Engineered T cells

Abstract

The present disclosure provides improved compositions for adoptive T cell therapy for treating, preventing, or ameliorating at least one symptom of cancer, infectious diseases, autoimmune diseases, inflammatory diseases, and immunodeficiency, or conditions associated therewith.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 62 / 845,311, filed May 8, 2019, which is incorporated herein by reference in its entirety.

[0003] Statement regarding sequence listing

[0004] The sequence listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the text file containing the sequence listing is BLBD_122_01WO_ST25. The text file is 37KB, created on May 6, 2020, and submitted electronically via EFS-Web at the same time as this specification is submitted. Background Art Technical Field

[0006] The present disclosure relates to improved adoptive cell therapy. More specifically, the present disclosure relates to improved signaling molecules, cells, and methods of using the same. Summary of the Invention

[0007] The present disclosure generally relates in part to improved adoptive immunotherapy, and in certain embodiments, immune effector cells comprise: a polynucleotide encoding an αβ TCR that binds MAGEA4, preferably a human paired enhancer αβ TCR that binds MAGEA4, preferably the MAGEA4 peptide GVYDGREHTV (SEQ ID NO: 1) presented by an HLA-A*02:01 encoded molecule; and a chimeric TGFβ receptor (CTBR), compositions, and methods of use thereof.

[0008] In various embodiments, the cell comprises a first polynucleotide encoding an engineered αβTCR that binds MAGEA4 (MAGEA4 TCR); and a second polynucleotide encoding a fusion polypeptide comprising: a first polypeptide comprising the extracellular TGFβ1 binding domain, a transmembrane domain, and an immunoreceptor intracellular signaling domain of TGFβR2; a polypeptide cleavage signal; and a second polypeptide comprising the extracellular TGFβ1 binding domain, a transmembrane domain, and an immunoreceptor intracellular signaling domain of TGFβR1.

[0009] In various embodiments, the cell comprises a first polynucleotide encoding a human paired-enhanced αβTCR (MAGEA4 eTCR) that binds MAGEA4; and a second polynucleotide encoding a fusion polypeptide comprising: a first polypeptide comprising the extracellular TGFβ1 binding domain, a transmembrane domain, and an immunoreceptor intracellular signaling domain of TGFβR2; a polypeptide cleavage signal; and a second polypeptide comprising the extracellular TGFβ1 binding domain, a transmembrane domain, and an immunoreceptor intracellular signaling domain of TGFβR1.

[0010] In certain embodiments, the immune receptor intracellular signaling domain of the first polypeptide is isolated from a cytokine receptor, an interleukin receptor, a pattern recognition receptor, or a toll-like receptor.

[0011] In certain embodiments, the immune receptor intracellular signaling domain of the second polypeptide is isolated from a cytokine receptor, an interleukin receptor, a pattern recognition receptor, or a toll-like receptor.

[0012] In certain embodiments, the immune receptor intracellular signaling domain of the first polypeptide is an IL-12Rβ2 intracellular signaling domain, and the immune receptor intracellular signaling domain of the second polypeptide is an IL-12Rβ1 intracellular signaling domain.

[0013] In various embodiments, the transmembrane domain of the first polypeptide comprises an IL-12Rβ2 transmembrane domain.

[0014] In further embodiments, the transmembrane domain of the second polypeptide comprises the IL-12Rβ1 transmembrane domain.

[0015] In some embodiments, the immune receptor intracellular signaling domain of the first polypeptide is an IL-12Rβ1 intracellular signaling domain, and the immune receptor intracellular signaling domain of the second polypeptide is an IL-12Rβ2 intracellular signaling domain.

[0016] In various embodiments, the transmembrane domain of the first polypeptide comprises an IL-12Rβ1 transmembrane domain.

[0017] In certain embodiments, the transmembrane domain of the second polypeptide comprises an IL-12Rβ2 transmembrane domain.

[0018] In a specific embodiment, the immune receptor intracellular signaling domain of the first polypeptide is an IL-7Rα intracellular signaling domain, and the immune receptor intracellular signaling domain of the second polypeptide is an IL-2Rγ intracellular signaling domain.

