequencing(Janitz,2008 Wiley-VCH)、PCR Protocols(Methods in Molecular Biology)(Park,Ed.,3rd Edition,2010 Humana Press)、Immobilized Cells And Enzymes(IRL Press,1986)、the treatise,Methods In Enzymology(Academic Press,Inc.,N.Y.)、Gene Transfer Vectors For Mammalian Cells(J.H.Miller and M.P.Calos eds.For reference, see research papers in specialist journals such as Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987), Handbook Of Experimental Immunology, Volumes I-IV (DM Weir and CC Blackwell, eds., 1986), Roitt, Essential Immunology, 6th Edition, (Blackwell Scientific Publications, Oxford, 1988), Current Protocols in Immunology (QE Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach and W. Strober, eds., 1991), Annual Review of Immunology, and Advances in Immunology.

[0075] Methods and techniques for generating and modifying novel TCRs are also known in the art, see, e.g., Linnemann, C. et al., Nat. Med., 19, 1534-1541 (2013); Scheper, W. et al., Nat. Med., 25, 89-94 (2019); Yossef, R. et al., JCI Insight, 3, 122467 (2018); Hu, Z. et al., Blood, 132, 1911-1921 (2018); Li, Y. et al., Nat. Biotechnol, 23, 349-354 (2005); Wagner, E K et al. J. Biol. Chem, 294, 5790-5804 (2019); Guo, X.-ZJ et al., Mol. Ther. Methods See Clin.Dev,3,15054(2016); Azizi,E.et al.,Cell,174,1293-1308(2018); Kieke,MCet al.,Proc.Natl Acad.Sci.USA,96,5651-5656(1999); Smith,SNet al,1319,95-141(Springer,2015); Tsuji,T.et al.,Cancer Immunol.Res,6,594-604(2018); and Spindler,MJ,et al.,Nat Biotechnol,38,609-619(2020).

[0076] B. Definition Before describing the present disclosure in more detail, it may be helpful to an understanding thereof to provide definitions of certain terms to be used herein.

[0077] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used to practice or test specific embodiments, preferred compositions, methods and materials embodiments are disclosed herein. For purposes of this disclosure, the following terms are defined below.

[0078] The articles "a," "an," and "the" are used herein to refer to one or to more than one (i.e., to at least one or to more than one) of the grammatical object of the article. By way of example, "an element" means one element or one or more elements.

[0079] The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination of the alternatives.

[0080] The term "and / or" should be understood to mean either or both of the alternatives.

[0081] As used herein, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by up to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% relative to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the term "about" or "approximately" refers to a range of ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length relative to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0082] In one embodiment, ranges such as, for example, 1 to 5, about 1 to 5, or about 1 to about 5, refer to each value subsumed within the range. For example, in one non-limiting and merely exemplary embodiment, the range "1 to 5" is equivalent to the expressions 1, 2, 3, 4, 5, or 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0, or 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0.

[0083] As used herein, the term "substantially" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, "substantially the same" refers to an amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that produces about the same effect, e.g., a physiological effect, as the reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0084] Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" will be understood to imply the inclusion of the specified step or element or group of steps or elements, but not the exclusion of any other step or element or group of steps or elements. "Consisting of" means including and limited to everything preceding the word "consisting of". Thus, the phrase "consisting of" indicates that the recited elements are necessary or mandatory, and that no other elements may be present. "Consisting essentially of" means including any elements that are limited to any elements recited thereafter, and other elements that do not interfere with or contribute to the activity or function specified in this disclosure for the recited elements. Thus, the phrase "consisting essentially of" indicates that the recited elements are necessary or mandatory, but that there are no other elements that materially affect the activity or function of the recited elements.

[0085] References throughout this specification to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "an embodiment," "an additional embodiment," or "a further embodiment," or combinations thereof, mean that the particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. As such, the appearances of such phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. Additionally, it should be understood that the affirmative recitation of a feature in an embodiment serves as a basis for the exclusion of that feature in certain embodiments.

[0086] As used herein, the term "TCR complex" refers to a complex formed by the association of CD3 with a TCR. For example, a TCR complex may be composed of a CD3 gamma chain, a CD3 delta chain, two CD3 epsilon chains, a homodimer of a CD3 zeta chain, a TCR alpha chain, and a TCR beta chain. In some embodiments, a TCR complex may be composed of a CD3 gamma chain, a CD3 delta chain, two CD3 epsilon chains, a homodimer of a CD3 zeta chain, a TCR gamma chain, and a TCR delta chain.

[0087] As used herein, a "component of a TCR complex" refers to a TCR chain (i.e., TCRα, TCRβ, TCRγ, or TCRδ), a CD3 chain (i.e., CD3γ, CD3δ, CD3ε, or CD3ζ), or a complex formed by two or more TCR chains or CD3 chains (e.g., a complex of TCRα and TCRβ, a complex of TCRγ and TCRδ, a complex of CD3ε and CD3δ, a complex of CD3γ and CD3ε, or a sub-TCR complex of TCRα, TCRβ, CD3γ, CD3δ, and two CD3ε chains).

[0088] As used herein, the terms "binding domain", "extracellular domain", "antigen binding domain", "extracellular binding domain", "extracellular antigen binding domain", "antigen-specific binding domain", "extracellular antigen-specific binding domain", "binding agent" and "antigen-binding agent" are used interchangeably and provide a polypeptide that has the ability to specifically bind to a target antigen of interest. Binding domains may be derived from either natural, synthetic, semi-synthetic, or recombinant sources.

[0089] The term "antibody" refers to a binding agent that is a polypeptide comprising at least a light or heavy chain immunoglobulin variable region or a fragment thereof that specifically recognizes and binds to an epitope of one or more antigens, such as peptides, lipids, polysaccharides, or antigenic determinant-containing nucleic acids, such as those recognized by immune cells.

[0090] The term "antibody" encompasses any naturally occurring, recombinant, modified or engineered immunoglobulin-like structure or antigen-binding fragment or portion thereof, or derivatives thereof, as further described elsewhere herein. Thus, the term refers to an immunoglobulin molecule that specifically binds to a target antigen, including, for example, chimeric antibodies, humanized antibodies, fully human antibodies, and bispecific antibodies. An intact antibody generally contains at least two full-length heavy chains and two full-length light chains, but in some cases may contain fewer chains, such as antibodies naturally occurring in camelids that may contain only heavy chains. An antibody may be derived only from a single source, or may be "chimeric", i.e., different portions of the antibody may be derived from two different antibodies. An antibody or antigen-binding portion thereof may be produced in a hybridoma, by recombinant DNA technology, or by enzymatic or chemical cleavage of an intact antibody.

[0091] The term "antigen-binding fragment" or "antigen-binding portion" refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Antigen-binding fragments include, but are not limited to, any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen forming a complex. In some embodiments, an antigen-binding portion of an antibody can be derived from an intact antibody molecule using any suitable standard technique, such as, for example, proteolytic digestion or recombinant genetic engineering techniques, including the manipulation and expression of DNA encoding the antibody variable and optionally constant domains.

[0092] A "single-chain Fv" or "scFv" antibody fragment comprises the VH and VL domains of an antibody, where these domains are present in a single polypeptide chain in either orientation (e.g., VL-VH or VH-VL). For example, in some embodiments, the scFv variable light chain is located c-terminal to that of the variable heavy chain. In some embodiments, the scFv variable heavy chain is located c-terminal to that of the variable light chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. For a review of scFvs, see, e.g., Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Eds. Rosenburg and Moore, (Springer-Verlag, New York, 1994), pp. 269-315.

[0093] As used herein, "V H H," "V H H antibody" or "V H "H domain" refers to an antibody fragment that contains the smallest known antigen-binding unit of the variable region of a heavy chain antibody (Koch-Nolte, et al., FASEB J., 21:3490-3498 (2007)).

[0094] An "isolated antibody or antigen-binding fragment thereof" refers to an antibody or antigen-binding fragment thereof that has been identified and separated and / or recovered from a component of its natural environment.

[0095] "Antigen (Ag)", "target antigen" and "polypeptide antigen" are used interchangeably and broadly include any molecule that contains an antigenic determinant within the binding region to which a TCR or antibody or fragment specifically binds. In certain embodiments, "antigen (Ag)" refers to a compound, composition, or substance that can stimulate the production of antibodies or a T cell response in an animal, including compositions that are injected or absorbed into an animal (such as those that contain cancer-specific proteins). Antigens react with the products of specific humoral or cellular immunity, including, for example, those induced by heterologous antigens such as the disclosed antigens.

[0096] An antigen may be a single unit molecule (such as a protein monomer or fragment) or a complex composed of multiple components. An antigen provides an epitope, e.g., a molecule or a portion of a molecule, or a complex of molecules or portions of molecules, that can be bound by a selective binding agent, such as an antigen-binding protein (e.g., including an antibody and / or a TCR). Thus, a selective binding agent can specifically bind to an antigen formed by two or more components in a complex. In some embodiments, an antigen can be used in an animal to produce antibodies capable of binding to that antigen. An antigen can have one or more epitopes that can interact with different antigen-binding proteins, e.g., antibodies. In preferred TCR-related embodiments, the terms "antigen (Ag)", "target antigen", and "polypeptide antigen" collectively refer to naturally processed or synthetically produced antigenic protein moieties. For example, there are tumor-associated antigens (TAA) or tumor-specific antigens (TSA), which range in length from about 7 amino acids to about 15 amino acids and can form a complex with MHC (e.g., HLA) to form a target antigen:MHC (e.g., HLA) complex.

[0097] "Target antigen" or "target antigen of interest" refers to a molecule expressed on the cell surface of a target cell to which a binding domain contemplated herein is designed to bind. In certain embodiments, the target antigen is an epitope of a polypeptide expressed on the surface of a cancer cell. "Epitope" or "antigenic determinant" refers to the region of an antigen to which a binding agent binds. Epitopes can be formed both from contiguous or non-contiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. Epitopes typically include at least 3, more commonly at least 5, about 9, or about 8-10 amino acids in a unique spatial conformation.

[0098] As used herein, the terms "selectively bind" or "selectively bound" or "selectively binding" or "selectively targeting", "specific binding affinity" or "specifically bind" or "specifically bound" or "specific binding" or "specifically targeting" describe preferential binding of one molecule to a target molecule in the presence of multiple off-target molecules (on-target binding). In certain embodiments, the term refers to binding of a TCR, antibody, or antigen-binding fragment thereof to an antigen with a binding affinity higher than background binding. A binding domain may have a binding affinity of, for example, about 10 5 M -1 Affinity or K a (i.e., the equilibrium binding constant for a particular binding interaction, having units of 1 / M). In certain embodiments, the binding domain (or fusion protein) "specifically binds" to an antigen if it binds to or associates with the antigen with a binding constant of about 10 6 M -1 , 10 7 M -1 , 10 8 M -1 , 10 9 M -1 , 10 10 M -1 , 10 11 M-1 , 10 12 M -1 , or 10 13 M -1 A "high affinity" binding domain (or single-chain fusion protein thereof) binds to a target with a Ka of at least 10 7 M -1 , at least 10 8 M -1 , at least 10 9 M -1 , at least 10 10 M -1 , at least 10 11 M -1 , at least 10 12 M -1 , at least 10 13 M -1 or greater K a It refers to a binding domain having the following structure:

[0099] Alternatively, the affinity may be expressed in M ​​units (e.g., 10 -5 M~10 -13 The equilibrium dissociation constant (K d The affinity of a binding domain polypeptide according to the present disclosure to a CAR protein may be readily determined using conventional techniques, such as competitive ELISA (enzyme-linked immunosorbent assay), or binding association, or by displacement assays using labeled ligands, or using a surface plasmon resonance instrument such as the Biacore T100 available from Biacore, Piscataway, NJ, or optical biosensor technology such as the EPIC system or EnSpire available from Corning and Perkin Elmer, respectively (see, e.g., Scatchard et al. (1949) Ann. NY Acad. Sci. 51:660; and U.S. Pat. Nos. 5,283,173; 5,468,614, or equivalents).

[0100] In one embodiment, the affinity of specific binding is about 2-fold higher than background binding, about 5-fold higher than background binding, about 10-fold higher than background binding, about 20-fold higher than background binding, about 50-fold higher than background binding, about 100-fold higher than background binding, or about 1000-fold higher than background binding, or more.

[0101] In certain embodiments, the engineered / hybrid TCR comprises an antibody or an antigen-binding fragment thereof. In the context of an engineered TCR, "antibody" refers to a binding agent that is a polypeptide comprising at least a light or heavy chain immunoglobulin variable region that specifically recognizes and binds an epitope of an antigen, such as a peptide, lipid, polysaccharide, or nucleic acid that comprises an antigenic determinant such as one recognized by an immune cell.

[0102] As will be understood by one of skill in the art, and as described elsewhere herein, a complete antibody comprises two heavy chains and two light chains, each heavy chain consisting of a variable region and a first, second, and third constant region, while each light chain consists of a variable region and a constant region.

[0103] Light and heavy chain variable regions contain a "framework" region interrupted by three hypervariable regions, also called "complementarity determining regions" or "CDRs." CDRs can be defined or identified by conventional methods, such as by sequence according to Kabat et al. (Wu, TT and Kabat, EA, J Exp Med. 132(2):211-50, (1970); Borden, P. and Kabat, EA, PNAS, 84:2440-2443 (1987); (Kabat et al., Sequences of Protein of Immunological Interest, USDepartment of Health and Human Services, 1991, which is incorporated herein by reference) or by structure according to Chothia et al. (See Chothia, C. and Lesk, AM, J Mol. Biol., 196(4):901-917 (1987); Chothia, C. et al., Nature, 342:877-883 (1989)).

[0104] Other boundaries defining CDRs that overlap with Kabat CDRs are described in Padlan (1995) FASEB J. 9:133-139 and MacCallum (1996) J. Mol. Biol. 262(5):732-45. Still other CDR boundary definitions may not strictly follow one of the systems herein, but nevertheless overlap with Kabat CDRs, although they may be shortened or extended in light of predictions or experimental findings that certain residues or groups of residues, or even entire CDRs, do not significantly affect antigen binding. For example, the CDRs of an antibody can be determined according to the AbM numbering scheme used by Oxford Molecular's AbM antibody modeling software (Oxford Molecular Group, Inc.), with reference to the AbM hypervariable regions that represent a compromise between the Kabat CDRs and the Chothia structural loops.

[0105] Furthermore, the CDRs of an antibody can be determined according to the IMGT numbering system, as described in Lefranc MP, (1999) The Immunologist 7:132-136 and Lefranc MP et al., (1999) Nucleic Acids Res 27:209-212.

[0106] Further methods of CDR determination are disclosed in MacCallum RM et al. (1996) J Mol Biol 262:732-745. See also, for example, Martin A. "Protein Sequence and Structure Analysis of Antibody Variable Domain," in Antibody Engineering, Kontermann and Dubel eds., Vol. 31, pp. 422-439, Springer-Verlag, Berlin (2001). Proprietary and publicly available programs, such as abYsis (abysis.org / abysis / ) and IMGT / V-QUEST (imgt.org / IMGT_vquest), that can be used to determine CDRs based on any of the CDR definitions described herein are known to those skilled in the art.

[0107] Illustrative examples of rules for predicting light chain CDRs include CDRL1 starting at about residue 24, preceded by a Cys, about 10-17 residues, followed by a Trp (typically Trp-Tyr-Gln, but also Trp-Leu-Gln, Trp-Phe-Gln, Trp-Tyr-Leu). CDRL2 starts about 16 residues after the end of CDRL1, is generally preceded by Ile-Tyr, Val-Tyr, Ile-Lys, Ile-Phe, and is 7 residues. CDRL3 starts about 33 residues after the end of CDRL2, is preceded by a Cys, is 7-11 residues, and is followed by Phe-Gly-XXX-Gly (XXX is any amino acid).

[0108] Illustrative examples of rules for predicting heavy chain CDRs include: CDRH1 starts about 26 residues after the end of CDRH1, is preceded by Cys-XXX-XXX-XXX, 10-12 residues, followed by Trp (typically Trp-Val, but also Trp-Ile, Trp-Ala); CDRH2 starts about 15 residues after the end of CDRH1, is generally preceded by Leu-Glu-Trp-Ile-Gly (SEQ ID NO: 138) or some variation, is 16-19 residues, followed by Lys / Arg-Leu / Ile / Val / Phe / Thr / Ala-Thr / Ser / Ile / Ala, with the AbM definition ending 7 residues earlier; CDRH3 starts about 33 residues after the end of CDRH2, is preceded by Cys-XXX-XXX (typically Cys-Ala-Arg), is 3-25 residues, followed by Trp-Gly-XXX-Gly.

[0109] References to "VH" or "VH" refer to the variable region of an immunoglobulin heavy chain, including an antibody, Fv, scFv, dsFv, Fab, or other antibody fragment disclosed herein. References to "VL" or "VL" refer to the variable region of an immunoglobulin light chain, including an antibody, Fv, scFv, dsFv, Fab, or other antibody fragment disclosed herein.

[0110] Additional definitions are set forth throughout this disclosure.

[0111] C. Engineered T Cell Receptors T cell receptors (TCRs) recognize peptide fragments of target antigens when they are presented by major histocompatibility complex (MHC) molecules. There are two distinct classes of MHC molecules, MHC I and MHC II, which deliver peptides to the cell surface from different cellular compartments. Engagement of TCRs with antigens and MHC leads to the activation of immune effector cells through a series of biochemical events mediated by associated enzymes, co-receptors, and specialized accessory molecules.

[0112] A TCR contemplated herein is a heterodimeric complex comprising a TCR alpha (TCRα) polypeptide / chain and a TCR beta (TCRβ) polypeptide / chain; or a TCR gamma (TCRγ) polypeptide / chain and a TCR delta (TCRδ) polypeptide / chain.

[0113] The human TCR alpha locus is located on chromosome 14 (14q11.2). The mature TCR alpha chain comprises a variable domain derived from recombination of a variable (V) segment and a joining (J) segment, and a constant (C) domain. The term "variable TCR alpha region" or "TCR alpha variable region" or "variable TCR alpha chain" or "TCR alpha variable chain" or "variable TCR alpha domain" or "TCR alpha variable domain" refers to the variable region of the TCR alpha chain.

[0114] The human TCR beta locus is located on chromosome 7 (7q34). The mature TCR beta chain comprises a variable domain derived from recombination of variable (V), diversity (D), and joining (J) segments, and one of two constant (C) domains. The terms "variable TCR beta region" or "TCR beta variable region" or "variable TCR beta chain" or "TCR beta variable chain" or "variable TCR beta domain" or "TCR beta variable domain" refer to the variable region of the TCR beta chain.

[0115] The human TCR gamma locus is located on chromosome 7 (7p14.1). The mature TCR gamma chain comprises a variable domain derived from recombination of a variable (V) segment with a joining (J) segment, and a constant (C) domain. The terms "variable TCR gamma region" or "TCR gamma variable region" or "variable TCR gamma chain" or "TCR gamma variable chain" or "variable TCR gamma domain" or "TCR gamma variable domain" refer to the variable region of the TCR gamma chain.

[0116] The human TCR delta locus is located on chromosome 14 (14q11.2). A mature TCR delta chain comprises a variable domain derived from recombination of variable (V), diversity (D) and joining (J) segments and one of two constant (C) domains. The terms "variable TCR delta region" or "TCR delta variable region" or "variable TCR delta chain" or "TCR delta variable chain" or "variable TCR delta domain" or "TCR delta variable domain" refer to the variable region of the TCR delta chain.

[0117] The rearranged V(D)J regions of both TCRα, TCRβ, TCRγ, and TCRδ chains each contain three hypervariable regions known as complementarity determining regions (CDRs). CDR3 is the main CDR that recognizes processed antigens, but CDR1 of the alpha chain has been shown to interact with the N-terminal portion of antigenic peptides, while CDR1 of the beta chain has been shown to interact with the C-terminal portion of antigenic peptides. CDR2 is believed to recognize MHC molecules. Framework regions (FRs) are located between the CDRs. These regions form the structure of the TCR variable regions.

[0118] The constant domains or constant regions of the TCR chains also contribute to the TCR structure and consist of an extracellular domain, a transmembrane domain, and a short cytoplasmic domain. The TCR structure results in the formation of a TCR complex. The TCR complex includes the TCRα or TCRγ chain, the TCRβ or TCRδ chain, and the accessory molecules CD3γ, CD3δ, CD3ε, and CD3ζ. Signals from the T cell complex are enhanced by simultaneous binding to MHC molecules by specific co-receptors. CD4 is a co-receptor for MHC II molecules expressed on helper T cells, and CD8 is a co-receptor for MHC I molecules expressed on cytotoxic T cells. The co-receptors not only ensure the specificity of the TCR to the antigen, but also allow long-term engagement between the antigen-presenting cell and the T cell, and also recruit essential intracellular molecules (e.g., LCK) involved in the signal transduction of activated T lymphocytes.

[0119] The engineered TCRs contemplated herein can be used to redirect immune effector cells to target cells. Additionally, the TCRs contemplated herein are engineered to include a functional antigen binding domain. In certain embodiments, the engineered TCR includes both a functional TCR binding domain (e.g., a functional TCR variable region) linked to one or both of the TCR polypeptides / chains and one or more separate antigen binding domains. Thus, in some embodiments, the engineered TCR variable domain and the additional antigen binding domain can bind to the same antigen or two different antigens, or more. In some embodiments, the engineered TCR can bind to both an intracellular antigen presented on an MHC molecule and a second antigen (e.g., a receptor, a ligand, or a cancer antigen). In some embodiments, the engineered TCR can bind to three different antigens.

[0120] The TCRs contemplated herein, sometimes referred to as engineered TCRs, hybridized TCRs, dual targeting TCRs, multitargeting TCRs, or ATOMIC (Antibody Tethered Orthogonal Multi-Compatible), comprise one or more antigen binding domain components ("A" components) and one or more TCR components ("C" components), with or without one or more linkers ("B" components), each of which are described in more detail in the subsections below.

[0121] The data of the Examples show that the engineered TCRs and fusion proteins disclosed herein can comprise an antigen binding domain ("A" component) and / or a TCR component ("C" component) specific for any antigen(s). One of skill in the art will readily appreciate that antigen binding domain components and TCR components can be combined to produce an engineered TCR or fusion protein that meets the characteristics of the engineered TCRs disclosed herein, regardless of antigen specificity or any particular sequence, e.g., variable domain or CDR sequences thereof.

[0122] This is because it has been unexpectedly discovered that the disclosed engineered TCRs and fusion proteins, comprising an antigen binding domain ("A" component) linked to one or more TCR binding domains ("C" components), have an efficient and effective structure that allows for simultaneous TCR targeting and secondary antigen binder targeting in a manner that preserves the function of both components. The antigen specificity of the components, as well as the sequences of the components, e.g., variable domain sequences or CDR sequences, can be varied by one of skill in the art using the exemplary general engineered TCR formulas provided herein. Thus, while the present disclosure and examples provide a multitude of engineered TCRs and fusion proteins comprising (i) antigen binding domain components and TCR components directed to different antigens, and (ii) different antigen binding domains directed to the same antigen, one of skill in the art will recognize that the engineered TCRs and fusion proteins disclosed and claimed herein should not be limited by antigen specificity or sequence, e.g., variable region sequences or CDR sequences.

[0123] 1. Antigen-binding domain components ("A" components) Provided herein are engineered TCRs and related fusion polypeptides comprising: (a) a TCR alpha or TCR gamma polypeptide comprising a TCR alpha or TCR gamma variable domain; (b) a TCR beta or TCR delta polypeptide comprising a TCR beta or TCR delta variable domain; and (c) one or more antigen binding domains (the "A" component) linked to a TCR alpha, TCR beta, TCR gamma and / or TCR delta variable domain.

[0124] In various embodiments, the one or more antigen binding domains (also referred to herein as binding agents or antigen-binding agents) comprise one or more, two or more, or three or more antigen binding domains. In some embodiments, the one or more antigen binding domains comprise one or more first antigen binding domains linked to any one or more of a TCR alpha, TCR beta, TCR gamma, and / or TCR delta variable domain. In some embodiments, the one or more antigen binding domains comprise a first antigen binding domain linked to a TCR alpha variable domain. In some embodiments, the one or more antigen binding domains comprise a first antigen binding domain linked to a TCR beta variable domain. In some embodiments, the one or more antigen binding domains comprise a first antigen binding domain linked to a TCR gamma variable domain. In some embodiments, the one or more antigen binding domains comprise a first antigen binding domain linked to a TCR delta variable domain. In some embodiments, the one or more antigen binding domains comprise (i) a first antigen binding domain linked to a TCR alpha variable domain, and (ii) a first antigen binding domain linked to a TCR beta variable domain. In some embodiments, the one or more antigen binding domains comprise (i) a first antigen binding domain linked to a TCR gamma variable domain, and (ii) a first antigen binding domain linked to a TCR delta variable domain. In some embodiments, the first antigen binding domains are the same or different and / or bind to the same or different target antigens.

[0125] In various embodiments, the first antigen binding domain is linked to the N-terminus of the variable domain.

[0126] In various embodiments, the one or more antigen binding domains comprise a second antigen binding domain linked to a first antigen binding domain. In some embodiments, the second antigen binding domain is N-terminal to the first antigen binding domain. In some embodiments, the one or more antigen binding domains comprise a second antigen binding domain linked to a first antigen binding domain linked to a TCR alpha variable domain. In some embodiments, the one or more antigen binding domains comprise a second antigen binding domain linked to a first antigen binding domain linked to a TCR beta variable domain. In some embodiments, the one or more antigen binding domains comprise a second antigen binding domain linked to a first antigen binding domain linked to a TCR gamma variable domain. In some embodiments, the one or more antigen binding domains comprise a second antigen binding domain linked to a first antigen binding domain linked to a TCR delta variable domain.

