T cell receptor

Abstract

The present disclosure provides improved T cell receptors, polynucleotides, polypeptides, vectors, cells, and methods of use thereof. This disclosure generally relates, in part, to isolated T cell receptors, polynucleotides, compositions, medicaments, and uses thereof, that have been modified (engineered) for increased expression, stability, and functional avidity. In various embodiments, an isolated T cell receptor (TCR) is provided that includes a minimally murine TCR alpha chain and a minimally murine TCR beta chain, wherein the transmembrane domain of the TCR alpha chain includes hydrophobic amino acid substitutions.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 000,800, filed March 27, 2020, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing Description The sequence listing for this application has been submitted in text format in lieu of paper and is incorporated herein by reference. The name of the text file containing the sequence listing is BLUE-130_PC_ST25.txt. The text file is 70 KB, was created on March 25, 2021, and is being submitted electronically via EFS-Web concurrently with the filing of this application.

[0003] The present invention relates to T cell receptors (TCRs) engineered to improve expression and functional avidity. More specifically, the present invention relates to TCRs with amino acid substitutions that improve expression and functional avidity, nucleotides encoding the TCRs, vectors, cells, compositions, agents, and methods of using them. [Background technology]

[0004] Description of related fields Despite technological advances in cancer diagnosis and treatment, many cancer patients still have a poor prognosis.

[0005] Adoptive cell therapy (ACT) is a promising approach for the treatment of malignant tumors and viral infections. The adoptive transfer of T lymphocytes genetically modified with antigen-specific T cell receptors (TCRs) attempts to harness and amplify the tumor-killing capacity of a patient's own T cells, eliminating tumors without harming healthy tissue. In theory, T cells from the immune system have the ability to recognize tumor-cell-specific protein patterns and mediate tumor cell destruction through various effector mechanisms.

[0006] However, this approach is not new to the field of tumor immunology, and numerous drawbacks prevent adoptive T cell therapy from being widely used to treat cancer and other diseases. A significant obstacle facing TCR gene therapy is TCR mispairing, which is the inaccurate pairing between the introduced TCRα or TCRβ chain and the endogenous TCRβ or TCRα chain. This can result in low surface expression of the therapeutic αβ TCR and even generate T cells with unknown specificity and toxicity. Another significant limitation of TCR gene therapy is the unpredictability of TCR expression, which can lead to TCR instability and reduced functional avidity. Summary of the Invention [Means for solving the problem]

[0007] This disclosure relates generally in part to isolated T cell receptors that have been modified (engineered) for increased expression, stability, and functional avidity, polynucleotides thereof, compositions, medicaments, and uses thereof.

[0008] In various embodiments, an isolated T cell receptor (TCR) is provided that includes a minimally murine TCR alpha chain and a minimally murine TCR beta chain, wherein the transmembrane domain of the TCR alpha chain includes hydrophobic amino acid substitutions.

[0009] In various embodiments, an isolated T cell receptor (TCR) is provided, comprising a minimally murine TCR alpha chain and a minimally murine TCR beta chain, wherein the transmembrane domain of the TCR alpha chain comprises a hydrophobic amino acid substitution, and wherein the TCR does not bind to MAGEA4.

[0010] In certain embodiments, the isolated T cell receptor (TCR) comprises a TCR alpha chain comprising a constant domain comprising minimal murine amino acid substitutions at positions 90, 91, 92, and 93, and hydrophobic amino acid substitutions at positions 115, 118, and 119, and a TCR beta chain comprising a constant domain comprising minimal murine amino acid substitutions at positions 18, 22, 133, 136, and 139.

[0011] In certain embodiments, the isolated T cell receptor (TCR) comprises a TCR alpha chain comprising a constant domain comprising the following amino acid substitutions: P90S, E91D, S92V, S93P, S115L, G118V, and F119L, and a TCR beta chain comprising a constant domain comprising the following amino acid substitutions: E18K, S22A, F133I, E / V136A, and Q139H.

[0012] In certain embodiments, the isolated T cell receptors (TCRs) include a TCR alpha chain comprising a constant domain comprising at least four minimal murine amino acid substitutions and at least three hydrophobic amino acid substitutions in the TCR alpha chain transmembrane domain, wherein the TCR alpha chain constant domain comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:4, and a TCR beta chain comprising a constant domain comprising at least five minimal murine amino acid substitutions, wherein the TCR beta chain constant domain comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:6.

[0013] In some embodiments, the isolated TCRs include a TCR alpha chain comprising a constant domain comprising minimal murine amino acid substitutions at positions 90, 91, 92, and 93 of the constant region, and hydrophobic amino acid substitutions at positions 115, 118, and 119, wherein the amino acid sequence of the TCR alpha constant region is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:4, and a TCR beta chain comprising a constant region comprising minimal murine amino acid substitutions at positions 18, 22, 133, 136, and 139, wherein the amino acid sequence of the TCR beta constant region is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:6.

[0014] In certain embodiments, the isolated TCRs include a TCR alpha chain comprising a constant domain comprising the following minimal murine amino acid substitutions in the constant domain: P90S, E91D, S92V, and S93P, and the following hydrophobic amino acid substitutions in the transmembrane domain of the constant domain: S115L, G118V, and F119L, wherein the amino acid sequence of the TCR alpha constant domain is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:4, and a TCR beta chain comprising a constant domain comprising the following minimal murine amino acid substitutions: E18K, S22A, F133I, E / V136A, and Q139H, wherein the amino acid sequence of the TCR beta constant domain is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:6.

[0015] In certain embodiments, the isolated T cell receptor (TCR) comprises a TCR alpha chain comprising a constant domain comprising the amino acid sequence set forth in SEQ ID NO:4, and a TCR beta chain comprising a constant domain comprising the amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:6.

[0016] In certain embodiments, the TCR binds to a target antigen selected from the group consisting of alpha-fetoprotein (AFP), B Melanoma Antigen (BAGE) family, Brother of the regulator of imprinted sites (BORIS), cancer-testis antigen, cancer-testis antigen 83 (CT-83), carbonic anhydrase IX (CA1X), carcinoembryonic antigen (CEA), cytomegalovirus (CMV) antigen, cytotoxic T cell (CTL)-recognized antigen on melanoma (CAMEL), Epstein-Barr virus (EBV) antigen, G antigen 1 (GAGE-1), GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, GAGE-8, glycoprotein 100 (GP100). 100), hepatitis B virus (HBV) antigen, hepatitis C virus (HCV) nonstructural protein 3 (NS3), human epidermal growth factor receptor 2 (HER-2), human papillomavirus (HPV)-E6, HPV-E7, human telomerase reverse transcriptase (hTERT), latent membrane protein 2 (LMP2), melanoma antigen family A,1 (MAGE-A1), MAGE-A2, MAGE-A3, 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:P antigen) family, placenta-specific 1 (PLAC1), preferentially expressed antigen in melanoma (PRAME), survivin, synovial sarcoma X 1 (SSX1), synovial sarcoma X 2 (SSX2), synovial sarcoma X 3 (SSX3), synovial sarcoma X 4 (SSX4), synovial sarcoma X 5 (SSX5), Synovial sarcoma X 8 (SSX8), thyroglobulin, tyrosinase, tyrosinase-related protein (TRP) 1, TRP2, Wilms tumor protein (WT-1), X Antigen Family Member 1 (XAGE1), and X Antigen Family Member 2 (XAGE2).

[0017] In a further embodiment, the expression and avidity of the TCR is increased compared to a TCR comprising a minimally murine TCR alpha chain and a minimally murine TCR beta chain, but where the transmembrane domain of the TCR alpha chain does not contain hydrophobic amino acid substitutions.

[0018] In a further embodiment, the expression and avidity of the TCR is increased compared to a TCR that does not comprise a minimally murine TCR alpha chain and a minimally murine TCR beta chain, but in which the transmembrane domain of the TCR alpha chain comprises hydrophobic amino acid substitutions.

[0019] In certain preferred embodiments, the isolated TCRs contemplated herein do not bind to MAGEA4.

[0020] In certain embodiments, fusion proteins are provided that include the TCR alpha and beta chains contemplated herein.

[0021] In a specific embodiment, the fusion protein comprises a minimally murine TCR alpha chain, the transmembrane domain of which comprises hydrophobic amino acid substitutions, a polypeptide cleavage signal, and a minimally murine TCR beta chain.

[0022] In a specific embodiment, the fusion protein comprises a TCR alpha chain comprising a constant domain comprising minimal murine amino acid substitutions at positions 90, 91, 92, and 93, and hydrophobic amino acid substitutions at positions 115, 118, and 119; a polypeptide cleavage signal; and a TCR beta chain comprising a constant domain comprising minimal murine amino acid substitutions at positions 18, 22, 133, 136, and 139.

[0023] In certain embodiments, the fusion protein comprises a TCR alpha chain comprising a constant domain comprising the following amino acid substitutions: P90S, E91D, S92V, S93P, S115L, G118V, and F119L; a polypeptide cleavage signal; and a TCR beta chain comprising a constant domain comprising the following amino acid substitutions: E18K, S22A, F133I, E / V136A, and Q139H.

[0024] In certain embodiments, the fusion protein comprises a TCR alpha chain comprising a constant domain comprising at least four minimal murine amino acid substitutions and at least three hydrophobic amino acid substitutions in the TCR alpha chain transmembrane domain, wherein the TCR alpha chain constant domain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:4; a TCR beta chain comprising a polypeptide cleavage signal and a constant domain comprising at least five minimal murine amino acid substitutions, wherein the TCR beta chain constant domain comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:6.

[0025] In certain embodiments, the fusion protein comprises a TCR alpha chain comprising a constant domain comprising minimal murine amino acid substitutions at positions 90, 91, 92, and 93 of the constant region and hydrophobic amino acid substitutions at positions 115, 118, and 119, wherein the amino acid sequence of the TCR alpha constant region is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:4; or a TCR beta chain comprising a constant region comprising a polypeptide cleavage signal and minimal murine amino acid substitutions at positions 18, 22, 133, 136, and 139, wherein the amino acid sequence of the TCR beta constant region is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:6.

[0026] In certain embodiments, the fusion protein comprises a TCR alpha chain comprising a constant domain comprising the following minimal murine amino acid substitutions in the constant domain: P90S, E91D, S92V, and S93P, and the following hydrophobic amino acid substitutions in the transmembrane domain of the constant domain: S115L, G118V, and F119L, wherein the amino acid sequence of the TCR alpha constant domain is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:4; or a TCR beta chain comprising a constant domain comprising a polypeptide cleavage signal and the following minimal murine amino acid substitutions: E18K, S22A, F133I, E / V136A, and Q139H, wherein the amino acid sequence of the TCR beta constant domain is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:6.

[0027] In certain embodiments, the fusion protein comprises a TCR alpha chain comprising a constant domain comprising the amino acid sequence set forth in SEQ ID NO:4, a polypeptide cleavage signal, and a TCR beta chain comprising a constant domain comprising the amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:6.

[0028] In some embodiments, the polypeptide cleavage signal is a viral self-cleaving peptide or a ribosomal skipping sequence.

[0029] In certain embodiments, the polypeptide cleavage signal is a viral 2A peptide.

[0030] In further embodiments, the polypeptide cleavage signal is an aphthovirus 2A peptide, a potyvirus 2A peptide, or a cardiovirus 2A peptide.

[0031] In additional 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 signa virus (TaV) 2A peptide, a porcine teschovirus-1 (PTV-1) 2A peptide, a tylovirus 2A peptide, and an encephalomyocarditis virus 2A peptide.

[0032] In certain preferred embodiments, the fusion proteins contemplated herein do not bind to MAGEA4.

[0033] In other embodiments, the nucleic acid encodes a TCR or fusion protein contemplated herein.

[0034] In some embodiments, the nucleic acid comprises a first polynucleotide encoding a minimally murine TCR alpha chain, wherein the TCR alpha chain transmembrane domain comprises a hydrophobic amino acid substitution, an internal ribosome entry site (IRES), and a second polynucleotide encoding a minimally murine TCR beta chain.

[0035] In certain embodiments, the nucleic acid comprises a first polynucleotide encoding a TCR alpha chain comprising a constant domain comprising minimal murine amino acid substitutions at positions 90, 91, 92, and 93, and hydrophobic amino acid substitutions at positions 115, 118, and 119; an IRES; and a second polynucleotide encoding a TCR beta chain comprising a constant domain comprising minimal murine amino acid substitutions at positions 18, 22, 133, 136, and 139.

[0036] In certain embodiments, the nucleic acid comprises a first polynucleotide encoding a TCR alpha chain comprising a constant domain comprising the following amino acid substitutions: P90S, E91D, S92V, S93P, S115L, G118V, and F119L; an IRES; and a second polynucleotide encoding a TCR beta chain comprising a constant domain comprising the following amino acid substitutions: E18K, S22A, F133I, E / V136A, and Q139H.

[0037] In some embodiments, the nucleic acid comprises a first polynucleotide encoding a TCR alpha chain comprising a constant domain comprising at least four minimal murine amino acid substitutions and at least three hydrophobic amino acid substitutions in the TCR alpha chain transmembrane domain, wherein the TCR alpha chain constant domain comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:4; and a second polynucleotide encoding a TCR beta chain comprising an IRES and a constant domain comprising at least five minimal murine amino acid substitutions, wherein the TCR beta chain constant domain comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:6.

[0038] In a preferred embodiment, the nucleic acid comprises a first polynucleotide encoding a TCR alpha chain comprising a constant domain comprising minimal murine amino acid substitutions at positions 90, 91, 92, and 93 of the constant region and hydrophobic amino acid substitutions at positions 115, 118, and 119, wherein the amino acid sequence of the TCR alpha constant region is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:4; and a second polynucleotide encoding a TCR beta chain comprising an IRES and a constant region comprising minimal murine amino acid substitutions at positions 18, 22, 133, 136, and 139, wherein the amino acid sequence of the TCR beta constant region is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:6.

[0039] In certain embodiments, the nucleic acid comprises a first polynucleotide encoding a TCR alpha chain comprising a constant domain comprising the following minimal murine amino acid substitutions in the constant domain: P90S, E91D, S92V, and S93P, and the following hydrophobic amino acid substitutions in the transmembrane domain of the constant domain: S115L, G118V, and F119L, wherein the amino acid sequence of the TCR alpha constant domain is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:4; and a second polynucleotide encoding a TCR beta chain comprising an IRES and a constant domain comprising the following minimal murine amino acid substitutions: E18K, S22A, F133I, E / V136A, and Q139H, wherein the amino acid sequence of the TCR beta constant domain is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:6.

[0040] In certain embodiments, the nucleic acid comprises a first polynucleotide encoding a TCR alpha chain comprising a constant domain comprising the amino acid sequence set forth in SEQ ID NO:4, an IRES, and a second polynucleotide encoding a TCR beta chain comprising a constant domain comprising the amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:6.

[0041] In certain preferred embodiments, the nucleic acids contemplated herein do not encode isolated TCRs or fusion proteins that bind MAGEA4.

[0042] In some embodiments, the vector comprises a nucleic acid encoding a TCR or fusion protein as contemplated herein.

[0043] In certain embodiments, the vector comprises a nucleic acid as contemplated herein.

[0044] In some embodiments, the vector is an expression vector.

[0045] In other embodiments, the vector is a retroviral or lentiviral vector.

[0046] In certain embodiments, the cells are modified to express a TCR as contemplated herein.

[0047] In certain embodiments, the cells are modified to express the anticipated fusion protein.

[0048] In certain embodiments, the cells are modified to express a nucleic acid as contemplated herein.

[0049] In certain embodiments, the cells comprise a vector as contemplated herein.

[0050] In certain embodiments, the cell is an immune effector cell.

[0051] In further embodiments, the cell is an immune effector cell selected from the group consisting of a T cell, a natural killer (NK) cell, or a natural killer T (NKT) cell.

[0052] In various embodiments, the composition comprises a TCR, a fusion protein, a nucleic acid, a vector, or a cell as contemplated herein.

[0053] In various embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and a TCR, fusion protein, nucleic acid, vector or cell contemplated herein.

[0054] A TCR, fusion protein, nucleic acid, vector, cell, composition or pharmaceutical composition as contemplated herein for use as a pharmaceutical.