[0019] In further embodiments, the transmembrane domain of the first polypeptide comprises an IL-7Rα transmembrane domain.

[0020] In further embodiments, the transmembrane domain of the second polypeptide comprises an IL-2Rγ transmembrane domain.

[0021] In various embodiments, the immune receptor intracellular signaling domain of the first polypeptide is an IL-2Rγ intracellular signaling domain, and the immune receptor intracellular signaling domain of the second polypeptide is an IL-7Rα intracellular signaling domain.

[0022] In some embodiments, the transmembrane domain of the first polypeptide comprises an IL-2Rγ transmembrane domain.

[0023] In certain embodiments, the transmembrane domain of the second polypeptide comprises an IL-7Rα transmembrane domain.

[0024] In certain embodiments, the immune receptor intracellular signaling domain of the first polypeptide is an IL-2Rβ intracellular signaling domain, and the immune receptor intracellular signaling domain of the second polypeptide is an IL-2Rγ intracellular signaling domain.

[0025] In various embodiments, the transmembrane domain of the first polypeptide comprises an IL-2Rβ transmembrane domain.

[0026] In further embodiments, the transmembrane domain of the second polypeptide comprises an IL-2Rγ transmembrane domain.

[0027] In certain embodiments, the immune receptor intracellular signaling domain of the first polypeptide is an IL-2Rγ intracellular signaling domain, and the immune receptor intracellular signaling domain of the second polypeptide is an IL-2Rβ intracellular signaling domain.

[0028] In certain embodiments, the transmembrane domain of the first polypeptide comprises an IL-2Rγ transmembrane domain.

[0029] In some embodiments, the transmembrane domain of the second polypeptide comprises an IL-2Rβ transmembrane domain.

[0030] In additional embodiments, the immune receptor intracellular signaling domain of the first polypeptide is an IL-21R intracellular signaling domain, and the immune receptor intracellular signaling domain of the second polypeptide is an IL-2Rγ intracellular signaling domain.

[0031] In various embodiments, the transmembrane domain of the first polypeptide comprises an IL-21R transmembrane domain.

[0032] In certain embodiments, the transmembrane domain of the second polypeptide comprises an IL-2Rγ transmembrane domain.

[0033] In certain embodiments, the immune receptor intracellular signaling domain of the first polypeptide is an IL-2Rγ intracellular signaling domain, and the immune receptor intracellular signaling domain of the second polypeptide is an IL-21R intracellular signaling domain.

[0034] In further embodiments, the transmembrane domain of the first polypeptide comprises an IL-2Rγ transmembrane domain.

[0035] In some embodiments, the transmembrane domain of the second polypeptide comprises an IL-21R transmembrane domain.

[0036] In additional embodiments, the immune receptor intracellular signaling domain of the first polypeptide is an IL-18R1 intracellular signaling domain, and the immune receptor intracellular signaling domain of the second polypeptide is an IL-18RAP intracellular signaling domain.

[0037] In various embodiments, the transmembrane domain of the first polypeptide comprises an IL-18R1 transmembrane domain.

[0038] In various embodiments, the transmembrane domain of the second polypeptide comprises an IL-18 RAP transmembrane domain.

[0039] In a specific embodiment, the immune receptor intracellular signaling domain of the first polypeptide is an IL-18RAP intracellular signaling domain, and the immune receptor intracellular signaling domain of the second polypeptide is an IL-18R1 intracellular signaling domain.

[0040] In certain embodiments, the transmembrane domain of the first polypeptide comprises an IL-18 RAP transmembrane domain.

[0041] In further embodiments, the transmembrane domain of the second polypeptide comprises an IL-18R1 transmembrane domain.

[0042] In certain embodiments, the immune receptor intracellular signaling domain of the first polypeptide is an IL-1R1 intracellular signaling domain, and the immune receptor intracellular signaling domain of the second polypeptide is an IL-1RAP intracellular signaling domain.

[0043] In various embodiments, the transmembrane domain of the first polypeptide comprises an IL-1R1 transmembrane domain.

[0044] In some embodiments, the transmembrane domain of the second polypeptide comprises an IL-1 RAP transmembrane domain.