[0127] In various embodiments, the one or more antigen binding domains comprise (i) a second antigen binding domain linked to a first antigen binding domain linked to a TCR alpha variable domain, and (ii) a second antigen binding domain linked to a first antigen binding domain linked to a TCR beta variable domain.

[0128] In various embodiments, the one or more antigen binding domains comprise (i) a second antigen binding domain linked to a first antigen binding domain linked to a TCR gamma variable domain, and (ii) a second antigen binding domain linked to a first antigen binding domain linked to a TCR delta variable domain.

[0129] In various embodiments, the second antigen-binding domains are the same or different and / or bind to the same or different target antigens. In some embodiments, the second antigen-binding domains are the same. In some embodiments, the second antigen-binding domains are different.

[0130] In various embodiments, one or more antigen binding domains (e.g., the first and / or second antigen binding domains) bind to the alpha folate receptor (FRα), α v β 6Integrins, ADGRE2, BACE2, B cell maturation antigen (BCMA), B7-H3 (CD276), B7-H4, B7-H6, CA19.9, carbonic anhydrase IX (CAIX), CCR1, CD7, CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD133, CD138, CD171, CD244, carcinoembryonic antigen (CEA), C-type lectin-like molecule-1 (CLL-1), CD2 subset 1 (CS-1), CLDN6, cMET, chondroitin sulfate proteoglycan 4 (CSPG4), CLDN18.2, cutaneous T-cell lymphoma-associated antigen 1 (CTAGE1), DLL3, epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), EGFR806, epithelial glycoprotein 2 (EGP2), epithelial glycoprotein 40 (EGP40), EPHB2, ERBB4, epithelial cell adhesion molecule (EPCAM), ephrin type A receptor 2 (EPHA2), fibroblast activation protein (FAP), Fc receptor-like 5 (FCRL5), fetal acetylcholinesterase receptor (AchR), FLT3, FN, FN-EDB, FRBeta, ganglioside G2 (GD2), ganglioside G3 (GD3), glypican-3 (GPC3), EGFR family including ErbB2 (HER2), HER2p95, EGFRv3, IL-10Rα, IL-13Rα2, kappa, cancer / testis antigen 2 (LAGE-1A), K-Ras, K-Ras G12C, K-Ras G12D, K-Ras G12V, lambda, Lewis-Y (LeY), L1 cell adhesion molecule (L1-CAM), LILRB2, LY6G6GD, melanoma antigen recognized by T cells 1 (MelanA or MART1), mesothelin (MSLN), MMP10, MUC1, MUC16, MHC class I chain-related protein A (MICA), MHC class I chain-related protein B (MICB), neural cell adhesion molecule (NCAM), prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), The present invention binds to a target antigen selected from the group consisting of synovial sarcoma, X-breakpoint 2 (SSX2), survivin, tumor-associated glycoprotein 72 (TAG72), transmembrane activator and CAML interactor (TACI), tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), TIM3, trophoblast glycoprotein (TPBG), UL16-binding protein (ULBP) 1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, and vascular endothelial growth factor receptor 2 (VEGFR2).

[0131] In various embodiments, the one or more antigen binding domains (e.g., the first and / or second antigen binding domains) are selected from the group consisting of alpha-fetoprotein (AFP), ASCL2, B melanoma antigen (BAGE) family members, regulator of imprinted sites Brother (BORIS), cancer-testis antigen, cancer-testis antigen 83 (CT-83), carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), cytomegalovirus (CMV) antigen, cytotoxic T cell (CTL)-recognized antigen on melanoma (CAMEL), epidermal growth factor receptor 1 (EGFR), epidermal growth factor receptor 2 (EGFR), epidermal growth factor receptor 3 (EGFR), epidermal growth factor receptor 4 (EGFR), epidermal growth factor receptor 5 (EGFR), epidermal growth factor receptor 6 (EGFR), epidermal growth factor receptor 7 (EGFR), epidermal growth factor receptor 8 (EGFR), epidermal growth factor receptor 9 (EGFR), epidermal growth factor receptor 10 (EGFR), epidermal growth factor receptor 11 (EGFR), epidermal growth factor receptor 12 (EGFR), epidermal growth factor receptor 13 (EGFR), epidermal growth factor receptor 14 (EGFR), epidermal growth factor receptor 15 (EGFR), epidermal growth factor receptor 16 (EGFR), epidermal growth factor receptor 17 (EGFR), epidermal growth factor receptor 18 (EGFR), epidermal growth factor receptor 19 (EGFR), epidermal growth factor receptor 20 (EGFR), epidermal growth factor receptor 21 (EGFR), epidermal growth factor receptor 22 (EGFR), epidermal growth factor receptor 23 (EGFR), epidermal growth factor receptor 24 (EGFR), epidermal growth factor receptor 25 (EGFR), epidermal growth factor receptor 26 (EGFR), epidermal growth factor receptor 27 (EGFR), epidermal growth factor receptor 28 (EGFR), epidermal growth factor Stein-Barr virus (EBV) antigen, EPHB2, G antigen 1 (GAGE-1), GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, GAGE-8, glycoprotein 100 (GP100), Hepatitis B virus (HBV) antigen, Hepatitis C virus (HCV) nonstructural protein 3 (NS3), Human papillomavirus (HPV)-E6, HPV-E7, human telomerase reverse transcriptase (hTERT), IGF2BP3 / A3, IGF2BP1, K-Ras, K-Ras G12C, K-Ras G12D, K-Ras G12V, latent membrane protein 2 (LMP2), LY6G6D, melanoma antigen family A,1 (MAGE-A1), MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, melanoma antigen recognized by T cells (MART-1), mesothelin (MSLN), mucin 1 (MUC1), mucin 16 (MUC16), New York esophageal squamous cell carcinoma-1 (NYESO-1), P53, P antigen (PAGE) family member, PAP, PIK3CA, PIK3K The present invention binds to a target polypeptide derived from a protein selected from the group consisting of H1047R, placenta specific 1 (PLAC1), preferentially expressed antigen in melanoma (PRAME), prostate specific antigen PSA, survivin, synovial sarcoma X1 (SSX1), synovial sarcoma X2 (SSX2), synovial sarcoma X3 (SSX3), synovial sarcoma X4 (SSX4), synovial sarcoma X5 (SSX5), synovial sarcoma X8 (SSX8), thyroglobulin, TP53 R175H, tyrosinase, tyrosinase-related protein (TRP) 1, TRP2, UBD, Wilms tumor protein (WT-1), Wnt10A, X antigen family member 1 (XAGE1), and X antigen family member 2 (XAGE2).

[0132] In various embodiments, the one or more antigen binding domains bind to CD33, CLL1, CD19, CD20, CD22, CD79A, CD79B, or BCMA. In some embodiments, the one or more antigen binding domains bind to CD19, CD20, CD22, CD33, CD79A, CD79B, B7H3, Muc16, Her2, EGFR, FN-EDB, CLDN18.2, DLL3, FLT3, CLL1, CD123, or BCMA.

[0133] In various embodiments, the one or more antigen binding domains comprise an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 1-32. In various embodiments, the one or more antigen binding domains comprise an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 1-32. In various embodiments, the one or more antigen binding domains comprise an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 1-32. In some embodiments, the one or more antigen binding domains comprise an amino acid sequence that is at least 96% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 1-32. In some embodiments, the one or more antigen binding domains comprise an amino acid sequence that is at least 97% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 1-32. In some embodiments, the one or more antigen binding domains comprise an amino acid sequence that is at least 98% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 1-32. In some embodiments, the one or more antigen binding domains comprise an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 1-32. In some embodiments, the one or more antigen binding domains comprise an amino acid sequence set forth in any one of SEQ ID NOs: 1-32.

[0134] In various embodiments, the one or more antigen binding domains are selected from the group consisting of camel Ig, llama Ig, alpaca Ig, Ig NAR, Fab' fragments, F(ab') 2 Fragments, bispecific Fab dimers (Fab2), trispecific Fab trimers (Fab3), Fv, single chain Fv proteins ("scFv"), bis-scFv, (scFv) 2 , a minibody, a diabody, a triabody, a tetrabody, a disulfide-stabilized Fv protein ("dsFv"), and a single domain antibody (sdAb, camelid VHH, nanobody), or an antigen-binding fragment thereof.

[0135] In various embodiments, the one or more antigen binding domains comprise one or more single chain variable fragments (scFv).

[0136] In various embodiments, the one or more antigen binding domains comprise one or more single domain antibodies (sdAbs). In some embodiments, the sdAbs are camelid VHHs, nanobodies, or heavy chain only antibodies (HcAbs). In certain embodiments, the sdAbs are camelid VHHs.

[0137] In various embodiments, the antibody or antigen-binding fragment thereof is human or humanized.

[0138] Numerous methods can be used to obtain antibodies, or antigen-binding fragments thereof. For example, antibodies can be produced using recombinant DNA methods. Monoclonal antibodies can also be produced by hybridoma generation (see, for example, Kohler and Milstein (1975) Nature, 256:495-499) according to known methods. Hybridomas formed in this manner are then screened using standard methods, such as enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (e.g., OCTET or BIACORE) analysis, to identify one or more hybridomas that produce antibodies that specifically bind to a particular antigen. Any form of a particular antigen, such as recombinant antigen, naturally occurring form, any variant or fragment thereof, as well as antigenic peptides thereof (e.g., any of the epitopes described herein as linear epitopes or within a scaffold as a conformational epitope) may be used as an immunogen. One exemplary method of generating antibodies includes screening a protein expression library, such as a phage or ribosome display library, that expresses antibodies or fragments thereof (e.g., scFv). Phage display is described, for example, in Ladner et al., U.S. Pat. No. 5,223,409; Smith (1985) Science 228:1315-1317; Clackson et al. (1991 ) Nature, 352:624-628; Marks et al. (1991) J. Mol. Biol., 222:581-597; WO 92 / 18619; WO 91 / 17271; WO 92 / 20791; WO 92 / 15679; WO 93 / 01288; WO 92 / 01047; WO 92 / 09690; and WO 90 / 02809.

[0139] In some embodiments, monoclonal antibodies are obtained from non-human animals and then modified, for example, into chimeras using suitable recombinant DNA technology.Various approaches to make chimeric antibodies have been described.See, for example, Morrison et al., Proc.Natl.Acad.Sci.USA 81:6851, 1985; Takeda et al., Nature 314:452, 1985; Cabilly et al., U.S. Patent No. 4,816,567; Boss et al., U.S. Patent No. 4,816,397; Tanaguchi et al., European Patent Publication No. 171496; European Patent Publication No. 0173494; and British Patent No. 2177096B.

[0140] For additional antibody production techniques, see Antibodies: A Laboratory Manual, eds. Harlow et al., Cold Spring Harbor Laboratory, 1988. This disclosure is not necessarily limited to any particular source, method of production, or other special characteristics of the antibodies.

[0141] In various embodiments, the one or more antigen binding domains comprise a ligand.

[0142] 2. Linker (the "B" component) As contemplated herein, an engineered TCR may or may not include linker residues ("B" components) between the various domains, e.g., added for proper spacing and conformation of the molecule. In particular, an engineered TCR includes a linker between one or more antigen binding domains and a TCR component, e.g., a TCR variable domain. In various embodiments, one or more antigen binding domains are linked to a TCR component, e.g., a TCR variable domain, by one or more polypeptide linkers. In some embodiments, a TCR includes two or more linkers between an antigen binding domain and a TCR variable domain. In some embodiments, an engineered TCR does not include a polypeptide linker ("B" component) between an antigen binding domain and a TCR component.

[0143] A "linker" or "polypeptide linker" or "linker polypeptide" is an amino acid sequence that connects adjacent domains of a polypeptide or fusion polypeptide. An example of a linker is a glycine polymer (G). n , glycine-serine polymer (G 1-5 S 1-5 ) n (wherein n is an integer of at least 1, 2, 3, 4, or 5), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine has access to much more phi-psi space than alanine and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Linkers can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids in length.

[0144] In certain embodiments, the engineered TCR and / or antigen binding domain comprises one, two, three, four, or five or more linkers. The linker can be between the TCR variable domain and the antigen binding domain, between two or more antigen binding domains, or between the VH and VL sequences within an antigen binding domain (e.g., scFv). In certain embodiments, the linker is about 2 to about 25 amino acids in length, about 5 to about 20 amino acids, or about 10 to about 20 amino acids, or any intervening length of amino acids. In some embodiments, the linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more amino acids in length.

[0145] In various embodiments, the one or more polypeptide linkers comprise a linker that is about 2 to about 25 amino acids in length. In some embodiments, the one or more polypeptide linkers comprise a linker that is about 3 to about 20 amino acids in length. In some embodiments, the one or more polypeptide linkers comprise a linker that is about 4 to about 15 amino acids in length. In some embodiments, the one or more polypeptide linkers comprise a linker that is about 4 to about 10 amino acids in length. In some embodiments, the one or more polypeptide linkers comprise a linker that is about 4 amino acids in length. In some embodiments, the one or more polypeptide linkers comprise a linker that is about 5 amino acids in length. In some embodiments, the one or more polypeptide linkers comprise a linker that is about 6 amino acids in length. In some embodiments, the one or more polypeptide linkers comprise a linker that is about 7 amino acids in length. In some embodiments, the one or more polypeptide linkers comprise a linker that is about 8 amino acids in length. In some embodiments, the one or more polypeptide linkers comprise a linker that is about 9 amino acids in length. In some embodiments, the one or more polypeptide linkers comprise a linker that is about 10 amino acids in length. In some embodiments, the one or more polypeptide linkers comprise a linker about 11 amino acids in length. In some embodiments, the one or more polypeptide linkers comprise a linker about 12 amino acids in length. In some embodiments, the one or more polypeptide linkers comprise a linker about 13 amino acids in length. In some embodiments, the one or more polypeptide linkers comprise a linker about 14 amino acids in length. In some embodiments, the one or more polypeptide linkers comprise a linker about 15 amino acids in length.

[0146] An example of a linker is a glycine polymer (G) n , glycine-serine polymer (G 1-5 S 1-5 ) n(where n is an integer of at least 1, 2, 3, 4 or 5), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and therefore can function as neutral tethers between domains of fusion proteins such as the engineered / hybridized TCRs described herein. Glycine has access to much more phi-psi space than alanine and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Those skilled in the art will recognize that the design of engineered / hybridized TCRs in certain embodiments can include linkers that are fully or partially flexible, such that the linker can include a flexible linker as well as one or more moieties that confer a less flexible structure to provide the desired TCR / hybrid structure.

[0147] Other exemplary linkers include the following amino acid sequences: DGGGS (SEQ ID NO:40); TGEKP (SEQ ID NO:41) (see, e.g., Liu et al., PNAS 5525-5530 (1997)); GGRR (SEQ ID NO:42) (Pomerantz et al. 1995, supra); (GGGGS) n (wherein n=1, 2, 3, 4 or 5 (SEQ ID NO: 35-39) (Kim et al., PNAS 93, 1156-1160 (1996); EGKSSGSGSESKVD (SEQ ID NO: 43) (Chaudhary et al., 1990, Proc. Natl. Acad. Sci. USA 87: 1066-1070); KESGSVSEQLAQFRSLD (SEQ ID NO: 44) (Bird et al., 1988, Science 242: 423-426), GGRRGGGS (SEQ ID NO: 45); LQRDGERP (SEQ ID NO: 46); LRQKDGGGSERP (SEQ ID NO: 47); LRQKD(GGGS) 2Examples of suitable flexible linkers include, but are not limited to, ERP (SEQ ID NO: 48). Alternatively, the flexible linker can be rationally designed using computer programs that can model both the DNA binding site and the peptide itself (Desjarlais & Berg, PNAS90:2256-2260 (1993), PNAS91:11099-11103 (1994)) or by phage display methods. In certain embodiments, the linker comprises the amino acid sequence: GSTSGSGKPGSGEGSTKG (SEQ ID NO: 49) or GSTSGSGKSSEGSGSTKG (SEQ ID NO: 50) (Cooper et al., Blood, 101(4):1637-1644 (2003) and Whitlow et al., Protein Eng., 6(8):989-95 (1993)). Other linkers include GSTSGSGKSSEGKG (SEQ ID NO:51), GSTSGSGKPGSGEGS (SEQ ID NO:52), or GGGS (SEQ ID NO:53).

[0148] In various embodiments, the one or more polypeptide linkers are selected from the group consisting of GG, GS, SG, SS, GSS, SSG, GSG, SGS, SGG, GGS, GGGS (SEQ ID NO:53), (GGGGS) 1~5 The polypeptide (SEQ ID NOs: 35-39), a linker selected from the group consisting of a marsupial γμ TCR derived linker (e.g., LEKT; SEQ ID NO: 33), and any combination thereof.

[0149] In various embodiments, the one or more polypeptide linkers comprise a linker derived from a marsupial γμ TCR. In certain embodiments, the marsupial γμ TCR linker is μLNK comprising the amino acid sequence set forth in SEQ ID NO: 33. In various embodiments, the one or more polypeptide linkers comprise a marsupial γμ TCR linker and a G4S linker set forth in SEQ ID NO: 34.

[0150] In various embodiments, the one or more polypeptide linkers comprise a GGGGS (SEQ ID NO: 35) linker (G4S). In various embodiments, the one or more polypeptide linkers comprise two GGGGS linkers (2 x G4S) (SEQ ID NO: 36). In various embodiments, the one or more polypeptide linkers comprise three GGGGS linkers (3 x G4S) (SEQ ID NO: 37). In various embodiments, the one or more polypeptide linkers comprise four GGGGS linkers (4 x G4S) (SEQ ID NO: 38). In various embodiments, the one or more polypeptide linkers comprise five GGGGS linkers (5 x G4S) (SEQ ID NO: 39).

[0151] In various embodiments, the one or more polypeptide linkers comprise an amino acid sequence set forth in any one of SEQ ID NOs:33-53.

[0152] In certain embodiments, the first and second antigen-binding domains are separated by a second polypeptide linker. In some embodiments, the second polypeptide linker comprises a linker about 2 to about 25 amino acids in length. In some embodiments, the second polypeptide linker comprises a linker about 3 to about 20 amino acids in length. In some embodiments, the second polypeptide linker comprises a linker about 4 to about 15 amino acids in length. In some embodiments, the second polypeptide linker comprises a linker about 4 to about 10 amino acids in length. In some embodiments, the second polypeptide linker comprises a linker about 4 amino acids in length. In some embodiments, the second polypeptide linker comprises a linker about 5 amino acids in length. In some embodiments, the second polypeptide linker comprises a linker about 6 amino acids in length. In some embodiments, the second polypeptide linker comprises a linker about 7 amino acids in length. In some embodiments, the second polypeptide linker comprises a linker about 8 amino acids in length. In some embodiments, the second polypeptide linker comprises a linker about 9 amino acids in length. In some embodiments, the second polypeptide linker comprises a linker about 10 amino acids in length. In some embodiments, the second polypeptide linker comprises a linker about 11 amino acids in length. In some embodiments, the second polypeptide linker comprises a linker about 12 amino acids in length. In some embodiments, the second polypeptide linker comprises a linker about 13 amino acids in length. In some embodiments, the second polypeptide linker comprises a linker about 14 amino acids in length. In some embodiments, the second polypeptide linker comprises a linker about 15 amino acids in length.

[0153] In various embodiments, the second polypeptide linker is selected from the group consisting of GG, GS, SG, SS, GSS, SSG, GSG, SGS, SGG, GGS, GGGS (SEQ ID NO: 53), (GGGGS) 1~5In some embodiments, the second polypeptide linker comprises a linker selected from the group consisting of a GGGGS (SEQ ID NO: 35-39) linker (G4S). In some embodiments, the one or more polypeptide linkers comprise two GGGGS linkers (2×G4S) (SEQ ID NO: 36). In some embodiments, the second polypeptide linker comprises three GGGGS linkers (3×G4S) (SEQ ID NO: 37). In some embodiments, the second polypeptide linker comprises four GGGGS linkers (4×G4S) (SEQ ID NO: 38). In some embodiments, the second polypeptide linker comprises five GGGGS linkers (5×G4S) (SEQ ID NO: 39).

[0154] In various embodiments, the second polypeptide linker comprises an amino acid sequence set forth in any one of SEQ ID NOs: 33-53, or a combination thereof.

[0155] 3. T cell receptor components ("C" components) The engineered T cell receptors (TCRs) contemplated herein bind to polypeptide antigens presented by major histocompatibility complex (MHC) class I molecules or MHC class II molecules, preferably to polypeptide antigens presented by MHC class I molecules.

[0156] "Major histocompatibility complex" (MHC) refers to glycoproteins that deliver peptide antigens to the cell surface. MHC class I molecules are heterodimers with a membrane-spanning α chain (containing three α domains) and noncovalently associated β2-microglobulin. MHC class II molecules are composed of two transmembrane glycoproteins, α and β, both of which also span the membrane. Each chain has two domains. MHC class I molecules deliver peptides originating from the cytoplasm to the cell surface, where the peptide:MHC complex mediates the expression of CD8 +They are recognized by T cells. MHC class II molecules deliver peptides originating from the vesicle system to the cell surface, where the peptide:MHC complex is bound to CD4 + Recognized by T cells. Human MHC is also called human leukocyte antigen (HLA).

[0157] The principles of antigen processing by antigen-presenting cells (APCs) (e.g., dendritic cells, macrophages, lymphocytes, or other cell types) and presentation of antigens by APCs to T cells are well established, including major histocompatibility complex (MHC)-restricted presentation between APCs and T cells that are immunocompatible (e.g., share at least one allele type of MHC gene suitable for antigen presentation) (see, e.g., Murphy, Janeway's Immunobiology (8th Ed.) 2011 Garland Science, NY, Chapters 6, 9, and 16). For example, antigenic peptides that originate from the cytoplasm and are processed (e.g., tumor antigens, intracellular pathogens, etc.) are often about 7 amino acids to about 11 amino acids in length and are associated with class I MHC molecules, whereas peptides processed in the vesicular system (e.g., bacterial peptides, viral peptides) often vary in length from about 10 amino acids to about 25 amino acids and are associated with class II MHC molecules.

[0158] In certain embodiments, the engineered TCRs contemplated herein bind to tumor antigens, such as, for example, TAAs and TSAs. "Tumor-associated antigens" or "TAAs" include, but are not limited to, oncofetal antigens, overexpressed antigens, lineage-restricted antigens, and cancer-testis antigens. TAAs are relatively restricted to tumor cells. TAAs are expressed at elevated levels in tumor cells, but at low levels in healthy cells. "Tumor-specific antigens" or "TSAs" include, but are not limited to, neoantigens and oncoviral antigens. TSAs are unique to tumor cells. TSAs are expressed in cancer cells and not in normal cells.

[0159] In certain embodiments, the engineered TCRs contemplated herein are directed to alpha-fetoprotein (AFP), ASCL2, B melanoma antigen (BAGE) family members, regulator of imprinted sites Brother (BORIS), cancer-testis antigen, cancer-testis antigen 83 (CT-83), carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), cytomegalovirus (CMV) antigen, cytotoxic T cell (CTL) recognition antigen on melanoma (CAMEL), Epstein-Barr virus, (EBV) antigen, EPHB2, G antigen 1 (GAGE-1), GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, GAGE-8, glycoprotein 100 (GP100), hepatitis B virus (HBV) antigen, hepatitis C virus (HCV) nonstructural protein 3 (NS3), human papillomavirus (HPV)-E6, HPV-E7, human telomerase reverse transcriptase (hTERT), IGF2BP3 / A3, IGF2BP1, K-Ras, K-Ras G12C, K-Ras G12D, K-Ras G12V, latent membrane protein 2 (LMP2), LY6G6D, melanoma antigen family A,1 (MAGE-A1), MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, melanoma antigen recognized by T cells (MART-1), mesothelin (MSLN), mucin 1 (MUC1), mucin 16 (MUC16), New York esophageal squamous cell carcinoma-1 (NYESO-1), P53, P antigen (PAGE) family member, PAP, PIK3CA, PIK3CA The polypeptide binds to an antigen portion of a polypeptide selected from the group consisting of H1047R, placenta specific 1 (PLAC1), preferentially expressed antigen in melanoma (PRAME), prostate specific antigen PSA, survivin, synovial sarcoma X1 (SSX1), synovial sarcoma X2 (SSX2), synovial sarcoma X3 (SSX3), synovial sarcoma X4 (SSX4), synovial sarcoma X5 (SSX5), synovial sarcoma X8 (SSX8), thyroglobulin, TP53 R175H, tyrosinase, tyrosinase-related protein (TRP) 1, TRP2, UBD, Wilms tumor protein (WT-1), Wnt10A, X antigen family member 1 (XAGE1), and X antigen family member 2 (XAGE2).In some embodiments, the TCR variable domain binds to a target polypeptide derived from MAGE-A4, PRAME, K-Ras, TP53R175H, PSA, or IGF2BP3. In some embodiments, the TCR variable domain binds to a target polypeptide derived from MAGE-A4.

[0160] As contemplated herein, an engineered TCR comprises a TCR component (a "C" component). In some embodiments, the TCR component comprises a TCR alpha polypeptide comprising a TCR alpha variable domain. In some embodiments, the TCR component comprises a TCR beta polypeptide comprising a TCR beta variable domain. In some embodiments, the TCR component comprises a TCR gamma polypeptide comprising a TCR gamma variable domain. In some embodiments, the TCR component comprises a TCR delta polypeptide comprising a TCR delta variable domain.

[0161] In one embodiment, the TCR components ("C" components) comprise a TCR alpha polypeptide comprising a TCR alpha variable domain; and a TCR beta polypeptide comprising a TCR beta variable domain. In certain embodiments, the TCR components comprise a TCR alpha polypeptide comprising a TCR alpha variable domain; a TCR beta polypeptide comprising a TCR beta variable domain; and one or more antigen binding domains linked to a TCR gamma variable domain and / or a TCR delta variable domain.