[0055] A TCR, fusion protein, nucleic acid, vector, cell, composition or pharmaceutical composition as contemplated herein for use in the treatment of cancer, wherein the cancer is preferably a hematological cancer or a solid tumour, more preferably the cancer is selected from the group consisting of sarcoma, prostate cancer, uterine cancer, thyroid cancer, testicular cancer, renal cancer, pancreatic cancer, ovarian cancer, esophageal cancer, non-small cell lung cancer, non-Hodgkin's lymphoma, multiple myeloma, melanoma, hepatocellular carcinoma, head and neck cancer, gastric cancer, endometrial cancer, colorectal cancer, bile duct cancer, breast cancer, bladder cancer, myeloid leukemia and acute lymphoblastic leukemia, most preferably the cancer is selected from the group consisting of NSCLC, SCLC, breast cancer, ovarian cancer or colorectal cancer, sarcoma or osteosarcoma. In an embodiment of the present invention, for example, the following items are provided: (Item 1) 1. An isolated T cell receptor (TCR) comprising a minimally murine TCR alpha chain and a minimally murine TCR beta chain, wherein the transmembrane domain of the TCR alpha chain comprises a hydrophobic amino acid substitution, and wherein the TCR does not bind to MAGEA4. (Item 2) 1. An isolated T cell receptor (TCR), comprising: (a) a TCR α chain comprising a constant domain comprising minimal murine amino acid substitutions at positions 90, 91, 92, and 93, and hydrophobic amino acid substitutions at positions 115, 118, and 119; and (b) an isolated TCR comprising a TCR β chain comprising a constant domain containing minimal murine amino acid substitutions at positions 18, 22, 133, 136, and 139. (Item 3) 1. An isolated T cell receptor (TCR), comprising: (a) a TCR α chain comprising a constant domain containing the amino acid substitutions P90S, E91D, S92V, S93P, S115L, G118V, and F119L; and b) An isolated TCR comprising a TCR β chain comprising a constant domain comprising the amino acid substitutions E18K, S22A, F133I, E / V136A, and Q139H. (Item 4) 1. An isolated T cell receptor (TCR), comprising: (a) a TCR alpha chain comprising a constant domain comprising at least four minimal murine amino acid substitutions and at least three hydrophobic amino acid substitutions in the TCR alpha chain transmembrane domain, wherein the TCR alpha chain constant domain comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:4; and (b) An isolated TCR comprising a TCR β chain comprising a constant domain comprising at least five minimal murine amino acid substitutions, wherein the TCR β chain constant domain comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 6. (Item 5) 1. An isolated T cell receptor (TCR), comprising: (a) a TCR alpha chain comprising a constant domain comprising the amino acid sequence set forth in SEQ ID NO: 4; and (b) an isolated TCR comprising a TCR beta chain comprising a constant domain comprising the amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:6. (Item 6) The TCR may bind to α-fetoprotein (AFP), B Melanoma Antigen (BAGE) family members, Brother of the regulator of imprinted sites (BORIS), cancer-testis antigen, cancer-testis antigen 83 (CT-83), carbonic anhydrase IX (CA1X), carcinoembryonic antigen (CEA), cytomegalovirus (CMV) antigen, cytotoxic T cell (CTL)-recognized antigen on melanoma (CAMEL), Epstein-Barr virus (EBV) antigen, G antigen 1 (GAGE-1), GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, GAGE-8, glycoprotein 100 (GP100), and / or other antigens. 100), hepatitis B virus (HBV) antigen, hepatitis C virus (HCV) nonstructural protein 3 (NS3), human epidermal growth factor receptor 2 (HER-2), human papillomavirus (HPV)-E6, HPV-E7, human telomerase reverse transcriptase (hTERT), latent membrane protein 2 (LMP2), melanoma antigen family A,1 (MAGE-A1), MAGE-A2, MAGE-A3, 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:P antigen) family, placenta-specific 1 (PLAC1), preferentially expressed antigen in melanoma (PRAME), survivin, synovial sarcoma X 1 (SSX1), synovial sarcoma X 2 (SSX2), synovial sarcoma X 3 (SSX3), synovial sarcoma X 4 (SSX4), synovial sarcoma 6. The isolated TCR of any one of items 1 to 5, which binds to a target antigen selected from the group consisting of X5 (SSX5), Synovial sarcoma X8 (SSX8), thyroglobulin, tyrosinase, tyrosinase-related protein (TRP) 1, TRP2, Wilms tumor protein (WT-1), X Antigen Family Member 1 (XAGE1), and X Antigen Family Member 2 (XAGE2). (Item 7) 7. The isolated TCR of any one of items 1 to 6, comprising a minimally murine TCR α chain and a minimally murine TCR β chain, but wherein the expression and avidity of the TCR is increased compared to a TCR that does not comprise a hydrophobic amino acid substitution in the transmembrane domain of the TCR α chain. (Item 8) 7. The isolated TCR of any one of items 1 to 6, wherein the expression and avidity of the TCR is increased compared to a TCR that does not comprise a minimally murine TCR α chain and a minimally murine TCR β chain, but wherein the transmembrane domain of the TCR α chain comprises hydrophobic amino acid substitutions. (Item 9) A fusion protein comprising the TCR α chain and the TCR β chain according to any one of items 1 to 8. (Item 10) A fusion protein comprising a minimally murine TCR alpha chain, a polypeptide cleavage signal, and a minimally murine TCR beta chain, wherein the transmembrane domain of the TCR alpha chain contains a hydrophobic amino acid substitution, and the fusion protein does not bind to MAGEA4. (Item 11) A fusion protein comprising: (a) a TCR α chain comprising a constant domain comprising minimal murine amino acid substitutions at positions 90, 91, 92, and 93, and hydrophobic amino acid substitutions at positions 115, 118, and 119; (b) a polypeptide cleavage signal, and (c) a TCR β chain comprising a constant domain comprising minimal murine amino acid substitutions at positions 18, 22, 133, 136, and 139; The fusion protein does not bind to MAGEA4. (Item 12) A fusion protein comprising: (a) a TCR α chain comprising a constant domain containing the amino acid substitutions P90S, E91D, S92V, S93P, S115L, G118V, and F119L; (b) a polypeptide cleavage signal, and (c) a TCR β chain comprising a constant domain comprising the following amino acid substitutions: E18K, S22A, F133I, E / V136A, and Q139H; The fusion protein does not bind to MAGEA4. (Item 13) A fusion protein comprising: (a) a TCR alpha chain comprising a constant domain comprising at least four minimal murine amino acid substitutions and at least three hydrophobic amino acid substitutions in the TCR alpha chain transmembrane domain, wherein the TCR alpha chain constant domain comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:4; (b) a polypeptide cleavage signal, and (c) a TCR β chain comprising a constant domain comprising at least five minimal murine amino acid substitutions, wherein the TCR β chain constant domain comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:6; The fusion protein does not bind to MAGEA4. (Item 14) A fusion protein comprising: (a) a TCR α chain comprising a constant domain comprising the amino acid sequence set forth in SEQ ID NO: 4; (b) a polypeptide cleavage signal, and (c) a TCR beta chain comprising a constant domain comprising the amino acid sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 6; The fusion protein does not bind to MAGEA4. (Item 15) 15. The fusion polypeptide according to any one of items 9 to 14, wherein the polypeptide cleavage signal is a viral self-cleaving peptide or a ribosomal skipping sequence. (Item 16) 16. The fusion polypeptide of any one of items 9 to 15, wherein the polypeptide cleavage signal is a viral 2A peptide. (Item 17) 17. The fusion polypeptide according to any one of items 9 to 16, wherein the polypeptide cleavage signal is an aphthovirus 2A peptide, a potyvirus 2A peptide, or a cardiovirus 2A peptide. (Item 18) 18. The fusion polypeptide of any one of items 9 to 17, wherein 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, Thosea asigna virus (TaV) 2A peptide, porcine teschovirus-1 (PTV-1) 2A peptide, tylovirus 2A peptide, and encephalomyocarditis virus 2A peptide. (Item 19) A nucleic acid encoding the TCR according to any one of items 1 to 8, or the fusion protein according to any one of items 9 to 18. (Item 20) A nucleic acid comprising a first polynucleotide encoding a minimally murine TCR alpha chain, wherein the transmembrane domain of the TCR alpha chain comprises a hydrophobic amino acid substitution, an internal ribosome entry site (IRES), and a second polynucleotide encoding a minimally murine TCR beta chain, wherein the fusion protein does not bind to MAGEA4. (Item 21) A nucleic acid, (a) a first polynucleotide encoding a TCR α chain comprising a constant domain comprising minimal murine amino acid substitutions at positions 90, 91, 92, and 93, and hydrophobic amino acid substitutions at positions 115, 118, and 119; (b) an IRES, and (c) a second polynucleotide encoding a TCR β chain comprising a constant domain comprising minimal murine amino acid substitutions at positions 18, 22, 133, 136, and 139; A nucleic acid wherein the fusion protein does not bind to MAGEA4. (Item 22) A nucleic acid, (a) a first polynucleotide encoding a TCR α chain comprising a constant domain comprising the following amino acid substitutions: P90S, E91D, S92V, S93P, S115L, G118V, and F119L; (b) an IRES, and (c) a second polynucleotide encoding a TCR β chain comprising a constant domain comprising the following amino acid substitutions: E18K, S22A, F133I, E / V136A, and Q139H; A nucleic acid wherein the fusion protein does not bind to MAGEA4. (Item 23) A nucleic acid, (a) a first polynucleotide encoding a TCR alpha chain comprising a constant domain comprising at least four minimal murine amino acid substitutions and at least three hydrophobic amino acid substitutions in the TCR alpha chain transmembrane domain, wherein the TCR alpha chain constant domain comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:4; (b) an IRES, and (c) a second polynucleotide encoding a TCR β chain comprising a constant domain comprising at least five minimal murine amino acid substitutions, wherein the TCR β chain constant domain comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:6; A nucleic acid wherein the fusion protein does not bind to MAGEA4. (Item 24) A nucleic acid, (a) a first polynucleotide encoding a TCR α chain comprising a constant domain comprising the amino acid sequence set forth in SEQ ID NO:4; (b) an IRES, and (c) a second polynucleotide encoding a TCR β chain comprising a constant domain comprising the amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:6; A nucleic acid wherein the fusion protein does not bind to MAGEA4. (Item 25) A vector comprising a nucleic acid encoding the TCR according to any one of Items 1 to 8 or the fusion protein according to any one of Items 9 to 18. (Item 26) 25. A vector comprising the nucleic acid according to any one of items 19 to 24, wherein the vector is preferably an expression vector, more preferably a retroviral vector, or even more preferably a lentiviral vector. (Item 27) A cell expressing the TCR according to any one of items 1 to 8. (Item 28) A cell expressing the fusion protein according to any one of items 9 to 18. (Item 29) A cell comprising the nucleic acid according to any one of items 19 to 24. (Item 30) A cell comprising the vector according to item 25 or 26. (Item 31) 31. The cell according to any one of items 27 to 30, wherein the cell is an immune effector cell. (Item 32) 32. The cell of any one of items 27 to 31, wherein the cell is an immune effector cell selected from the group consisting of a T cell, a natural killer (NK) cell, or a natural killer T (NKT) cell. (Item 33) A composition comprising the TCR according to any one of Items 1 to 8, the fusion protein according to any one of Items 9 to 18, the nucleic acid according to any one of Items 19 to 24, the vector according to Item 25 or 26, or the cell according to any one of Items 27 to 32. (Item 34) A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the TCR according to any one of Items 1 to 8, the fusion protein according to any one of Items 9 to 18, the nucleic acid according to any one of Items 19 to 24, the vector according to Item 25 or 26, or the cell according to any one of Items 27 to 32. (Item 35) 34. The TCR according to any one of Items 1 to 8, the fusion protein according to any one of Items 9 to 18, the nucleic acid according to any one of Items 19 to 24, the vector according to Item 25 or 26, or the cell according to any one of Items 27 to 32, the composition according to Item 33, or the pharmaceutical composition according to Item 34, for use as a pharmaceutical. (Item 36) 34. The TCR according to any one of Items 1 to 8, the fusion protein according to any one of Items 9 to 18, the nucleic acid according to any one of Items 19 to 24, the vector according to Item 25 or 26, or the cell according to any one of Items 27 to 32, the composition according to Item 33, or the pharmaceutical composition according to Item 34, for use in the treatment of cancer, wherein the cancer is preferably a blood cancer or a solid tumor, more preferably the cancer is selected from the group consisting of sarcoma, prostate cancer, uterine cancer, thyroid cancer, testicular cancer, kidney cancer, pancreatic cancer, ovarian cancer, esophageal cancer, non-small cell lung cancer, non-Hodgkin's lymphoma, multiple myeloma, melanoma, hepatocellular carcinoma, head and neck cancer, gastric cancer, endometrial cancer, colorectal cancer, bile duct cancer, breast cancer, bladder cancer, myeloid leukemia and acute lymphoblastic leukemia, most preferably the cancer is selected from the group consisting of NSCLC, SCLC, breast cancer, ovarian cancer, or colorectal cancer, sarcoma, or osteosarcoma. [Brief explanation of the drawings]

[0056] [Figure 1] Figure 1 shows the effect of various amino acid substitutions on MAGEA4 TCR expression. Donor cells (n = 2) were transduced with lentiviral constructs encoding TCRTM (hydrophobic mutations in the transmembrane domain), TCRMM (minimal murine mutations), TCRTM / MM (both mutation sets), or TCRWT (human TCR) and cultured for 10 days. Untransduced (UTD) T cells served as controls. Expression was verified by labeling with the MAGEA4 peptide (pentameric structure) on day 10. [Figure 2] Figure 2A shows that TCR expression was increased in T cells transduced with a lentiviral vector encoding TCR™ / MM compared to T cells transduced with a lentiviral vector encoding TCRWT under two different transduction (Tdxn) conditions (left panel). Figure 2B shows that the transduction efficiency of donor cells (n = 2) with the lentiviral TCR constructs TCR™ / MM and TCRWT was comparable under two different Tdxn conditions (right panel). [Figure 3]Figure 3A shows TCR mispairing in T cells expressing TCRWT. Pairing is expressed as the percentage of positive cells detected by v-beta staining and tetramer antigen staining. Equal percentages of positive cells detected by v-beta staining and tetramer antigen staining suggest specific TCR pairing. Figure 3B shows TCR mispairing in T cells expressing TCRTM / MM (right panel). Pairing is expressed as the percentage of positive cells detected by v-beta staining and tetramer antigen staining. Equal percentages of positive cells detected by v-beta staining and tetramer antigen staining suggest specific TCR pairing. [Figure 4] Figure 4A shows IFNγ production from donor T cells transduced with lentiviral vectors encoding TCRWT and TCRTM / MM. UTD T cells, TCRWT, or TCRTM / MM T cells were cocultured with MAGEA4-expressing tumor cells at an E:T ratio of 1:1, and IFNγ expression was measured 24 hours later (left panel). Figure 4B shows the cytotoxic activity of donor T cells transduced with lentiviral vectors encoding TCRWT and TCRTM / MM. UTD T cells, TCRWT, or TCRTM / MM T cells were cocultured with MAGEA4-expressing tumor cells at an E:T ratio of 1:1, and cytotoxic activity was measured over 3 days (right panel). [Figure 5A] Figures 5A and 5B show the effect of various mutations on NY-ESO-1 TCR expression. Donor cells (n = 2) were transduced with lentiviral constructs encoding TCRWT (human TCR), TCRMM (minimal murine mutations), or TCRTM / MM (hydrophobic mutations in the transmembrane domain and minimal murine mutations) and cultured for 10 days. Untransduced (UTD) T cells served as controls. Figure 5A shows NY-ESO-1 TCR expression verified by labeling with the NY-ESO peptide (pentameric structure) on day 10. [Figure 5B] Figure 5B shows the mean fluorescence intensity (MFI) of NY-ESO-1 TCR expression for each donor. [Figure 6]Figure 6 shows mispairing of NY-ESO-1 TCR in UTD T cells, TCRWT T cells, and TCRTM / MM T cells. Pairing is expressed as the percentage of positive cells detected by v-beta staining and tetramer antigen staining. DETAILED DESCRIPTION OF THE INVENTION