[0045] In various embodiments, the immune receptor intracellular signaling domain of the first polypeptide is an IL-1 RAP intracellular signaling domain, and the immune receptor intracellular signaling domain of the second polypeptide is an IL-1 R1 intracellular signaling domain.

[0046] In some embodiments, the transmembrane domain of the first polypeptide comprises an IL-1 RAP transmembrane domain.

[0047] In certain embodiments, the transmembrane domain of the second polypeptide comprises the IL-1R1 transmembrane domain.

[0048] In certain embodiments, the immune receptor intracellular signaling domain of the first polypeptide is an IL-1 RAP intracellular signaling domain, and the immune receptor intracellular signaling domain of the second polypeptide is an IL-1 RL2 intracellular signaling domain.

[0049] In various embodiments, the transmembrane domain of the first polypeptide comprises an IL-1 RAP transmembrane domain.

[0050] In further embodiments, the transmembrane domain of the second polypeptide comprises an IL-1RL2 transmembrane domain.

[0051] In additional embodiments, the immune receptor intracellular signaling domain of the first polypeptide is an IL-1RL2 intracellular signaling domain, and the immune receptor intracellular signaling domain of the second polypeptide is an IL-1RAP intracellular signaling domain.

[0052] In various embodiments, the transmembrane domain of the first polypeptide comprises an IL-1RL2 transmembrane domain.

[0053] In certain embodiments, the transmembrane domain of the second polypeptide comprises the IL-1 RAP transmembrane domain.

[0054] In additional embodiments, the immune receptor intracellular signaling domain of the first polypeptide is an IFNAR1 intracellular signaling domain, and the immune receptor intracellular signaling domain of the second polypeptide is an IFNAR2 intracellular signaling domain.

[0055] In various embodiments, the transmembrane domain of the first polypeptide comprises an IFNAR1 transmembrane domain.

[0056] In certain embodiments, the transmembrane domain of the second polypeptide comprises an IFNAR2 transmembrane domain.

[0057] In certain embodiments, the immune receptor intracellular signaling domain of the first polypeptide is an IFNAR2 intracellular signaling domain, and the immune receptor intracellular signaling domain of the second polypeptide is an IFNAR1 intracellular signaling domain.

[0058] In some embodiments, the transmembrane domain of the first polypeptide comprises an IFNAR2 transmembrane domain.

[0059] In various embodiments, the transmembrane domain of the second polypeptide comprises an IFNAR1 transmembrane domain.

[0060] In additional embodiments, the immune receptor intracellular signaling domain of the first polypeptide is a TLR1 intracellular signaling domain, and the immune receptor intracellular signaling domain of the second polypeptide is a TLR1 intracellular signaling domain.

[0061] In certain embodiments, the transmembrane domain of the first polypeptide comprises a TLR1 transmembrane domain.

[0062] In certain embodiments, the transmembrane domain of the second polypeptide comprises a TLR1 transmembrane domain.

[0063] In some embodiments, the immune receptor intracellular signaling domain of the first polypeptide is a TLR2 intracellular signaling domain, and the immune receptor intracellular signaling domain of the second polypeptide is a TLR2 intracellular signaling domain.

[0064] In various embodiments, the transmembrane domain of the first polypeptide comprises a TLR2 transmembrane domain.

[0065] In further embodiments, the transmembrane domain of the second polypeptide comprises a TLR2 transmembrane domain.

[0066] In various embodiments, the immune receptor intracellular signaling domain of the first polypeptide is a TLR3 intracellular signaling domain, and the immune receptor intracellular signaling domain of the second polypeptide is a TLR3 intracellular signaling domain.

[0067] In certain embodiments, the transmembrane domain of the first polypeptide comprises a TLR3 transmembrane domain.

[0068] In certain embodiments, the transmembrane domain of the second polypeptide comprises a TLR3 transmembrane domain.

[0069] In additional embodiments, the immune receptor intracellular signaling domain of the first polypeptide is a TLR4 intracellular signaling domain, and the immune receptor intracellular signaling domain of the second polypeptide is a TLR4 intracellular signaling domain.

[0070] In various embodiments, the transmembrane domain of the first polypeptide comprises a TLR4 transmembrane domain.