[0162] In one embodiment, the TCR components ("C" components) comprise a TCR gamma polypeptide comprising a TCR gamma variable domain; and a TCR delta polypeptide comprising a TCR delta variable domain. In certain embodiments, the TCR components comprise a TCR gamma polypeptide comprising a TCR gamma variable domain; a TCR delta polypeptide comprising a TCR delta variable domain; and one or more antigen binding domains linked to the TCR gamma variable domain and / or the TCR delta variable domain.

[0163] In various embodiments, the TCR components ("C" components) comprise a TCR constant domain. One of skill in the art will appreciate that a given TCR variable domain may be paired with any one of several different constant domains. For example, any one of the TCR alpha, TCR beta, TCR gamma, or TCR delta variable domains may be paired with any one of the TCR alpha, TCR beta, TCR gamma, or TCR delta constant domains. In some embodiments, the TCR alpha polypeptide comprises a TCR alpha constant domain. In some embodiments, the TCR beta polypeptide comprises a TCR beta constant domain. In some embodiments, the TCR gamma polypeptide comprises a TCR gamma constant domain. In some embodiments, the TCR delta polypeptide comprises a TCR delta constant domain. In some embodiments, the TCR alpha variable domain is paired with a TCR delta constant domain. In some embodiments, the TCR alpha variable domain is paired with a TCR delta constant domain. In some embodiments, the TCR beta variable domain is paired with a TCR gamma constant domain. In some embodiments, the TCR beta variable domain is paired with a TCR gamma constant domain. In some embodiments, the TCR beta variable domain is paired with a TCR delta constant domain. In some embodiments, the TCR gamma variable domain is paired with a TCR alpha constant domain. In some embodiments, the TCR gamma variable domain is paired with a TCR beta constant domain. In some embodiments, the TCR delta variable domain is paired with a TCR alpha constant domain. In some embodiments, the TCR delta variable domain is paired with a TCR beta constant domain.

[0164] The constant domains may be derived from naturally occurring constant domains or may be mutated to enhance pairing with each other or increase stability when expressed over pairing with the native TCR. Such pairing and stability enhanced TCRs are known, see, e.g., WO2021195503A1 and WO2018102795A1, which are incorporated by reference in their entireties.

[0165] In various embodiments, the TCR alpha constant domain comprises an amino acid sequence at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 82 or 88. In some embodiments, the TCR alpha constant domain comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 82 or 88. In some embodiments, the TCR alpha constant domain comprises an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 82 or 88. In some embodiments, the TCR alpha constant domain comprises an amino acid sequence at least 96% identical to the amino acid sequence set forth in SEQ ID NO: 82 or 88. In some embodiments, the TCR alpha constant domain comprises an amino acid sequence at least 97% identical to the amino acid sequence set forth in SEQ ID NO: 82 or 88. In some embodiments, the TCR alpha constant domain comprises an amino acid sequence at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 82 or 88. In some embodiments, the TCR alpha constant domain comprises an amino acid sequence at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 82 or 88. In some embodiments, the TCR alpha constant domain comprises the amino acid sequence set forth in SEQ ID NO: 82 or 88.

[0166] In various embodiments, the TCR β constant domain comprises an amino acid sequence at least 85% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 80, 81, 86, or 87. In some embodiments, the TCR β constant domain comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 80, 81, 86, or 87. In some embodiments, the TCR β constant domain comprises an amino acid sequence at least 95% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 80, 81, 86, or 87. In some embodiments, the TCR β constant domain comprises an amino acid sequence at least 96% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 80, 81, 86, or 87. In some embodiments, the TCR β constant domain comprises an amino acid sequence at least 97% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 80, 81, 86, or 87. In some embodiments, the TCR β constant domain comprises an amino acid sequence at least 98% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 80, 81, 86, or 87. In some embodiments, the TCR β constant domain comprises an amino acid sequence at least 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 80, 81, 86, or 87. In some embodiments, the TCR β constant domain comprises the amino acid sequence set forth in any one of SEQ ID NOs: 80, 81, 86, or 87.

[0167] In various embodiments, the TCR gamma constant domain comprises an amino acid sequence at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 83 or 84. In some embodiments, the TCR gamma constant domain comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 83 or 84. In some embodiments, the TCR gamma constant domain comprises an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 83 or 84. In some embodiments, the TCR gamma constant domain comprises an amino acid sequence at least 96% identical to the amino acid sequence set forth in SEQ ID NO: 83 or 84. In some embodiments, the TCR gamma constant domain comprises an amino acid sequence at least 97% identical to the amino acid sequence set forth in SEQ ID NO: 83 or 84. In some embodiments, the TCR gamma constant domain comprises an amino acid sequence at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 83 or 84. In some embodiments, the TCR gamma constant domain comprises an amino acid sequence at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 83 or 84. In some embodiments, the TCR gamma constant domain comprises the amino acid sequence set forth in SEQ ID NO: 83 or 84.

[0168] In various embodiments, the TCR δ constant domain comprises an amino acid sequence at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 85. In some embodiments, the TCR δ constant domain comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 85. In some embodiments, the TCR δ constant domain comprises an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 85. In some embodiments, the TCR δ constant domain comprises an amino acid sequence at least 96% identical to the amino acid sequence set forth in SEQ ID NO: 85. In some embodiments, the TCR δ constant domain comprises an amino acid sequence at least 97% identical to the amino acid sequence set forth in SEQ ID NO: 85. In some embodiments, the TCR δ constant domain comprises an amino acid sequence at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 85. In some embodiments, the TCR δ constant domain comprises an amino acid sequence at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 85. In some embodiments, the TCR δ constant domain comprises the amino acid sequence set forth in SEQ ID NO: 85.

[0169] In various embodiments, the TCR alpha polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 105-111. In some embodiments, the TCR alpha polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 105. In some embodiments, the TCR alpha polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 106. In some embodiments, the TCR alpha polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 107. In some embodiments, the TCR alpha polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 108. In some embodiments, the TCR alpha polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 109. In some embodiments, the TCR alpha polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 110. In some embodiments, the TCR alpha polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 111.

[0170] In various embodiments, the TCR alpha polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 62, 64, 66, 68, 70, 72, 74, and 76. In some embodiments, the TCR alpha polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 62. In some embodiments, the TCR alpha polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 64. In some embodiments, the TCR alpha polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 66. In some embodiments, the TCR alpha polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 68. In some embodiments, the TCR alpha polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 70. In some embodiments, the TCR alpha polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 72. In some embodiments, the TCR alpha polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 74. In some embodiments, the TCR alpha polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 76.

[0171] In various embodiments, the TCR gamma polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs:78.

[0172] In various embodiments, the TCR beta polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 103 or 104. In some embodiments, the TCR beta polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 103. In some embodiments, the TCR beta polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 104.

[0173] In various embodiments, the TCR β polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 63, 65, 67, 69, 71, 73, 75, and 77. In some embodiments, the TCR β polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 63. In some embodiments, the TCR β polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 65. In some embodiments, the TCR β polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 67. In some embodiments, the TCR β polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 69. In some embodiments, the TCR β polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 71. In some embodiments, the TCR β polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 73. In some embodiments, the TCR β polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 75. In some embodiments, the TCR β polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 77.

[0174] In various embodiments, the TCR delta polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs:79.

[0175] As discussed herein, one or more antigen binding domains are linked to one or both TCR variable domains of the TCR component. For example, one or more antigen binding domains may be linked to any one or more of the TCR alpha, TCR beta, TCR gamma, or TCR delta variable domains, as the case may be. Various antigen binding domain / TCR component arrangements are contemplated herein. For example, a first antigen binding domain may be linked to the N-terminus of one or both TCR polypeptides (e.g., TCR alpha / beta or TCR gamma / delta variable regions). Additionally, a second antigen binding domain may be linked to the N-terminus of the first antigen binding domain, thereby generating a tandem antigen binding domain. The first and second antigen binding domains may be targeted to bind to the same or different antigens. Similarly, multiple first binding domains may be targeted to bind to the same or different antigens, and multiple second binding domains may be targeted to bind to the same or different antigens.

[0176] In various embodiments, the TCR component further comprises a signal sequence / peptide. In some embodiments, the TCR alpha, beta, gamma, or delta polypeptide comprises an N-terminal signal sequence.

[0177] In some embodiments, the TCR alpha polypeptide comprises an N-terminal TCR alpha, TCR beta, TCR gamma, TCR delta, CD8 alpha, or IgK signal sequence. In some embodiments, the TCR alpha polypeptide comprises an N-terminal TCR alpha signal sequence. In some embodiments, the TCR alpha polypeptide comprises an N-terminal IgK signal sequence. In some embodiments, the TCR alpha polypeptide comprises an N-terminal CD8 alpha signal sequence.

[0178] In some embodiments, the TCR β polypeptide comprises an N-terminal TCR α, TCR β, TCR γ, TCR δ, CD8 α, or IgK signal sequence. In some embodiments, the TCR β polypeptide comprises an N-terminal TCR β signal sequence. In some embodiments, the TCR β polypeptide comprises an N-terminal IgK signal sequence. In some embodiments, the TCR β polypeptide comprises an N-terminal CD8 α signal sequence.

[0179] In some embodiments, the TCRγ polypeptide comprises an N-terminal TCRα, TCRβ, TCRγ, TCRδ, CD8α, or IgK signal sequence. In some embodiments, the TCRγ polypeptide comprises an N-terminal TCRγ signal sequence. In some embodiments, the TCRγ polypeptide comprises an N-terminal IgK signal sequence. In some embodiments, the TCRγ polypeptide comprises an N-terminal CD8α signal sequence.

[0180] In some embodiments, the TCR δ polypeptide comprises an N-terminal TCR alpha, TCR beta, TCR gamma, TCR delta, CD8 alpha, or IgK signal sequence. In some embodiments, the TCR δ polypeptide comprises an N-terminal TCR delta signal sequence. In some embodiments, the TCR δ polypeptide comprises an N-terminal IgK signal sequence. In some embodiments, the TCR δ polypeptide comprises an N-terminal CD8 alpha signal sequence.

[0181] D. Exemplary Engineered TCR Polypeptides and Complexes A variety of engineered TCR polypeptides and their associated variants and complexes are contemplated herein. As discussed above, engineered TCRs surprisingly have multispecificity, the ability to simultaneously target both intracellular and extracellular targets, and increased sensitivity to non-MHC-presented targets.

[0182] Engineered TCRs can be constructed in multiple formats and can be designed and constructed using known components (e.g., antigen binding domains, polypeptide linkers, and TCR alpha and beta chains) and techniques. For example, one or more antigen binding domains (e.g., one or more "A" components) can be linked to one or more TCR components (e.g., one or more "C" components) with or without one or more polypeptide linkers (e.g., with or without one or more "B" components) using standard cloning techniques. The "A" components can optionally be linked to either the TCR alpha or TCR beta polypeptides / chains, or both, or to the TCR gamma or TCR delta, or both, of the "C" components. Exemplary engineered TCR formulas are provided below: AC A.B.C.

[0183] Exemplary antigen binding domains, linkers, and TCRs can be found in Tables 3-5 below. Additionally, Table 6 provides an exemplary list of engineered TCR / ATOMIC polypeptides and complexes based on the antigen binding domains, linkers, and TCRs provided in Tables 3, 4, and 5 (see Example 10). One of skill in the art will appreciate that other combinations are possible, including combinations using other antigen binding domains, linkers, and TCRs known to or newly developed by those of skill in the art.

[0184] In various embodiments, the TCR alpha polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 105. In various embodiments, the TCR alpha polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 106. In various embodiments, the TCR alpha polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 107. In various embodiments, the TCR alpha polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 108. In various embodiments, the TCR alpha polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 109. In various embodiments, the TCR alpha polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 110. In various embodiments, the TCR alpha polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 111.

[0185] In various embodiments, the TCR β polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 103. In various embodiments, the TCR β polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 104.

[0186] E. Polypeptides A variety of polypeptides, fusion polypeptides, and polypeptide variants are contemplated herein, including, but not limited to, TCR polypeptides, TCR alpha chain polypeptides, TCR beta chain polypeptides, TCR fusion polypeptides, and fragments thereof.

[0187] "Polypeptide," "peptide," and "protein" are used interchangeably and follow their conventional meanings unless specified to the contrary, i.e., as amino acid sequences. A polypeptide is not limited to a particular length, e.g., a polypeptide may contain a full-length polypeptide or a polypeptide fragment, and a polypeptide may include one or more post-translational modifications of a polypeptide, e.g., glycosylation, acetylation, phosphorylation, and other modifications, both natural and non-natural, known in the art.

[0188] As used herein, "isolated polypeptide" and the like refers to the in vitro synthesis, isolation and / or purification of a peptide or polypeptide molecule from the cellular environment and from association with other components of a cell, i.e., not substantially associated with in vivo substances. In certain embodiments, an isolated polypeptide is a synthetic, recombinant, or semi-synthetic polypeptide, or a polypeptide obtained or derived from a recombinant source.

[0189] Polypeptides include "polypeptide variants." Polypeptide variants may differ from naturally occurring polypeptides in one or more amino acid substitutions, deletions, additions, and / or insertions. Such variants may be natural or synthetically produced by modifying one or more of the polypeptide sequences contemplated herein. For example, in certain embodiments, it may be desirable to improve the binding affinity, stability, expression, specific pairing, functional avidity, and / or biological properties of the TCRs by introducing one or more substitutions, deletions, additions, and / or insertions into any one or more of the TCR alpha, TCR beta, TCR gamma, and / or TCR delta polypeptides, variable domains, and / or constant regions. In certain embodiments, polypeptides include those having at least about 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to any of the polypeptide sequences contemplated herein, and typically such variants retain at least one biological activity of the reference sequence.

[0190] Polypeptides include "polypeptide fragments." A polypeptide fragment may be monomeric or multimeric and refers to a biologically active polypeptide having an amino-terminal deletion, a carboxyl-terminal deletion, and / or an internal deletion or substitution of a naturally occurring or recombinantly produced polypeptide. As used herein, the term "biologically active fragment" or "minimal biologically active fragment" refers to a polypeptide fragment that retains at least 100%, at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% of the naturally occurring polypeptide activity. In certain embodiments, a polypeptide fragment may contain an amino acid chain of at least 5 to about 500 amino acids in length. In certain embodiments, the fragment is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids in length.

[0191] As mentioned above, in certain embodiments, polypeptides may be modified in various ways, including amino acid substitution, deletion, truncation and insertion. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of reference polypeptides can be generated by mutations in DNA. Methods for mutagenesis and nucleotide sequence modification are known in the art. See, for example, Kunkel (1985, Proc.Natl.Acad.Sci.USA.82:488-492), Kunkel et al. (1987, Methods in Enzymol, 154:367-382), U.S. Patent No. 4,873,192, Watson, JD et al. (Molecular Biology of the Gene, Fourth Edition, Benjamin / Cummings, Menlo Park, Calif., 1987), and references cited therein. Guidance regarding appropriate amino acid substitutions that do not affect the biological activity of the protein of interest can be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC).

[0192] In some embodiments, a polypeptide variant contains one or more conservative substitutions. A "conservative substitution" is one in which an amino acid is replaced with another amino acid with similar properties, and one skilled in the art of peptide chemistry would predict that such a substitution will not substantially change the secondary structure and hydrophobicity of the polypeptide. Modifications may be made to the polynucleotide and polypeptide structures contemplated in certain embodiments, and still result in functional molecules that encode variant or derivative polypeptides with desired characteristics. When it is desired to change the amino acid sequence of a polypeptide to generate an equivalent or improved variant polypeptide, one skilled in the art can change one or more of the codons of the encoding DNA sequence, for example, according to Table 1. [Table 1]

[0193] Guidance for determining which amino acid residues may be substituted, inserted or deleted without abolishing biological activity can be found using computer programs known in the art, such as DNASTAR, DNA Strider, Geneious, Mac Vector, or Vector NTI software. The amino acid changes in the protein variants disclosed herein are preferably conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes include substitutions of one of a family of amino acids that are related at their side chains. Naturally occurring amino acids are generally divided into four families: acidic amino acids (aspartic acid, glutamic acid), basic amino acids (lysine, arginine, histidine), nonpolar amino acids (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar amino acids (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan and tyrosine are sometimes classified jointly as aromatic amino acids. In peptides or proteins, suitable conservative substitutions of amino acids are known to those skilled in the art and can generally be made without altering the biological activity of the resulting molecule. Those skilled in the art recognize that single amino acid substitutions in non-essential regions of a polypeptide generally do not significantly alter biological activity (see, for example, Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p. 224).

[0194] As discussed above, amino acid substitutions may be based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.

[0195] Polypeptide variants also include glycosylated forms, aggregate conjugates with other molecules, and covalent conjugates with unrelated chemical moieties (e.g., pegylated molecules). Covalent variants can be prepared by linking functionalities to groups present in the amino acid chain or in the N- or C-terminal residues, as known in the art. Variants also include allelic variants, species variants, and mutant proteins. Truncation or deletion of regions that do not affect the functional activity of the protein are also variants.

[0196] In certain embodiments, expression of both TCR alpha and beta, or both TCR gamma and delta polypeptides in the same cell is desired. Polynucleotide sequences encoding the TCRs may be separated by an IRES sequence, as discussed elsewhere herein.

[0197] In preferred embodiments, fusion polypeptides are contemplated herein. Fusion polypeptides and fusion proteins refer to polypeptides having at least two, three, four, five, six, seven, eight, nine, or ten or more polypeptide segments. Fusion polypeptides are typically linked C-terminus to N-terminus, but can also be linked C-terminus to C-terminus, N-terminus to N-terminus, or M-terminus to C-terminus. In certain embodiments, the polypeptides of a fusion protein can be in any order or in a specified order.

[0198] In particularly preferred embodiments, TCR polypeptides (i.e., TCR alpha, TCR beta, TCR gamma, and / or TCR delta polypeptides) may be expressed as fusion polypeptides that include one or more self-cleaving polypeptide sequences separating the TCR polypeptides.

[0199] In certain embodiments, a TCR contemplated herein (e.g., an engineered TCR) is expressed as a fusion polypeptide comprising a TCR alpha polypeptide, a polypeptide linker (e.g., a self-cleaving polypeptide), and a TCR beta polypeptide. In certain embodiments, a TCR contemplated herein (e.g., an engineered TCR) is expressed as a fusion polypeptide comprising a TCR gamma polypeptide, a polypeptide linker (e.g., a self-cleaving polypeptide), and a TCR delta polypeptide.

[0200] In some embodiments, a TCR (e.g., an engineered TCR) is expressed as a fusion protein that includes, from N-terminus to C-terminus, a TCR alpha polypeptide, a polypeptide linker (e.g., a self-cleaving polypeptide), and a TCR beta polypeptide. In some embodiments, a TCR (e.g., an engineered TCR) is expressed as a fusion protein that includes, from N-terminus to C-terminus, a TCR beta polypeptide, a polypeptide linker (e.g., a self-cleaving polypeptide), and a TCR alpha polypeptide.

[0201] In some embodiments, a TCR (e.g., an engineered TCR) is expressed as a fusion protein that includes, from N-terminus to C-terminus, a TCRγ polypeptide, a polypeptide linker (e.g., a self-cleaving polypeptide), and a TCRδ polypeptide. In some embodiments, a TCR (e.g., an engineered TCR) is expressed as a fusion protein that includes, from N-terminus to C-terminus, a TCRδ polypeptide, a polypeptide linker (e.g., a self-cleaving polypeptide), and a TCRγ polypeptide.

[0202] In certain embodiments, an engineered TCR (e.g., an engineered TCR complex) contemplated herein is expressed as a fusion polypeptide comprising: (a) a TCR β polypeptide comprising a TCR β variable domain; (b) a polypeptide cleavage signal; and (c) a TCR α polypeptide comprising one or more antigen binding domains, a polypeptide linker, and a TCR α variable domain.

[0203] In certain embodiments, an engineered TCR (e.g., an engineered TCR complex) contemplated herein is expressed as a fusion polypeptide comprising: (a) a TCR β polypeptide comprising one or more antigen binding domains, a polypeptide linker, and a TCR β variable domain; (b) a polypeptide cleavage signal; and (c) a TCR α polypeptide comprising a TCR α variable domain.

[0204] In certain embodiments, an engineered TCR (e.g., an engineered TCR complex) contemplated herein is expressed as a fusion polypeptide comprising: (a) a TCR β polypeptide comprising one or more antigen binding domains, a polypeptide linker, and a TCR β variable domain; (b) a polypeptide cleavage signal; and (c) a TCR α polypeptide comprising one or more antigen binding domains, a polypeptide linker, and a TCR α variable domain.

[0205] In certain embodiments, an engineered TCR (e.g., an engineered TCR complex) contemplated herein is expressed as a fusion polypeptide comprising: (a) a TCRγ polypeptide comprising a TCRγ variable domain; (b) a polypeptide cleavage signal; and (c) a TCRδ polypeptide comprising one or more antigen binding domains, a polypeptide linker, and a TCRδ variable domain.

[0206] In certain embodiments, an engineered TCR (e.g., an engineered TCR complex) contemplated herein is expressed as a fusion polypeptide comprising: (a) a TCRγ polypeptide comprising one or more antigen binding domains, a polypeptide linker, and a TCRγ variable domain; (b) a polypeptide cleavage signal; and (c) a TCRδ polypeptide comprising a TCRδ variable domain.

[0207] In certain embodiments, an engineered TCR (e.g., an engineered TCR complex) contemplated herein is expressed as a fusion polypeptide comprising: (a) a TCRγ polypeptide comprising one or more antigen binding domains, a polypeptide linker, and a TCRγ variable domain; (b) a polypeptide cleavage signal; and (c) a TCRδ polypeptide comprising one or more antigen binding domains, a polypeptide linker, and a TCRδ variable domain.

[0208] The fusion polypeptide may comprise any of the TCR polypeptides contemplated herein.

[0209] Fusion proteins contemplated herein also include a polypeptide cleavage signal between the TCR polypeptides. Examples of polypeptide cleavage signals include polypeptide cleavage recognition sites, such as protease cleavage sites, nuclease cleavage sites (e.g., rare restriction enzyme recognition sites, self-cleaving ribozyme recognition sites), and self-cleaving viral oligopeptides (see deFelipe and Ryan, 2004. Traffic, 5(8); 616-26).

[0210] Suitable protease cleavage sites and autocleaving peptides are known to those of skill in the art (see, for example, Ryan et al., 1997. J. Gener. Virol. 78, 699-722; Scymczak et al. (2004) Nature Biotech. 5, 589-594). Examples of protease cleavage sites include, but are not limited to, cleavage sites for potyvirus NIa protease (e.g., tobacco etch virus protease), potyvirus HC protease, potyvirus P1 (P35) protease, byovirus NIa protease, biovirus RNA-2-encoded protease, aphthovirus L protease, enterovirus 2A protease, rhinovirus 2A protease, picorna 3C protease, comovirus 24K protease, nepovirus 24K protease, RTSV (Waika virus) 3C-like protease, PYVF (parsnip yellow mottle virus) 3C-like protease, heparin, thrombin, factor Xa, and enterokinase. Due to its high cleavage stringency, in one embodiment the TEV (Tobacco Etch Virus) protease cleavage site, EXXYXQ(G / S), e.g., ENLYFQG (SEQ ID NO:114) and ENLYFQS (SEQ ID NO:115), is preferred, where X represents any amino acid (TEV cleavage occurs between Q and G or between Q and S).

[0211] In certain embodiments, the polypeptide cleavage signal is a viral autocleaving peptide or a ribosomal skipping sequence.

[0212] Illustrative examples of ribosomal skipping sequences include, but are not limited to, 2A or 2A-like sites, sequences or domains (Donnelly et al., 2001 J. Gen. Virol. 82:1027-1041).

[0213] In certain embodiments, the viral 2A peptide is an aphthovirus 2A peptide, a potyvirus 2A peptide, or a cardiovirus 2A peptide. In one embodiment, the viral 2A peptide is selected from the group consisting of a foot and mouth disease virus (FMDV) 2A peptide, an equine rhinitis A virus (ERAV) 2A peptide, a zosea signavirus (TaV) 2A peptide, a porcine teschovirus-1 (PTV-1) 2A peptide, a tylovirus 2A peptide, and an encephalomyocarditis virus 2A peptide.

[0214] Examples of 2A sites are provided in Table 1. [Table 2]

[0215] In certain embodiments, the fusion protein comprises a polypeptide cleavage signal, which is a viral autocleaving peptide or a ribosomal skipping sequence.

[0216] In certain embodiments, the fusion protein comprises a polypeptide cleavage signal, the polypeptide cleavage signal is a viral 2A peptide. In certain embodiments, the fusion protein comprises a polypeptide cleavage signal, the polypeptide cleavage signal is an aphthovirus 2A peptide, a potyvirus 2A peptide, or a cardiovirus 2A peptide. In certain embodiments, the fusion protein comprises a polypeptide cleavage signal, the polypeptide cleavage signal is a viral 2A peptide, the viral 2A peptide is selected from the group consisting of a foot and mouth disease virus (FMDV) 2A peptide, an equine rhinitis A virus (ERAV) 2A peptide, a zosea signa virus (TaV) 2A peptide, a porcine teschovirus-1 (PTV-1) 2A peptide, a tylovirus 2A peptide, and an encephalomyocarditis virus 2A peptide.

[0217] In certain embodiments, the polypeptide cleavage signal is a viral autocleavage peptide or a ribosomal skipping sequence. In some embodiments, the polypeptide cleavage signal is a viral 2A peptide. In some embodiments, the polypeptide cleavage signal is an aphthovirus 2A peptide, a potyvirus 2A peptide, or a cardiovirus 2A peptide. In some embodiments, the polypeptide cleavage signal is a viral 2A peptide, selected from the group consisting of foot and mouth disease virus (FMDV) 2A peptide, equine rhinitis A virus (ERAV) 2A peptide, zosea signa virus (TaV) 2A peptide, porcine teschovirus-1 (PTV-1) 2A peptide, tylovirus 2A peptide, and encephalomyocarditis virus 2A peptide.