[0057] A brief explanation of sequence numbers SEQ ID NO: 1 sets forth the amino acid sequence of the human TCR alpha constant region. SEQ ID NO: 2 sets forth the amino acid sequence of human TCR beta constant region 1. SEQ ID NO: 3 sets forth the amino acid sequence of human TCR beta constant region 2. SEQ ID NO: 4 sets forth the amino acid sequence of the human TCR alpha constant region with minimal murine amino acid substitutions and hydrophobic amino acid substitutions in the transmembrane domain. SEQ ID NO: 5 sets forth the amino acid sequence of human TCR beta constant region 1 containing minimal murine amino acid substitutions. SEQ ID NO: 6 sets forth the amino acid sequence of human TCR beta constant region 2 containing minimal murine amino acid substitutions. SEQ ID NO: 7 sets forth the amino acid sequence of the human MART-1 TCR alpha chain, including the constant region with minimal murine amino acid substitutions, and hydrophobic amino acid substitutions in the transmembrane domain. SEQ ID NO: 8 sets forth the amino acid sequence of the human MART-1 TCR β chain, including the constant region containing minimal murine amino acid substitutions. SEQ ID NO: 9 sets forth the amino acid sequence of the human MART-1 TCR alpha chain, including the constant region with minimal murine amino acid substitutions, and hydrophobic amino acid substitutions in the transmembrane domain. SEQ ID NO: 10 sets forth the amino acid sequence of the human MART-1 TCR β chain, including the constant region containing minimal murine amino acid substitutions. SEQ ID NO: 11 sets forth the amino acid sequence of the human WT-1 TCR α chain, including the constant region with minimal murine amino acid substitutions and hydrophobic amino acid substitutions in the transmembrane domain. SEQ ID NO: 12 sets forth the amino acid sequence of the human WT-1 TCR β chain, including the constant region containing minimal murine amino acid substitutions. SEQ ID NO: 13 sets forth the amino acid sequence of the human HPV16 E6 TCR alpha chain, including the constant region with minimal murine amino acid substitutions, and hydrophobic amino acid substitutions in the transmembrane domain. SEQ ID NO: 14 sets forth the amino acid sequence of the human HPV16 E6 TCR β chain, including the constant region containing minimal murine amino acid substitutions. SEQ ID NO: 15 sets forth the amino acid sequence of the human NY-ESO-1 TCR α chain, including the constant region with minimal murine amino acid substitutions and hydrophobic amino acid substitutions in the transmembrane domain. SEQ ID NO: 16 sets forth the amino acid sequence of the human NY-ESO-1 TCR β chain, including the constant region containing minimal murine amino acid substitutions. SEQ ID NO: 17 sets forth the amino acid sequence of the human NY-ESO-1 TCR α chain, including the constant region with minimal murine amino acid substitutions and hydrophobic amino acid substitutions in the transmembrane domain. SEQ ID NO: 18 sets forth the amino acid sequence of the human NY-ESO-1 TCR β chain, including the constant region containing minimal murine amino acid substitutions. SEQ ID NO: 19 sets forth the amino acid sequence of the human NY-ESO-1 TCR α chain, including the constant region with minimal murine amino acid substitutions and hydrophobic amino acid substitutions in the transmembrane domain. SEQ ID NO: 20 sets forth the amino acid sequence of the human NY-ESO-1 TCR β chain, including the constant region containing minimal murine amino acid substitutions. SEQ ID NO: 21 sets forth the amino acid sequence of the human NY-ESO-1 TCR α chain, including the constant region with minimal murine amino acid substitutions and hydrophobic amino acid substitutions in the transmembrane domain. SEQ ID NO: 22 sets forth the amino acid sequence of the human NY-ESO-1 TCR β chain, including the constant region containing minimal murine amino acid substitutions. SEQ ID NO: 23 sets forth the amino acid sequence of the human HPV16 E7 TCR alpha chain, including the constant region with minimal murine amino acid substitutions, and hydrophobic amino acid substitutions in the transmembrane domain. SEQ ID NO: 24 sets forth the amino acid sequence of the human HPV16 E7 TCR β chain, including the constant region containing minimal murine amino acid substitutions. SEQ ID NO: 25 sets forth the amino acid sequence of the human GP100 TCR alpha chain, including the constant region with minimal murine amino acid substitutions, and hydrophobic amino acid substitutions in the transmembrane domain. SEQ ID NO: 26 sets forth the amino acid sequence of the human GP100 TCR β chain, including the constant region containing minimal murine amino acid substitutions. SEQ ID NOs: 27 to 37 list the amino acid sequences of various linkers. SEQ ID NOs: 38 to 62 describe the amino acid sequences of the protease cleavage site and the cleavage site of the self-cleaving polypeptide. SEQ ID NO: 63 sets forth the polynucleotide sequence of the consensus Kozak sequence. Throughout this disclosure, reference will be made to amino acid positions using the constant regions of TCR alpha and TCR beta, with amino acid positions numbered with reference to SEQ ID NOS: 1 and 4 for TCR alpha and SEQ ID NOS: 2, 3, 5, and 6 for TCR beta.

[0058] In the above sequences, X refers to any amino acid, if present, or to the absence of an amino acid.

[0059] A. Overview This disclosure generally relates to modified T cell receptors with increased expression, stability, and functional avidity. TCR avidity is determined by the affinity of the TCR for its target peptide and the expression of the TCR. The affinity of a TCR for a target peptide is generally in the range of 1 μM to 10 μM. However, if the affinity of a TCR is too high, it may result in either thymic rejection or undesirable off-target activity. TCR avidity can also be improved by increasing the number of TCR molecules expressed on the cell surface, potentially through codon optimization or chain orientation optimization to achieve balanced expression. TCR stability may also play a role in TCR expression.

[0060] Positively charged residues in the TCR transmembrane region can destabilize the TCR and reduce its expression. Altering the composition of the transmembrane domain can reduce destabilization and increase expression, but the chain cannot be dramatically altered because some charged residues are essential for interaction with the CD3 complex.

[0061] In addition to TCR chain instability, competition with endogenous TCR chains also contributes to low expression. Mispairing of transgenic (exogenous) chains with endogenous chains results in reduced TCR expression, reduced TCR functional avidity, and potentially off-target toxicity. Previous approaches to address this issue have included knocking out the endogenous TCR locus or replacing transgenic constant domains (e.g., human) with constant domains from a different species (e.g., mouse). However, these strategies may result in incomplete inactivation of the endogenous TCR locus and increase the risk of immunogenicity due to the presence of the foreign constant domain.

[0062] The present inventors have made the unexpected discovery that engineered TCRs using a combination of minimal mouse amino acid substitutions and hydrophobic amino acid substitutions in the transmembrane domain of TCRα synergistically increase TCR stability, expression, specific pairing, and functional avidity. Furthermore, the present inventors have surprisingly found that by engineering the TCR constant domain, many TCRs (both high-affinity and low-affinity) can possess the aforementioned properties, thereby providing a tractable immunotherapy strategy. Furthermore, the engineered TCRs contemplated herein offer other advantages over engineered TCR T cells in the art, including a simple manufacturing process, reduced TCR T cell numbers to meet dose requirements, and the potential for further engineering without reducing TCR expression.

[0063] In various embodiments, T cell receptors (TCRs) are provided that have been engineered to increase stability, expression, and functional avidity. The TCRs contemplated herein include one or more amino acid substitutions to minimally murine the TCR and one or more hydrophobic amino acid substitutions in the transmembrane domain. In certain embodiments, the TCR includes a TCR alpha chain with a constant region that is minimally murine and includes hydrophobic amino acid substitutions in the transmembrane domain, and a TCR alpha chain with a minimally murine constant region.

[0064] In specific embodiments, the TCRs contemplated herein comprise one, two, three, or four amino acid substitutions in the TCR alpha constant region to minimally murine the TCR alpha chain, one, two, or three hydrophobic amino acid substitutions in the TCR alpha transmembrane domain, and one, two, three, four, or five amino acid substitutions in the TCR beta constant region to minimally murine the TCR beta chain. In a preferred embodiment, the TCRs contemplated herein comprise four amino acid substitutions in the TCR alpha constant region to minimally murine the TCR alpha chain, three hydrophobic amino acid substitutions in the TCR alpha transmembrane domain, and five amino acid substitutions in the TCR beta constant region to minimally murine the TCR beta chain.

[0065] The TCRs contemplated herein typically bind to target antigens presented by major histocompatibility complex (MHC) molecules. In certain embodiments, the TCRs contemplated herein bind to target antigens expressed on cancer cells, i.e., tumor antigens, including but not limited to tumor-associated antigens (TAA) and tumor-specific antigens (TSA).

[0066] In certain embodiments, one or more polynucleotides encoding the engineered TCR are contemplated. The TCR alpha and TCR beta chains may be encoded by different polynucleotides, or may be encoded by a single polynucleotide as a polycistronic protein or as a fusion polypeptide, where the chains are optionally separated by a polynucleotide encoding a linker polypeptide, such as a self-cleaving polypeptide. In certain embodiments, the polynucleotide encodes the TCR alpha chain, a self-cleaving polypeptide, and a TCR beta polypeptide. In other specific embodiments, the polynucleotide encodes the TCR beta chain, a self-cleaving polypeptide, and a TCR alpha polypeptide.

[0067] In certain embodiments, it is further contemplated that the TCR polynucleotides are introduced into immune effector cells. Immune effector cells expressing the TCRs contemplated herein may be formulated as compositions or pharmaceutical compositions and used in the manufacture of a medicament for the treatment of cancer and / or in methods of treating cancer.

[0068] In preferred embodiments, the TCRs contemplated herein do not bind to MAGEA4, including but not limited to primate or human MAGEA4.

[0069] 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 generally be performed as described in various general and more specific references in microbiology, molecular biology, biochemistry, molecular genetics, cell biology, virology and immunology, cited and discussed throughout this specification. For example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd 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., 1987, Cold Spring Harbor Laboratory), 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 (D.M. Weir and C.C. Blackwell, eds., 1986), Roitt, Essential Immunology, 6th Edition, (Blackwell Scientific Publications, Oxford, 1988), Current Protocols in Immunology (Q.E. Coligigan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach and W. Strober, eds., 1991), Annual Review of Immunology, and research articles in such journals as Advances in Immunology.

[0070] B. Definition Before describing this disclosure in more detail, it may be helpful to an understanding thereof to provide definitions of certain terms to be used herein.

[0071] Unless otherwise specified, 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 particular embodiments, preferred compositions, methods and materials embodiments are disclosed herein. For purposes of this disclosure, the following terms are defined below.

[0072] 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 one or more) of the grammatical object of the article. By way of example, "an element" means one element or one or more elements.

[0073] The use of the alternative (eg, "or") should be understood to mean either one, both, or any combination of the alternatives.

[0074] The term "and / or" should be understood to mean either one or both of the alternatives.

[0075] 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 the quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length relative to the reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0076] In one embodiment, a range, for example, 1 to 5, about 1 to 5, or about 1 to about 5, refers 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.

[0077] 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.

[0078] 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 component or group of steps or components, but not the exclusion of any other step or component or group of steps or components. "Consisting of" means including and limited to everything that follows the word "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are necessary or mandatory, and that no other elements may be present. "Consisting essentially of" means including any elements listed after the phrase, and any elements limited to other elements that do not interfere with or contribute to the activity or function specified in this disclosure for the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are necessary or mandatory, but that there are no other elements that materially affect the activity or function of the listed elements.

[0079] 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 that embodiment is included in at least one embodiment. Thus, the appearances of such phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. Furthermore, it should be understood that the affirmative recitation of a feature in one embodiment serves as grounds for the exclusion of that feature in certain embodiments.

[0080] A "foreign" molecule is a molecule that is not normally present in a cell but is introduced into a cell by one or more genetic, biochemical, or other methods. Exemplary foreign molecules include, but are not limited to, small organic molecules, proteins, nucleic acids, carbohydrates, lipids, glycoproteins, lipoproteins, polysaccharides, modified derivatives of any of the above molecules, or any complex containing one or more of the above molecules. Methods for introducing foreign molecules into cells are known to those skilled in the art and include, but are not limited to, lipid-mediated delivery (i.e., liposomes containing neutral and cationic lipids), electroporation, direct injection, cell fusion, biolistics, biopolymer nanoparticles, calcium phosphate co-precipitation, DEAE-dextran-mediated delivery, and viral vector-mediated delivery.

[0081] An "endogenous" molecule is one that is normally present in a particular cell at a particular developmental stage under particular environmental conditions. Additional endogenous molecules can include proteins.

[0082] Additional definitions are set forth throughout this disclosure.

[0083] CT cell receptor T cell receptors (TCRs) recognize peptide fragments of target antigens when they are presented by major histocompatibility complex (MHC) molecules. Two distinct classes of MHC molecules, MHC I and MHC II, 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.

[0084] The TCRs contemplated herein are heterodimeric complexes comprising a TCR alpha (TCRα) chain and a TCR beta (TCRβ) chain. The human TCRα locus is located on chromosome 14 (14q11.2). The mature TCRα chain comprises a variable domain derived from recombination of a variable (V) segment and a joining (J) segment, and a constant (C) domain. The terms "variable TCRα region" or "TCRα variable chain" or "TCRα variable domain" refer to the variable region of the TCRα chain. The human TCRβ locus is located on chromosome 7 (7q34). The mature TCRβ chain comprises a variable domain derived from recombination of a variable (V) segment, a diversity (D) segment, and a joining (J) segment, and one of two constant (C) domains. The terms "variable TCRβ region" or "TCRβ variable chain" or "TCRβ variable domain" refer to the variable region of the TCRβ chain.

[0085] The rearranged V(D)J regions of both the TCR α and TCR β 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 thought to recognize MHC molecules. Framework regions (FR) are located between the CDRs. These regions form the structure of the TCR variable region.

[0086] 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.

[0087] The TCR structure forms the TCR complex, which includes the TCRα chain, TCRβ chain, and accessory molecules CD3γ, CD3δ, CD3ε, and CD3ζ. Signals from the T cell complex are enhanced by simultaneous binding to MHC molecules by specific coreceptors. CD4 is a coreceptor for MHC II molecules expressed on helper T cells, and CD8 is a coreceptor for MHC I molecules expressed on cytotoxic T cells. Coreceptors not only ensure the specificity of the TCR for antigen, but also enable long-term engagement between the antigen-presenting cell and the T cell and recruit essential intracellular molecules (e.g., LCK) involved in signal transduction of activated T lymphocytes.

[0088] The TCRs contemplated herein can be used to redirect immune effector cells to target cells. The TCRs contemplated herein are engineered to enhance TCR stability, TCR expression, specific TCR pairing, and functional avidity.

[0089] In certain embodiments, the constant domains of the TCR alpha and TCR beta chains are engineered or modified to increase TCR stability, TCR expression, specific TCR pairing, and functional avidity.

[0090] To efficiently enhance correct pairing of engineered TCR sequences and avoid mispairing with endogenous TCR chains, engineered TCR sequences are modified to minimize murinization of the TCRα and TCRβ constant domains. TCR murinization refers to replacing the human TCRα and TCRβ constant domains with their respective mouse counterparts. Nine amino acids have been identified that contribute to improved expression of murinized TCRs. "Minimal murinization" offers the advantages of enhancing cell surface expression while also reducing the number of "foreign" amino acid residues in the amino acid sequence, thereby reducing the risk of immunogenicity. Minimal murinization refers to the substitution of one, two, three, or four amino acids, preferably all four, in the TCRα constant domain, and one, two, three, four, or five amino acids, preferably all five, in the TCRβ constant domain, which contribute to improved expression of murinized TCRs. In a preferred embodiment, minimal murinization refers to the substitution of four amino acids in the constant domain of human TCR alpha and five amino acids in the human TCR beta constant domain, which amino acids contribute to improved expression of the murinized TCR.

[0091] The engineered or modified TCRs contemplated herein comprise minimally murine TCRα and TCRβ constant domains and further comprise hydrophobic amino acid substitutions in the TCRα transmembrane domain to enhance TCR stability, TCR expression, and functional avidity. The transmembrane domain of the TCRα chain has been shown to contribute to the overall lack of stability of the chain, thereby affecting the formation and surface expression of the overall TCR-CD3 complex. In certain embodiments, TCR stability, expression, and avidity are improved by substituting one, two, or three, and preferably all three, amino acids in the TCRα transmembrane domain with hydrophobic amino acids. In a preferred embodiment, the TCRα transmembrane domain contains three hydrophobic amino acid substitutions, thereby improving TCR stability, expression, and avidity.

[0092] Examples of hydrophobic amino acids suitable for use in certain embodiments include alanine (A), valine (V), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), tyrosine (Y), and tryptophan (W). In preferred embodiments, the hydrophobic amino acid is selected from the group consisting of alanine (A), valine (V), isoleucine (I), and leucine (L). In more preferred embodiments, the hydrophobic amino acid is selected from the group consisting of valine (V), isoleucine (I), and leucine (L). In even more preferred embodiments, the hydrophobic amino acid is valine (V) and leucine (L).

[0093] In certain embodiments, the engineered TCR comprises a minimally murine TCR alpha chain and a minimally murine TCR beta chain, wherein the transmembrane domain of the TCR alpha chain comprises hydrophobic amino acid substitutions.

[0094] In a preferred embodiment, the engineered TCR comprises a minimally murine TCR alpha chain comprising four amino acid substitutions in the TCR alpha constant region and a minimally murine TCR beta chain comprising five amino acid substitutions in the TCR beta constant region, wherein the transmembrane domain of the TCR alpha chain further comprises three hydrophobic amino acid substitutions.

[0095] In a preferred embodiment, the engineered TCR comprises a TCR α chain comprising a constant domain comprising minimal murine amino acid substitutions at positions 90, 91, 92, and 93 of the constant region, and hydrophobic amino acid substitutions at positions 115, 118, and 119, and a TCR β chain comprising a constant domain comprising minimal murine amino acid substitutions at positions 18, 22, 133, 136, and 139.

[0096] In a preferred embodiment, the engineered TCR comprises a TCR α chain comprising a constant domain with the following minimal murine amino acid substitutions: P90S, E91D, S92V, and S93P, and the following hydrophobic amino acid substitutions in the transmembrane domain of the constant region: S115L, G118V, and F119L, and a TCR β chain comprising a constant domain with the following minimal murine amino acid substitutions: E18K, S22A, F133I, E / V136A, and Q139H.