[0071] In some embodiments, the transmembrane domain of the second polypeptide comprises a TLR4 transmembrane domain.

[0072] In various embodiments, the immune receptor intracellular signaling domain of the first polypeptide is a TLR5 intracellular signaling domain, and the immune receptor intracellular signaling domain of the second polypeptide is a TLR5 intracellular signaling domain.

[0073] In certain embodiments, the transmembrane domain of the first polypeptide comprises a TLR5 transmembrane domain.

[0074] In further embodiments, the transmembrane domain of the second polypeptide comprises a TLR5 transmembrane domain.

[0075] In various embodiments, the immune receptor intracellular signaling domain of the first polypeptide is a TLR6 intracellular signaling domain, and the immune receptor intracellular signaling domain of the second polypeptide is a TLR6 intracellular signaling domain.

[0076] In various embodiments, the transmembrane domain of the first polypeptide comprises a TLR6 transmembrane domain.

[0077] In certain embodiments, the transmembrane domain of the second polypeptide comprises a TLR6 transmembrane domain.

[0078] In specific embodiments, the immune receptor intracellular signaling domain of the first polypeptide is a TLR7 intracellular signaling domain, and the immune receptor intracellular signaling domain of the second polypeptide is a TLR7 intracellular signaling domain.

[0079] In various embodiments, the transmembrane domain of the first polypeptide comprises a TLR7 transmembrane domain.

[0080] In further embodiments, the transmembrane domain of the second polypeptide comprises a TLR7 transmembrane domain.

[0081] In some embodiments, the immune receptor intracellular signaling domain of the first polypeptide is a TLR8 intracellular signaling domain, and the immune receptor intracellular signaling domain of the second polypeptide is a TLR8 intracellular signaling domain.

[0082] In further embodiments, the transmembrane domain of the first polypeptide comprises a TLR8 transmembrane domain.

[0083] In various embodiments, the transmembrane domain of the second polypeptide comprises a TLR8 transmembrane domain.

[0084] In certain embodiments, the immune receptor intracellular signaling domain of the first polypeptide is a TLR9 intracellular signaling domain, and the immune receptor intracellular signaling domain of the second polypeptide is a TLR9 intracellular signaling domain.

[0085] In certain embodiments, the transmembrane domain of the first polypeptide comprises a TLR9 transmembrane domain.

[0086] In certain embodiments, the transmembrane domain of the second polypeptide comprises a TLR9 transmembrane domain.

[0087] In various embodiments, the immune receptor intracellular signaling domain of the first polypeptide is a TLR10 intracellular signaling domain, and the immune receptor intracellular signaling domain of the second polypeptide is a TLR10 intracellular signaling domain.

[0088] In some embodiments, the transmembrane domain of the first polypeptide comprises a TLR10 transmembrane domain.

[0089] In certain embodiments, the transmembrane domain of the second polypeptide comprises a TLR10 transmembrane domain.

[0090] In certain embodiments, the polypeptide cleavage signal is a viral self-cleaving polypeptide.

[0091] In various embodiments, the polypeptide cleavage signal is a viral autolytic 2A polypeptide.

[0092] In a specific embodiment, the polypeptide cleavage signal is a viral self-cleaving polypeptide selected from the group consisting of: foot-and-mouth disease virus (FMDV) (F2A) peptide, equine rhinitis virus A (ERAV) (E2A) peptide, cleaved beta-tetrasomal virus (TaV) (T2A) peptide, porcine teschovirus-1 (PTV-1) (P2A) peptide, Theiler virus 2A peptide and encephalomyocarditis virus 2A peptide.

[0093] In various embodiments, the fusion polypeptide comprises the amino acid sequence set forth in SEQ ID NO:5.

[0094] In various embodiments, the cell comprises a first polynucleotide encoding a MAGEA4 TCR; and a second polynucleotide encoding a fusion polypeptide comprising: a TGFβR2 polypeptide comprising an extracellular TGFβ1 binding domain of TGFβR2, an IL-12Rβ2 transmembrane domain; and an IL-12Rβ2 intracellular signaling domain; a viral self-cleaving 2A peptide; and a TGFβR1 polypeptide comprising an extracellular TGFβ1 binding domain of TGFβR1; an IL-12Rβ1 transmembrane domain, and an IL-12Rβ1 intracellular signaling domain.