[0218] In various embodiments, the polypeptide cleavage signal comprises a self-cleaving peptide (eg, a 2A peptide) and a GSG amino acid sequence immediately upstream (ie, N-terminal) of the 2A peptide.

[0219] In various embodiments, the polypeptide cleavage signal further comprises a furin recognition site (e.g., a self-cleaving 2A peptide) upstream of the polypeptide cleavage signal. In certain embodiments, the furin recognition site comprises the amino acid sequence set forth in SEQ ID NO:112.

[0220] In various embodiments, the polypeptide cleavage signal comprises an amino acid sequence set forth in any one of SEQ ID NOs: 113-137. In some embodiments, the polypeptide cleavage signal comprises an amino acid sequence set forth in SEQ ID NO: 113. In some embodiments, the polypeptide cleavage signal comprises an amino acid sequence set forth in SEQ ID NO: 114. In some embodiments, the polypeptide cleavage signal comprises an amino acid sequence set forth in SEQ ID NO: 115. In some embodiments, the polypeptide cleavage signal comprises an amino acid sequence set forth in SEQ ID NO: 116. In some embodiments, the polypeptide cleavage signal comprises an amino acid sequence set forth in SEQ ID NO: 117. In some embodiments, the polypeptide cleavage signal comprises an amino acid sequence set forth in SEQ ID NO: 118. In some embodiments, the polypeptide cleavage signal comprises an amino acid sequence set forth in SEQ ID NO: 119. In some embodiments, the polypeptide cleavage signal comprises an amino acid sequence set forth in SEQ ID NO: 120. In some embodiments, the polypeptide cleavage signal comprises an amino acid sequence set forth in SEQ ID NO: 121. In some embodiments, the polypeptide cleavage signal comprises an amino acid sequence set forth in SEQ ID NO: 122. In some embodiments, the polypeptide cleavage signal comprises an amino acid sequence set forth in SEQ ID NO: 123. In some embodiments, the polypeptide cleavage signal comprises an amino acid sequence set forth in SEQ ID NO: 124. In some embodiments, the polypeptide cleavage signal comprises the amino acid sequence set forth in SEQ ID NO: 125. In some embodiments, the polypeptide cleavage signal comprises the amino acid sequence set forth in SEQ ID NO: 126. In some embodiments, the polypeptide cleavage signal comprises the amino acid sequence set forth in SEQ ID NO: 127. In some embodiments, the polypeptide cleavage signal comprises the amino acid sequence set forth in SEQ ID NO: 128. In some embodiments, the polypeptide cleavage signal comprises the amino acid sequence set forth in SEQ ID NO: 129. In some embodiments, the polypeptide cleavage signal comprises the amino acid sequence set forth in SEQ ID NO: 130. In some embodiments, the polypeptide cleavage signal comprises the amino acid sequence set forth in SEQ ID NO: 131. In some embodiments, the polypeptide cleavage signal comprises the amino acid sequence set forth in SEQ ID NO: 132.In some embodiments, the polypeptide cleavage signal comprises the amino acid sequence set forth in SEQ ID NO: 133. In some embodiments, the polypeptide cleavage signal comprises the amino acid sequence set forth in SEQ ID NO: 134. In some embodiments, the polypeptide cleavage signal comprises the amino acid sequence set forth in SEQ ID NO: 135. In some embodiments, the polypeptide cleavage signal comprises the amino acid sequence set forth in SEQ ID NO: 136. In some embodiments, the polypeptide cleavage signal comprises the amino acid sequence set forth in SEQ ID NO: 137.

[0221] In various embodiments, the TCR β or TCR δ polypeptide is N-terminal to the TCR α or TCR γ polypeptide.

[0222] In various embodiments, the TCR alpha or gamma polypeptide is N-terminal to the TCR beta or TCR delta polypeptide.

[0223] In certain embodiments, the fusion polypeptide comprises an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 91-97, 100, and 102. In some embodiments, the fusion polypeptide comprises an amino acid sequence at least 85% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 91-97, 100, and 102. In some embodiments, the fusion polypeptide comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 91-97, 100, and 102. In some embodiments, the fusion polypeptide comprises an amino acid sequence at least 95% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 91-97, 100, and 102. In some embodiments, the fusion polypeptide comprises an amino acid sequence at least 96% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 91-97, 100, and 102. In some embodiments, the fusion polypeptide comprises an amino acid sequence that is at least 97% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 91-97, 100, and 102. In some embodiments, the fusion polypeptide comprises an amino acid sequence that is at least 98% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 91-97, 100, and 102. In some embodiments, the fusion polypeptide comprises an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 91-97, 100, and 102.

[0224] In certain embodiments, the fusion polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 91-97, 100, and 102. In some embodiments, the fusion polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 91. In some embodiments, the fusion polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 92. In some embodiments, the fusion polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 93. In some embodiments, the fusion polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 94. In some embodiments, the fusion polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 95. In some embodiments, the fusion polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 96. In some embodiments, the fusion polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 97. In some embodiments, the fusion polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 100. In some embodiments, the fusion polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 102.

[0225] F. Polynucleotides In certain embodiments, one or more polynucleotides are provided that encode one or more TCR polypeptides, TCR alpha polypeptides, TCR beta polypeptides, TCR gamma polypeptides, TCR delta polypeptides, TCR fusion polypeptides, and fragments thereof. As used herein, the term "polynucleotide" or "nucleic acid" refers to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and DNA / RNA hybrids. Polynucleotides may be monocistronic or polycistronic, single-stranded or double-stranded, and may be recombinant, synthetic, or isolated. Polynucleotides include, but are not limited to, pre-messenger RNA (pre-mRNA), messenger RNA (mRNA), RNA, genomic DNA (gDNA), PCR-amplified DNA, complementary DNA (cDNA), synthetic DNA, or recombinant DNA. Polynucleotide refers to a polymeric form of nucleotides of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 1000, at least 5000, at least 10000, or at least 15000 or more nucleotides in length, including ribonucleotides or deoxyribonucleotides, or modified forms of either type of nucleotide, as well as all intermediate lengths. In this context, "intermediate length" will be readily understood to mean any length between the cited values, e.g., 6, 7, 8, 9, etc., 101, 102, 103, etc., 151, 152, 153, etc., 201, 202, 203, etc. In certain embodiments, a polynucleotide or variant has at least or about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a reference sequence.

[0226] As used herein, an "isolated polynucleotide" refers to a polynucleotide that has been purified from adjacent sequences in nature, e.g., a DNA fragment that has been removed from sequences that normally flank the fragment. In certain embodiments, an "isolated polynucleotide" also refers to a complementary DNA (cDNA), recombinant DNA, or other polynucleotide that does not occur in nature and is produced by man. In certain embodiments, an isolated polynucleotide is a synthetic polynucleotide, a recombinant polynucleotide, a semi-synthetic polynucleotide, or a polynucleotide obtained or derived from a recombinant source.

[0227] In various embodiments, the polynucleotide comprises an mRNA that encodes a polypeptide contemplated herein, hi some embodiments, the mRNA comprises a cap, one or more nucleotides, and a poly(A) tail.

[0228] In various embodiments, the polynucleotide is an mRNA that is introduced into a cell to transiently express a desired polypeptide.

[0229] As used herein, "transient" refers to expression of a non-integrated transgene over a period of hours, days, or weeks, which is shorter than the period of expression of a polynucleotide when integrated into the genome of a cell or contained within a stable plasmid replicon.

[0230] In certain embodiments, the mRNA encoding the polypeptide is an in vitro transcribed mRNA.As used herein, "in vitro transcribed RNA" refers to an RNA, preferably an mRNA, synthesized in vitro.Generally, the in vitro transcribed RNA is generated from an in vitro transcription vector.The in vitro transcription vector comprises a template that is used to generate the in vitro transcribed RNA.

[0231] In certain embodiments, an mRNA may further comprise a 5' cap or modified 5' cap, and / or a poly(A) sequence. As used herein, a 5' cap (also referred to as an RNA cap, an RNA 7-methylguanosine cap, or an RNA m 7GA 5' cap (also called a cap) is a modified guanine nucleotide added to the "front" or 5' end of a eukaryotic messenger RNA immediately after the initiation of transcription. The 5' cap is linked to the first transcribed nucleotide and contains a terminal group that is recognized by the ribosome and protected from RNases. The capping moiety can be modified to modulate the function of the mRNA, such as translation stability or efficiency. In certain embodiments, the mRNA comprises a poly(A) sequence of about 50 to about 5000 adenines. In one embodiment, the mRNA comprises a poly(A) sequence of about 100 to about 1000 bases, about 200 to about 500 bases, or about 300 to about 400 bases. In one embodiment, the mRNA comprises a poly(A) sequence of about 65 bases, about 100 bases, about 200 bases, about 300 bases, about 400 bases, about 500 bases, about 600 bases, about 700 bases, about 800 bases, about 900 bases, or about 1000 bases or more. The poly(A) sequence may be chemically or enzymatically modified to modulate mRNA function, such as localization, stability, or efficiency of translation. In certain embodiments, the polynucleotide may be codon-optimized. As used herein, the term "codon optimization" refers to the replacement of codons in a polynucleotide encoding a polypeptide to increase the expression, stability, and / or activity of the polypeptide. Factors that influence codon optimization include, but are not limited to, one or more of the following: (i) variation in codon bias between two or more organisms or genes or a synthetically constructed bias table; (ii) variation in the degree of codon bias within an organism, gene or set of genes; (iii) systematic variation of a codon with its context; (iv) variation of a codon with its decoding tRNA; (v) variation of a codon with its GC % either in the entire triplet or in one position of the triplet; (vi) variation in similarity to a reference sequence, e.g., a natural sequence; (vii) variation in codon frequency cutoff; (viii) structural properties of the mRNA transcribed from the DNA sequence; (ix) prior knowledge of the function of the DNA sequence on which the design of the codon substitution set is based; (x) synthetic variation of the codon set for each amino acid; and / or (xi) isolated removal of incorrect translation start positions.

[0232] As used herein, terms such as "polynucleotide variant" and "variant" refer to a polynucleotide that exhibits a comparable sequence identity with a reference polynucleotide sequence, or hybridizes with a reference sequence under stringent conditions as defined herein.These terms include polynucleotides in which one or more nucleotides are added or deleted or replaced with different nucleotides compared to the reference polynucleotide.In this regard, it is understood in the art that certain modifications, including mutations, additions, deletions and substitutions, can be made to the reference polynucleotide, and the modified polynucleotide can retain the biological function or biological activity of the reference polynucleotide.

[0233] Polynucleotide variants include polynucleotide fragments that encode biologically active polypeptide fragments or variants. As used herein, the term "polynucleotide fragment" refers to a polynucleotide fragment that encodes a polypeptide that retains at least 100%, at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% of the activity of a naturally occurring polypeptide, including at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, It refers to a polynucleotide fragment having a length of 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700 or more nucleotides. A polynucleotide fragment refers to a polynucleotide that encodes a polypeptide having an amino-terminal deletion, a carboxyl-terminal deletion, a carboxy-terminal deletion, and / or an internal deletion or substitution of one or more amino acids of a naturally-occurring or recombinantly produced polypeptide.

[0234] "Sequence identity", or phrases such as "50% identical sequence to", as used herein, refer to the degree to which sequences are identical nucleotide-by-nucleotide or amino acid-by-amino acid over a comparison window.Thus, "percentage of sequence identity" may be calculated by: comparing two optimally aligned sequences over a comparison window; determining the number of positions where identical nucleobases (e.g., A, T, C, G, I) or identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) are present in both sequences; calculating the number of matching positions; dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size); and multiplying the result by 100 to calculate the percentage of sequence identity. Included are nucleotides and polypeptides having at least about 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the reference sequences described herein, and typically the polypeptide variants retain at least one biological activity of the reference polypeptide.

[0235] Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include "reference sequence," "comparison window," "sequence identity," "percentage of sequence identity," and "substantial identity." A "reference sequence" comprises nucleotides and amino acid residues that are at least 12 monomeric units in length, and often 15-18 monomeric units, and often at least 25 monomeric units in length. Two polynucleotides may each contain (1) sequences that are similar between the two polynucleotides (i.e., only a portion of the complete polynucleotide sequence), and (2) sequences that diverge between the two polynucleotides, and sequence comparison between two (or more) polynucleotides is typically performed by comparing the sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of at least 6 contiguous positions, usually about 50 to about 100, more commonly about 100 to about 150, and a sequence is compared to a reference sequence over the same number of contiguous positions after the two sequences are optimally aligned. The comparison window may contain about 20% or less additions or deletions (i.e., gaps) compared to the reference sequence (not including additions or deletions) for optimal alignment of two sequences. Optimal alignment of sequences for aligning the comparison window can be performed by computerized implementation of algorithms (GAP, BESTFIT, FASTA, and TFASTA) in Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA, or by inspection and best alignment (i.e., resulting in the highest homology over the comparison window) generated by any of the various methods selected. For example, see the BLAST family of programs disclosed by Altschul et al., 1997, Nucl.Acids Res.25:3389.A detailed discussion of sequence analysis can be found in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons Inc., 1994-1998, Chapter 15, Unit 19.3.

[0236] Terms describing the orientation of a polynucleotide include 5' (usually the end of a polynucleotide having a free phosphate group) and 3' (usually the end of a polynucleotide having a free hydroxyl (OH) group). A polynucleotide sequence may be annotated in a 5' to 3' orientation or a 3' to 5' orientation. For DNA and mRNA, the 5' to 3' strand is designated the "sense", "plus" or "coding" strand because its sequence is identical to that of the pre-messenger (pre-mRNA) [except for uracil (U) in RNA instead of thymine (T) in DNA]. For DNA and mRNA, the complementary 3' to 5' strand, which is the strand transcribed by RNA polymerase, is designated as the "template", "antisense", "minus" or "non-coding" strand. As used herein, the term "reverse" refers to a 5' to 3' sequence written in a 3' to 5' direction or a 3' to 5' sequence written in a 5' to 3' direction.

[0237] Furthermore, those skilled in the art will recognize that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that code for the polypeptides described herein or variant fragments thereof. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any native gene. Nevertheless, polynucleotides that vary due to differences in codon usage are contemplated, and in certain embodiments, for example, polynucleotides optimized for human and / or primate codon preferences are contemplated. Furthermore, alleles of genes comprising the polynucleotide sequences provided herein may also be used. An allele is an endogenous gene that is altered as a result of one or more mutations, such as deletions, additions and / or substitutions of nucleotides.

[0238] As used herein, the term "nucleic acid cassette" or "expression cassette" refers to a genetic sequence in a vector capable of expressing an RNA and subsequently expressing a polypeptide. In one embodiment, the nucleic acid cassette contains a gene of interest, e.g., a polynucleotide of interest. In another embodiment, the nucleic acid cassette contains one or more expression control sequences, e.g., a promoter, an enhancer, a poly(A) sequence, and a gene of interest, e.g., a polynucleotide of interest. A vector may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleic acid cassettes. The nucleic acid cassettes are positionally and sequentially oriented within the vector so that the nucleic acid in the cassette can be transcribed into RNA, translated into a protein or polypeptide if necessary, subjected to appropriate post-translational modifications required for activity in the transformed cell, targeted to an appropriate intracellular compartment for transfer to a compartment suitable for biological activity, or secreted into an extracellular compartment. The cassette preferably has a 3' and 5' end adapted for immediate insertion into the vector, e.g., a restriction endonuclease site at each end. In a preferred embodiment, the nucleic acid cassette encodes one or more chains of a TCR. The cassette can be extracted and inserted as a single unit into a plasmid or viral vector.

[0239] Polynucleotides include polynucleotides of interest. As used herein, the term "polynucleotide of interest" refers to a polynucleotide that encodes a polypeptide, a polypeptide variant, or a fusion polypeptide. A vector may include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 polynucleotides of interest. In certain embodiments, the polynucleotide of interest encodes a polypeptide that provides a therapeutic effect in the treatment or prevention of a disease or injury. Polynucleotides of interest, and the polypeptides encoded therefrom, include both polynucleotides that encode wild-type polypeptides, as well as functional variants and fragments. In certain embodiments, functional variants have at least 80%, at least 90%, at least 95%, or at least 99% identity to the corresponding wild-type reference polynucleotide or polypeptide sequence. In certain embodiments, functional variants or fragments have at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the biological activity of the corresponding wild-type polypeptide.

[0240] The polynucleotides contemplated herein, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters and / or enhancers, untranslated regions (UTRs), signal sequences, Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosome entry sites (IRES), recombinase recognition sites (e.g., LoxP, FRT, and Att sites), stop codons, transcription termination signals, and polynucleotides encoding self-cleaving polypeptides, epitope tags, etc., disclosed elsewhere herein or known in the art, so that their overall length may vary significantly. Thus, polynucleotide fragments of almost any length may be used in certain embodiments, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.

[0241] Polynucleotides may be prepared, manipulated, and / or expressed using any of a variety of established techniques known and available in the art. To express a desired polypeptide, a nucleotide sequence encoding the polypeptide can be inserted into an appropriate vector, which is discussed further below.

[0242] Examples of vectors include, but are not limited to, plasmids, autonomously replicating sequences, and transposable elements, such as piggyBac, Sleeping Beauty, Mos1, Tc1 / mariner, Tol2, mini-Tol2, Tc3, MuA, Himar I, Frog Prince and derivatives thereof.

[0243] Additional examples of vectors include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages, such as lambda phage or M13 phage, and animal viruses.

[0244] Examples of viruses useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), pox viruses, baculoviruses, papilloma viruses, and papova viruses (e.g., SV40).

[0245] Exemplary expression vectors include, but are not limited to, pClneo vector (Promega) for expression in mammalian cells, pLenti4 / V5-DEST™, pLenti6 / V5-DEST™, and pLenti6.2 / V5-GW / lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. In certain embodiments, coding sequences for the polypeptides disclosed herein may be ligated into such expression vectors for expression of the polypeptides in mammalian cells.

[0246] In certain embodiments, the vector is an episomal vector or a vector that is maintained extrachromosomally. As used herein, the term "episomal" refers to a vector that can replicate without being integrated into the chromosomal DNA of the host, and does not decay over time with the division of the host cell, which also means that the vector replicates extrachromosomally or episomally.

[0247] "Control elements," "control sequences," present in an expression vector are non-translated regions of the vector, including origins of replication, selection cassettes, promoters, enhancers, translation initiation signals (Shine Dalgarno or Kozak sequences), introns, polyadenylation sequences, 5' and 3' non-translated regions, which interact with host cellular proteins to effect transcription and translation. Such factors may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation factors may be used, including ubiquitous and inducible promoters.

[0248] In certain embodiments, vectors include, but are not limited to, expression vectors and viral vectors, which contain exogenous, endogenous, or heterologous control sequences, such as promoters and / or enhancers. An "endogenous" control sequence is a sequence that is naturally linked to a given gene in the genome. An "exogenous" control sequence is one that is placed in juxtaposition to a gene by means of genetic engineering (i.e., molecular biology techniques) such that transcription of that gene is directed by the linked enhancer / promoter. A "heterologous" control sequence is an exogenous sequence that originates from a different species than the cell being genetically engineered.

[0249] As used herein, the term "promoter" refers to a recognition site in a polynucleotide (DNA or RNA) to which an RNA polymerase binds. The RNA polymerase initiates and transcribes a polynucleotide operably linked to the promoter. In certain embodiments, promoters that operate in mammalian cells contain an AT-rich region located approximately 25-30 bases upstream from the site where transcription is initiated, and / or a separate sequence, a CNCAAT region, located 70-80 bases upstream from the transcription initiation site, where N can be any nucleotide.

[0250] The term "enhancer" refers to a DNA segment that contains a sequence that can provide enhanced transcription, and in some cases can function regardless of orientation relative to another control sequence. Enhancers can function in concert with or additively with a promoter and / or other enhancer elements. The term "promoter / enhancer" refers to a DNA segment that contains a sequence that can provide both promoter and enhancer functions.

[0251] The term "operably linked" refers to a juxtaposition where the described components are in a relationship permitting them to function in their intended manner. In one embodiment, the term refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter and / or enhancer) and a second polynucleotide sequence, e.g., a polynucleotide of interest, where the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.

[0252] As used herein, the term "structural expression control sequence" refers to a promoter, enhancer, or promoter / enhancer that allows for continuous or sequential transcription of an operably linked sequence. A structural expression control sequence may be a "ubiquitous" promoter, enhancer, or promoter / enhancer that allows for expression in a variety of cell and tissue types, or it may be a "cell-specific," "cell type-specific," "cell line-specific," or "tissue-specific" promoter, enhancer, or promoter / enhancer that allows for expression in restricted cell and tissue types, respectively.

[0253] Exemplary ubiquitous expression control sequences suitable for use in certain embodiments include, but are not limited to, the cytomegalovirus (CMV) immediate early promoter, the viral Simian Virus 40 (SV40) (e.g., early or late), Moloney Murine Leukemia Virus (MoMLV) LTR promoter, Rous Sarcoma Virus (RSV) LTR, Herpes Simplex Virus (HSV) (thymidine kinase) promoter, the H5, P7.5 and P11 promoters from vaccinia virus, elongation factor 1 alpha (EF1a) promoter, early growth response protein 1 (EGR1), ferritin H (FerH), ferritin L (FerL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70 kDa protein 5 (HSPA5), heat shock protein 90 kDa, and the like. beta, member 1 (HSP90B1), heat shock protein 70 kDa (HSP70), beta-kinesin (β-KIN), human ROSA 26 locus (Irions et al., Nature Biotechnology 25, 1477-1482 (2007)), ubiquitin C promoter (UBC), phosphoglycerate kinase-1 (PGK) promoter, cytomegalovirus enhancer / chicken beta-actin (CAG) promoter, beta-actin promoter and myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer binding site substitution (MND) U3 promoter (Haas et al. Journal of Virology. 2003; 77 (17): 9439-9450).

[0254] In one embodiment, the vector contains the MNDU3 promoter.

[0255] In one embodiment, the vector contains the EF1a promoter including the first intron of the human EF1a gene.

[0256] In one embodiment, the vector contains the EF1a promoter lacking the first intron of the human EF1a gene.

[0257] In certain embodiments, it may be desirable to express a polynucleotide comprising an engineered TCR from a T cell specific promoter.

[0258] As used herein, "conditional expression" may refer to any type of conditional expression, including inducible expression, repressible expression, expression in cells or tissues having a particular physiological, biological or disease state, and the like. This definition is not intended to exclude cell type-specific or tissue-specific expression. Certain embodiments provide for conditional expression of a polynucleotide of interest, e.g., expression is controlled by exposing a cell, tissue, organism to a treatment or condition in which the polynucleotide is expressed or in which expression of a polynucleotide encoded by the polynucleotide of interest is increased or decreased.

[0259] Examples of inducible promoters / systems include, but are not limited to, steroid-inducible promoters, such as promoters of genes encoding glucocorticoid receptors or estrogen receptors (inducible by treatment with the corresponding hormones), metallothionein promoters (inducible by treatment with various heavy metals), MX-1 promoters (inducible by interferon), the "GeneSwitch" mifepristone-regulated system (Sirin et al., 2003, Gene, 323:67), cumate-inducible gene switches (WO2002 / 088346), tetracycline-dependent regulatory systems, and the like.

[0260] Conditional expression can also be achieved by using site-specific DNA recombinases. According to certain embodiments, the vector contains at least one (typically two) site for recombination mediated by a site-specific recombinase. As used herein, the term "recombinase" or "site-specific recombinase" includes excisive or integrative proteins, enzymes, cofactors or associated proteins involved in a recombination reaction involving one or more recombination sites (e.g., two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.), which may be wild-type proteins (see Landy, Current Opinion in Biotechnology 3:699-707 (1993)), or mutants, derivatives (e.g., fusion proteins comprising the recombination protein sequence or fragments thereof), fragments and variants thereof. Illustrative examples of recombinases suitable for use in certain embodiments include, but are not limited to, Cre, Int, IHF, Xis, Flp, Fis, Hin, Gin, ΦC31, Cin, Tn3 resolvase, TndX, XerC, XerD, TnpX, Hjc, Gin, SpCCE1, and ParA.

[0261] A vector may contain one or more recombination sites for any of a wide range of site-specific recombinases. It should be understood that the target site for the site-specific recombinase is in addition to any site(s) required for integration of the vector, e.g., a retroviral or lentiviral vector. As used herein, the term "recombination sequence," "recombination site," or "site-specific recombination site" refers to a specific nucleic acid sequence that a recombinase recognizes and binds to.

[0262] For example, one recombination site for Cre recombinase is loxP, a 34 base pair sequence that contains two 13 base pair inverted repeats (which remain as recombinase binding sites) flanking an 8 base pair core sequence (see Figure 1 in Sauer, B., Current Opinion in Biotechnology 5:521-527 (1994)). Other exemplary loxP sites include, but are not limited to, lox511 (Hoess et al., 1996; Bethke and Sauer, 1997), lox5171 (Lee and Saito, 1998), lox2272 (Lee and Saito, 1998), m2 (Langer et al., 2002), lox71 (Albert et al., 1995), and lox66 (Albert et al., 1995).

[0263] Suitable recognition sites for the FLP recombinase include, but are not limited to, FRT (McLeod et al., 1996), F 1 , F 2 , F 3 (Schlake and Bode, 1994), F 4 , F 5 (Schlake and Bode, 1994), FRT(LE) (Senecoff et al., 1988), and FRT(RE) (Senecoff et al., 1988).