[0097] In a preferred embodiment, the engineered TCR includes a TCR alpha chain comprising a constant domain comprising minimal murine amino acid substitutions at positions 90, 91, 92 and 93 of the constant region and hydrophobic amino acid substitutions at positions 115, 118 and 119, wherein the amino acid sequence of the TCR alpha constant region is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:4, and a TCR beta chain comprising a constant region comprising minimal murine amino acid substitutions at positions 18, 22, 133, 136, and 139, wherein the amino acid sequence of the TCR beta constant region is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:6.

[0098] In certain embodiments, engineered TCRs include a TCR alpha chain comprising a constant domain comprising the following minimal murine amino acid substitutions in the constant domain: P90S, E91D, S92V, and S93P, and the following hydrophobic amino acid substitutions in the transmembrane domain of the constant domain: S115L, G118V, and F119L, wherein the amino acid sequence of the TCR alpha constant domain is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:4, and a TCR beta chain comprising a constant domain comprising the following minimal murine amino acid substitutions: E18K, S22A, F133I, E / V136A, and Q139H, wherein the amino acid sequence of the TCR beta constant domain is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:6.

[0099] In certain preferred embodiments, the engineered TCR comprises a TCR alpha chain comprising a constant domain comprising the amino acid sequence set forth in SEQ ID NO:4, and a TCR beta chain comprising a constant domain comprising the amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:6.

[0100] In certain embodiments, the engineered TCR comprises a variable domain that binds to an antigen. In a preferred embodiment, the antigen is not MAGEA4.

[0101] D. Target antigen 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.

[0102] "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 in the cytoplasm to the cell surface, where the peptide:MHC complex mediates CD8 expression. + Recognition by T cells. MHC class II molecules deliver peptides originating from the vesicular system to the cell surface, where the peptide:MHC complex binds to CD4 + Recognized by T cells. Human MHC is also called human leukocyte antigen (HLA).

[0103] In preferred embodiments, "antigen (Ag)," "target antigen," and "polypeptide antigen" are used interchangeably and collectively refer to naturally processed or synthetically produced antigenic protein moieties, such as tumor-associated antigens (TAAs) or tumor-specific antigens (TSAs), which range in length from about 7 amino acids to about 15 amino acids and can complex with MHC (e.g., HLA) to form target antigen:MHC (e.g., HLA) complexes.

[0104] The principles of antigen processing by antigen-presenting cells (APCs) (e.g., dendritic cells, macrophages, lymphocytes, or other cell types) and presentation to T cells by APCs 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 of an 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 originating from the cytoplasm and processed (e.g., tumor antigens, intracellular pathogens, etc.) are often about 7 to about 11 amino acids in length and are associated with class I MHC molecules. On the other hand, peptides processed in the vesicular system (e.g., bacterial peptides, viral peptides) often vary in length from about 10 to about 25 amino acids and are associated with class II MHC molecules.

[0105] In certain embodiments, the engineered TCRs contemplated herein bind to tumor antigens, such as 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 but not in normal cells.

[0106] In certain embodiments, an engineered TCR contemplated herein binds to an antigenic portion of a polypeptide selected from the group consisting of alpha-fetoprotein (AFP), B Melanoma Antigen (BAGE) family, Brother of the regulator of imprinted sites (BORIS), cancer-testis antigen, cancer-testis antigen 83 (CT-83), carbonic anhydrase IX (CA1X), carcinoembryonic antigen (CEA), cytomegalovirus (CMV) antigen, cytotoxic T cell (CTL)-recognized antigen on melanoma (CAMEL), Epstein-Barr virus (EBV) antigen, G antigen. GAGE-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 epidermal growth factor receptor 2 (HER-2), human papillomavirus (HPV)-E6, HPV-E7, human telomerase reverse transcriptase (hTERT), latent membrane protein 2 (LMP2), melanoma antigen family A,1 (MAGE-A1), MAGE-A2, MAGE-A3, 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:P antigen) family, placenta-specific 1 (PLAC1), preferentially expressed antigen in melanoma (PRAME), survivin, synovial sarcoma X 1 (SSX1), synovial sarcoma X 2 (SSX2), synovial sarcoma X 3 (SSX3), synovial sarcoma X 4 (SSX4), synovial sarcoma X 5 (SSX5), Synovial sarcoma X 8 (SSX8), thyroglobulin, tyrosinase, tyrosinase-related protein (TRP) 1, TRP2, Wilms tumor protein (WT-1), X Antigen Family Member 1 (XAGE1), and X Antigen Family Member 2 (XAGE2).

[0107] In certain embodiments, the engineered TCRs contemplated herein bind to an antigenic portion of a polypeptide selected from the group consisting of CT-83, MAGE-A3, MART-1, MUC16, NY-ESO-1, PLAC-1, PRAME, SSX2, survivin, and WT-1.

[0108] In certain embodiments, the engineered TCRs contemplated herein bind to an antigenic portion of NY-ESO-1.

[0109] E. Polypeptides A variety of polypeptides, fusion polypeptides, and polypeptide variants are contemplated herein, including, but not limited to, TCR polypeptides, TCR α chain polypeptides, TCR β chain polypeptides, TCR fusion polypeptides, and fragments thereof. In certain embodiments, exemplary polypeptides contemplated herein include polypeptides comprising the amino acid sequence set forth in any one of SEQ ID NOs: 4-26.

[0110] "Polypeptide," "peptide," and "protein" are used interchangeably and follow their conventional meanings, i.e., amino acid sequences, unless otherwise specified. 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, such as glycosylation, acetylation, phosphorylation, and other modifications, both natural and non-natural, known in the art.

[0111] 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 its cellular environment and from association with other components of a cell, i.e., not being substantially associated with substances in vivo. In certain embodiments, an isolated polypeptide is a synthetic, recombinant, or semi-synthetic polypeptide, or a polypeptide obtained or derived from a recombinant source.

[0112] 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 a TCR by introducing one or more substitutions, deletions, additions, and / or insertions into the TCR α and / or TCR β chains. 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%, 86%, 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.

[0113] Polypeptides include "polypeptide fragments." Polypeptide fragments may be monomeric or multimeric and refer to biologically active polypeptides having amino-terminal deletions, carboxyl-terminal deletions, and / or internal deletions or substitutions in naturally occurring or recombinantly produced polypeptides. 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 activity of a naturally occurring polypeptide. In certain embodiments, a polypeptide fragment may contain an amino acid chain 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.

[0114] As mentioned above, in certain embodiments, polypeptides may be modified in various ways, including amino acid substitution, deletion, truncation, and insertion. Methods for such manipulation are generally known in the art. For example, amino acid sequence variants of a reference polypeptide can be created 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 the 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 Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC).

[0115] In preferred embodiments, fusion polypeptides are contemplated herein. Fusion polypeptides and fusion proteins refer to polypeptides having at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more polypeptide segments. Fusion polypeptides are typically linked C-terminally to N-terminally, but can also be linked C-terminally to C-terminally, N-terminally to N-terminally, or M-terminally to C-terminally. In certain embodiments, the polypeptides of a fusion protein can be in any order or in a specified order.

[0116] In certain embodiments, a TCR contemplated herein is expressed as a fusion polypeptide comprising a TCR alpha chain, a polypeptide linker, and a TCR beta chain. In some embodiments, a TCR is expressed as a fusion protein, the fusion protein comprising, from 5' to 3', a TCR alpha chain, a polypeptide linker, and a TCR beta chain. In some embodiments, a TCR is expressed as a fusion protein, the fusion protein comprising, from 5' to 3', a TCR beta chain, a polypeptide linker, and a TCR alpha chain.

[0117] A "linker" is an amino acid sequence that connects adjacent domains of a polypeptide or fusion polypeptide. Examples of linkers include glycine polymers (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 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. Other exemplary linkers include, but are not limited to, the following amino acid sequences: DGGGS (SEQ ID NO: 27); TGEKP (SEQ ID NO: 28) (see, e.g., Liu et al., PNAS 5525-5530 (1997)), GGRR (SEQ ID NO: 29) (Pomerantz et al. 1995, supra), (GGGGS) n(wherein n=1, 2, 3, 4, or 5) (SEQ ID NO: 30) (Kim et al., PNAS 93, 1156-1160 (1996)), EGKSSGSGSESKVD (SEQ ID NO: 31) (Chaudhary et al., 1990, Proc. Natl. Acad. Sci. USA 87:1066-1070), KESGSVSSEQLAQFRSLD (SEQ ID NO: 32) (Bird et al., 1988, Science 242:423-426), GGRRGGGS (SEQ ID NO: 33), LQRDGERP (SEQ ID NO: 34), LRQKDGGGSERP (SEQ ID NO: 35), LRQKD(GGGS)2ERP (SEQ ID NO: 36). Alternatively, flexible linkers can be modeled using a computer program capable of modeling both the DNA binding site and the peptide itself (Desjarlais & Berg, PNAS 90:2256-2260 (1993), PNAS 91:11099-11103 (1994)) or rationally designed using phage display methods. In certain embodiments, the linker comprises the amino acid sequence: GSTSGSGKPGSGEGSTKG (SEQ ID NO: 37) (Cooper et al., Blood, 101(4):1637-1644 (2003)).

[0118] In certain embodiments, the fusion polypeptide comprises a minimally murine TCR alpha chain, a polypeptide linker, and a minimally murine TCR beta chain, wherein the transmembrane domain of the TCR alpha chain comprises a hydrophobic amino acid substitution. In some embodiments, the fusion protein comprises, from 5' to 3', a minimally murine TCR alpha chain, a polypeptide linker, and a minimally murine TCR beta chain, wherein the transmembrane domain of the TCR alpha chain comprises a hydrophobic amino acid substitution. In some embodiments, the fusion protein comprises, from 5' to 3', a minimally murine TCR beta chain, a polypeptide linker, and a minimally murine TCR alpha chain, wherein the transmembrane domain of the TCR alpha chain comprises a hydrophobic amino acid substitution.

[0119] In certain embodiments, the fusion polypeptide comprises a minimally murine TCR α chain comprising four amino acid substitutions in the TCR α constant region, a polypeptide linker, and a minimally murine TCR β chain comprising five amino acid substitutions in the TCR β constant region, wherein the transmembrane domain of the TCR α chain further comprises three hydrophobic amino acid substitutions. In some embodiments, the fusion protein comprises, from 5' to 3', a minimally murine TCR α chain comprising four amino acid substitutions in the TCR α constant region, a polypeptide linker, and a minimally murine TCR β chain comprising five amino acid substitutions in the TCR β constant region, wherein the transmembrane domain of the TCR α chain further comprises three hydrophobic amino acid substitutions. In some embodiments, the fusion protein comprises, from 5' to 3', a minimally murine TCR β chain comprising five amino acid substitutions in the TCR β constant region, a polypeptide linker, and a minimally murine TCR α chain comprising four amino acid substitutions in the TCR α constant region, wherein the transmembrane domain of the TCR α chain further comprises three hydrophobic amino acid substitutions.

[0120] In certain embodiments, the fusion polypeptide comprises a minimally murinized TCR alpha chain (e.g., SEQ ID NOs: 7, 9, 11, 13, 15, 17, 19, 21, 23, and 25) comprising the following minimal murinized amino acid substitutions in the TCR alpha constant domain: P90S, E91D, S92V, and S93P, and the following hydrophobic amino acid substitutions in the transmembrane domain of the TCR alpha constant domain: S115L, G118V, and F119L, a polypeptide linker, and a TCR beta chain (e.g., SEQ ID NOs: 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26) comprising a constant domain comprising the following minimal murinized amino acid substitutions: E18K, S22A, F133I, E / V136A, and Q139H. In some embodiments, the fusion protein comprises, from 5' to 3', a TCR alpha chain comprising a constant domain comprising the following minimal murine amino acid substitutions in the TCR alpha constant domain: P90S, E91D, S92V, and S93P, and the following hydrophobic amino acid substitutions in the transmembrane domain of the TCR alpha constant domain: S115L, G118V, and F119L; a polypeptide linker; and a TCR beta chain comprising a constant domain comprising the following minimal murine amino acid substitutions: E18K, S22A, F133I, E / V136A, and Q139H. In some embodiments, a fusion protein comprising: a TCR beta chain comprising a constant domain comprising, from 5' to 3', the following minimal murine amino acid substitutions: E18K, S22A, F133I, E / V136A, and Q139H; a polypeptide linker; and a TCR alpha chain comprising a constant domain comprising the following minimal murine amino acid substitutions in the TCR alpha constant domain: P90S, E91D, S92V, and S93P; and the following hydrophobic amino acid substitutions in the transmembrane domain of the TCR alpha constant domain: S115L, G118V, and F119L.

[0121] In certain embodiments, the fusion polypeptide comprises a TCR alpha chain comprising a constant domain comprising minimal murine amino acid substitutions at positions 90, 91, 92, and 93 of the TCR alpha constant domain and hydrophobic amino acid substitutions at positions 115, 118, and 119, wherein the amino acid sequence of the TCR alpha constant domain is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:4; a polypeptide linker; and a TCR beta chain comprising a constant domain comprising minimal murine amino acid substitutions at positions 18, 22, 133, 136, and 139, wherein the amino acid sequence of the TCR beta constant domain is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:6. In some embodiments, the fusion protein comprises, from 5' to 3', a TCR alpha chain comprising a constant domain comprising minimal murine amino acid substitutions at positions 90, 91, 92, and 93 of the TCR alpha constant domain and hydrophobic amino acid substitutions at positions 115, 118, and 119, wherein the amino acid sequence of the TCR alpha constant domain is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:4; a polypeptide linker; and a TCR beta chain comprising a constant domain comprising minimal murine amino acid substitutions at positions 18, 22, 133, 136, and 139, wherein the amino acid sequence of the TCR beta constant domain is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:6.In some embodiments, the fusion protein comprises: a TCR β chain comprising a constant domain comprising, from 5' to 3', minimal murine amino acid substitutions at positions 18, 22, 133, 136, and 139, wherein the amino acid sequence of the TCR β constant domain is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 6; a polypeptide linker; and a TCR α chain comprising a constant domain comprising minimal murine amino acid substitutions at positions 90, 91, 92, and 93 of the TCR α constant domain and hydrophobic amino acid substitutions at positions 115, 118, and 119, wherein the amino acid sequence of the TCR α constant domain is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 4.

[0122] In certain embodiments, the fusion polypeptide comprises a TCR alpha chain comprising a constant domain comprising the following minimal murine amino acid substitutions in the constant domain: P90S, E91D, S92V, and S93P, and the following hydrophobic amino acid substitutions in the transmembrane domain of the constant domain: S115L, G118V, and F119L, wherein the amino acid sequence of the TCR alpha constant domain is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:4; or a TCR beta chain comprising a polypeptide linker and a constant domain comprising the following minimal murine amino acid substitutions: E18K, S22A, F133I, E / V136A, and Q139H, wherein the amino acid sequence of the TCR beta constant domain is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:6. In some embodiments, the fusion protein comprises a TCR alpha chain comprising, from 5' to 3', a constant domain comprising the following minimal murine amino acid substitutions in the constant domain: P90S, E91D, S92V, and S93P, and the following hydrophobic amino acid substitutions in the transmembrane domain of the constant domain: S115L, G118V, and F119L, wherein the amino acid sequence of the TCR alpha constant domain is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:4; a polypeptide linker; and a TCR beta chain comprising a constant domain comprising the following minimal murine amino acid substitutions: E18K, S22A, F133I, E / V136A, and Q139H, wherein the amino acid sequence of the TCR beta constant domain is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:6.In some embodiments, the amino acid sequence of the TCR β constant domain comprises, from 5' to 3', the following minimal murine amino acid substitutions: E18K, S22A, F133I, E / V136A, and Q139H, wherein the amino acid sequence of the TCR β constant domain is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:6; and a TCR α chain comprising a polypeptide linker and a constant domain comprising the following minimal murine amino acid substitutions in the constant domain: P90S, E91D, S92V, and S93P, and the following hydrophobic amino acid substitutions in the transmembrane domain of the constant domain: S115L, G118V, and F119L, wherein the amino acid sequence of the TCR α constant domain is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:4.

[0123] In a preferred embodiment, the polypeptide linker is a polypeptide cleavage signal. 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).

[0124] Suitable protease cleavage sites and self-cleaving peptides are known to those skilled 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 their high cleavage stringency, in one embodiment TEV (tobacco etch virus) protease cleavage sites, e.g., EXXYXQ(G / S) (SEQ ID NO: 38), such as ENLYFQG (SEQ ID NO: 39) and ENLYFQS (SEQ ID NO: 40), are preferred, where X represents any amino acid (TEV cleavage occurs between Q and G or between Q and S).

[0125] In certain embodiments, the polypeptide cleavage signal is a viral self-cleaving peptide or a ribosomal skipping sequence.

[0126] Examples of ribosomal skipping sequences include, but are not limited to, 2A or 2A-like sites, sequences, or domains (see Donnelly et al., 2001, J. Gen. Virol. 82:1027-1041). In certain embodiments, the viral 2A peptide is an aphthovirus 2A peptide, a potyvirus 2A peptide, or a cardiovirus 2A peptide.