[0264] Other examples of recognition sequences are the attB, attP, attL, and attR sequences, which are recognized by the recombinase enzyme λ integrase, e.g., phi-c31. The φC31 SSR mediates recombination only between the heterotypic sites attB (34 bp long) and attP (approximately 39 bp long) (Groth et al., 2000). attB and attP are named after the attachment sites of the phage integrase on the bacterial and phage genomes, respectively, but both contain imperfect inverted repeats that are likely bound by φC31, approximately 100 nm in diameter (Groth et al., 2000). The product sites, attL and attR, are furthermore effectively inactive to φC31-mediated recombination (Belteki et al., 2003), rendering the reaction irreversible. To catalyze insertion, it has been found that attB-bearing DNA inserts into genomic attP sites more easily than attP sites insert into genomic attB sites (Thyagarajan et al., 2001; Belteki et al., 2003). Thus, a typical strategy is to position an attP-bearing "docking site" at a defined locus by homologous recombination, which is then joined with an attB-bearing incoming sequence for insertion.

[0265] As used herein, "internal ribosome entry site" or "IRES" refers to an element that promotes direct entry of an internal ribosome into an initiation codon, such as ATG, of a cistron (protein coding region), resulting in cap-independent gene translation. See, e.g., Jackson et al., 1990. Trends Biochem Sci 15(12):477-83) and Jackson and Kaminski. 1995. RNA1(10):985-1000. In certain embodiments, a vector comprises one or more polynucleotides of interest that encode one or more polypeptides. In certain embodiments, to achieve efficient translation of each of the multiple polypeptides, the polynucleotide sequences may be separated by one or more IRES sequences or polynucleotide sequences that encode self-cleaving polypeptides. In one embodiment, the IRES used in the polynucleotides contemplated herein is the EMCV IRES.

[0266] As used herein, the term "Kozak sequence" refers to a short nucleotide sequence that greatly promotes initial binding of mRNA to the small ribosomal subunit and increases translation. The consensus Kozak sequence is (GCC)RCCATGG (SEQ ID NO: 139), where R is a purine (A or G) (Kozak, 1986. Cell. 44(2):283-92, and Kozak, 1987. Nucleic Acids Res. 15(20):8125-48). In certain embodiments, the vector comprises a polynucleotide, which has a consensus Kozak sequence and encodes a desired polypeptide, such as a TCR.

[0267] Factors that induce efficient termination and polyadenylation of heterologous nucleic acid transcripts increase expression of heterologous genes. Transcription termination signals are generally found downstream of polyadenylation signals. In certain embodiments, the vector includes a polyadenylation sequence 3' of the polynucleotide encoding the expressed polypeptide. As used herein, the term "polyA site" or "polyA sequence" refers to a DNA sequence that directs both the termination and polyadenylation of the nascent RNA transcript by RNA polymerase II. Polyadenylation sequences can promote mRNA stability by adding a polyA tail to the 3' end of the coding sequence, thus contributing to improved translation efficiency. Cleavage and polyadenylation are directed by poly(A) sequences in the RNA. The core poly(A) sequence of mammalian pre-mRNA has two recognition elements flanking the cleavage polyadenylation site. Typically, a nearly invariant AAUAAA hexamer is present 20-50 nucleotides upstream of a more variable element rich in U or GU residues. Cleavage of the initial transcript occurs between these two elements, adding up to 250 adenosines to the 5' cleavage product. In certain embodiments, the core poly(A) sequence is a polyA sequence of choice (e.g., AATAAA, ATTAAA, AGTAAA). In certain embodiments, the poly(A) sequence is the SV40 polyA sequence, bovine growth hormone polyA sequence (BGHpA), rabbit β-globin polyA sequence (rβgpA), variants thereof, or another suitable heterologous or endogenous polyA sequence known in the art.

[0268] In some embodiments, the polynucleotide, or the cell carrying the polynucleotide, utilizes a suicide gene, including an inducible suicide gene, to reduce the risk of direct toxicity and / or uncontrolled amplification. In certain aspects, the suicide gene is not immunogenic to the host or the cell carrying the polynucleotide. Certain examples of suicide genes that can be used are caspase-9 or caspase-8 or cytosine deaminase. Caspase-9 can be activated using a specific chemical inducer of dimerization (CID).

[0269] In certain embodiments, polycistronic polynucleotides encoding fusion proteins encoding TCRs are contemplated herein. In some embodiments, polycistronic polynucleotides encoding TCRs comprising TCR alpha polypeptides / chains and TCR beta polypeptides / chains are introduced into cells. In some embodiments, polycistronic polynucleotides encoding TCRs comprising TCR gamma polypeptides / chains and TCR delta polypeptides / chains are introduced into cells.

[0270] In certain embodiments, the polycistronic polynucleotide comprises a TCR alpha polypeptide / chain 5' to a TCR beta polypeptide / chain. In other embodiments, the polycistronic polynucleotide comprises a TCR beta polypeptide / chain 5' to a TCR alpha polypeptide / chain. In other embodiments, the polycistronic polynucleotide comprises a TCR delta polypeptide / chain 5' to a TCR gamma polypeptide / chain. In other embodiments, the polycistronic polynucleotide comprises a TCR gamma polypeptide / chain 5' to a TCR delta polypeptide / chain.

[0271] G. Vector In certain embodiments, one or more polynucleotides encoding the TCR alpha and / or beta polypeptides / chains are introduced into cells (eg, immune effector cells) by a non-viral or viral vector.

[0272] The term "vector" is used herein to refer to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule. The transferred nucleic acid is generally inserted, for example, into a vector nucleic acid molecule. The vector may contain sequences that direct autonomous replication in a cell, or may contain sequences sufficient to allow integration into host cell DNA. In certain embodiments, non-viral vectors are used to deliver one or more polynucleotides contemplated herein to T cells.

[0273] Examples of non-viral vectors include, but are not limited to, mRNA, plasmids (e.g., DNA or RNA plasmids), transposons, cosmids, and bacterial artificial chromosomes. Other non-viral vectors are discussed above.

[0274] Exemplary non-viral methods of delivery of polynucleotides contemplated in certain embodiments include, but are not limited to, electroporation, sonoporation, lipofection, microinjection, biolistec, virosomes, liposomes, immunoliposomes, nanoparticles, polycation or lipid:nucleic acid complexes, naked DNA, artificial virions, DEAE-dextran mediated transfer, gene guns, and heat shock.

[0275] Examples of non-viral / polynucleotide delivery systems suitable for use in certain contemplated embodiments include, but are not limited to, systems provided by Amaxa Biosystems, Maxcyte, Inc., BTX Molecular Delivery Systems, and Copernicus Therapeutics Inc. Lipofection reagents are commercially available (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids suitable for efficient receptor-recognition lipofection of polynucleotides are described in the literature. See, for example, Liu et al. (2003) Gene Therapy. 10:180-187; and Balazs et al. (2011) Journal of Drug Delivery. 2011:1-12. Antibody-targeted delivery, bacteria-guided delivery, and non-biological nanocell-based delivery are also contemplated in certain embodiments.

[0276] In various embodiments, the polynucleotide is the mRNA that is introduced into cell to transiently express desired polypeptide.As used herein, "transient" refers to the expression of non-integrated transgene over a period of hours, days, or weeks, and the expression period is shorter than the expression period of polynucleotide when it is integrated into the genome of cell or contained in stable plasmid replicon.

[0277] In certain embodiments, one or more of the polynucleotides contemplated herein are delivered to T cells using a viral vector.

[0278] In certain embodiments, contemplated polynucleotide-containing viral vectors may be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous or intracranial infusion) or local application as described below. Alternatively, vectors may be delivered ex vivo to cells, such as, for example, cells explanted from an individual patient (e.g., mobilized peripheral blood, lymphocytes, bone marrow aspirates, tissue biopsies, etc.) or hematopoietic stem cells of a universal donor, followed by reimplantation of the cells into the patient.

[0279] In one embodiment, viral vectors containing nuclease variants and / or donor repair templates are administered directly to an organism for transduction of cells in vivo. Alternatively, naked DNA may be administered. Administration is by any route normally used to introduce molecules into final contact with blood or tissue cells, including, but not limited to, injection, infusion, topical application, and electroporation. Suitable methods of administering such nucleic acids are available and well known to those skilled in the art, and although a particular composition can be administered using more than one route, a particular route may often provide a more immediate and more effective response than another route.

[0280] Examples of viral vector systems suitable for use in certain contemplated embodiments include, but are not limited to, adeno-associated virus (AAV), retrovirus, herpes simplex virus, adenovirus, and vaccinia virus vectors.

[0281] In certain embodiments, a polycistronic polynucleotide encoding a TCR comprising a TCR alpha polypeptide / chain and a TCR beta polypeptide / chain is introduced into a cell by a non-viral or viral vector. In certain embodiments, the polycistronic polynucleotide comprises a TCR alpha polypeptide / chain 5' to the TCR beta polypeptide / chain. In other embodiments, the polycistronic polynucleotide comprises a TCR beta polypeptide / chain 5' to the TCR alpha polypeptide / chain.

[0282] In some embodiments, a polycistronic polynucleotide encoding a TCR comprising a TCR alpha polypeptide / chain and a TCR beta polypeptide / chain is introduced into a cell by a non-viral or viral vector. In some embodiments, a polycistronic polynucleotide encoding a TCR comprising a TCR gamma polypeptide / chain and a TCR delta polypeptide / chain is introduced into a cell by a non-viral or viral vector.

[0283] In certain embodiments, the polycistronic polynucleotide comprises a TCR alpha polypeptide / chain 5' to a TCR beta polypeptide / chain. In other embodiments, the polycistronic polynucleotide comprises a TCR beta polypeptide / chain 5' to a TCR alpha polypeptide / chain. In other embodiments, the polycistronic polynucleotide comprises a TCR delta polypeptide / chain 5' to a TCR gamma polypeptide / chain. In other embodiments, the polycistronic polynucleotide comprises a TCR gamma polypeptide / chain 5' to a TCR delta polypeptide / chain.

[0284] In various embodiments, the one or more polynucleotides are introduced into immune effector cells, e.g., T cells, by transducing the cells with a recombinant adeno-associated virus (rAAV) containing the one or more polynucleotides.

[0285] AAV is a small (about 26 nm), replication-deficient, mostly episomal, non-enveloped virus. AAV can infect both dividing and non-dividing cells and can integrate its genome into the genome of a host cell. Recombinant AAV (rAAV) typically consists of a minimal transgene and its control sequences, as well as 5' and 3' AAV inverted terminal repeats (ITRs). The ITR sequences are about 145 bp in length. In certain embodiments, rAAV contains the ITRs and capsid sequences isolated from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10.

[0286] In some embodiments, chimeric rAAVs are used. The ITR sequences are isolated from one AAV serotype, and the capsid sequences are isolated from another AAV serotype. For example, a rAAV that includes ITR sequences from AAV2 and capsid sequences from AAV6 is called AAV2 / AAV6. In certain embodiments, a rAAV vector can include ITRs from AAV2 and capsid proteins from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10. In a preferred embodiment, the rAAV includes ITR sequences from AAV2 and capsid sequences from AAV6. In a preferred embodiment, the rAAV includes ITR sequences from AAV2 and capsid sequences from AAV2.

[0287] In some embodiments, methods of engineering and selection may be performed on AAV capsids to increase the probability that they will transduce cells of interest.

[0288] The construction, production, and purification of rAAV vectors are disclosed, for example, in U.S. Pat. Nos. 9,169,494, 9,169,492, 9,012,224, 8,889,641, 8,809,058, and 8,784,799, each of which is incorporated by reference in its entirety.

[0289] In various embodiments, one or more polynucleotides are introduced into immune effector cells, e.g., T cells, comprising one or more polynucleotides by transducing the cells with a retrovirus, e.g., a lentivirus.

[0290] As used herein, the term "retrovirus" refers to an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy and then covalently integrates its genomic DNA into the host genome. Exemplary retroviruses suitable for use in certain embodiments include, but are not limited to, Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemia virus, murine stem cell virus (MSCV) and Rous sarcoma virus (RSV), and lentiviruses.

[0291] As used herein, the term "lentivirus" refers to a group (or genus) of complex retroviruses. Exemplary lentiviruses include, but are not limited to, HIV (including human immunodeficiency virus, HIV type 1 and HIV type 2), Visnamaedivirus (VMV), Caprine Arthritis Encephalitis Virus (CAEV), Equine Infectious Anemia Virus (EIAV), Feline Immunodeficiency Virus (FIV), Bovine Immunodeficiency Virus (BIV), and Simian Immunodeficiency Virus (SIV). In one embodiment, an HIV-based vector backbone (i.e., HIV cis-acting sequence elements) is preferred.

[0292] In various embodiments, lentiviral vectors contemplated herein contain one or more LTRs and one or more or all of the following accessory elements: cPPT / FLAP, Psi (Ψ) packaging signal, export element, poly(A) sequence, and may optionally contain a WPRE or HPRE, an insulator element, a selection marker, and a cell suicide gene, as otherwise discussed herein.

[0293] In certain embodiments, the lentivirus vector contemplated herein may be an integrative, or non-integrative, or integration-defective lentivirus.As used herein, the term "integration-defective lentivirus" or "IDLV" refers to a lentivirus that has an integrase that is deficient in integrating viral genome into the genome of a host cell.Integration-incompetent viral vectors are described in patent application WO2006 / 010834, which is incorporated herein by reference in its entirety.

[0294] Exemplary mutations in the HIV-1 pol gene suitable for reducing integrase activity include, but are not limited to, H12N, H12C, H16C, H16V, S81 R, D41A, K42A, H51A, Q53C, D55V, D64E, D64V, E69A, K71A, E85A, E87A, D116 N, D1161, D116A, N120G, N1201, N120E, E152G, E152A, D35E, K156E, K156A, E 157A, K159E, K159A, K160A, R166A, D167A, E170A, H171A, K173A, K186Q, K1 86T, K188T, E198A, R199c, R199T, R199A, D202A, K211A, Q214L, Q216L, Q221 L, W235F, W235E, K236S, K236A, K246A, G247W, D253A, R262A, R263A, and K264H.

[0295] In one embodiment, the HIV-1 integrase deleted pol gene comprises D64V, D116I, D116A, E152G, or E152A mutations, D64V, D116I, and E152G mutations, or D64V, D116A, and E152A mutations.

[0296] In one embodiment, the HIV-1 integrase defective pol gene comprises a D64V mutation.

[0297] The term "long terminal repeat (LTR)" refers to the domains of base pairs located at the ends of retroviral DNA; in the native sequence, the LTRs are direct repeats and contain the U3, R and U5 regions.

[0298] As used herein, the term "FLAP element" or "cPPT / FLAP" refers to a nucleic acid whose sequence includes the central polypurine tract and central termination sequences (cPPT and CTS) of retroviruses, such as HIV-1 and HIV-2. Suitable FLAP elements are described in U.S. Pat. No. 6,682,907 and Zennou, et al., 2000, Cell, 101:173. In another embodiment, the lentiviral vector contains a FLAP element having one or more mutations in the cPPT and / or CTS elements. In yet another embodiment, the lentiviral vector contains either the cPPT or CTS elements. In yet another embodiment, the lentiviral vector does not contain a cPPT or CTS element.

[0299] As used herein, the term "packaging signal" or "packaging sequence" refers to the psi [Ψ] sequence located in the retroviral genome, which is required for inserting viral RNA into the viral capsid or viral particle.See, for example, Clever et al., 1995. J. of Virology, Vol. 69, No. 4, pp. 2101-2109.

[0300] The term "export factor" refers to a cis-acting post-transcriptional regulator that controls the transport of RNA transcripts from the nucleus to the cytoplasm of a cell. Examples of RNA export factors include, but are not limited to, the rev-responsive factor (RRE) of human immunodeficiency virus (HIV) (see, e.g., Cullen et al., 1991. J. Virol. 65:1053; and Cullen et al., 1991. Cell 58:423) and the post-transcriptional regulator (HPRE) of hepatitis B virus.

[0301] In certain embodiments, the expression of heterologous sequence in viral vector is increased by incorporating post-transcriptional regulators, efficient polyadenylation sites, and optionally transcription termination signals into the vector.Various post-transcriptional regulatory elements can increase the expression of heterologous nucleic acid in protein, for example, Woodchuck Hepatitis Virus post-transcriptional regulatory element (WPRE; Zufferey et al., 1999 J.Virol., 73:2886); Post-transcriptional regulatory element present in Hepatitis B virus (HPRE) (Huang et al., Mol.Cell.Biol., 5:3864); and the like (Liu et al., 1995 Genes Dev., 9:1766).

[0302] Lentiviral vectors preferably include several safety enhancements as a result of the modification of the LTR. A "self-inactivating" (SIN) vector refers to a vector that lacks replication capacity, such as a retroviral or lentiviral vector, in which the right (3') LTR enhancer-promoter region, known as the U3 region, is modified (e.g., by deletion or substitution) to inhibit viral transcription beyond the first round of viral replication. Self-inactivation is preferably achieved by introducing a deletion in the U3 region of the 3'LTR of the vector DNA, i.e., the DNA used to generate the vector RNA. This deletion is then transferred to the 5'LTR of the proviral DNA during reverse transcription. In certain embodiments, it is desirable to remove enough of the U3 sequence to greatly reduce or completely abolish the transcriptional activity of the LTR, thereby greatly reducing or abolishing the production of full-length vector RNA in transduced cells. In the case of HIV-based lentivectors, it has been shown that such vectors can tolerate large U3 deletions, including removal of the LTR TATA box (e.g., deletion of -418 to -18), without significant loss of vector titer.

[0303] Additional safety enhancement is provided by replacing the U3 region of the 5'LTR with a heterologous promoter that drives transcription of the viral genome during production of viral particles. Examples of heterologous promoters that can be used include, for example, Simian Virus 40 (SV40) (e.g., early or late), Cytomegalovirus (CMV) (e.g., immediate early), Moloney Murine Leukemia Virus (MoMLV), Rous Sarcoma Virus (RSV), and Herpes Simplex Virus (HSV) (thymidine kinase) promoters.

[0304] As used herein, the term "pseudotype" or "pseudotyped" refers to a virus in which the viral envelope protein has been replaced with the envelope of another virus that has favorable properties. For example, the HIV envelope protein (encoded by the env gene) is normally encoded by the CD4+ Although targeting the virus to presentation cells, pseudotyping HIV with the vesicular stomatitis virus G-protein (VSV-G) envelope protein allows HIV to infect a broad range of cells.

[0305] In one embodiment, lentiviral vectors are produced by known methods, see, e.g., Kutner et al., BMC Biotechnol. 2009;9:10.doi:10.1186 / 1472-6750-9-10; Kutner et al., Nat. Protoc. 2009;4(4):495-505.doi:10.1038 / nprot.2009.22.

[0306] According to certain embodiments contemplated herein, most or all of the backbone sequences of the viral vectors are derived from lentiviruses, such as HIV-1. However, it should be understood that many different sources of retroviral and / or lentiviral sequences can be used, or that a number of combined substitutions and modifications of a particular lentiviral sequence can be accommodated without impairing the ability of the transfer vector to perform the functions described herein. Furthermore, various lentiviral vectors are known in the art, see Naldini et al. (1996a, 1996b and 1998); Zufferey et al., (1997), Dull et al., 1998, U.S. Patent Nos. 6,013,516 and 5,994,136, many of which can be adapted to produce the viral vectors or transfer plasmids contemplated herein.

[0307] In various embodiments, the one or more polynucleotides are introduced into the immune effector cells by transducing the cells with an adenovirus containing the one or more polynucleotides.

[0308] Adenovirus-based vectors are capable of extremely high transduction efficiency in many cell types and do not require cell division. High titers and high levels of expression have been obtained using such vectors. The vectors can be produced in large quantities in a relatively simple system. Most adenovirus vectors are engineered such that a transgene replaces the Ad E1a, E1b, and / or E3 genes, and the replication-deficient vectors are then propagated in human 293 cells that supply the deleted gene functions in trans. Ad vectors can transduce multiple types of tissues in vivo, including non-dividing cells, differentiated cells, such as those found in the liver, kidney, and muscle. Traditional Ad vectors have a large carrying capacity.

[0309] The creation and amplification of current replication-deficient adenoviral vectors may utilize a unique helper cell line called 293. This cell line was transformed with Ad5 DNA fragments from human embryonic kidney cells and constitutively expresses E1 protein (Graham et al., 1977). Because the E3 region is dispensable for the adenoviral genome (Jones & Shenk, 1978), current adenoviral vectors utilize 293 cells to deliver foreign DNA in either the E1, D3, or both regions (Graham & Prevec, 1991). Adenoviral vectors have been used for eukaryotic gene expression (Levrero et al., 1991; Gomez-Foix et al., 1992) and vaccine development (Grunhaus & Horwitz, 1992; Graham & Prevec, 1992). Experiments in administering recombinant adenoviruses to various tissues include tracheal instillation (Rosenfeld et al., 1991; Rosenfeld et al., 1992), intramuscular injection (Ragot et al., 1993), peripheral intravenous injection (Herz & Gerard, 1993), and stereotactic inoculation into the brain (Le Gal La Salle et al., 1993). Examples of the use of Ad vectors in clinical trials include polynucleotide therapy for antitumor immunization using intramuscular injection (Sterman et al., Hum. Gene Ther. 7:1083-9 (1998)).

[0310] In various embodiments, the one or more polynucleotides are introduced into immune effector cells by transducing the cells with a herpes simplex virus, eg, HSV-1, HSV-2, to contain the one or more polynucleotides.

[0311] Mature HSV virions consist of an enveloped icosahedral capsid containing a viral genome consisting of a linear double-stranded DNA molecule that is 152 kb. In one embodiment, the HSV-based viral vector is defective in one or more essential or non-essential HSV genes. In one embodiment, the HSV-based viral vector is replication-defective. Most replication-defective HSV vectors contain deletions to remove one or more immediate early, early, or late HSV genes to prevent replication. For example, the HSV vector may be defective in an immediate early gene selected from the group consisting of ICP4, ICP22, ICP27, ICP47, and combinations thereof. The advantages of HSV vectors are their ability to enter latency, which can result in long-term DNA expression, and their large viral DNA genome, which can accommodate up to 25 kb of exogenous DNA. HSV-based vectors are described, for example, in U.S. Pat. Nos. 5,837,532, 5,846,782 and 5,804,413, and International Patent Applications WO91 / 02788, WO96 / 04394, WO98 / 15637 and WO99 / 06583, which are incorporated herein by reference in their entireties.

[0312] H. Genetically Modified Cells In various embodiments, cells genetically modified to express engineered TCRs are contemplated herein. In some embodiments, immune effector cells genetically modified to express engineered TCRs as contemplated herein are used in the preparation or manufacture of medicaments for the treatment of cancer.

[0313] As used herein, the term "genetically engineered" or "genetically modified" refers to the addition of additional genetic material in the form of DNA or RNA to the total genetic material in a cell. The terms "genetically modified cell", "modified cell", and "redirected cell" are used interchangeably. As used herein, the term "gene therapy" refers to the introduction of extra genetic material in the form of DNA or RNA to the total genetic material in a cell to restore, correct, or alter the expression of a gene, or to introduce extra genetic material in the form of DNA or RNA for the purpose of expressing a therapeutic polypeptide, e.g., an engineered TCR.

[0314] In certain embodiments, a polynucleotide encoding an engineered TCR contemplated herein is introduced into an immune effector cell to express the engineered TCR and to redirect the immune effector cell to a target cell expressing a target antigen. An "immune effector cell" is any cell of the immune system that has one or more effector functions (e.g., cytotoxic cell killing activity, cytokine secretion, induction of ADCC and / or CDC, etc.). Exemplary immune effector cells contemplated herein include, but are not limited to, cytotoxic T cells (CTL; ​​CD8 + T cells), TILs, and helper T cells (HTLs; CD4 + In certain embodiments, the cells include T lymphocytes, including αβ T cells. In certain embodiments, the cells include γδ T cells engineered to express an αβ TCR. In one embodiment, the immune effector cells include natural killer (NK) cells. In one embodiment, the immune effector cells include natural killer T (NKT) cells.

[0315] Immune effector cells may be self or non-self (e.g., allogeneic, syngeneic, or xenogeneic). As used herein, "autologous" refers to cells derived from the same subject. As used herein, "allogeneic" refers to cells of the same species that are genetically different from the compared cells. As used herein, "syngeneic" refers to cells of a different subject that are genetically identical to the compared cells. As used herein, "xenogeneic" refers to cells of a different species from the compared cells. In a preferred embodiment, the cells are autologous.

[0316] Examples of immune effector cells for use with the engineered TCRs in certain embodiments include T lymphocytes. The term "T cell" or "T lymphocyte" is art-recognized and is intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. T cells can be T helper (Th) cells, e.g., T helper 1 (Th1) or T helper 2 (Th2) cells. T cells can be T helper cells (HT1; CD4 + T cell) CD4 + T cells, cytotoxic T cells (CTL:CD8 + T cells), CD4 + CD8 + T cells, CD4 - CD8 - T cells, or any other subset of T cells. Other exemplary populations of T cells suitable for use in certain embodiments include naive T cells (T N ), T memory stem cells (T SCM ), central memory T cells (T CM ), effector memory T cells (T EM ), and effector T cells (T EFF ) are mentioned.

[0317] As will be appreciated by those skilled in the art, other cells may also be used with the engineered TCR contemplated herein as immune effector cells. In particular, immune effector cells also include NK cells, NKT cells, neutrophils, and macrophages. Immune effector cells also include precursors of effector cells, and such precursor cells may be induced to differentiate into immune effector cells in vivo or in vitro. Thus, in certain embodiments, immune effector cells include precursors of immune effector cells, such as hematopoietic stem cells (HSCs), contained within the CD34+ population of cells derived from, for example, umbilical cord blood, bone marrow, or mobilized peripheral blood, which differentiate into mature immune effector cells when administered to a subject, or may be induced to differentiate in vitro into mature immune effector cells.

[0318] As used herein, the term "CD34+ cell" refers to a cell that expresses CD34 protein on its cell surface.As used herein, "CD34" refers to a cell surface glycoprotein (e.g., sialomucin protein) that often acts as a cell-cell adhesion factor and is involved in the entry of T cells into lymph nodes.CD34+ cell population contains hematopoietic stem cells (HSCs), which differentiate when administered to patients and give rise to all hematopoietic lineages, including T cells, NK cells, NKT cells, neutrophils, and cells of monocyte / macrophage lineage.

[0319] A method for producing immune effector cells expressing the engineered TCR contemplated herein is provided in certain embodiments. In one embodiment, the method comprises transfecting or transducing immune effector cells isolated from an individual such that the immune effector cells express a polynucleotide or polycistronic message encoding the engineered TCR contemplated herein or a fusion protein encoding the engineered TCR contemplated herein. In certain embodiments, the transduced cells are then cultured and expanded before being administered to a subject.

[0320] In some embodiments, immune effector cells are isolated from an individual and genetically modified without further manipulation in vitro.These cells may then be directly re-administered to the individual.In further embodiments, immune effector cells are first activated and stimulated to proliferate in vitro before being genetically modified to express the engineered TCR contemplated herein.In this regard, immune effector cells may be cultured before and / or after being genetically modified.

[0321] In certain embodiments, a source of cells is obtained from a subject prior to in vitro manipulation or genetic modification of immune effector cells as described herein. In certain embodiments, the modified immune effector cells comprise T cells.

[0322] In certain embodiments, PBMCs may be directly genetically modified to express an engineered TCR as contemplated herein. In certain embodiments, following isolation of PBMCs, T lymphocytes may be further isolated, and in certain embodiments, both cytotoxic and helper T lymphocytes may be preserved into naive, memory and effector T cell subpopulations, either before or after genetic modification and / or expansion.

[0323] Immune effector cells, such as T cells, may be isolated using known methods and then genetically modified, or immune effector cells may be activated and expanded (or differentiated in the case of progenitor cells) in vitro and then genetically modified. In certain embodiments, immune effector cells, such as T cells, are activated, stimulated for expression, and then genetically modified with a TCR contemplated herein (e.g., transduced with a viral vector comprising a nucleic acid encoding a polycistronic message encoding an engineered TCR contemplated herein). In various embodiments, the T cells may be activated and expanded before or after genetic modification using, for example, the methods described in U.S. Patent Nos. 6,352,694, 6,534,055, 6,905,680, 6,692,964, 5,858,358, 6,887,466, 6,905,681, 7,144,575, 7,067,318, 7,172,869, 7,232,566, 7,175,843, 5,883,223, 6,905,874, 6,797,514, 6,867,041, and U.S. Patent Application Publication No. 20060121005.

[0324] In one embodiment, CD34 + The cells are transduced with a nucleic acid construct as contemplated herein. In a particular embodiment, the transduced CD34 + The cells are differentiated in vivo into mature immune effector cells, generally after administration to a subject from whom the cells were originally isolated. + Cells may be stimulated in vitro prior to exposure to, or after being genetically modified with, one or more of the following cytokines: Flt-3 ligand (FLT3), stem cell factor (SCF), megakaryocyte growth and differentiation factor (TPO), IL-3, and IL-6, according to previously reported methods (Asheuer et al., 2004; Imren, et al., 2004).

[0325] In certain embodiments, the population of modified immune effector cells for the treatment of cancer comprises the engineered TCR contemplated herein.For example, the population of modified immune effector cells is prepared from peripheral blood mononuclear cells (PBMCs) obtained from a patient (autologous donor) diagnosed with a B-cell malignancy as described herein.PBMCs form a heterogeneous population of T lymphocytes that can be CD4+, CD8+, or CD4+ and CD8+.

[0326] PBMCs may also contain other cytotoxic lymphocytes such as NK cells or NKT cells. An expression vector carrying the coding sequence of an engineered TCR contemplated in certain embodiments is introduced into a population of human donor T cells, NK cells, or NKT cells. In certain embodiments, successfully transduced T cells carrying the expression vector can be further expanded to increase the number of these CAR protein expressing T cells by isolating CD3 positive T cells using flow cytometry, followed by cell activation using anti-CD3 antibodies and / or anti-CD28 antibodies and IL-2 or any other method known in the art as described elsewhere herein. Standard procedures are used for cryopreservation of T cells expressing CAR protein T cells for storage and / or preparation for use in human subjects. In one embodiment, in vitro transduction, culture, and / or expansion of T cells is performed in the absence of non-human animal derived products such as fetal calf serum and fetal bovine serum. Because a heterogeneous population of PBMCs are genetically modified, the resulting transduced cells are a heterogeneous population of modified cells that contain a BCMA targeting CAR as contemplated herein.

[0327] In further embodiments, a mixture of, for example, one, two, three, four, five, or more different expression vectors can be used in genetically modifying a donor population of immune effector cells, each vector encoding a different chimeric antigen receptor protein as contemplated herein, and the resulting modified immune effector cells form a mixed population of modified cells.

[0328] Genetically engineered cells, including T cells, can be produced using a variety of methods known in the art, see, for example, WO 2016 / 094304, which is incorporated by reference in its entirety.

[0329] I. Compositions and Formulations Compositions contemplated herein may include one or more engineered TCR polypeptides, TCR alpha polypeptides, TCR beta polypeptides, TCR gamma polypeptides, TCR delta polypeptides, TCR fusion polypeptides, polynucleotides, vectors containing the same genetically modified immune effector cells contemplated herein. Compositions include, but are not limited to, pharmaceutical compositions. In a preferred embodiment, the composition comprises one or more cells modified to express an engineered TCR contemplated herein.

[0330] A "pharmaceutical composition" refers to a composition formulated in a pharma- ceutically or physiologically acceptable solution for administration to a cell or animal, alone or in combination with one or more other therapeutic modalities. If desired, it is understood that the composition may be administered in combination with other agents as well, such as, for example, cytokines, growth factors, hormones, small molecules, chemotherapeutic agents, prodrugs, drugs, antibodies, or a variety of other pharma- ceutical active agents. There is virtually no limit to the other components that may be included in the composition, provided that the added agents do not adversely affect the ability of the composition to deliver the intended therapy. In a preferred embodiment, the pharmaceutical composition comprises a pharma- ceutically acceptable carrier, diluent, or excipient, as well as one or more cells modified to express an engineered TCR as contemplated herein.

[0331] As used herein, the phrase "pharmacologically acceptable" is employed to refer to those compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0332] As used herein, a "pharmaceutically acceptable carrier, diluent, or excipient" includes, but is not limited to, an adjuvant, carrier, excipient, lubricant, sweetener, diluent, preservative, dye / colorant, flavoring agent, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, surface active agent, or emulsifier approved by the U.S. Food and Drug Administration as acceptable for human or veterinary use. Exemplary pharma- ceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; tragacanth; malt; gelatin; talc; cocoa butter, waxes, animal and vegetable fats, paraffin, silicone, bentonite, silicic acid, zinc oxide; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, phosphate buffers, and any other compatible substance employed in pharmaceutical formulations.

[0333] In certain embodiments, the formulation of pharma- ceutically acceptable carrier solutions is known in the art, as is the development of appropriate dosing and treatment regimens for use of the particular compositions described herein in a variety of treatment regimens, including, for example, enteral and parenteral, e.g., intravascular, intravenous, intraarterial, intra-arterial, intraosseous, intraventricular, intracerebral, intracranial, intrathecal, intrathecal, and intramedullary administration and formulations. Those of skill in the art will appreciate that certain embodiments contemplated herein are known, for example, in the pharmaceutical arts, and can be found, for example, in Remington: The Science and Practice of Pharmacy, volume I and volume II.22, incorporated herein by reference in their entireties. nd It will be understood that the present invention may include other formulations such as those described in the 2012 Edition. Edited by Loyd V. Allen Jr. Philadelphia, PA: Pharmaceutical Press;

[0334] In certain embodiments, the compositions include an amount of immune effector cells expressing an engineered TCR as contemplated herein. As used herein, the term "amount" refers to an "effective amount" or "effective amount" of genetically modified therapeutic cells, such as T cells, to achieve a beneficial or desired prophylactic or therapeutic result, including a clinical result.

[0335] "Prophylactically effective amount" refers to an amount of genetically modified therapeutic cells effective to achieve a desired prophylactic result. Typically, but not necessarily, a prophylactic dose is used before or in subjects at an early stage of disease, so the prophylactically effective amount is less than the therapeutically effective amount.

[0336] A "therapeutically effective amount" of genetically modified therapeutic cells may vary depending on factors such as the disease state, age, sex, and weight of the individual, as well as the ability of the stem and progenitor cells to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the virus or transduced therapeutic cells are outweighed by the therapeutically beneficial effects. The term "therapeutically effective amount" includes an amount effective to "treat" a subject (e.g., a patient). When a therapeutic amount is indicated, the exact amount of the composition to be administered can be determined by a physician, taking into account individual differences in the age, weight, tumor size, degree of infection or metastasis, and condition of the patient (subject).

[0337] Generally, the pharmaceutical compositions comprising the T cells described herein are 6 ~10 13 Cells / kg body weight, preferably 10 8 ~10 13 It can be said that a dose of cells / kg body weight can be administered, including all integer values ​​within the range. The number of cells depends on the intended end use of the composition and the type of cells contained in the composition. For the uses presented herein, the cells are generally in a volume of liters or less, and may be 500 mL or less, or even 250 mL or 100 mL or less. Thus, the desired cell density is often less than 10 6 cells / ml, typically greater than 10 7 >10 cells / ml, typically 8 Clinically relevant immune cell counts may be divided into multiple infusions and cumulatively reach 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 or 10 13The compositions may be administered multiple times at doses within these ranges. The cells may be allogeneic, syngeneic, xenogeneic, or autologous to the patient undergoing therapy. If desired, the treatment may also include administration of mitogens (e.g., PHA) or lymphokines, cytokines, and / or chemokines (e.g., IFN-γ, IL-2, IL-12, TNF-alpha, IL-18, and TNF-beta, GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MIP1α, etc.) contemplated herein to enhance induction of an immune response.

[0338] In general, compositions comprising cells activated and expanded as contemplated herein may be utilized for the treatment and prevention of diseases occurring in immunocompromised individuals. In certain embodiments, compositions comprising immune effector cells modified to express engineered TCRs as contemplated herein are used to treat cancer. The modified immune effector cells may be administered alone or as a pharmaceutical composition in combination with carriers, diluents, excipients, and / or other components, such as IL-2 or other cytokines or other cell populations. In certain embodiments, the pharmaceutical composition comprises an amount of genetically modified T cells in combination with one or more pharma- ceutical or physiologically acceptable carriers, diluents, or excipients.

[0339] Pharmaceutical compositions comprising immune effector cell populations (e.g., T cells) modified to express an engineered TCR may include buffers, such as neutral buffered saline, phosphate buffered saline, and the like; carbohydrates, such as glucose, mannose, sucrose, or dextran, mannitol; proteins; amino acids, such as polypeptides or glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0340] The compositions are preferably formulated for parenteral administration, eg, intravascular (intravenous or intraarterial), intraperitoneal, or intramuscular administration.

[0341] Liquid pharmaceutical compositions, whether in solution, suspension, or other similar form, may contain one or more of the following: water for injection, saline, preferably saline, Ringer's solution, or isotonic saline, sterile diluents such as fixed oils, polyethylene glycol, glycerin, propylene glycol, or other solvents, such as synthetic mono- or diglycerides that may serve as a solvent or suspending medium; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium sulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates, and agents for adjusting isotonicity such as sodium chloride or dextrose. Parenteral preparations may be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic. Pharmaceutical compositions for injection are preferably sterile.

[0342] In one embodiment, the T cell compositions contemplated herein are formulated in a pharma- ceutically acceptable cell culture medium. Such compositions are suitable for administration to a human subject. In a particular embodiment, the pharma-ceutically acceptable cell culture medium is a serum-free medium.

[0343] Serum-free media have several advantages over serum-containing media, including a simpler and more transparent composition, lower amounts of contaminants, elimination of potential sources of infectious entities, and lower costs. In various embodiments, serum-free media may be animal-free and optionally protein-free. Optionally, the media may contain biopharmaceutical acceptable recombinant proteins. "Animal-free" media refers to media whose components are derived from non-animal sources. Recombinant proteins replace natural animal proteins in animal-free media, whose nutrients are derived from synthetic, plant, or microbial sources. "Protein-free" media, in contrast, are defined as being substantially protein-free.

[0344] Examples of serum-free media for use in certain compositions include, but are not limited to, QBSF-60 (Quality Biological, Inc.), StemPro-34 (Life Technologies), and X-VIVO 10.

[0345] In a preferred embodiment, a composition comprising immune effector cells contemplated herein is formulated in a solution comprising PlasmaLyte A.

[0346] In another preferred embodiment, the composition comprising the immune effector cells contemplated herein is formulated in a solution comprising a cryopreservation medium. For example, a cryopreservation medium comprising a cryopreservative may be used to maintain high cell activity after thawing. Exemplary cryopreservation media used in certain compositions include, but are not limited to, CryoStor CS10, CryoStor CS5, and CryoStor CS2.

[0347] In a more preferred embodiment, compositions comprising immune effector cells contemplated herein are formulated in a solution comprising 50:50 PlasmaLyte A to CryoStor CS10.

[0348] In certain embodiments, the composition comprises an effective amount of genome-edited immune effector cells modified to express an engineered TCR as contemplated herein. Thus, the immune effector cell composition may be administered alone or in combination with other known cancer treatments, such as radiation therapy, chemotherapy, transplantation, immunotherapy, hormonal therapy, photodynamic therapy, etc. The composition may be administered in combination with antibiotics. Such therapeutic agents may be accepted in the art as standard treatments for certain disease conditions described herein, such as, for example, certain cancers. Examples of contemplated therapeutic agents include, in certain embodiments, cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory agents, chemotherapeutic agents, radiation therapy, therapeutic antibodies, or other active adjunctive agents.

[0349] In certain embodiments, the composition comprising the genome-edited immune effector cells modified to express engineered TCR can be administered in combination with any number of chemotherapeutic agents.Various other therapeutic agents may be used in combination with the compositions contemplated herein.In one embodiment, the composition comprising the immune effector cells expressing engineered TCR is administered in combination with an anti-inflammatory agent.

[0350] In certain embodiments, compositions comprising immune effectors modified to express an engineered TCR as contemplated herein are administered in conjunction with a therapeutic antibody (e.g., a mono- or bispecific antibody or fragment thereof) and / or an immune cell engager (NK engager). Exemplary therapeutic antibodies suitable for use in combination with the CAR-modified T cells contemplated in certain embodiments include, but are not limited to, atezolizumab, avelumab, bavituximab, bevacizumab (Avastin), bivatuzumab, blinatumomab, conatumumab, crizotinib, daratumumab, duligotumab, dacetuzumab, dalotuzumab, dutuzumab, teufeliz ... These include ruvalumab, elotuzumab (HuLuc63), gemtuzumab, ibritumomab, indatuximab, inotuzumab, ipilimumab, lorvotuzumab, lucatumumab, milatuzumab, moxetumomab, nivolumab, ocaratuzumab, ofatumumab, pembrolizumab, rituximab, siltuximab, teprotumumab, and ublituximab.

[0351] J. Treatment method The genetically modified immune effector cells expressing the engineered TCRs contemplated herein provide improved methods of adoptive immunotherapy for use in the prevention, treatment, and amelioration of cancer, or for use in the prevention, treatment, or amelioration of at least one symptom associated with cancer.

[0352] In various embodiments, the genetically modified immune effector cells contemplated herein provide improved methods of adoptive immunotherapy used to increase cytotoxicity in cancer cells in a subject or to reduce the number of cancer cells in a subject.

[0353] In certain embodiments, the specificity of a primary immune effector cell is redirected to a cell expressing a particular antigen, e.g., a cancer cell, by genetically modifying the primary immune effector cell with an engineered TCR, as contemplated herein. In various embodiments, a viral vector is used to genetically modify the immune effector cell with a particular polynucleotide encoding the engineered TCR. In some embodiments, the engineered TCR comprises (a) a TCR alpha polypeptide comprising a TCR alpha variable domain; (b) a TCR beta polypeptide comprising a TCR beta variable domain; and (c) one or more antigen binding domains linked to the TCR alpha variable domain and / or the TCR beta variable domain. In some embodiments, the engineered TCR comprises (a) a TCR gamma polypeptide comprising a TCR gamma variable domain; (b) a TCR delta polypeptide comprising a TCR delta variable domain; and (c) one or more antigen binding domains linked to the TCR gamma variable domain and / or the TCR delta variable domain. In certain embodiments, the linker is a polypeptide linker. In certain embodiments, the polypeptide linker comprises an amino acid sequence set forth in any one or more of SEQ ID NOs: 33-53.

[0354] In one embodiment, a type of cell therapy is provided in which T cells are genetically modified to express the engineered TCR contemplated herein, and are infused into recipients in need thereof.The infused cells can kill disease-causing cells in recipients.Unlike antibody therapy, T cell therapy can replicate in vivo, resulting in long-term persistence that can provide sustained cancer treatment.

[0355] In one embodiment, T cells expressing engineered TCRs contemplated herein can undergo robust in vivo T cell expansion and can persist for extended periods of time, hi another embodiment, T cells expressing engineered TCRs contemplated herein evolve into specific memory T cells or stem cell memory T cells that can be reactivated to inhibit any further tumor formation or growth.

[0356] In certain embodiments, the modified immune effector cells expressing the engineered TCRs contemplated herein are used to treat solid tumors or cancers.

[0357] In certain embodiments, the engineered immune effector cells contemplated herein are selected from the group consisting of, but not limited to, adrenal carcinoma, adrenocortical carcinoma, anal carcinoma, appendix carcinoma, astrocytoma, atypical teratoid tumor / atypical rhabdomyoid tumor, basal cell carcinoma, bile duct carcinoma, bladder carcinoma, bone cancer, brain / CNS cancer, breast cancer, bronchial tumor, cardiac tumor, cervical cancer, bile duct carcinoma, chondrosarcoma, chordoma, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma in situ (DCIS), endometrial cancer, epithelial carcinoma, esophageal carcinoma, nasal neuroblastoma, Ewing's sarcoma, cranial Extragonadal germ cell tumor, extragonadal germ cell tumor, eye cancer, fallopian tube cancer, fibrous tissue sarcoma, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, glioma, glioblastoma, head and neck cancer, hemangioblastoma, hepatocellular carcinoma, hypopharyngeal carcinoma, intraocular melanoma, Kaposi's sarcoma, renal cancer, laryngeal cancer, leiomyosarcoma, tongue cancer, liposarcoma, hepatic cancer, lung cancer, non-small cell lung cancer, embryonal carcinoid tumor, malignant mesothelioma, medullary carcinoma, medulloblastoma, meningioma, melanoma, Merkel cell carcinoma, midline carcinoma, oral cancer used to treat solid tumors or cancers including myxosarcoma, myelodysplastic syndrome, myeloproliferative neoplasms, cancer of the nasal cavity and paranasal sinuses, nasopharyngeal cancer, neuroblastoma, oligodendroglioma, oral cancer, oral cavity cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, islet cell tumor, papillary carcinoma, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pinealoma, pituitary tumor, pleuropulmonary blastoma, primary pleurima, prostate cancer, rectal cancer, retinoblastoma, renal cell carcinoma, cancer of the renal pelvis and ureter, rhabdomyosarcoma, salivary gland cancer, sebaceous gland cancer, skin cancer, soft tissue sarcoma, squamous cell carcinoma, small cell lung cancer, small intestine cancer, gastric cancer, sweat gland carcinoma, synovial tumor, testicular cancer, pharyngeal cancer, thymic cancer, thyroid cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vascular cancer, vulvar cancer, and Wilms' tumor

[0358] In certain embodiments, the engineered immune effector cells contemplated herein are used in the treatment of solid tumors or cancers, including, but not limited to, non-small cell lung cancer, squamous cell carcinoma of the head and neck, colorectal cancer, pancreatic cancer, breast cancer, thyroid cancer, bladder cancer, cervical cancer, esophageal cancer, ovarian cancer, gastric cancer, endometrial cancer, glioma, glioblastoma, and oligodendroglioma.

[0359] In certain embodiments, the engineered immune effector cells contemplated herein are used in the treatment of solid tumors or cancers, including, but not limited to, non-small cell lung cancer, metastatic colorectal cancer, glioblastoma, head and neck cancer, pancreatic cancer, and breast cancer.

[0360] In certain embodiments, the engineered immune effector cells contemplated herein are used in the treatment of glioblastoma.

[0361] In certain embodiments, modified immune effector cells expressing engineered TCRs as contemplated herein are used to treat liquid or hematological cancers.

[0362] In certain embodiments, the engineered immune effector cells contemplated herein are used to treat B-cell malignancies, including, but not limited to, leukemia, lymphoma, and multiple myeloma.

[0363] In certain embodiments, the engineered immune effector cells contemplated herein are directed to, but are not limited to, leukemias, lymphomas, and multiple myelomas: acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, erythroleukemia, hairy cell leukemia (HCL), chronic lymphocytic leukemia (CLL), and chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia (CMML), and polycythemia vera, Hodgkin's lymphoma, nodular lymphocyte-predominant Hodgkin's lymphoma, It is used to treat liquid cancers including Burkitt's lymphoma, small lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, mantle cell lymphoma, marginal zone lymphoma, mycosis fungoides, anaplastic large cell lymphoma, Sézary syndrome, precursor T-lymphoblastic lymphoma, multiple myeloma, overt multiple myeloma, smoldering multiple myeloma, plasma cell leukemia, nonsecretory myeloma, IgD myeloma, osteosclerotic myeloma, solitary bone plasmacytoma, and extramedullary plasmacytoma.

[0364] In certain embodiments, the liquid or hematological cancer is selected from the group consisting of acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), multiple myeloma (MM), acute myeloid leukemia (AML), or chronic myelogenous leukemia (CML).

[0365] In a preferred embodiment, the liquid or hematological cancer is multiple myeloma (MM).

[0366] In a preferred embodiment, the liquid or hematological cancer is relapsed / refractory multiple myeloma (MM).

[0367] In certain embodiments, the engineered immune effector cells contemplated herein are used to treat acute myeloid leukemia (AML).

[0368] In certain embodiments, the engineered immune effector cells contemplated herein are used in the treatment of lymphoma (eg, non-Hodgkin's lymphoma or DLBCL).

[0369] As used herein, the terms "individual" and "subject" are often used interchangeably and refer to any animal that exhibits symptoms of a disease, injury, or condition that can be treated with the gene therapy vectors, cell-based therapeutic agents, and methods otherwise contemplated herein. In a preferred embodiment, a subject includes any animal that exhibits symptoms of a cancer-related disease, injury, or condition that can be treated with the gene therapy vectors, cell-based therapeutic agents, and methods otherwise contemplated herein. Suitable subjects (e.g., patients) include laboratory animals (e.g., mice, rats, rabbits, or guinea pigs), farm animals, domestic animals, or pets (e.g., cats or dogs). Non-human primates, preferably human patients, are also included.

[0370] As used herein, the term "patient" refers to a subject who has been diagnosed with a particular disease, injury or condition that can be treated using the gene therapy vectors, cell-based therapeutic agents, and methods disclosed elsewhere herein.

[0371] As used herein, "treatment" or "treating" includes any beneficial or desired effect on the symptoms or pathology of a disease or condition, and may include even a minimal reduction in one or more measurable markers of the disease or condition being treated. Treatment may optionally include either a reduction in the disease or condition, or a delay in the progression of the disease or condition. "Treatment" does not necessarily indicate a complete elimination or cure of the disease or condition, or its associated symptoms.

[0372] As used herein, "prevent" and similar terms, e.g., "prevented," "preventing," refer to an approach aimed at preventing, inhibiting, or reducing the likelihood of occurrence or recurrence of a disease or condition. It also refers to delaying the occurrence or recurrence of a disease or condition, or delaying the occurrence or recurrence of symptoms of a disease or condition. As used herein, "prevention" and similar terms also include reducing the intensity, effects, symptoms, and / or burden of a disease or condition prior to the occurrence or recurrence of the disease or condition.

[0373] As used herein, "amelioration of at least one symptom of" refers to a reduction in one or more symptoms of the disease or condition that the subject is being treated for. In certain embodiments, the disease or condition that is being treated is cancer, in which case the one or more symptoms that are improved include, but are not limited to, weakness, fatigue, shortness of breath, easy bruising and bleeding, frequent infections, enlarged lymph nodes, abdominal distension or pain (caused by distended abdominal organs), bone or joint pain, bone fractures, unexpected weight loss, loss of appetite, night sweats, persistent low-grade fever, and decreased urination (caused by impaired kidney function).

[0374] "Enhancing" or "promoting" or "increasing" or "expanding" generally refers to the ability of a composition contemplated herein, e.g., a genetically modified T cell expressing an engineered TCR contemplated herein, to produce, induce, or cause a greater physiological response (i.e., downstream effect) compared to a response caused by either a vehicle or a control molecule / composition. Measurable physiological responses include, among others, increased T cell expansion, activation, persistence, and / or cancer cell killing capacity, as will be apparent from an understanding of the art and the present disclosure. An "increased" or "enhanced" amount is typically a "statistically significant" amount and may include an increase that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more (e.g., 500-fold, 1000-fold) (including all integers and decimals in between and above 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) over the response provided by the vehicle or control composition.

[0375] "Reduce" or "lower" or "lower" or "reduce" or "attenuate" generally refers to the ability of the compositions contemplated herein to produce, elicit or cause a physiological response (i.e., downstream effect) that is smaller than the response caused by a vehicle or control molecule / composition. The amount of "reduction" or "reduction" is typically a "statistically significant" amount and may include a reduction of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more (e.g., 500-fold, 1000-fold) (including all integers and decimals between and above 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) of the response caused by a vehicle, a control composition, or a response in a particular cell line.

[0376] "Maintain" or "preserve" or "maintain" or "no change" or "no substantial change" or "no substantial decrease" generally refers to the compositions contemplated herein producing, eliciting, or causing a similar or equivalent physiological response in a cell (i.e., a downstream effect) compared to the response caused by a vehicle, a control molecule / composition, or in a particular cell line. An equivalent response is one that is not substantially different, or not measurably different, from the reference response.