[0127] 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.

[0128] Examples of 2A sites are provided in Table 1. [Table 1]

[0129] In certain embodiments, the fusion polypeptide comprises a minimally murine TCR alpha chain, a polypeptide cleavage signal, and a minimally murine TCR beta chain, wherein the transmembrane domain of the TCR alpha chain comprises a hydrophobic amino acid substitution. In some embodiments, the fusion protein comprises, from 5' to 3', a minimally murine TCR alpha chain, a polypeptide cleavage signal, and a minimally murine TCR beta chain, wherein the transmembrane domain of the TCR alpha chain comprises a hydrophobic amino acid substitution. In some embodiments, the fusion protein comprises, from 5' to 3', a minimally murine TCR beta chain, a polypeptide cleavage signal, and a minimally murine TCR alpha chain, wherein the transmembrane domain of the TCR alpha chain comprises a hydrophobic amino acid substitution.

[0130] In certain embodiments, the fusion polypeptide comprises a minimally murine TCR α chain comprising four amino acid substitutions in the TCR α constant region, a polypeptide cleavage signal, and a minimally murine TCR β chain comprising five amino acid substitutions in the TCR β constant region, wherein the transmembrane domain of the TCR α chain further comprises three hydrophobic amino acid substitutions. In some embodiments, the fusion protein comprises, from 5' to 3', a minimally murine TCR α chain comprising four amino acid substitutions in the TCR α constant region, a polypeptide cleavage signal, and a minimally murine TCR β chain comprising five amino acid substitutions in the TCR β constant region, wherein the transmembrane domain of the TCR α chain further comprises three hydrophobic amino acid substitutions. In some embodiments, the fusion protein comprises, from 5' to 3', a minimally murine TCR β chain comprising five amino acid substitutions in the TCR β constant region, a polypeptide cleavage signal, and a minimally murine TCR α chain comprising four amino acid substitutions in the TCR α constant region, wherein the transmembrane domain of the TCR α chain further comprises three hydrophobic amino acid substitutions.

[0131] In a specific embodiment, the fusion polypeptide comprises a minimally murinized TCR alpha chain comprising the following minimal murinized amino acid substitutions in the TCR alpha constant domain: P90S, E91D, S92V, and S93P, and the following hydrophobic amino acid substitutions in the transmembrane domain of the TCR alpha constant domain: S115L, G118V, and F119L, a polypeptide cleavage signal, and a TCR beta chain comprising a constant domain comprising the following minimal murinized amino acid substitutions: E18K, S22A, F133I, E / V136A, and Q139H. In some embodiments, the fusion protein comprises, from 5' to 3', a TCR alpha chain comprising a constant domain comprising the following minimal murine amino acid substitutions in the TCR alpha constant domain: P90S, E91D, S92V, and S93P, and the following hydrophobic amino acid substitutions in the transmembrane domain of the TCR alpha constant domain: S115L, G118V, and F119L; a polypeptide cleavage signal; and a TCR beta chain comprising a constant domain comprising the following minimal murine amino acid substitutions: E18K, S22A, F133I, E / V136A, and Q139H. In some embodiments, a fusion protein comprising: a TCR beta chain comprising a constant domain comprising, from 5' to 3', the following minimal murine amino acid substitutions: E18K, S22A, F133I, E / V136A, and Q139H; a polypeptide cleavage signal; and a TCR alpha chain comprising a constant domain comprising the following minimal murine amino acid substitutions in the TCR alpha constant domain: P90S, E91D, S92V, and S93P; and the following hydrophobic amino acid substitutions in the transmembrane domain of the TCR alpha constant domain: S115L, G118V, and F119L.

[0132] In certain embodiments, the fusion polypeptide comprises a TCR alpha chain comprising a constant domain comprising minimal murine amino acid substitutions at positions 90, 91, 92, and 93 of the TCR alpha constant domain and hydrophobic amino acid substitutions at positions 115, 118, and 119 of the TCR alpha constant domain, wherein the amino acid sequence of the TCR alpha constant domain is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:4; or a TCR beta chain comprising a constant domain comprising a polypeptide cleavage signal and minimal murine amino acid substitutions at positions 18, 22, 133, 136, and 139, wherein the amino acid sequence of the TCR beta constant domain is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:6. In some embodiments, the fusion protein comprises a TCR alpha chain comprising, from 5' to 3', a constant domain comprising minimal murine amino acid substitutions at positions 90, 91, 92, and 93 of the TCR alpha constant domain and hydrophobic amino acid substitutions at positions 115, 118, and 119 of the TCR alpha constant domain, wherein the amino acid sequence of the TCR alpha constant domain is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:4; a polypeptide cleavage signal; and a constant domain comprising minimal murine amino acid substitutions at positions 18, 22, 133, 136, and 139, wherein the amino acid sequence of the TCR beta constant domain is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:6.In some embodiments, the fusion protein comprises: a TCR β chain comprising a constant domain comprising, from 5' to 3', minimal murine amino acid substitutions at positions 18, 22, 133, 136, and 139, wherein the amino acid sequence of the TCR β constant domain is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 6; a TCR α chain comprising a constant domain comprising a polypeptide cleavage signal and minimal murine amino acid substitutions at positions 90, 91, 92, and 93 of the TCR α constant domain and hydrophobic amino acid substitutions at positions 115, 118, and 119 of the TCR α constant domain, wherein the amino acid sequence of the TCR α constant domain is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 4.

[0133] In certain embodiments, the fusion polypeptides include a TCR alpha chain comprising a constant domain comprising the following minimal murine amino acid substitutions in the constant domain: P90S, E91D, S92V, and S93P, and the following hydrophobic amino acid substitutions in the transmembrane domain of the constant domain: S115L, G118V, and F119L, wherein the amino acid sequence of the TCR alpha constant domain is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:4; or a TCR beta chain comprising a constant domain comprising a polypeptide cleavage signal and the following minimal murine amino acid substitutions: E18K, S22A, F133I, E / V136A, and Q139H, wherein the amino acid sequence of the TCR beta constant domain is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:6. In some embodiments, the fusion protein comprises a TCR alpha chain comprising, from 5' to 3', a constant domain comprising the following minimal murine amino acid substitutions in the constant domain: P90S, E91D, S92V, and S93P, and the following hydrophobic amino acid substitutions in the transmembrane domain of the constant domain: S115L, G118V, and F119L, wherein the amino acid sequence of the TCR alpha constant domain is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:4; a TCR beta chain comprising a constant domain comprising a polypeptide cleavage signal and the following minimal murine amino acid substitutions: E18K, S22A, F133I, E / V136A, and Q139H, wherein the amino acid sequence of the TCR beta constant domain is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:6.In some embodiments, the amino acid sequence of the TCR β constant domain comprises, from 5' to 3', the following minimal murine amino acid substitutions: E18K, S22A, F133I, E / V136A, and Q139H, wherein the amino acid sequence of the TCR β constant domain is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:6; and a TCR α chain comprising a polypeptide cleavage signal and a constant domain comprising the following minimal murine amino acid substitutions: P90S, E91D, S92V, and S93P in the constant domain, and the following hydrophobic amino acid substitutions: S115L, G118V, and F119L in the transmembrane domain of the constant domain, wherein the amino acid sequence of the TCR α constant domain is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:4.

[0134] In certain embodiments, the fusion protein comprises a polypeptide cleavage signal, which is a viral self-cleaving peptide or a ribosomal skipping sequence.

[0135] In a specific embodiment, the fusion protein comprises a polypeptide cleavage signal, wherein the polypeptide cleavage signal is a viral 2A peptide.

[0136] In certain embodiments, the fusion protein comprises a polypeptide cleavage signal, wherein the polypeptide cleavage signal is an aphthovirus 2A peptide, a potyvirus 2A peptide, or a cardiovirus 2A peptide.

[0137] In certain embodiments, the fusion protein comprises a polypeptide cleavage signal, wherein the polypeptide cleavage signal is a viral 2A peptide 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.

[0138] F. Polynucleotides In certain embodiments, one or more polynucleotides are provided that encode one or more TCR polypeptides, TCR α chain polypeptides, TCR β chain 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 having a length 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, 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 recited values, such as 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.

[0139] Examples of polynucleotides include, but are not limited to, polynucleotides encoding SEQ ID NOs: 4-26.

[0140] As used herein, an "isolated polynucleotide" refers to a polynucleotide that has been purified from sequences that naturally flank it, e.g., a DNA fragment that has been removed from sequences that normally flank it. In certain embodiments, an "isolated polynucleotide" also refers to a complementary DNA (cDNA), recombinant DNA, or other polynucleotide that is not found in nature and has been created 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.

[0141] In various embodiments, the polynucleotide comprises an mRNA encoding a polypeptide contemplated herein, hi some embodiments, the mRNA comprises a cap, one or more nucleotides, and a poly(A) tail.

[0142] In certain embodiments, a polynucleotide may be codon-optimized. As used herein, the term "codon optimization" refers to substituting 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 synthetically constructed bias tables; (ii) variation in the degree of codon bias within an organism, gene, or set of genes; (iii) systematic variation of codons with their context; (iv) variation of codons with their decoding tRNAs; (v) variation of codons with GC % either across triplets or at a single position in a triplicate; (vi) variation in similarity to a reference sequence, e.g., a natural sequence; (vii) variation in codon frequency cutoffs; (viii) structural properties of mRNA transcribed from a DNA sequence; (ix) prior knowledge of the function of the DNA sequence underlying the design of the codon substitution set; (x) synthetic variation of the codon set for each amino acid; and / or (xi) isolated removal of incorrect translation start positions.

[0143] As used herein, terms such as " polynucleotide variant " and " variant " refer to a polynucleotide that exhibits substantial 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 a reference polynucleotide.In this regard, it is understood in the art that certain modifications, including mutations, additions, deletions and substitutions, can be made to a reference polynucleotide, and the modified polynucleotide can retain the biological function or biological activity of the reference polynucleotide.

[0144] 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. It refers to polynucleotide fragments 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.

[0145] As used herein, "sequence identity," or phrases such as "a sequence 50% identical to," refers 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" can be calculated by: comparing two optimally aligned sequences over a comparison window; determining the number of positions where the same nucleic acid base (e.g., A, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) is 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., 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%, 86%, 97%, 98%, or 99% sequence identity to any of the reference sequences described herein; typically, the polypeptide variant retains at least one biological activity of the reference polypeptide.

[0146] 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, often 15-18 monomeric units, and often at least 25 monomeric units in length. Two polynucleotides may each contain (1) similar sequences between the two polynucleotides (i.e., only a portion of the complete polynucleotide sequence) and (2) divergent sequences between the two polynucleotides. 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 six contiguous positions, usually about 50 to about 100, more commonly about 100 to about 150, and a sequence is compared to the reference sequence over the same number of contiguous positions after the two sequences are optimally aligned. The comparison window may contain no more than about 20% additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. Optimal alignment of the 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 across the comparison window) generated by any of the various methods selected. See, for example, 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.

[0147] 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). Polynucleotide sequences can be annotated in the 5' to 3' or 3' to 5' direction. 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 the "template," "antisense," "minus," or "non-coding" strand. As used herein, the term "reverse" refers to a 5' to 3' sequence written in the 3' to 5' direction or a 3' to 5' sequence written in the 5' to 3' direction.

[0148] Furthermore, those skilled in the art will recognize that, as a result of the degeneracy of the genetic code, there are numerous nucleotide sequences that encode the polypeptides described herein or variant fragments thereof. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any native gene. However, polynucleotides that vary due to differences in codon usage are contemplated, and in certain embodiments, polynucleotides optimized for, for example, human and / or primate codon preferences are contemplated. Furthermore, alleles of genes comprising the polynucleotide sequences provided herein may also be used. Alleles are endogenous genes that are altered as a result of one or more mutations, such as deletions, additions, and / or substitutions of nucleotides.

[0149] As used herein, the term "nucleic acid cassette" or "expression cassette" refers to a genetic sequence in a vector capable of expressing RNA and subsequently 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, 10, or more nucleic acid cassettes. The nucleic acid cassettes are positionally and sequentially oriented within the vector, allowing the nucleic acid in the cassette to be transcribed into RNA, translated into a protein or polypeptide if necessary, subjected to appropriate post-translational modifications required for activity in transformed cells, targeted to an appropriate intracellular compartment for transport to a compartment suitable for biological activity, or secreted into an extracellular compartment. The cassette preferably has 3' and 5' ends adapted for immediate insertion into a vector, e.g., restriction endonuclease sites 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.

[0150] Polynucleotides include polynucleotides of interest. As used herein, the term "polynucleotide of interest" refers to a polynucleotide encoding a polypeptide, polypeptide variant, or fusion polypeptide. A vector may contain 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 encoding wild-type polypeptides and 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.

[0151] Regardless of the length of the coding sequence itself, the polynucleotides contemplated herein may be combined with other DNA sequences disclosed elsewhere herein or known in the art, 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., resulting in a wide variation in their overall length. 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.

[0152] 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 that polypeptide may be inserted into an appropriate vector.

[0153] 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.

[0154] 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.

[0155] 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 virus), poxviruses, baculoviruses, papilloma viruses, and papovaviruses (e.g., SV40).

[0156] Exemplary expression vectors include, but are not limited to, pClneo vector (Promega) for expression in mammalian cells, and pLenti4 / V5-DEST™, pLenti6 / V5-DEST™, and pLenti6.2 / V5-GW / lacZ (Invitrogen) for lentiviral-mediated gene transfer and expression in mammalian cells. In certain embodiments, the coding sequences for the polypeptides disclosed herein may be ligated into such expression vectors for expression of the polypeptides in mammalian cells.

[0157] 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 a host, and is not gradually reduced with the division of the host cell, which also means that the vector replicates extrachromosomally or episomally.

[0158] "Control elements" or "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, and 5' and 3' non-translated regions, which interact with host cellular proteins to effect transcription and translation. Such elements may vary in strength and specificity. Depending on the vector system and host utilized, any number of appropriate transcription and translation factors, including ubiquitous promoters and inducible promoters, may be used.

[0159] In certain embodiments, vectors include, but are not limited to, expression vectors and viral vectors, which contain exogenous, endogenous, or heterologous regulatory sequences, such as promoters and / or enhancers. An "endogenous" regulatory sequence is a sequence that is naturally linked to a given gene in the genome. An "exogenous" regulatory sequence is one that is placed in juxtaposition to a gene by means of genetic engineering (i.e., molecular biological techniques) such that transcription of that gene is directed by the linked enhancer / promoter. A "heterologous" regulatory sequence is an exogenous sequence that originates from a species different from the cell being genetically engineered.

[0160] 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 transcription of 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 start site, where N can be any nucleotide.

[0161] The term "enhancer" refers to a DNA segment containing a sequence that can provide enhanced transcription, and in some cases can function regardless of orientation relative to another regulatory sequence. Enhancers can function cooperatively or additively with promoter elements and / or other enhancer elements. The term "promoter / enhancer" refers to a DNA segment containing a sequence that can provide both promoter and enhancer functions.

[0162] The term "operably linked" refers to a juxtaposition wherein the described components are in a relationship permitting them to function in their intended manner. In one embodiment, the term refers to the 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.

[0163] As used herein, the term "structural expression control sequence" refers to a promoter, enhancer, or promoter / enhancer that permits continuous or sequential transcription of an operably linked sequence. A structural expression control sequence may be a "ubiquitous" promoter, enhancer, or promoter / enhancer that permits 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 permits expression in restricted cell and tissue types, respectively.

[0164] Examples of 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).

[0165] In one embodiment, the vector contains the MNDU3 promoter.

[0166] In one embodiment, the vector contains the EF1a promoter including the first intron of the human EF1a gene.

[0167] In one embodiment, the vector contains the EF1a promoter lacking the first intron of the human EF1a gene.

[0168] As used herein, "conditional expression" can refer to any type of conditional expression, including, but not limited to, inducible expression, repressible expression, expression in cells or tissues having a particular physiological, biological, or disease state, etc. This definition is not intended to exclude cell-type- or tissue-specific expression. Certain embodiments provide for conditional expression of a polynucleotide of interest, e.g., expression controlled by exposing a cell, tissue, or organism to a treatment or condition that results in expression of the polynucleotide or that increases or decreases expression of a polynucleotide encoded by the polynucleotide of interest.

[0169] 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.

[0170] As used herein, "internal ribosome entry site" or "IRES" refers to a factor 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. RNA 1(10):985-1000. In certain embodiments, a vector contains one or more polynucleotides of interest encoding one or more polypeptides. In certain embodiments, to achieve efficient translation of each of multiple polypeptides, the polynucleotide sequences may be separated by one or more IRES sequences or polynucleotide sequences encoding self-cleaving polypeptides. In one embodiment, the IRES used in the polynucleotides contemplated herein is the EMCV IRES.