[0377] In one embodiment, a method of treating cancer in a subject in need thereof comprises administering an effective amount, e.g., a therapeutically effective amount, of a composition comprising a genetically modified immune effector cell as contemplated herein. The amount and frequency of administration will be determined by factors such as the condition of the patient, the type and severity of the patient's disease, although appropriate dosages may be determined by clinical trials.

[0378] In one embodiment, the amount of immune effector cells, e.g., T cells expressing an engineered TCR, in the composition administered to a subject is at least 1×10 7 Cells, at least 0.5 x 10 8 Cells, at least 1 x 10 8 Cells, at least 0.5 x 10 9 Cells, at least 1 x 10 9 Cells, at least 1 x 10 10 Cells, at least 1 x 10 11 Cells, at least 1 x 10 12 Cells, at least 5 x 10 12 cells, or at least 1 x 10 13 It is a cell.

[0379] In certain embodiments, about 1 x 10 7 T cells ~ approx. 1x10 13 T cells, approximately 1x10 8 T cells ~ approx. 1x10 13 T cells, approximately 1x10 9 T cells ~ approx. 1x10 13 T cells, approximately 1x10 10T cells ~ approx. 1x10 13 T cells, approximately 1x10 11 T cells ~ approx. 1x10 13 T cells, or approximately 1x10 12 T cells ~ approx. 1x10 13 The T cells are administered to a subject.

[0380] In one embodiment, the amount of immune effector cells, e.g., T cells expressing an engineered TCR, in the composition administered to a subject is at least 0.1 x 10 4 At least 0.5 × 10 cells / kg body weight 4 At least 1 x 10 cells / kg body weight 4 Cells / kg body weight, at least 5 × 10 4 At least 1x10 cells / kg body weight 5 At least 0.5x10 cells / kg body weight 6 At least 1x10 cells / kg body weight 6 At least 0.5x10 cells / kg body weight 7 At least 1x10 cells / kg body weight 7 At least 0.5x10 cells / kg body weight 8 At least 1x10 cells / kg body weight 8 At least 2x10 cells / kg body weight 8 Cells / kg body weight, at least 3x10 8 Cells / kg body weight, at least 4x10 8 Cells / kg body weight, at least 5x10 8 cells / kg body weight or at least 1x10 9 cells / kg body weight.

[0381] In certain embodiments, about 1×10 6 1x10 T cells / kg body weight 8 T cells / kg body weight, approximately 2x10 6 0.9x10 T cells / kg body weight 8 T cells / kg body weight, 3x10 6 T cells / kg ~ approx. 0.8x10 8 T cells / kg body weight, approximately 4x10 6 Approximately 0.7x10 T cells / kg body weight 8T cells / kg body weight, approximately 5x10 6 Approximately 0.6 x 10 T cells / kg body weight 8 T cells / kg, or approximately 5x10 6 Approximately 0.5 x 10 T cells / kg body weight 8 T cells / kg are administered to the subject.

[0382] One of skill in the art will recognize that multiple administrations of the compositions contemplated herein may be required to achieve the desired therapeutic effect. For example, the compositions may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times over a period of 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 5 years, 10 years or more.

[0383] In certain embodiments, it may be desirable to administer activated immune effector cells to a subject, then draw blood from the subject (or perform apheresis), activate the immune effector cells derived therefrom, and reinfuse these activated and expanded immune effector cells back into the patient. This process may be performed multiple times, every few weeks. In certain embodiments, the immune effector cells may be activated from a blood draw of 10cc to 400cc. In certain embodiments, the immune effector cells are activated from a blood draw of 20cc, 30cc, 40cc, 50cc, 60cc, 70cc, 80cc, 90cc, 100cc, 150cc, 200cc, 250cc, 300cc, 350cc, or 400cc or more. Without being bound by theory, the use of a multiple blood draw / multiple reinfusion protocol may aid in the selection of certain populations of immune effector cells.

[0384] Administration of the compositions contemplated herein may be performed in any convenient manner, including aerosol inhalation, injection, ingestion, transfusion, implantation, or transplantation. In a preferred embodiment, the compositions are administered parenterally. As used herein, the phrases "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravascular, intravenous, intramuscular, intraarterial, intrathecal, intraarticular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion. In one embodiment, the compositions contemplated herein are administered to a subject by direct injection into a tumor, lymph node, or site of infection.

[0385] In one embodiment, an effective amount of the composition is administered to a subject in need thereof to increase the cellular immune response to the subject's B cell-related condition. The immune response may include cellular immune responses mediated by cytotoxic T cells, regulatory T cells, and helper T cell responses that can kill infected cells. Humoral immune responses may also be induced that are mediated primarily by helper T cells that can activate B cells, thus resulting in antibody production. Various techniques may be used to analyze the type of immune response induced by the composition, and these techniques are well described in the art, for example, in Current Protocols in Immunology, Edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober (2001) John Wiley & Sons, NY, NY.

[0386] In one embodiment, a method is provided for treating a subject diagnosed with cancer comprising removing immune effector cells from the subject, genetically modifying said immune effector cells with a vector comprising a nucleic acid encoding an engineered TCR as contemplated herein, thereby producing a population of modified immune effector cells, and administering the population of modified immune effector cells to the subject. In a preferred embodiment, the immune effector cells comprise T cells.

[0387] In certain embodiments, a method is provided for stimulating immune effector cells that mediate an immune modulator response against a target cell population in a subject, comprising administering to the subject a population of immune effector cells expressing a nucleic acid construct encoding an engineered TCR as contemplated herein.

[0388] Methods of administering the cell compositions contemplated in certain embodiments include any method that is effective to result in the reintroduction of ex vivo genetically modified immune effector cells, either directly expressing an engineered TCR in the subject, or reintroducing genetically modified precursors of immune effector cells that differentiate into mature immune effector cells expressing a CAR when introduced into the subject. One method involves transducing peripheral blood T cells ex vivo with a nucleic acid construct contemplated herein and returning the transduced cells to the subject.

[0389] All publications, patent applications, and issued patents cited in this specification are herein incorporated by reference to the same extent as if each individual publication, patent application, or issued patent was specifically and individually indicated to be incorporated by reference.

[0390] The foregoing embodiments have been described in detail with reference to figures and examples for purposes of clarity and understanding, but in light of the teachings contemplated herein, it will be readily apparent to those skilled in the art that certain changes and modifications may be made without departing from the spirit or scope of the appended claims. The following examples are provided for illustrative purposes only and not for purposes of limitation. Those skilled in the art will readily recognize various non-critical parameters that can be changed or modified to produce essentially similar results. [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4] [Table 8-5] [Table 8-6] [Table 8-7] [Table 8-8] [Table 8-9] [Table 8-10] [Table 8-11] [Table 8-12] [Table 8-13] [Table 8-14] [Table 8-15] [Table 8-16] [Table 8-17] [Table 8-18] EXAMPLES

[0391] Example 1 Evaluation of engineered TCRs A MAGEA4-reactive HLA-A2-restricted T-cell receptor (TCR) was embedded into a VHH targeting human CD33 to generate an engineered dual-targeting TCR ("VHH-TCR") (SEQ ID NO: 93) (Figures 1A and 1B). This was assessed for expression and function in comparison to a TCR targeting MAGEA4 (SEQ ID NO: 89) and DARIC (Dimerizer-Controlled Immunoreceptor Complex, a Controllable and Adaptable Antigen Recognition System) targeting human CD33 (SEQ ID NO: 90) ("Comparison") (Figure 1A). Dual-targeting TCR T cells were generated in a 10-day process using G-REX® flasks. Briefly, peripheral blood mononuclear cells (PBMCs) were cultured in medium containing IL-2 (CellGenix, GmbH) and antibodies specific for CD3 and CD28 (Miltenyi Biotec, Inc.). Lentiviruses encoding the test constructs were added one day after the start of culture. On day 3, CAR T cells were transferred from the 24-well plate to a 24-well G-REX flask, where the cells were maintained until harvest on day 10.

[0392] T cells were examined for cell surface VHH expression using flow cytometry. T cells were stained using iFlour488-labeled anti-camelid VHH antibody (Genscript). Surface VHH expression was higher in VHH-TCR compared to CD33DARIC (Figure 2A). Furthermore, biological activity of T cells was evaluated for interferon gamma production in co-culture with a CD33-positive tumor cell line (adherent A549 cell line stably transduced with CD33). As shown in Figure 2B, interferon gamma production of VHH-TCR was >3-fold higher than CD33-DARIC, which was activated by including 1 nm of rapamycin in the co-culture. Live cell imaging by IncuCyte was used to analyze tumor cell proliferation of A549.CD33 stably transduced with the red reporter. A549 cells proliferated normally in the presence of UTD T cells and MAGEA4 TCR-T cells. Coculture with either VHH-TCR or CD33-DARIC resulted in elimination of tumor cells, with VHH-TCR achieving elimination more rapidly than DARIC (Figure 2C).

[0393] Flow cytometry was performed to evaluate the higher MAGEA4 tetramer / HLA-multimer binding in VHH-TCR compared to MAGEA4 TCR (Figure 3A). Furthermore, the biological activity of T cells was evaluated for interferon gamma production in co-culture with a MAGEA4-positive tumor cell line (adherent A549 cell line stably transduced with MAGEA4 and HLA-A2). As shown in Figure 3B, interferon gamma production of VHH-TCR is approximately 3-fold higher than MAGEA4 TCR. Live cell imaging by IncuCyte was used to analyze tumor cell growth of adherent A549.MAGEA4.HLA-A cell line stably transduced with red reporter. A549 cells proliferated normally in the presence of UTD T cells and CD33-DARIC cells. Co-culture of MAGEA4 TCR resulted in complete elimination of tumor cells, whereas VHH-TCR resulted in complete and more rapid elimination of tumor cells (Figure 3C).

[0394] The sensitivity of VHH-TCR was compared to MAGEA4 TCR by setting up co-cultures with A549 cells, which do not express MAGEA4, pulsed with a wide range of MAGEA4 peptide concentrations. As shown in Figure 4A, VHH-TCR shows similar kinetics in co-cultures with a wide range of MAGEA4 peptide expression, but superior interferon gamma release compared to MAGEA4 TCR. The sensitivity of VHH-TCR was compared to CD33 DARIC by setting up co-cultures with A549 cells, which do not express CD33, electroporated with a wide range of CD33 mRNA concentrations. As shown in Figures 4B and 4C, VHH-TCR shows similar kinetics in co-cultures with a wide range of CD33 mRNA expression, but superior interferon gamma production compared to CD33 DARIC activated with 1 nM rapamycin. Additionally, co-cultures were set up using cell lines that endogenously express various levels of CD33; HL-60 has high CD33 expression, Kasumi1 has moderate CD33 expression, and OCI-AML3 has low CD33 expression. As shown in Figures 5A-5C, VHH-TCR exhibited superior interferon gamma expression upon co-culture, most evident in OCI-AML3, which expresses low levels of CD33.

[0395] Example 2 Evaluation of engineered TCR configurations Four configurations were evaluated: 1) VHH added to TRB (T cell receptor beta chain) separated by a marsupial mu linker (LEKT) (SEQ ID NO: 91), 2) VHH added to TRB separated by mu linker + G4S (SEQ ID NO: 92), 3) VHH added to TRA (T cell receptor alpha chain) separated by mu linker (SEQ ID NO: 93), and 4) VHH added to TRA separated by mu linker + G4S (SEQ ID NO: 94) (Figure 6). TCR T cells were generated as described in Example 1.

[0396] T cells were examined for cell surface VHH expression using flow cytometry. T cells were stained using iFlour488 labeled T cells (Genscript). Surface VHH expression was higher in VHH-TCR than CD33 DARIC (SEQ ID NO: 90) and was comparable in all orientations tested (Figure 7A). Furthermore, biological activity of T cells was evaluated for interferon gamma production in co-culture with a CD33 positive tumor cell line (adherent A549 cell line stably transduced with CD33). As shown in Figure 7B, interferon gamma production of all VHH-TCRs was >2-fold greater than CD33-DARIC activated with 1 nm rapamycin, and VHH-TCRs with VHH added to TRA separated by mu linker + G4S were superior for all constructs evaluated. Live cell imaging by IncuCyte was used to analyze tumor cell proliferation of A549.CD33 stably transduced with red reporter. A549 cells proliferated normally in the presence of UTD and MAGEA4 TCR T cells. Coculture with all VHH-TCR or activated CD33-DARIC led to the elimination of tumor cells, with VHH-TCR achieving elimination more rapidly than DARIC (Figure 7C).

[0397] Flow cytometry was performed to evaluate MAGEA4 tetramer / HLA-multimer binding, which was comparable for all VHH TCRs and MAGEA4 TCRs (Figure 8A). Furthermore, T cell biological activity was evaluated for interferon gamma production in co-culture with a MAGEA4-positive tumor cell line (adherent A549 cell line stably transduced with MAGEA4 and HLA-A2). As shown in Figure 8B, compared to MAGEA4 TCR, interferon gamma production of VHH-TCR with VHH embedded in TRB is lower, but slightly greater when VHH was added to TRA separated by mu linker and approximately 3-fold greater when VHH was added to TRA separated by mu linker+G4S. Tumor cell growth of adherent A549.MAGEA4.HLA-A cell line stably transduced with red reporter was analyzed using live cell imaging by IncuCyte. A549 cells proliferated normally in the presence of UTD T cells and CD33-DARIC cells. Co-culture with TCR with VHH embedded in TRB had incomplete elimination of tumor cells. Co-culture with MAGEA4 TCR resulted in complete elimination of tumor cells, and co-culture with VHH embedded in TRA resulted in complete and more rapid elimination of tumor cells (Figure 8C).

[0398] Example 3 Further evaluation of linkers in engineered TCRs The significance of the linker on the VHH was further evaluated by comparing constructs where 1) VHH was added to TRA separated by mu linker (LEKT)+G4S (SEQ ID NO: 94), 2) VHH was added to TRA separated by 1xG4S (SEQ ID NO: 95), and 3) VHH was added to TRA separated by 2xG4S (SEQ ID NO: 96) (Figure 9). TCR T cells were generated as described in Example 1.

[0399] T cells were examined for cell surface CD33 expression using flow cytometry. T cells were stained using His-tagged CD33-Fc reagent (Acros) with secondary staining with APC-labeled streptavidin. Surface CD33 expression was comparable in all three evaluated formats (Figure 10A). Furthermore, biological activity of T cells was evaluated for interferon gamma production in co-culture with a CD33-positive tumor cell line (adherent A549 cell line stably transduced with CD33). As shown in Figure 10B, interferon gamma production of VHH-TCR with mu-linker+1G4S and 1G4S was comparable and highest for VHH-TCR with 2G4S. Tumor cell proliferation of A549.CD33 stably transduced with red reporter was analyzed using live cell imaging by IncuCyte. A549 cells proliferated normally in the presence of UTD T cells. Co-culture with all VHH-TCRs led to elimination of tumor cells (Figure 10C).

[0400] Flow cytometry was performed to assess MAGEA4 tetramer / HLA-multimer binding, which was comparable in all three formats evaluated (Figure 11A). Furthermore, T cell biological activity was assessed for interferon gamma production in co-culture with MAGEA4-positive tumor cell lines (adherent A549 cell lines stably transduced with MAGEA4 and HLA-A2). As shown in Figure 11B, interferon gamma production of mu-linker+1G4S and VHH-TCR with one G4S was comparable and highest in VHH-TCR with two G4S. Live cell imaging by IncuCyte was used to analyze tumor cell growth of adherent A549.MAGEA4.HLA-A cell lines stably transduced with red reporter. A549 cells proliferated normally in the presence of UTD T cells. Coculture with all VHH-TCRs resulted in complete elimination of tumor cells, the most rapid with the VHH-TCR bearing two G4S (Figure 11C).

[0401] Example 4 Evaluation of engineered multitargeting TCRs with tandem binders MAGEA4-reactive HLA-A2-restricted T-cell receptors (TCRs) were embedded with tandem VHHs (SEQ ID NO: 97) targeting human CD33 and CLL1 (Figures 12A and 12B). They were evaluated for expression and function in comparison with known TCRs targeting MAGEA4 and human CD33 (SEQ ID NO: 90), CLL1 (SEQ ID NO: 98), and DARIC (Dimerizer-Controlled Immune Receptor Complex, a Controllable and Adaptable Antigen Recognition System) targeting both CD33 and CLL1 in tandem (SEQ ID NO: 99) ("Comparison") (Figure 12A). Dual-targeting TCR T cells were produced using G-REX® flasks in a 10-day process using the same protocol as in Example 1.

[0402] T cells were examined for cell surface CD33 expression using flow cytometry. T cells were stained using His-tagged CD33-Fc reagent (Acros) and secondary stained with APC-tagged streptavidin. Surface CD33 expression was higher in CD33-CLL1-TCR compared to CD33 DARIC and CD33-CLL1 DARIC (Figure 13A). Furthermore, the biological activity of T cells was evaluated for interferon gamma production in co-culture with a CD33-positive tumor cell line (adherent A549 cell line stably transduced with CD33). As shown in Figure 13B, interferon gamma production of CD33-CLL1-TCR is comparable to CD33-DARIC and CD33-CLL1 DARIC (the latter two were activated by the addition of 1 nm rapamycin).

[0403] T cells were examined for cell surface CLL1 expression using flow cytometry. T cells were stained using PE-labeled CLL1-Fc reagent (Creative Biomart). Surface CLL1 expression was higher in CD33-CLL1-TCR compared to CLL1 DARIC and CD33-CLL1 DARIC (Figure 14A). Furthermore, the biological activity of T cells was evaluated for interferon gamma production in co-culture with a CLL1-positive tumor cell line (adherent A549 cell line stably transduced with CLL1). As shown in Figure 14B, CD33-CLL1 TCR produces robust interferon gamma in co-culture with CLL1-expressing cell lines.

[0404] Flow cytometry was performed to assess higher MAGEA4 tetramer / HLA-multimer binding in CD33-CLL1-TCR compared to MAGEA4 TCR (Figure 15A). Furthermore, the biological activity of T cells was assessed for interferon gamma production in co-culture with MAGEA4-positive tumor cell lines (adherent A549 cell lines stably transduced with MAGEA4 and HLA-A2). As shown in Figure 15B, interferon gamma production of CD33-CLL1-TCR is comparable to MAGEA4 TCR.

[0405] Example 5 Evaluation of VHH-based engineered TCRs Two engineered TCRs were constructed using MAGEA4-reactive HLA-A2-restricted T cell receptors (TCRs) embedded with one of two anti-BCMA VHHs, respectively. The same anti-BCMA VHHs were also formatted in CAR format. These were evaluated for expression and function in comparison with a TCR targeting MAGEA4 and a known scFv-based CAR targeting BCMA ("Comparison"). Using the same protocol as in Example 1, T cells were produced in a 10-day process using G-REX® flasks.

[0406] T cells were examined for cell surface CAR and TCR expression using flow cytometry and assessed for MAGEA4 tetramer / HLA-multimer binding. T cells were stained using PE-labeled BCMA Fc reagent (AcroBio). Surface BCMA binder expression was detectable on all constructs with BCMA binders (Figure 16). Both VHH TCRs were robustly detected by MAGEA4 tetramers and comparable to MAGE-A4 TCRs.

[0407] The biological activity of the T cells was evaluated for interferon gamma production in co-culture with a tumor cell line positive for MAGEA4 (an adherent A375 cell line that endogenously expresses MAGEA4 and HLA-A2). As shown in Figure 17, the VHH TCR expressed very robust levels of interferon gamma, with expression comparable to that of the MAGEA4 TCR.

[0408] The biological activity of T cells was also evaluated for interferon gamma production in co-culture with a BCMA-positive tumor cell line (Toledo suspension cell line endogenously expresses low levels of BCMA). As shown in Figure 18A, VHH TCRs produced similar or more interferon gamma than their respective VHH CARs. The biological activity of T cells in co-culture with Toledo cells was further evaluated for interleukin 2 (IL2) production, a more sensitive assay. As shown in Figure 18B, none of the VHH CARs produced detectable amounts of IL2, while both VHH TCRs produced robust IL2. Antigen-independent signaling of T cells was evaluated by interferon gamma production in co-culture without tumor cell lines. As shown in Figure 19, VHH CARs had detectable levels of interferon gamma production, while VHH TCRs had low or no detectable interferon gamma production in the absence of tumor cells.

[0409] Example 6 Evaluation of scFV-based engineered TCRs A MAGEA4-reactive HLA-A2-restricted T-cell receptor (TCR) was embedded with a human BCMA-targeting scFv (SEQ ID NO: 100) and a 2xG4S linker between the scFv and Va (Figures 20A and 20B). It was evaluated for expression and function in comparison with a MAGEA4-targeting TCR (SEQ ID NO: 89) and a human BCMA-targeting scFv-based CAR (SEQ ID NO: 101) ("Comparison") (Figure 20A). Dual-targeting TCR T cells were generated in the same manner as in Example 1.

[0410] T cells were examined for cell surface CAR expression using flow cytometry. T cells were stained using PE-labeled BCMA Fc reagent (AcroBio). Surface BCMA binder expression was comparable between scFv-TCR and anti-BCMA CAR (Figure 21A). Furthermore, biological activity of T cells was evaluated for interferon gamma production in co-culture with tumor cell lines expressing various levels of BCMA (HT1080 engineered to overexpress high levels of BCMA, RPMI-8226: moderate endogenous BCMA expression, Toledo: low endogenous expression). As shown in Figure 21B, interferon gamma production of scFv-TCR was comparable to anti-BCMA CAR in high BCMA expressing cell lines and more in moderate and low expressing cell lines. IL2 secretion of scFv-TCR was more than anti-BCMA CAR in media and co-culture with low BCMA expressing cell lines (Figure 21C). Another sensitive assay, tumor necrosis factor a secretion, was assessed in Figure 21D, which was higher in RPMI-8226 and Toledo, medium and low expressing BCMA cell lines. Live cell imaging by IncuCyte was used to analyze tumor cell proliferation of HT1080.BCMA stably transduced with a red reporter. HT1080.BCMA cells were grown in the presence of UTD T cells and MAGEA4 TCR-T cells. Co-culture with either scFv-TCR or anti-BCMA CAR resulted in tumor cell elimination, with scFv-TCR achieving elimination more rapidly than CAR (Figure 21E).

[0411] Flow cytometry was performed to assess MAGEA4 tetramer / HLA-multimer binding, which was comparable for scFv-TCR and MAGEA4 TCR (Figure 22A). Furthermore, T cell biological activity was assessed for interferon gamma production, IL2 and tumor necrosis factor a in MAGEA4-positive tumor cell lines (adherent A375 cell lines that endogenously express MAGEA4 and HLA-A2). As shown in Figure 22B, interferon gamma and tumor necrosis factor a production of VHH TCR is comparable to MAGEA4 TCR.

[0412] Example 7 Evaluation of engineered TCRs In a CD33 antigen-only positive tumor model A MAGEA4-reactive HLA-A2-restricted T-cell receptor (TCR) was embedded into a VHH targeting human CD33 to generate an engineered dual-targeting TCR ("VHH-TCR") (SEQ ID NO: 93) (Figures 1A and 1B). Dual-targeting TCR T cells were generated in the same manner as in Example 1. Engineered T cells were evaluated for expression and in vitro function in the same manner as in Example 1.

[0413] The ability of VHH-TCRs to recognize and function in the presence of VHH antigens was assessed in vivo using a systemic luciferase-tagged HL-60 tumor model in NSG mice. The HL-60 model expresses CD33 but not MAGEA4, so any observed antitumor activity is the result of VHH-TCR signaling following VHH recognition of CD33. Luciferase-tagged HL-60 cells were implanted intravenously into naive female NSG mice and allowed to establish for five days. Mice were randomized into groups of five animals using a similar procedure on study day-1 (D-1). On D0, animals were administered intravenously either untransduced T cells, MAGE4 TCR T cells, CD33-DARIC T cells, or VHH-TcR T cells. T cell doses were normalized to 10E6 receptor-positive cells / mouse; untransduced T cell doses were normalized to match the highest total T cell dose. Animals treated with CD33-DARIC T cells were maintained on a Monday / Wednesday / Friday 0.1 mg / kg rapamycin schedule starting on DO. As shown in Figure 23, tumor growth remains unchecked in animals treated with either untransduced T cells or MAGEA4 TCR T cells. Both CD33-DARIC and VHH-TCR T cells show comparable tumor control.

[0414] Example 8 Evaluation of engineered TCRs In a TCR antigen-only positive tumor model A MAGEA4-reactive HLA-A2-restricted T-cell receptor (TCR) was embedded into a VHH targeting human CD33 to generate an engineered dual-targeting TCR ("VHH-TCR") (SEQ ID NO: 93) (Figures 1A and 1B). Dual-targeting TCR T cells were generated in the same manner as in Example 1. Engineered T cells were evaluated for expression and in vitro function in the same manner as in Example 1.

[0415] The ability of VHH-TCR to recognize and function in the presence of TCR antigen was evaluated in vivo using a subcutaneous NCI-H2023 tumor model in NSG mice. The NCI-H2023 model expresses MAGEA4 but not CD33, so any observed antitumor activity is the result of VHH-TCR signaling following TCR recognition of MAGEA4. NCI-H2023 cells were implanted subcutaneously into naive female NSG mice and allowed to establish for 21 days. Mice were randomized into groups of 5 animals using a similar procedure on study day-1 (D-1). On D0, animals were administered intravenously either untransduced T cells, MAGE4 TCR T cells, CD33-DARIC T cells, or VHH-TCR T cells. T cell doses were normalized to 10E6 receptor positive cells / mouse; untransduced T cell doses were normalized to match the highest total T cell dose. Animals treated with CD33-DARIC T cells were maintained on a Monday / Wednesday / Friday 0.1 mg / kg rapamycin schedule starting on DO. As shown in Figure 24, tumor growth remains unchecked in animals treated with either non-transduced T cells or CD33-DARIC T cells. Both MAGEA4 TCR and VHH-TCR T cells initially show comparable tumor control. Loss of tumor control occurs more quickly in animals treated with VHH-TCR T cells.