[0171] 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, increasing translation. The consensus Kozak sequence is (GCC)RCCATGG (SEQ ID NO: 63), 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 having a consensus Kozak sequence and encoding a desired polypeptide, such as a TCR.

[0172] Factors that induce efficient termination and polyadenylation of heterologous nucleic acid transcripts increase heterologous gene expression. Transcription termination signals are generally located downstream of polyadenylation signals. In certain embodiments, vectors contain a polyadenylation sequence 3' to the polynucleotide encoding the polypeptide to be expressed. As used herein, the terms "polyA site" or "polyA sequence" refer to a DNA sequence that induces both 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-mRNAs 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 poly(A) sequence of choice (e.g., AATAAA, ATTAAA, AGTAAA). In certain embodiments, the poly(A) sequence is SV40 poly(A), bovine growth hormone poly(A) sequence (BGHpA), rabbit β-globin poly(A) sequence (rβgpA), variants thereof, or another suitable heterologous or endogenous poly(A) sequence known in the art.

[0173] In some embodiments, the polynucleotide or cells harboring the polynucleotide utilize 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 cells harboring the polynucleotide. Examples of suicide genes that can be used include caspase-9, caspase-8, or cytosine deaminase. Caspase-9 can be activated using a specific chemical inducer of dimerization (CID).

[0174] In certain embodiments, one or more polynucleotides encoding the TCR α and TCR β chains are introduced into a cell (e.g., an immune effector cell) by a non-viral or viral vector. 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 within the cell or may contain sequences sufficient to allow integration into host cell DNA. In certain embodiments, a non-viral vector is used to deliver one or more polynucleotides contemplated herein to a T cell.

[0175] Examples of non-viral vectors include, but are not limited to, mRNA, plasmids (eg, DNA or RNA plasmids), transposons, cosmids, and bacterial artificial chromosomes.

[0176] Non-viral methods of polynucleotide delivery contemplated in certain embodiments include, but are not limited to, electroporation, sonoporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, nanoparticles, polycation or lipid:nucleic acid complexes, naked DNA, artificial virions, DEAE-dextran mediated transfer, gene guns, and heat shock.

[0177] Examples of 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 have been described in the literature. See, e.g., 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.

[0178] In various embodiments, the polynucleotide is the mRNA that is introduced into cells to transiently express desired polypeptide.As used herein, " transient " refers to the expression of non-integrated transgene for 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 cells or contained in a stable plasmid replicon.

[0179] In certain embodiments, viral vectors are used to deliver one or more polynucleotides contemplated herein to T cells.

[0180] Examples of viral vector systems suitable for use in certain embodiments contemplated herein include, but are not limited to, adeno-associated viral (AAV), retroviral (including lentiviral), herpes simplex viral, adenoviral, and vaccinia viral vectors.

[0181] In certain embodiments, a polycistronic polynucleotide encoding a TCR comprising a TCR alpha chain and a TCR beta chain is introduced into a cell by a non-viral or viral vector. In certain embodiments, a polycistronic polynucleotide encodes a fusion protein encoding a TCR as contemplated herein comprising a minimally murine TCR alpha chain and a hydrophobic amino acid substitution in the TCR alpha transmembrane domain, a polypeptide cleavage signal, and a minimally murine TCR beta chain.

[0182] In certain embodiments, a polycistronic polynucleotide encoding a TCR comprising a TCR alpha chain and a TCR beta chain is introduced into a cell by a non-viral or viral vector. In certain embodiments, a polycistronic polynucleotide encodes a fusion protein encoding a TCR contemplated herein comprising a minimally murine TCR alpha chain and a hydrophobic amino acid substitution in the TCR alpha transmembrane domain, an IRES, and a minimally murine TCR beta chain.

[0183] In certain embodiments, the polycistronic polynucleotide comprises a TCR alpha chain 5' to a TCR beta chain, hi other embodiments, the polycistronic polynucleotide comprises a TCR alpha chain 3' to a TCR beta chain.

[0184] G. Genetically Modified Cells In various embodiments, cells genetically modified to express a TCR contemplated herein, comprising a minimally murine TCR alpha chain, hydrophobic amino acid substitutions in the TCR alpha transmembrane domain, and a minimally murine TCR beta chain, for use in the treatment of cancer are provided. In various embodiments, immune effector cells genetically modified to express a TCR contemplated herein, comprising a minimally murine TCR alpha chain, hydrophobic amino acid substitutions in the TCR alpha transmembrane domain, and a minimally murine TCR beta chain, are used in the preparation or manufacture of a medicament for the treatment of cancer.

[0185] In certain embodiments, polynucleotides encoding the TCRs contemplated herein are introduced into immune effector cells to express the TCRs contemplated herein and to redirect the immune effector cells to target cells expressing a target antigen. In certain embodiments, one or more polynucleotides encoding a TCR contemplated herein comprising a minimally murine TCR α chain, a hydrophobic amino acid substitution in the TCR α transmembrane domain, and a minimally murine TCR β chain are introduced into one or more immune effector cells. In certain embodiments, a polynucleotide encoding a fusion protein comprising a TCR comprising a minimally murine TCR α chain, a hydrophobic amino acid substitution in the TCR α transmembrane domain, a polypeptide linker, e.g., a polypeptide cleavage signal, and a minimally murine TCR β chain is introduced into one or more immune effector cells.

[0186] 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 comprise αβ T cells. In certain embodiments, the cells comprise γδ T cells engineered to express an αβ TCR. In one embodiment, the immune effector cells comprise natural killer (NK) cells. In one embodiment, the immune effector cells comprise natural killer T (NKT) cells.

[0187] Immune effector cells may be self or non-self (e.g., allogeneic, syngeneic, or xenogeneic). As used herein, "self" 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.

[0188] Examples of immune effector cells for use with TCRs contemplated in certain embodiments include T lymphocytes. The terms "T cell" or "T lymphocyte" are art-recognized and are intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. T cells may be T helper (Th) cells, such as T helper 1 (Th1) cells or T helper 2 (Th2) cells. T cells may be helper T 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.

[0189] As will be understood by those skilled in the art, other cells can also be used as immune effector cells with the TCRs contemplated herein. 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 into mature immune effector cells in vitro.

[0190] As used herein, the term "CD34+ cells" refers to cells that express CD34 protein on their 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 populations contain hematopoietic stem cells (HSCs), which, when administered to patients, differentiate and give rise to all hematopoietic lineages, including T cells, NK cells, NKT cells, neutrophils, and cells of the monocyte / macrophage lineage.

[0191] In certain embodiments, methods of generating immune effector cells expressing a TCR contemplated herein are provided. In one embodiment, the method comprises transfecting or transducing immune effector cells isolated from an individual such that the immune effector cells express a polycistronic message encoding a TCR comprising a modified TCR α chain and a modified TCR β chain, or a fusion protein encoding a TCR contemplated herein comprising a minimally murine TCR α chain and hydrophobic amino acid substitutions in the TCR α transmembrane domain, a polypeptide linker, and a minimally murine TCR β chain.

[0192] In a preferred embodiment, the method comprises transfecting or transducing immune effector cells isolated from an individual such that the immune effector cells express a polycistronic message encoding a TCR as contemplated herein, comprising a minimally murine TCR alpha chain and a hydrophobic amino acid substitution in the TCR alpha transmembrane domain, a 2A self-cleaving polypeptide, and a minimally murine TCR beta chain. In certain embodiments, the transduced cells are then cultured and expanded before being administered to the subject.

[0193] In some embodiments, immune effector cells are isolated from an individual and genetically modified without further in vitro manipulation. These cells may then be directly re-administered to the individual. In a further embodiment, immune effector cells are first activated and stimulated to expand in vitro, and then genetically modified to express a TCR as contemplated herein, comprising a minimally murine TCR alpha chain and a hydrophobic amino acid substitution in the TCR alpha transmembrane domain, a linker, and a minimally murine TCR beta chain. In this regard, immune effector cells may be cultured before and / or after genetic modification.

[0194] In certain embodiments, a source of cells is obtained from a subject prior to in vitro manipulation or genetic modification of the immune effector cells described herein, hi certain embodiments, the modified immune effector cells comprise T cells.

[0195] In certain embodiments, PBMCs may be directly genetically modified using the methods contemplated herein to express a polycistronic message encoding a TCR contemplated herein, comprising a minimally murine TCR α chain and hydrophobic amino acid substitutions in the TCR α transmembrane domain, a polypeptide linker, and a minimally murine TCR β chain. In certain embodiments, after isolation of PBMCs, T lymphocytes are further isolated, and in certain embodiments, both cytotoxic and helper T lymphocytes can be preserved into naive, memory, and effector T cell subpopulations, either before or after genetic modification and / or expansion.

[0196] Immune effector cells, e.g., T cells, may be isolated using known methods and then genetically modified, or the 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, e.g., T cells, are activated and stimulated for expansion 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 a TCR contemplated herein, comprising a minimally murine TCR alpha chain and a hydrophobic amino acid substitution in the TCR alpha transmembrane domain, a polypeptide linker, and a minimally murine TCR beta chain), and then activated and expanded in vitro. In various embodiments, the T cells may be activated and expanded before or after genetic modification using, for example, 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.

[0197] In one embodiment, CD34 + The cells are transduced with a nucleic acid construct as contemplated herein. In certain embodiments, the transduced CD34 + The cells are differentiated in vivo into mature immune effector cells after administration to a subject, generally the 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).

[0198] In certain embodiments, the population of modified immune effector cells for cancer treatment comprises the CAR and CCR contemplated herein. For example, the population of modified immune effector cells is prepared from peripheral blood mononuclear cells (PBMCs) obtained from patients (autologous donors) diagnosed with the B-cell malignancies described herein. PBMCs form a heterogeneous population of T lymphocytes, which may be CD4+, CD8+, or CD4+ and CD8+.

[0199] H. Compositions and Formulations Compositions contemplated herein may comprise one or more TCR polypeptides, TCR α chain polypeptides, TCR β chain polypeptides, TCR fusion polypeptides, polynucleotides, vectors comprising the same, genetically modified immune effector cells, etc., contemplated herein. Compositions include, but are not limited to, pharmaceutical compositions. In preferred embodiments, compositions comprise one or more cells engineered to express an engineered TCR comprising a minimally murine TCR α chain and a hydrophobic amino acid substitution in the TCR α transmembrane domain and a minimally murine TCR β chain, or one or more cells engineered to express a fusion protein comprising a TCR comprising a minimally murine TCR α chain and a hydrophobic amino acid substitution in the TCR α transmembrane domain, a polypeptide linker such as a polypeptide cleavage signal, and a minimally murine TCR β chain. In preferred embodiments, compositions comprise one or more cells engineered to express a fusion protein comprising a minimally murine TCR α chain and a TCR comprising a hydrophobic amino acid substitution in the TCR α transmembrane domain, a 2A self-cleaving polypeptide, and a minimally murine TCR β chain.

[0200] A "pharmaceutical composition" refers to a composition formulated in a pharmaceutically or physiologically acceptable solution for administration to a cell or animal, alone or in combination with one or more other therapeutic modalities. It should also be understood that, if desired, 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 various other pharmaceutically 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 its intended therapy. In a preferred embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, diluent or excipient and one or more cells modified to express an engineered TCR comprising a minimally murine TCR alpha chain and hydrophobic amino acid substitutions in the TCR alpha transmembrane domain and a minimally murine TCR beta chain, or one or more cells modified to express a fusion protein comprising a TCR comprising a minimally murine TCR alpha chain and hydrophobic amino acid substitutions in the TCR alpha transmembrane domain, a polypeptide linker, and a minimally murine TCR beta chain.

[0201] As used herein, the phrase "pharmaceutically acceptable" is employed to refer to those compounds, substances, 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.

[0202] As used herein, "pharmaceutically acceptable carrier, diluent, or excipient" includes, but is not limited to, isotonic saline, Ringer's solution, ethyl alcohol, phosphate buffer, and any other compatible substance employed in pharmaceutical formulations.

[0203] In certain embodiments, the composition comprises an amount of immune effector cells, which express a TCR comprising a minimally murine TCR α chain and hydrophobic amino acid substitutions in the TCR α transmembrane domain, and a minimally murine TCR β chain. As used herein, the term "amount" refers to an "effective amount" or "effective quantity" of genetically modified therapeutic cells, such as T cells, to achieve a beneficial or desired prophylactic or therapeutic result, including a clinical result.

[0204] A "prophylactically effective amount" refers to an amount of genetically modified therapeutic cells effective to achieve a desired prophylactic result. Typically, but not necessarily, a prophylactically effective amount is less than a therapeutically effective amount, since a prophylactic dose is used in subjects prior to or at an early stage of disease.

[0205] A "therapeutically effective amount" of genetically modified therapeutic cells can vary depending on factors such as the individual's condition, age, sex, and weight, 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 harmful 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 patient's (subject's) age, weight, tumor size, extent of infection or metastasis, and condition.

[0206] Generally, pharmaceutical compositions comprising the T cells described herein are administered in a dose of 10 6 ~10 13 cells / kg body weight, preferably 10 8 ~10 13It 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 liter or less, and can be 500 mL or less, or even 250 mL or 100 mL or less. Therefore, the desired cell density is often 10 6 cells / ml, typically greater than 10 7 cells / ml, typically >10 8 Clinically relevant immune cell counts may be divided into multiple infusions, cumulatively exceeding 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 or 10 13 The 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.

[0207] The compositions are preferably formulated for parenteral administration, eg, intravascular (intravenous or intraarterial), intraperitoneal, or intramuscular administration.

[0208] Liquid pharmaceutical compositions, whether in solution, suspension, or other similar form, may contain one or more sterile diluents such as water for injection, saline, preferably saline, Ringer's solution, or isotonic saline. 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.

[0209] In one embodiment, the T cell compositions contemplated herein are formulated in a pharmaceutically acceptable cell culture medium. Such compositions are suitable for administration to a human subject. In a specific embodiment, the pharmaceutically acceptable cell culture medium is a serum-free medium.

[0210] Serum-free media have several advantages over serum-containing media, including a simpler and more transparent composition, reduced contaminant load, elimination of potential sources of infectious agents, and reduced cost. In various embodiments, serum-free media are 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, and the nutrients are obtained from synthetic, plant, or microbial sources. "Protein-free" media, in contrast, are defined as being substantially protein-free.

[0211] 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.

[0212] In a preferred embodiment, compositions comprising immune effector cells contemplated herein are formulated in a solution comprising PlasmaLyte A.

[0213] In another preferred embodiment, the compositions comprising the immune effector cells contemplated herein are formulated in a solution containing a cryopreservation medium. For example, a cryopreservation medium containing a cryopreservative may be used to maintain high cell viability after thawing. Examples of cryopreservation media for use in certain compositions include, but are not limited to, CryoStor CS10, CryoStor CS5, and CryoStor CS2.

[0214] 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.

[0215] In certain embodiments, the composition comprises an effective amount of genome-edited immune effector cells, alone or in combination with one or more therapeutic agents, wherein the immune effector cells have been modified to express a TCR comprising a minimally murine TCR α chain, a hydrophobic amino acid substitution in the TCR α transmembrane domain, and a minimally murine TCR β chain. 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, or photodynamic therapy. The composition may also be administered in combination with antibiotics. Such therapeutic agents may be art-recognized as standard treatments for certain disease states described herein, such as certain cancers. Examples of therapeutic agents contemplated in certain embodiments include cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory agents, chemotherapeutic agents, radiation therapy, therapeutic antibodies, or other active adjunctive agents.

[0216] In certain embodiments, compositions comprising genome-edited immune effector cells may be administered in combination with any number of chemotherapeutic agents, wherein the immune effector cells have been engineered to express a TCR comprising a minimally murine TCR alpha chain and hydrophobic amino acid substitutions in the TCR alpha transmembrane domain, and a minimally murine TCR beta chain.

[0217] In certain embodiments, the composition comprising immune effector cells is administered in conjunction with a therapeutic antibody, wherein the immune effector cells have been engineered to express a TCR comprising a minimally murine TCR alpha chain, hydrophobic amino acid substitutions in the TCR alpha transmembrane domain, and a minimally murine TCR beta chain. Exemplary therapeutic antibodies suitable for use in combination with contemplated CAR-modified T cells in certain embodiments include, but are not limited to, atezolizumab, avelumab, bavituximab, bevacizumab (Avastin), bivatuzumab, blinatumomab, conatumumab, crizotinib, daratumumab, durigotumab, dacetuzumab, dalotuzumab, and dutuzumab. These include ruvalumab, elotuzumab (HuLuc63), gemtuzumab, ibritumomab, indatuximab, inotuzumab, ipilimumab, lorvotuzumab, lucatumumab, milatuzumab, moxetumomab, nivolumab, ocaratuzumab, ofatumumab, pembrolizumab, rituximab, siltuximab, teprotumumab, and ublituximab.