[0416] Example 9 Evaluation of engineered TCR constructs containing CD19 scFv MAGEA4-reactive HLA-A2-restricted T-cell receptors (TCRs) were embedded with a scFv targeting human CD19 (SEQ ID NO: 102) and evaluated for expression and function in comparison to MAGEA4-reactive HLA-A2-restricted T-cell receptors (TCRs) embedded with a scFv targeting human BCMA (SEQ ID NO: 100). Dual-targeted TCR T cells were generated as described in Example 1.

[0417] T cells were examined for cell surface TCR expression using flow cytometry. T cells were stained using PE-labeled anti-TCR Vb1 antibody (Miltenyi Biotech). Surface expression of the engineered constructs was comparable (Figure 25A). Furthermore, biological activity of T cells was assessed by measuring interferon gamma production in co-culture with the suspension tumor cell line RPMI-8226 (endogenous BCMA expression, undetectable CD19 expression), and the suspension tumor cell line K562.CD19 (undetectable BCMA expression, stably transduced with CD19). As shown in Figures 25B and 25C, CD19 ScFv TCR T cells produce interferon gamma in response to tumor cell lines positive for surface CD19 at levels comparable to those produced by BCMA ScFv TCR T cells in response to tumor cell lines positive for surface BCMA.

[0418] Example 10 Exemplary Engineered TCR Constructs As contemplated herein, antigen binding domains (also referred to herein as "binding agents" or "antigen binders"), polypeptide linkers, and TCRs can surprisingly be combined to produce engineered TCRs with multispecificity. In other words, the components can be combined without destroying the functionality of either the antigen binding domain(s) or the TCR(s). Thus, the engineered TCRs contemplated herein surprisingly provide (1) multispecificity, (2) increased sensitivity to non-MHC-presented targets, and (3) the ability to simultaneously target both intracellular and extracellular targets.

[0419] Engineered TCRs can be constructed in multiple formats and can be designed and constructed using known components (e.g., antigen binding domains, polypeptide linkers, and TCR alpha and beta chains) and techniques. For example, one or more antigen binding domains (e.g., one or more "A" components) can be linked to one or more TCR components (e.g., one or more "C" components) with or without one or more polypeptide linkers (e.g., with or without one or more "B" components) using standard cloning techniques. The "A" components can optionally be linked to either the TCR alpha or TCR beta polypeptides / chains, or both, or to the TCR gamma or TCR delta, or both, of the "C" components. An exemplary general engineered TCR formula is provided below: AC A.B.C.

[0420] The engineered TCRs contemplated herein can be designed and constructed using known components (e.g., TCR alpha and beta chains, linkers, and antigen binding domains) and techniques. Table 3 provides an exemplary list of known antigen binding domains. Table 4 provides an exemplary list of known polypeptide linkers. Table 5 provides an exemplary list of known TCRs. However, other known antigen binding domains, linkers, and TCRs can be found in, for example, U.S. Patent No. 20120082661, WO 2016014789, WO 2022046730, WO 2016033570, U.S. Patent No. 8147832B2, WO 2014026054, WO 2018145649, WO 2014065961, WO 2015065962, WO 2016033570, WO 2015065962 ... Publication No. 2020123947, International Publication No. 2013049254, International Publication No. 2019241685, International Publication No. 2019241688, International Publication No. 2016049214, International Publication No. 2018236870, International Publication No. 2020102240, International Publication No. 2018183888, U.S. Patent No. 6217866B1, International Publication No. 2008119566, International Publication No. 2003055917, International Publication No. 2018073 680, International Publication No. 2014146672, International Publication No. 2019200007, International Publication No. 2016016859, International Publication No. 2018119279, International Publication No. 2020227072, International Publication No. 2020227073, International Publication No. 2020227071, International Publication No. 2017153402, International Publication No. 2007042289, International Publication No. 2018028647, International Publication No. 2005113595, U.S. Patent No. No. 20180273602, International Publication No. 2019067242, International Publication No. 2020193767, U.S. Patent No. 10538572B2, US11078252B2, International Publication No. 2019140100, International Publication No. 2015009606, International Publication No. 2021195503, International Publication No. 2007131092, U.S. Patent No. 20190169260 can be found throughout the literature.The present invention is not intended to be limited to the exemplary components disclosed in Tables 3-5, as other known antigen binding domains, linkers, and TCRs are well known in the literature. [Table 3] [Table 4] [Table 5]

[0421] As one example of an engineered TCR contemplated herein, an antigen binding domain of Table 3 (e.g. an antigen binding domain selected from component A1) can be combined with one or more polypeptide linkers of Table 4 (e.g. component B1) and one or both TCR variable domains of a TCR of Table 5 (e.g. component C1) to produce a novel engineered TCR construct (e.g. ATOMIC construct #1, see below). Additionally, as further demonstrated and contemplated herein, multiple "A" components can be combined to produce multispecific antigen binding domains / regions (e.g. tandem antigen binding domains) and multiple polypeptide linkers can be combined to produce a functional linker.

[0422] Table 6 provides an exemplary, non-limiting list of engineered TCRs (i.e., ATOMIC constructs) based on the antigen binding domains, linkers, and TCRs provided in Tables 3, 4, and 5. One of skill in the art will understand that other combinations are possible, including combinations using other antigen binding domains, linkers, and TCRs known to or developed de novo by one of skill in the art. [Table 6-1] [Table 6-2] [Table 6-3]

Table 6-4

Table 6-5

Table 6-6

Table 6-7

Table 6-8

Table 6-9

Table 6-10

Table 6-11

Table 6-12

Table 6-13

Table 6-14

Table 6-15

Table 6-16

Table 6-17

Table 6-18

Table 6-19

Table 6-20

Table 6-21

Table 6-22

Table 6-23

Table 6-24

Table 6-25

Table 6-26

Table 6-27

Table 6-28

Table 6-29

Table 6-30

Table 6-31

Table 6-32

Table 6-33

Table 6-34

Table 6-35

Table 6-36

Table 6-37

[0423] As will be apparent to one of skill in the art, certain engineered TCR constructs containing multiple A and B components (ATOMIC) are contemplated and are surprisingly effective (see Examples 2-9).

[0424] Furthermore, the engineered TCR (ATOMIC) contemplated herein may also comprise a natural or engineered TCR constant domain. For example, the constant domain may be a natural or engineered TCR alpha, TCR beta, TCR gamma, or TCR delta constant domain. Furthermore, any TCR variable domain may be combined with any TCR constant domain. For example, a TCR alpha variable domain may be combined with any one of a TCR alpha, TCR beta, TCR gamma, or TCR delta constant domain, a TCR beta variable domain may be combined with any one of a TCR alpha, TCR beta, TCR gamma, or TCR delta constant domain, a TCR gamma variable domain may be combined with any one of a TCR alpha, TCR beta, TCR gamma, or TCR delta constant domain, and a TCR delta variable domain may be combined with any one of a TCR alpha, TCR beta, TCR gamma, or TCR delta constant domain. Exemplary natural and pairing-enhanced TCR constant domains are provided in Table 7 below. For other examples of TCR constant domains, see also WO2021195503A1, which is incorporated by reference in its entirety. [Table 7]

[0425] In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by this disclosure.

Claims

**Claim 1** An engineered T cell receptor (TCR) comprising: (i) a) A TCRα polypeptide comprising a TCRα variable domain; b) A TCRβ polypeptide comprising a TCRβ variable domain; and c) One or more antigen-binding domains linked to said TCRα variable domain and / or TCRβ variable domain; or (ii) a) A TCRγ polypeptide comprising a TCRγ variable domain; b) A TCRδ polypeptide comprising a TCRδ variable domain; and c) One or more antigen-binding domains linked to said TCRγ variable domain and / or TCRδ variable domain An engineered T cell receptor (TCR). **Claim 2** The engineered TCR according to claim 1, wherein the TCRα polypeptide comprises a TCRα constant domain, the TCRβ polypeptide comprises a TCRβ constant domain, or The TCRγ polypeptide comprises a TCRγ constant domain, the TCRδ polypeptide comprises a TCRδ constant domain The engineered TCR according to claim 1. **Claim 3** The engineered TCR according to claim 1, wherein the one or more antigen-binding domains comprise a first antigen-binding domain linked to said TCRα, TCRγ, TCRβ and / or TCRδ variable domain **Claim 4** The engineered TCR according to claim 3, wherein the one or more antigen-binding domains comprise a second antigen-binding domain linked to the N-terminus of the first antigen-binding domain linked to the N-terminus of said TCRα, TCRγ, TCRβ and / or TCRδ variable domain **Claim 5** The one or more antigen-binding domains comprise (i) A first antigen-binding domain linked to the N-terminus of said TCRα or TCRγ variable domain, and (ii) A first antigen-binding domain linked to the N-terminus of said TCRβ or TCRδ variable domain; and / or (i) A second antigen-binding domain linked to the N-terminus of the first antigen-binding domain linked to the N-terminus of said TCRα or TCRγ variable domain, and (ii) A second antigen-binding domain linked to the N-terminus of the first antigen-binding domain linked to the N-terminus of said TCRβ or TCRδ variable domain The engineered TCR according to claim 3. **Claim 6** The engineered TCR according to claim 1 or claim 2, wherein the one or more antigen-binding domains are the same or different and / or bind to the same or different target antigens. **Claim 7** The one or more antigen-binding domains are alpha-folate receptor (FRα), ανβ6 integrin, ADGRE2, BACE2, B-cell maturation antigen (BCMA), B7-H3 (CD276), B7-H4, B7-H6, CA19.9, carbonic anhydrase IX (CAIX), CCR1, CD7, CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD133, CD138, CD171, CD244, carcinoembryonic antigen (CEA), C-type lectin-like molecule-1 (CLL-1), CD2 subset 1 (CS-1), CLDN6, cMET, chondroitin sulfate proteoglycan 4 (CSPG4), CLDN18.2, cutaneous T-cell lymphoma-associated antigen 1 (CTAGE1), DLL3, epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), EGFR806, epithelial glycoprotein 2 (EGP2), epithelial glycoprotein 40 (EGP40), EphB2, ErbB4, epithelial cell adhesion molecule (EpCAM), ephrin A-type receptor 2 (EPHA2), fibroblast activation protein (FAP), Fc receptor-like 5 (FCRL5), fetal acetylcholine esterase receptor (AchR), FLT3, FN, FN-EDB, FRBeta, ganglioside G2 (GD2), ganglioside G3 (GD3), glypican-3 (GPC3), the EGFR family including ErbB2 (HER2), HER2p95, EGFRv3, IL-10Rα, IL-13Rα2, copper, cancer / testis antigen 2 (LAGE-1A), K-Ras, K-Ras G12C, K-Ras G12D, K-RasIt binds to a target antigen selected from the group consisting of G12V, lambda, Lewis-Y (LeY), L1 cell adhesion molecule (L1-CAM), LILRB2, LY6G6GD, melanoma antigen recognized by T cells 1 (MelanA or MART1), mesothelin (MSLN), MMP10, MUC1, MUC16, MHC class I chain-related protein A (MICA), MHC class I chain-related protein B (MICB), neural cell adhesion molecule (NCAM), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), synovial sarcoma, X breakpoint 2 (SSX2), survivin, tumor-associated glycoprotein 72 (TAG72), transmembrane activator and CAML interactor (TACI), tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), TIM3, trophoblast glycoprotein (TPBG), UL16-binding protein (ULBP) 1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, and vascular endothelial growth factor receptor 2 (VEGFR2); and / or wherein the one or more antigen-binding domains bind to a target polypeptide derived from a protein selected from the group consisting of alpha-fetoprotein (AFP), ASCL2, B melanoma antigen (BAGE) family members, regulator of replication origin Brother (BORIS), cancer-testis antigen, cancer-testis antigen 83 (CT-83), carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), cytomegalovirus (CMV) antigen, cytotoxic T lymphocyte (CTL)-recognized antigen on melanoma (CAMEL), Epstein-Barr virus (EBV) antigen, EPHB2, G antigen 1 (GAGE-1), GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, GAGE-8, glycoprotein 100 (GP100), hepatitis B virus (HBV) antigen, hepatitis C virus (HCV) nonstructural protein 3 (NS3), human papillomavirus (HPV)-E6, HPV-E7, human telomerase reverse transcriptase (hTERT), IGF2BP3 / A3, IGF2BP1, K-Ras, K-Ras G12C, K-Ras G12D, K-Ras G12V, latent membrane protein 2 (LMP2), LY6G6D, melanoma antigen family A, 1 (MAGE-A1), MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, melanoma antigen recognized by T cells (MART-1), mesothelin (MSLN), mucin 1 (MUC1), mucin 16 (MUC16), New York esophageal squamous cell carcinoma-1 (NY-ESO-1), P53, P antigen (PAGE) family members, PAP, PIK3CA, PIK3CA H1047R, placenta-specific 1 (PLAC1), preferentially expressed antigen in melanoma (PRAME), prostate-specific antigen PSA, survivin, synovial sarcoma X1 (SSX1), synovial sarcoma X2 (SSX2), synovial sarcoma X3 (SSX3), synovial sarcoma X4 (SSX4), synovial sarcoma X5 (SSX5), synovial sarcoma X8 (SSX8), thyroglobulin, TP53 R175H, tyrosinase, tyrosinase-related protein (TRP) 1, TRP2, UBD, Wilms tumor protein (WT-1), Wnt10A, X antigen family member 1 (XAGE1), and X antigen family member 2 (XAGE2). The engineered TCR according to claim 1 or claim 2.

8. The engineered TCR according to claim 1 or claim 2, wherein the one or more antigen-binding domains comprise an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 32.

9. The engineered TCR according to claim 1 or claim 2, wherein the one or more antigen-binding domains comprise an antibody or an antigen-binding fragment thereof selected from the group consisting of camel Ig, llama Ig, alpaca Ig, Ig NAR, Fab’ fragment, F(ab’)2 fragment, bispecific Fab dimer (Fab2), trispecific Fab trimer (Fab3), Fv, single-chain Fv protein (“scFv”), bis-scFv, (scFv)2, minibody, diabody, triabody, tetrabody, disulfide-stabilized Fv protein (“dsFv”), and single-domain antibody (sdAb, camelid VHH, nanobody).

10. The engineered TCR according to claim 1, wherein the one or more antigen-binding domains are linked to the TCR variable domain by one or more polypeptide linkers that are about 2 to about 25 amino acids in length.

11. The engineered TCR according to claim 4, wherein the first and second antigen-binding domains are separated by one or more polypeptide linkers that are about 2 to about 25 amino acids in length.

12. The engineered TCR according to claim 10 or claim 11, wherein the one or more polypeptide linkers comprise a linker selected from the group consisting of GG, GS, SG, SS, GSS, SSG, GSG, SGS, SGG, GGS, GGGS (SEQ ID NO: 53), (GGGGGS)1-5 polypeptides (SEQ ID NOs: 35-39), a linker derived from marsupial γμ TCR (e.g., LEKT; SEQ ID NO: 33), the amino acid sequence set forth in any one of SEQ ID NOs: 34 and 40-52, and any combination thereof.

13. The engineered TCR according to claim 10 or claim 11, wherein the one or more polypeptide linkers comprise a linker derived from marsupial γμ TCR that comprises the amino acid sequence set forth in SEQ ID NO:

33.

14. The engineered TCR according to claim 10 or claim 11, wherein the one or more polypeptide linkers comprise a GGGGS (SEQ ID NO: 35) linker (G4S).

15. The engineered TCR of claim 10 or claim 11, wherein the one or more polypeptide linkers comprise a marsupial γμ TCR linker and the G4S linker set forth in SEQ ID NO:

34.

16. The engineered TCR of claim 10 or claim 11, wherein the one or more polypeptide linkers comprise two GGGGS linkers (2×G4S) (SEQ ID NO: 36).

17. The engineered TCR of claim 11, wherein the first and second antigen-binding domains are separated by a second polypeptide linker.

18. The second polypeptide linker is about 2 to about 25 amino acids in length; The second polypeptide linker is about 4 to about 15 amino acids in length; The second polypeptide linker comprises a linker selected from the group consisting of GG, GS, SG, SS, GSS, SSG, GSG, SGS, SGG, GGS, GGGGS (SEQ ID NO: 53), (GGGGGS)1-5 polypeptides (SEQ ID NOs: 35-39), and any combination thereof; and / or The second polypeptide linker comprises an amino acid sequence set forth in any one of SEQ ID NOs: 33-53, The engineered TCR of claim 17.

19. The engineered TCR of claim 1 or claim 2, wherein the TCR variable domain binds to a target polypeptide presented by an MHC complex.

20. The TCR variable domain binds to a target polypeptide derived from a protein selected from the group consisting of alpha-fetoprotein (AFP), ASCL2, B melanoma antigen (BAGE) family member, regulator of imprinted sites Brother (BORIS), cancer-testis antigen, cancer-testis antigen 83 (CT-83), carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), cytomegalovirus (CMV) antigen, cytotoxic T lymphocyte (CTL) recognition antigen on melanoma (CAMEL), Epstein-Barr virus (EBV) antigen, EPHB2, G antigen 1 (GAGE-1), GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, GAGE-8, glycoprotein 100 (GP100), hepatitis B virus (HBV) antigen, hepatitis C virus (HCV) non-structural protein 3 (NS3), human papillomavirus (HPV)-E6, HPV-E7, human telomerase reverse transcriptase (hTERT), IGF2BP3 / A3, IGF2BP1, K-Ras, K-Ras G12C, K-Ras G12D, K-Ras G12V, latent membrane protein 2 (LMP2), LY6G6D, melanoma antigen family A, 1 (MAGE-A1), MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, melanoma antigen recognized by T cells (MART-1), mesothelin (MSLN), mucin 1 (MUC1), mucin 16 (MUC16), New York esophageal squamous cell carcinoma-1 (NY-ESO-1), P53, P antigen (PAGE) family member, PAP, PIK3CA, PIK3CA H1047R, placenta-specific 1 (PLAC1), preferentially expressed antigen in melanoma (PRAME), prostate-specific antigen PSA, survivin, synovial sarcoma X1 (SSX1), synovial sarcoma X2 (SSX2), synovial sarcoma X3 (SSX3), synovial sarcoma X4 (SSX4), synovial sarcoma X5 (SSX5), synovial sarcoma X8 (SSX8), thyroglobulin, TP53 R175H, tyrosinase, tyrosinase-related protein (TRP) 1, TRP2, UBD, Wilms tumor protein (WT-1), Wnt10A, X antigen family member 1 (XAGE1), and X antigen family member 2 (XAGE2); or The TCR variable domain binds to a target polypeptide derived from MAGE-A4, PRAME, K-Ras, TP53R175H, PSA, or IGF2BP3; or The TCR variable domain binds to a target polypeptide derived from MAGE-A4, The engineered TCR according to claim 1 or claim 2.

21. The TCR α constant domain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 82 or 88; The TCR β constant domain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in any one of SEQ ID NO: 80, 81, 86, or 87; The TCR γ constant domain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 83 or 84; and / or The TCR δ constant domain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in any one of SEQ ID NO: 85, The engineered TCR according to claim 1 or claim 2.

22. a) The TCR α or TCR γ polypeptide comprises (i) an amino acid sequence set forth in any one of SEQ ID NO: 105-111, or (ii) a TCR α or TCR γ variable domain comprising an amino acid sequence set forth in any one of SEQ ID NO: 62, 64, 66, 68, 70, 72, 74, 76, and 78; or b) The TCR β or TCR δ polypeptide comprises (i) an amino acid sequence set forth in SEQ ID NO: 103 or 104, or (ii) a TCR β or TCR δ variable domain comprising an amino acid sequence set forth in any one of SEQ ID NO: 63, 65, 67, 69, 71, 73, 75, 77, and 79, The engineered TCR according to claim 1 or claim 2.

23. A fusion polypeptide comprising (i) a) A TCR β polypeptide comprising a TCR β variable domain; and b) A TCR α polypeptide comprising one or more antigen-binding domains, a polypeptide linker, and a TCR α variable domain; or (ii) a) A TCR β polypeptide comprising one or more antigen-binding domains, a polypeptide linker, and a TCR β variable domain; and b) A TCR α polypeptide comprising a TCR α variable domain; or (iii) a) A TCRβ polypeptide comprising one or more antigen-binding domains, a polypeptide linker, and a TCRβ variable domain; and b) A TCRα polypeptide comprising one or more antigen-binding domains, a polypeptide linker, and a TCRα variable domain; or (iv) a) A TCRγ polypeptide comprising a TCRγ variable domain; and b) A TCRδ polypeptide comprising one or more antigen-binding domains, a polypeptide linker, and a TCRδ variable domain; or (v) a) A TCRγ polypeptide comprising one or more antigen-binding domains, a polypeptide linker, and a TCRγ variable domain; and b) A TCRδ polypeptide comprising a TCRδ variable domain; or (vi) a) A TCRγ polypeptide comprising one or more antigen-binding domains, a polypeptide linker, and a TCRγ variable domain; and b) A TCRδ polypeptide comprising one or more antigen-binding domains, a polypeptide linker, and a TCRδ variable domain; or (vii) The TCRβ polypeptide and TCRα polypeptide of the engineered TCR according to claim 1 or claim 2; or (viii) The TCRγ polypeptide and TCRδ polypeptide of the engineered TCR according to claim 1 or claim 2 A fusion polypeptide comprising.

24. The fusion polypeptide according to claim 23, wherein the fusion polypeptide further comprises a polypeptide cleavage signal.

25. The polypeptide cleavage signal is a) A viral self-cleaving peptide or a ribosome-skipping sequence; b) A viral 2A peptide; c) An aphthovirus 2A peptide, a potyvirus 2A peptide, or a cardiovirus 2A peptide; d) A viral 2A peptide selected from the group consisting of foot-and-mouth disease virus (FMDV) 2A peptide, equine rhinitis A virus (ERAV) 2A peptide, Thosea asigna virus (TaV) 2A peptide, porcine teschovirus-1 (PTV-1) 2A peptide, Theiler's virus 2A peptide, and encephalomyocarditis virus 2A peptide; e) Comprising a furin recognition site upstream of the self-cleaving peptide, and optionally, the furin recognition site comprises the amino acid sequence set forth in SEQ ID NO: 112; f) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 113 to 137; or g) the polypeptide cleavage signal comprises the amino acid sequence set forth in any one of SEQ ID NOs: 113 to 137, The fusion polypeptide according to claim 24.

26. The fusion polypeptide according to claim 23, wherein the TCRα, TCRβ, TCRγ and TCRδ polypeptides each comprise an N-terminal signal sequence selected from the group consisting of an IgK signal sequence and a TCRα signal sequence.

27. The fusion polypeptide according to claim 23, wherein the fusion polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 91 to 97, 100, and 102.

28. A polynucleotide encoding a TCR polypeptide of the engineered TCR according to claim 1 or claim 2, or a fusion polypeptide comprising the engineered TCR according to claim 1 or claim 2.

29. A vector comprising the polynucleotide according to claim 28.

30. The vector according to claim 29, wherein the vector is an expression vector, a retroviral vector, or a lentiviral vector.

31. A cell comprising the engineered TCR according to claim 1 or claim 2, or a fusion polypeptide comprising the engineered TCR according to claim 1 or claim 2.

32. The cell is a) hematopoietic cells; b) T cells, αβ-T cells, or γδ-T cells; c) CD3+, CD4+, and / or CD8+ cells; d) immune effector cells; e) cytotoxic T lymphocytes (CTLs), tumor-infiltrating lymphocytes (TILs), or helper T cells; or f) T cells, natural killer (NK) cells, or natural killer T (NKT) cells The cell according to claim 31.

33. The source of the cell is peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, spleen tissue, or a tumor, The cell according to claim 31.

34. The cell according to claim 31, wherein the cell is an isolated non-natural cell.

35. The cell according to claim 31, wherein the cell is obtained from a subject.

36. The cell according to claim 31, wherein the cell is a human cell.

37. A composition comprising the engineered TCR according to claim 1 or claim 2, or a fusion polypeptide comprising the engineered TCR according to claim 1 or claim 2.

38. The composition according to claim 37, for use in the treatment of solid cancer or hematological malignancy in a subject in need of treatment for solid cancer or hematological malignancy.

39. The composition for use according to claim 38, wherein the solid cancer or hematological malignancy is selected from the group consisting of lung cancer, squamous cell carcinoma, colorectal cancer, pancreatic cancer, breast cancer, thyroid cancer, bladder cancer, cervical cancer, esophageal cancer, ovarian cancer, gastric cancer, endometrial cancer, brain cancer, sarcoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck squamous cell carcinoma, glioma, glioblastoma, oligodendroglioma, osteosarcoma, or leukemia, lymphoma, multiple myeloma, non-Hodgkin lymphoma, acute myeloid leukemia (AML), or acute lymphoblastic leukemia (ALL).

40. A composition comprising the cells according to claim 31.

41. The composition according to claim 40, for use in the treatment of solid cancer or hematological malignancy in a subject in need of treatment for solid cancer or hematological malignancy.

42. The composition for use according to claim 41, wherein the solid cancer or hematological malignancy is selected from the group consisting of lung cancer, squamous cell carcinoma, colorectal cancer, pancreatic cancer, breast cancer, thyroid cancer, bladder cancer, cervical cancer, esophageal cancer, ovarian cancer, gastric cancer, endometrial cancer, brain cancer, sarcoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck squamous cell carcinoma, glioma, glioblastoma, oligodendroglioma, osteosarcoma, or leukemia, lymphoma, multiple myeloma, non-Hodgkin lymphoma, acute myeloid leukemia (AML), or acute lymphoblastic leukemia (ALL).

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