[0218] In certain embodiments, the formulation of pharmaceutically acceptable carrier solutions is known in the art, as is the development of appropriate dosing and treatment regimens for use of certain compositions described herein in a variety of treatment regimens, including, for example, enteral and parenteral, e.g., intravascular, intravenous, intraarterial, 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 well known, for example, in the pharmaceutical arts and are described in, for example, Remington: The Science and Practice of Pharmacy, volume I and volume II.22, which are 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; 2012.

[0219] I. Treatment method The genetically modified immune effector cells expressing a TCR comprising a minimally murine TCR alpha chain and hydrophobic amino acid substitutions in the TCR alpha transmembrane domain and a minimally murine TCR beta chain contemplated herein provide an improved method 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.

[0220] In one embodiment, a type of cell therapy in which T cells are genetically modified to express a TCR comprising a minimally murine TCR α chain, hydrophobic amino acid substitutions in the TCR α transmembrane domain, and a minimally murine TCR β chain is infused into a recipient in need thereof. The infused cells can kill disease-causing cells in the recipient. Unlike antibody therapy, T cell therapy can replicate in vivo, resulting in long-term persistence that can provide sustained cancer treatment.

[0221] In one embodiment, T cells expressing a TCR comprising a minimally murine TCR α chain, hydrophobic amino acid substitutions in the TCR α chain transmembrane domain, and a minimally murine TCR β chain can undergo stable in vivo T cell expansion and persist for long periods of time. In another embodiment, T cells expressing a TCR comprising a minimally murine TCR α chain, hydrophobic amino acid substitutions in the TCR α transmembrane domain, and a minimally murine TCR β chain can differentiate into specific memory T cells or stem cell memory T cells, which can be reactivated to inhibit the formation or growth of any additional tumors.

[0222] In certain embodiments, engineered immune effector cells expressing a TCR comprising a minimally murine TCR alpha chain and hydrophobic amino acid substitutions in the TCR alpha transmembrane domain and a minimally murine TCR beta chain as contemplated herein are used in the treatment of solid tumors or cancers.

[0223] In certain embodiments, the engineered immune effector cells contemplated herein are selected from, but are not limited to, adrenal carcinoma, adrenocortical carcinoma, anal cancer, appendiceal cancer, astrocytoma, atypical teratoid tumor / atypical rhabdomyoid tumor, basal cell carcinoma, bile duct carcinoma, bladder cancer, 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 cancer, 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 cancer, intraocular melanoma, Kaposi's sarcoma, kidney cancer, laryngeal cancer, leiomyosarcoma, tongue cancer, liposarcoma, liver 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 uterine cancer, myxosarcoma, myelodysplastic syndrome, myeloproliferative neoplasms, cancer of the nasal cavity and paranasal sinuses, nasopharyngeal carcinoma, neuroblastoma, oligodendroglioma, oral cancer (oral cavity cancer), oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, islet cell tumor, papillary carcinoma, paraganglioma, parathyroid carcinoma, penile cancer, pharyngeal carcinoma, 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 carcinoma, 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 carcinoma, thyroid cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vascular cancer, vulvar cancer, and Wilms' tumor

[0224] 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, 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, and oligodendroglioma.

[0225] 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.

[0226] In certain embodiments, the engineered immune effector cells contemplated herein are used in the treatment of glioblastoma.

[0227] In certain embodiments, engineered immune effector cells expressing a TCR comprising a minimally murine TCR alpha chain and hydrophobic amino acid substitutions in the TCR alpha transmembrane domain and a minimally murine TCR beta chain as contemplated herein are used in the treatment of liquid or hematological cancers.

[0228] 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.

[0229] In certain embodiments, the engineered immune effector cells contemplated herein are directed against leukemias, lymphomas, and multiple myeloma, including, but not limited to, 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, non-secretory myeloma, IgD myeloma, osteosclerotic myeloma, solitary bone plasmacytoma, and extramedullary plasmacytoma.

[0230] In certain embodiments, the engineered immune effector cells contemplated herein are used to treat acute myeloid leukemia (AML).

[0231] As used herein, the terms "individual" and "subject" are often used interchangeably and refer to any animal exhibiting 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 preferred embodiments, a subject includes any animal exhibiting 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.

[0232] 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.

[0233] 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.

[0234] As used herein, "prevent" and similar terms, such as "prevented" and "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 onset or recurrence of a disease or condition, or delaying the onset 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 before the onset or recurrence of the disease or condition.

[0235] As used herein, "amelioration of at least one symptom of" refers to a reduction in one or more symptoms of the disease or condition for which the subject is being treated. In certain embodiments, the disease or condition for which treatment 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, broken bones, unexpected weight loss, loss of appetite, night sweats, persistent low-grade fever, and decreased urination (caused by impaired kidney function).

[0236] "Enhancing," or "promoting," or "increasing," or "expanding" generally refers to a composition contemplated herein, e.g., a genetically modified T cell expressing a TCR comprising a minimally murine TCR alpha chain and hydrophobic amino acid substitutions in the TCR alpha transmembrane domain and a minimally murine TCR beta chain, producing, eliciting, or causing a greater physiological response (i.e., downstream effect) compared to the 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 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.) over the response produced by the vehicle or control composition.

[0237] "Decrease," or "lower," or "reduce," or "diminish," or "attenuate" generally refers to the ability of a composition contemplated herein to produce, elicit, or generate a physiological response (i.e., a downstream effect) that is smaller than the response induced by a vehicle or control molecule / composition. The amount of "reduction" or "decreased" is typically a "statistically significant" amount and can include a decrease 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 decimal points in between and above 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) of the response induced by a vehicle, a control composition, or a response in a particular cell line.

[0238] "Maintain," or "preserve," or "maintain," or "no change," or "no substantial change," or "no substantial decrease" generally refer to a composition contemplated herein producing, eliciting, or producing a similar or equivalent physiological response (i.e., downstream effect) in a cell compared to the response produced by a vehicle, a control molecule / composition, or the response in a particular cell line. An equivalent response is one that is not substantially different, or not measurably different, from the reference response.

[0239] 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 and the type and severity of the patient's disease, although appropriate dosages may be determined through clinical trials.

[0240] In one embodiment, the amount of immune effector cells, e.g., T cells, expressing a TCR comprising a minimally murine TCR α chain and hydrophobic amino acid substitutions in the TCR α transmembrane domain, and a minimally murine TCR β chain, in the composition administered to a subject is at least 1×10 7 cells, at least 0.5x10 8 Cells, at least 1x10 8 cells, at least 0.5x10 9 Cells, at least 1x10 9 Cells, at least 1x10 10 Cells, at least 1x10 11 Cells, at least 1x10 12 Cells, at least 5x10 12 cells, or at least 1x10 13 It is a cell.

[0241] 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 1x109 T cells ~ approx. 1x10 13 T cells, approximately 1x10 10 T cells ~ approx. 1x10 13 T cells, approximately 1x10 11 T cells ~ approx. 1x10 13 T cells, or approximately 1 x 10 12 T cells ~ approx. 1x10 13 The T cells are administered to the subject.

[0242] In one embodiment, the amount of immune effector cells, e.g., T cells, expressing a TCR comprising a minimally murine TCR α chain and hydrophobic amino acid substitutions in the TCR α transmembrane domain, and a minimally murine TCR β chain, in the composition administered to a subject is at least 0.1 x 10 4 At least 0.5x10 cells / kg body weight 4 At least 1x10 cells / kg body weight 4 Cells / kg body weight, at least 5x10 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.

[0243] In certain embodiments, about 1 x 10 6 T cells / kg body weight ~ approx. 1x10 8 T cells / kg body weight, approximately 2x10 6 T cells / kg body weight ~ approx. 0.9x10 8 T cells / kg body weight, approx. 3x106 T cells / kg body weight ~ approx. 0.8x10 8 T cells / kg body weight, approx. 4x10 6 T cells / kg body weight ~ approx. 0.7x10 8 T cells / kg body weight, approx. 5x10 6 T cells / kg body weight ~ approx. 0.6x10 8 T cells / kg body weight, or approximately 5x10 6 T cells / kg body weight ~ approx. 0.5x10 8 T cells / kg body weight are administered to the subject.

[0244] Those skilled 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.

[0245] In some embodiments, it may be desirable to administer activated immune effector cells to a subject, then withdraw blood from the subject (or perform apheresis), activate the immune effector cells 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 some embodiments, immune effector cells may be activated from a blood withdrawal of 10 cc to 400 cc. In some embodiments, immune effector cells are activated from a blood withdrawal of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, 100 cc, 150 cc, 200 cc, 250 cc, 300 cc, 350 cc, or 400 cc or more. Without being bound by theory, the use of multiple blood withdrawal / multiple reinfusion protocols may aid in the selection of certain populations of immune effector cells.

[0246] Administration of the compositions contemplated herein can be carried out in any convenient manner, including aerosol inhalation, injection, ingestion, transfusion, transplantation, or implantation. In a preferred embodiment, the compositions are administered parenterally. As used herein, the phrases "parenteral administration" and "parenterally administered" 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.

[0247] In one embodiment, an effective amount of a composition that increases a cellular immune response to a B cell-related condition in a subject is administered to a subject in need thereof. The immune response may include a cellular immune response mediated by cytotoxic T cells, regulatory T cells, and helper T cell responses, which can kill infected cells. A humoral immune response, primarily mediated by helper T cells that can activate B cells and thus lead to antibody production, may also be elicited. Various techniques may be used to analyze the type of immune response induced by the composition, and these techniques are fully 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.

[0248] In one embodiment, a method of treating a subject diagnosed with cancer is provided, comprising removing immune effector cells from the subject, genetically modifying the immune effector cells with a vector comprising a nucleic acid encoding a TCR comprising a minimally murine TCR alpha chain and a hydrophobic amino acid substitution in the TCR alpha transmembrane domain, a polypeptide linker, and a minimally murine TCR beta chain as contemplated herein, thereby generating 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.

[0249] In certain embodiments, a method of stimulating an immune effector cell-mediated immunomodulatory response against a target cell population in a subject is provided, the method comprising administering to the subject a population of immune effector cells expressing a nucleic acid construct encoding a TCR comprising a minimally murine TCR alpha chain and a hydrophobic amino acid substitution in the TCR alpha transmembrane domain, a polypeptide linker, and a minimally murine TCR beta chain.

[0250] In certain embodiments, contemplated methods of administering cell compositions include any method effective to result in the reintroduction of genetically modified immune effector cells ex vivo, wherein the genetically modified immune effector cells either directly express a TCR comprising a minimally murine TCR α chain and a hydrophobic amino acid substitution in the TCR α transmembrane domain, a polypeptide linker, and a minimally murine TCR β chain as contemplated herein, in the subject, or upon reintroduction of genetically modified progenitor cells of the immune effector cells, which differentiate into mature immune effector cells expressing the TCR upon introduction into the subject. One method includes transducing peripheral blood T cells ex vivo with a nucleic acid construct as contemplated herein and returning the transduced cells to the subject.

[0251] 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.

[0252] Although the foregoing embodiments have been described in detail in the figures and examples for purposes of clarity and understanding, it will be readily apparent to those skilled in the art in light of the teachings contemplated herein that certain changes and modifications can 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 a variety of non-critical parameters that can be changed or modified to yield essentially similar results. [Example]

[0253] Example 1 Amino acid substitutions in the T cell receptor (TCR) constant region synergistically increase TCR expression The MAGEA4 TCR sequence was cloned and engineered into a lentiviral vector using standard cloning techniques. WT The construct is the unmodified parent construct. TM The construct contains three hydrophobic amino acid substitutions in the TCR alpha chain transmembrane domain (S115L, G118V, F119L; numbered with reference to the TCR alpha constant region). MM The construct contains four murine amino acid substitutions in the TCR α chain constant region (P90S, E91D, S92V, S93P; numbered with reference to the TCR α chain constant region) and five murine amino acid substitutions in the TCR β chain constant region (E18K, S22A, F133I, E / V136A, Q139H; numbered with reference to the TCR β constant region). MM The construct contains nine murine amino acid substitutions in the constant regions of the TCR α and TCR β chains, as well as three hydrophobic amino acid substitutions in the TCR α chain transmembrane domain.

[0254] Peripheral blood mononuclear cells (PBMCs) from two normal donors were activated with CD3 and CD28 antibodies and expressed TCR. WT , TCR TM , TCR MM , or TCR TM / MM After 10 days, UTD T cells or TCR WT , TCR TM , TCR MM Or TCR TM / MM T cells transduced with lentiviral vectors encoding MAGEA4 were stained with a 1:20 dilution of MAGEA4 pentamer-peptide labeling reagent in flow staining buffer and analyzed by flow cytometry for PE fluorescence. WT Compared with T cells transduced with a lentiviral vector encoding a TCR TM and TCR MM TCR expression was increased two-fold in T cells transduced with the lentiviral vector. TM / MM T cells transduced with a lentiviral vector encoding IL-17 showed a synergistic 4-fold increase (Figure 1). T cells transduced with lentiviral vectors showed comparable vector copy numbers (VCN).

[0255] Example 2 T cells transduced with the modified TCR showed increased expression compared with T cells transduced with the unmodified TCR.

[0256] PBMCs from two normal donors were activated with CD3 and CD28 antibodies and TCR WT or TCR TM / MM T cells were transduced with a lentiviral vector encoding Tdxn1 using two different transduction conditions (Tdxn1 - enhanced tdxn process, Tdxn2 - basal tdxn process) and cultured for 10 days. Untransduced (UTD) T cells served as a control.

[0257] After 10 days, cells were stained with a 1:20 dilution of MAGEA4 pentamer-peptide labeling reagent in flow staining buffer and analyzed by flow cytometry for PE fluorescence. WT Compared with T cells transduced with a lentiviral vector encoding a TCR TM / MM A four-fold increase in T cells transduced with a lentiviral vector encoding IL-1 was observed under both transduction conditions (Figure 2A).

[0258] RT-PCR was used to measure vector copy number (VCN) and assess LVV integration under each transduction condition. VCN was comparable across transduction conditions (Figure 2B).

[0259] Example 3 T cells transduced with modified TCRs abolish TCR mispairing. PBMCs were activated with CD3 and CD28 antibodies and TCR WT or TCR TM / MM and cultured for 10 days.

[0260] After 10 days, TCR WT and TCR TM / MM Transduced T cells were assessed for specific TCR pairing using double staining with a 1:20 dilution of pentamer-peptide-conjugated PE reagent and a 1:100 dilution of v-beta chain FITC-conjugated fluorescent dye in flow staining buffer. Equal percentages of positive cells detected by v-beta staining and tetramer antigen staining suggest specific TCR pairing.

[0261] TCR TM / MM T cells transduced with TCR WT These data suggest that TCR transduction is a promising strategy for T cells with a specific TCR-specific pairing. TM / MM The alterations present in Figure 3A and Figure 3B indicate that TCR mispairing is abolished.

[0262] Example 4 T cells transduced with engineered TCRs have potent antitumor capabilities PBMCs from two normal donors were activated with CD3 and CD28 antibodies and TCR WT or TCR TM / MM and cultured for 10 days.

[0263] IFNγ-assay:UTD T cells, or TCR WT Or TCR TM / MM T cells transduced with lentiviral vectors encoding TCR were co-cultured with MAGEA4-positive A549 tumor Nuc-red cells at a 1:1 E:T ratio for 24 hours, and normalization was performed based on TCR expression. After 24 hours, supernatants were collected from these samples and analyzed using the Meso Scale Discovery (MSD) assay to measure cytokine production. TM / MM -T cells expressing TCR WT -expressing T cells showed a significant (4-fold) increase in IFNγ production compared to T cells expressing T cells (Figure 4A).

[0264] Cytotoxicity Assay: UTD T Cell, or TCR WT Or TCR TM / MM T cells transduced with lentiviral vectors encoding α-TCR were co-cultured with MAGEA4-positive A549 tumor Nuc-red cells at a 1:1 E:T ratio and normalized based on TCR expression. Cytotoxicity was monitored over a 3-day period using an Incucyte S3. TM / MM -T cells expressing TCR WT The killing curve was steeper than that of IgG-expressing or UTD T cells (Figure 4B).

[0265] Example 5 Amino acid substitutions in the T cell receptor (TCR) constant region synergistically increase TCR expression and specific TCR pairing in the NY-ESO-1 TCR The sequence of the NY-ESO-1 TCR (SEQ ID NOs: 15 and 16) was cloned and modified into a lentiviral vector using standard cloning techniques. MM The construct contains four murine amino acid substitutions in the TCR α chain constant region (P90S, E91D, S92V, S93P; numbered with reference to the TCR α chain constant region) and five murine amino acid substitutions in the TCR β chain constant region (E18K, S22A, F133I, E / V136A, Q139H; numbered with reference to the TCR β constant region). MM The construct contains nine murine amino acid substitutions in the constant regions of the TCR α and TCR β chains, as well as three hydrophobic amino acid substitutions in the TCR α chain transmembrane domain (S115L, G118V, F119L; numbered with reference to the TCR α constant region).

[0266] Peripheral blood mononuclear cells (PBMCs) from two normal donors were activated with CD3 and CD28 antibodies and expressed TCR. WT , TCR MM , or TCR TM / MM and cultured for 10 days.

[0267] Onset: 10 days later, UTD T cells, or TCR WT , TCR MM , or TCR TM / MM Cells transduced with lentiviral vectors encoding NY-ESO-1 pentamer-peptide labeling reagent at a 1:20 dilution in flow staining buffer were stained and analyzed by flow cytometry for PE fluorescence. Flow analysis revealed that NY-ESO-1 pentamer-peptide labeling reagents were ubiquitously expressed in UTD T cells, or TCR T cells. WT Or TCR MM Compared with T cells transduced with a lentiviral vector encoding a TCR TM / MM T cells transduced with lentiviral vectors encoding α-glucan-1 (α-glucan-1) showed increased TCR expression (Figure 5A). The mean fluorescence intensity of TCR staining for each donor is shown in Figure 5B.

[0268] Pairing: 10 days later, UTD T cells, or TCR WT , TCR MM , or TCR TM / MM Cells transduced with lentiviral vectors encoding NY-ESO-1 pentamer-peptide-conjugated PE reagent at a 1:20 dilution and v-beta chain FITC-conjugated fluorescent dye at a 1:100 dilution in flow staining buffer were assessed for specific TCR pairing. Equal percentages of positive cells detected by v-beta staining and tetramer antigen staining suggest specific TCR pairing. WT Or TCR MM Compared with T cells transduced with a lentiviral vector encoding a TCR TM / MM Transduced T cells showed an increase in specific pairing.

[0269] Example 6 Amino acid substitutions in the T cell receptor (TCR) constant region in the MART-1 TCR The sequences of the MART-1 TCR α and β chains (SEQ ID NOs: 7 and 8; SEQ ID NOs: 9 and 10) containing pairing-enhancing mutations were generated (TCR TM / MM ), which is cloned into a lentiviral vector using standard cloning techniques. TM / MM contains four murine amino acid substitutions (P90S, E91D, S92V, S93P; numbered with reference to the TCR α chain constant region) and three hydrophobic amino acid substitutions (S115L, G118V, F119L; numbered with reference to the TCR α constant region) in the constant region of the TCR α chain, and five murine amino acid substitutions (E18K, S22A, F133I, E / V136A, Q139H; numbered with reference to the TCR β constant region) in the constant region of the TCR β chain.

[0270] Peripheral blood mononuclear cells (PBMCs) from two normal donors were activated with CD3 and CD28 antibodies and expressed TCR. WT , or TCR TM / MMThe cells are transduced with a lentiviral vector encoding the .gamma.- VIII gene and cultured for 10 days.

[0271] Onset: 10 days later, UTD T cells, or TCR WT Or TCR TM / MM Cells transduced with lentiviral vectors encoding were stained with a 1:20 dilution of MART-1 pentamer-peptide labeling reagent in flow staining buffer and analyzed by flow cytometry for PE fluorescence.

[0272] Pairing: 10 days later, UTD T cells, or TCR WT Or TCR TM / MM Cells transduced with lentiviral vectors encoding MART-1 are assessed for specific TCR pairing using dual staining with a 1:20 dilution of MART-1 pentamer-peptide-labeled PE reagent and a 1:100 dilution of v-beta chain FITC-labeled fluorescent dye in flow staining buffer.

[0273] Example 7 Amino acid substitutions in the T cell receptor (TCR) constant region of WT-1 TCR The sequences of the WT-1 TCR α and β chains (SEQ ID NOs: 11 and 12) containing pairing-enhancing mutations were generated (TCR TM / MM ), which is cloned into a lentiviral vector using standard cloning techniques. TM / MM contains four murine amino acid substitutions (P90S, E91D, S92V, S93P; numbered with reference to the TCR α chain constant region) and three hydrophobic amino acid substitutions (S115L, G118V, F119L; numbered with reference to the TCR α constant region) in the constant region of the TCR α chain, and five murine amino acid substitutions (E18K, S22A, F133I, E / V136A, Q139H; numbered with reference to the TCR β constant region) in the constant region of the TCR β chain.

[0274] Peripheral blood mononuclear cells (PBMCs) from two normal donors were activated with CD3 and CD28 antibodies and expressed TCR. WT, or TCR TM / MM The cells are transduced with a lentiviral vector encoding the .gamma.- VIII gene and cultured for 10 days.

[0275] Onset: 10 days later, UTD T cells, or TCR WT Or TCR TM / MM Cells transduced with lentiviral vectors encoding were stained with a 1:20 dilution of WT-1 pentamer-peptide labeling reagent in flow staining buffer and analyzed by flow cytometry for PE fluorescence.

[0276] Pairing: 10 days later, UTD T cells, or TCR WT Or TCR TM / MM Cells transduced with lentiviral vectors encoding WT-1 are assessed for specific TCR pairing using dual staining with a 1:20 dilution of WT-1 pentamer-peptide-labeled PE reagent and a 1:100 dilution of v-beta chain FITC-labeled fluorescent dye in flow staining buffer.

[0277] Example 8 Amino acid substitutions in the T cell receptor (TCR) constant region in the HPV16 E6 TCR The HPV16 E6 TCR α and β chain sequences (SEQ ID NOs: 13 and 14) containing pairing-enhancing mutations were generated (TCR TM / MM ), which is cloned into a lentiviral vector using standard cloning techniques. TM / MM contains four murine amino acid substitutions (P90S, E91D, S92V, S93P; numbered with reference to the TCR α chain constant region) and three hydrophobic amino acid substitutions (S115L, G118V, F119L; numbered with reference to the TCR α constant region) in the constant region of the TCR α chain, and five murine amino acid substitutions (E18K, S22A, F133I, E / V136A, Q139H; numbered with reference to the TCR β constant region) in the constant region of the TCR β chain.

[0278] Peripheral blood mononuclear cells (PBMCs) from two normal donors were activated with CD3 and CD28 antibodies and expressed TCR. WT , or TCR TM / MM The cells are transduced with a lentiviral vector encoding the .gamma.- VIII gene and cultured for 10 days.

[0279] Onset: 10 days later, UTD T cells, or TCR WT Or TCR TM / MM Cells transduced with lentiviral vectors encoding were stained with a 1:20 dilution of HPV16 E6 pentamer-peptide labeling reagent in flow staining buffer and analyzed by flow cytometry for PE fluorescence.

[0280] Pairing: 10 days later, UTD T cells, or TCR WT Or TCR TM / MM Cells transduced with lentiviral vectors encoding HPV16 E6 pentamer-peptide-labeled PE reagent at a 1:20 dilution and v-beta chain FITC-labeled fluorescent dye at a 1:100 dilution in flow staining buffer are assessed for specific TCR pairing.

[0281] Example 9 Amino acid substitutions in the T cell receptor (TCR) constant region in the NY-ESO-1 TCR The NY-ESO-1 TCR α and β chain sequences (SEQ ID NOs: 17 and 18; SEQ ID NOs: 19 and 20; SEQ ID NOs: 21 and 22) containing pairing-enhancing mutations were generated (TCR TM / MM ), which is cloned into a lentiviral vector using standard cloning techniques. TM / MMcontains four murine amino acid substitutions (P90S, E91D, S92V, S93P; numbered with reference to the TCR α chain constant region) and three hydrophobic amino acid substitutions (S115L, G118V, F119L; numbered with reference to the TCR α constant region) in the constant region of the TCR α chain, and five murine amino acid substitutions (E18K, S22A, F133I, E / V136A, Q139H; numbered with reference to the TCR β constant region) in the constant region of the TCR β chain.

[0282] Peripheral blood mononuclear cells (PBMCs) from two normal donors were activated with CD3 and CD28 antibodies and expressed TCR. WT , or TCR TM / MM The cells are transduced with a lentiviral vector encoding the .gamma.- VIII gene and cultured for 10 days.

[0283] Onset: 10 days later, UTD T cells, or TCR WT Or TCR TM / MM Cells transduced with lentiviral vectors encoding NY-ESO-1 were stained with a 1:20 dilution of NY-ESO-1 pentamer-peptide labeling reagent in flow staining buffer and analyzed by flow cytometry for PE fluorescence.

[0284] Pairing: 10 days later, UTD T cells, or TCR WT Or TCR TM / MM Cells transduced with lentiviral vectors encoding NY-ESO-1 are assessed for specific TCR pairing using dual staining with a 1:20 dilution of NY-ESO-1 pentamer-peptide-labeled PE reagent and a 1:100 dilution of v-beta chain FITC-labeled fluorescent dye in flow staining buffer.

[0285] Example 10 Amino acid substitutions in the T cell receptor (TCR) constant region in the HPV16 E7 TCR The sequences of the HPV16 E7 TCR α and β chains (SEQ ID NOs: 23 and 24) containing pairing-enhancing mutations were generated (TCR TM / MM), which is cloned into a lentiviral vector using standard cloning techniques. TM / MM contains four murine amino acid substitutions (P90S, E91D, S92V, S93P; numbered with reference to the TCR α chain constant region) and three hydrophobic amino acid substitutions (S115L, G118V, F119L; numbered with reference to the TCR α constant region) in the constant region of the TCR α chain, and five murine amino acid substitutions (E18K, S22A, F133I, E / V136A, Q139H; numbered with reference to the TCR β constant region) in the constant region of the TCR β chain.

[0286] Peripheral blood mononuclear cells (PBMCs) from two normal donors were activated with CD3 and CD28 antibodies and expressed TCR. WT , or TCR TM / MM The cells are transduced with a lentiviral vector encoding the .gamma.- VIII gene and cultured for 10 days.

[0287] Onset: 10 days later, UTD T cells, or TCR WT Or TCR TM / MM Cells transduced with lentiviral vectors encoding were stained with a 1:20 dilution of HPV16 E7 pentamer-peptide labeling reagent in flow staining buffer and analyzed by flow cytometry for PE fluorescence.

[0288] Pairing: 10 days later, UTD T cells, or TCR WT Or TCR TM / MM Cells transduced with lentiviral vectors encoding HPV16 E7 are assessed for specific TCR pairing using dual staining with a 1:20 dilution of HPV16 E7 pentamer-peptide-labeled PE reagent and a 1:100 dilution of v-beta chain FITC-labeled fluorescent dye in flow staining buffer.

[0289] Example 11 Amino acid substitutions in the T cell receptor (TCR) constant region in the GP100 TCR The GP100 TCR α and β chain sequences (SEQ ID NOs: 25 and 26) containing pairing-enhancing mutations were generated (TCR TM / MM ), which is cloned into a lentiviral vector using standard cloning techniques. TM / MM contains four murine amino acid substitutions (P90S, E91D, S92V, S93P; numbered with reference to the TCR α chain constant region) and three hydrophobic amino acid substitutions (S115L, G118V, F119L; numbered with reference to the TCR α constant region) in the constant region of the TCR α chain, and five murine amino acid substitutions (E18K, S22A, F133I, E / V136A, Q139H; numbered with reference to the TCR β constant region) in the constant region of the TCR β chain.

[0290] Peripheral blood mononuclear cells (PBMCs) from two normal donors were activated with CD3 and CD28 antibodies and expressed TCR. WT , or TCR TM / MM The cells are transduced with a lentiviral vector encoding the .gamma.- VIII gene and cultured for 10 days.

[0291] Onset: 10 days later, UTD T cells, or TCR WT Or TCR TM / MM Cells transduced with lentiviral vectors encoding were stained with GP100 pentamer-peptide labeling reagent at a 1:20 dilution in flow staining buffer and analyzed by flow cytometry for PE fluorescence.

[0292] Pairing: 10 days later, UTD T cells, or TCR WT Or TCR TM / MM Cells transduced with lentiviral vectors encoding are assessed for specific TCR pairing using dual staining with a 1:20 dilution of GP100 pentamer-peptide-labeled PE reagent and a 1:100 dilution of v-beta chain FITC-labeled fluorescent dye in flow staining buffer.

[0293] 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 the present disclosure.

Claims

1. (a) a TCR alpha chain comprising a constant domain comprising the amino acid sequence set forth in SEQ ID NO: 4; and (b) a TCR β chain comprising a constant domain comprising the amino acid sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 6 An engineered T cell receptor (TCR), comprising:

2. The engineered TCR of claim 1, wherein the TCR does not bind to MAGE-A4.

3. The TCR may bind to α-fetoprotein (AFP), B melanoma antigen (BAGE) family members, brother of the regulator of imprinted sites (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), melanoma), Epstein-Barr virus (EBV) antigen, 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 epidermal growth factor receptor 2 (HER-2), human papillomavirus (HPV)-E6, HPV-E7, human telomerase reverse transcriptase (hTERT), latent membrane protein 2 (LMP2) 2), Melanoma antigen family A,1 (MAGE-A1), MAGE-A2, MAGE-A3, 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:P antigen) family, placenta-specific 1 (PLAC1), preferentially expressed antigen in melanoma (PRAME), survivin, synovial sarcoma X 1 (SSX1), synovial sarcoma X 2 (SSX2), synovial sarcoma X 3 (SSX3), synovial sarcoma X 4 (SSX4), synovial sarcoma X 5 (SSX5), synovial sarcoma X 8 (SSX8), thyroglobulin, tyrosinase, tyrosinase-related protein (TRP) 1, TRP2, Wilms tumor protein (WT-1), X Antigen The engineered TCR of claim 1 or claim 2, which binds to a target antigen selected from the group consisting of X Antigen Family Member 1 (XAGE1), and X Antigen Family Member 2 (XAGE2).

4. (a) the expression and avidity of the TCR is increased compared to a TCR comprising a minimally murine TCR α chain and a minimally murine TCR β chain, but the transmembrane domain of the TCR α chain does not contain hydrophobic amino acid substitutions; and / or (b) the expression and avidity of the TCR is increased compared to a TCR that does not contain a minimally murine TCR alpha chain and a minimally murine TCR beta chain, but in which the transmembrane domain of the TCR alpha chain contains hydrophobic amino acid substitutions; The engineered TCR of any one of claims 1 to 3.

5. A fusion protein comprising the TCRα chain and the TCRβ chain according to any one of claims 1 to 4.

6. A fusion protein comprising a TCR alpha chain according to any one of claims 1 to 4, a polypeptide cleavage signal, and a TCR beta chain according to any one of claims 1 to 4.

7. The fusion protein of claim 6, wherein the polypeptide cleavage signal is a viral self-cleaving peptide, a viral 2A peptide, or a ribosomal skipping sequence.

8. The fusion protein described in claim 7, wherein the polypeptide cleavage signal is an aphthovirus 2A peptide, a potyvirus 2A peptide, or a cardiovirus 2A peptide.

9. One or more nucleic acids encoding the TCR of any one of claims 1 to 4, or the fusion protein of any one of claims 5 to 8.

10. A vector comprising a nucleic acid encoding the TCR of any one of claims 1 to 4 or the fusion protein of any one of claims 5 to 8.

11. The vector of claim 10 , wherein the vector is an expression vector, a retroviral vector, or a lentiviral vector.

12. A cell expressing the TCR of any one of claims 1 to 4 or the fusion protein of any one of claims 5 to 8.

13. A cell comprising the nucleic acid described in claim 9, or the vector described in claim 10 or claim 11.

14. The cell of claim 12 or claim 13, wherein the cell is an immune effector cell.

15. The cell of claim 14, wherein the cell is an immune effector cell selected from the group consisting of a T cell, a natural killer (NK) cell, and a natural killer T (NKT) cell.

16. A composition comprising the TCR of any one of claims 1 to 4, the fusion protein of any one of claims 5 to 8, the nucleic acid of claim 9, the vector of claim 10 or claim 11, or the cell of any one of claims 12 to 15.

17. The composition of claim 16, wherein the composition further comprises a pharmaceutically acceptable carrier.

18. 18. A TCR according to any one of claims 1 to 4, a fusion protein according to any one of claims 5 to 8, a nucleic acid according to claim 9, a vector according to claim 10 or claim 11, a cell according to any one of claims 12 to 15, or a composition according to claim 16 or claim 17, for use in the treatment of cancer.

19. 19. The TCR, fusion protein, nucleic acid, vector, cell, or composition of claim 18, wherein the cancer is a blood cancer or a solid tumor.

20. 20. The TCR, fusion protein, nucleic acid, vector, cell, or composition of claim 18 or 19, wherein the cancer is selected from the group consisting of sarcoma, prostate cancer, uterine cancer, thyroid cancer, testicular cancer, renal cancer, pancreatic cancer, ovarian cancer, esophageal cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), non-Hodgkin's lymphoma, multiple myeloma, melanoma, hepatocellular carcinoma, head and neck cancer, gastric cancer, endometrial cancer, colorectal cancer, bile duct cancer, breast cancer, bladder cancer, myeloid leukemia, and acute lymphoblastic leukemia.

21. 21. The TCR, fusion protein, nucleic acid, vector, cell, or composition of claim 20, wherein the cancer is selected from the group consisting of NSCLC, SCLC, breast cancer, ovarian cancer, colon cancer, sarcoma, and osteosarcoma.

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