[0095] In certain embodiments, the cell comprises a first polynucleotide encoding a MAGEA4 TCR; and a second polynucleotide encoding a fusion polypeptide comprising: a TGFβR2 polypeptide comprising an extracellular TGFβ1 binding domain of TGFβR2, an IL-12Rβ1 transmembrane domain; and an IL-12Rβ1 intracellular signaling domain; a viral self-cleaving 2A peptide; and a TGFβR1 polypeptide comprising an extracellular TGFβ1 binding domain of TGFβR1; an IL-12Rβ2 transmembrane domain; and an IL-12Rβ2 intracellular signaling domain.

[0096] In certain embodiments, the cell comprises a first polynucleotide encoding a MAGEA4 TCR; and a second polynucleotide encoding a fusion polypeptide comprising: a TGFβR2 polypeptide comprising an extracellular TGFβ1 binding domain of TGFβR2, an IL-7Rα transmembrane domain; and an IL-7Rα intracellular signaling domain; a viral self-cleaving 2A peptide; and a TGFβR1 polypeptide comprising an extracellular TGFβ1 binding domain of TGFβR1; an IL-2Rγ transmembrane domain and an IL-2Rγ intracellular signaling domain.

[0097] In some embodiments, the cell comprises a first polynucleotide encoding a MAGEA4 TCR; and a second polynucleotide encoding a fusion polypeptide comprising: a TGFβR2 polypeptide comprising an extracellular TGFβ1 binding domain of TGFβR2, an IL-2Rγ transmembrane domain; and an IL-2Rγ intracellular signaling domain; a viral self-cleaving 2A peptide; and a TGFβR1 polypeptide comprising an extracellular TGFβ1 binding domain of TGFβR1; an IL-7Rα transmembrane domain; and an IL-7Rα intracellular signaling domain.

[0098] In additional embodiments, the cell comprises a first polynucleotide encoding a MAGEA4 TCR; and a second polynucleotide encoding a fusion polypeptide comprising: a TGFβR2 polypeptide comprising the extracellular TGFβ1 binding domain of TGFβR2, an IL-2Rβ transmembrane domain; and an IL-2Rβ intracellular signaling domain; a viral self-cleaving 2A peptide; and a TGFβR1 polypeptide comprising the extracellular TGFβ1 binding domain of TGFβR1; an IL-2Rγ transmembrane domain and an IL-2Rγ intracellular signaling domain.

[0099] In additional embodiments, the cell comprises a first polynucleotide encoding a MAGEA4 TCR; and a second polynucleotide encoding a fusion polypeptide comprising: a TGFβR2 polypeptide comprising the extracellular TGFβ1 binding domain of TGFβR2, the IL-2Rγ transmembrane domain; and the IL-2Rγ intracellular signaling domain; a viral self-cleaving 2A peptide; and a TGFβR1 polypeptide comprising the extracellular TGFβ1 binding domain of TGFβR1; the IL-2Rβ transmembrane domain; and the IL-2Rβ intracellular signaling domain.

[0100] In various embodiments, the cell comprises a first polynucleotide encoding a MAGEA4 TCR; and a second polynucleotide encoding a fusion polypeptide comprising: a TGFβR2 polypeptide comprising the extracellular TGFβ1 binding domain of TGFβR2, an IL-21R transmembrane domain; and an IL-21R intracellular signaling domain; a viral self-cleaving 2A peptide; and a TGFβR1 polypeptide comprising the extracellular TGFβ1 binding domain of TGFβR1; an IL-2Rγ transmembrane domain and an IL-2Rγ intracellular signaling domain.

[0101] In certain embodiments, the cell comprises a first polynucleotide encoding a MAGEA4 TCR; and a second polynucleotide encoding a fusion polypeptide comprising: a TGFβR2 polypeptide comprising an extracellular TGFβ1 binding domain of TGFβR2, an IL-2Rγ transmembrane domain; and an IL-2Rγ intracellular signaling domain; a viral self-cleaving 2A peptide; and a TGFβR1 polypeptide comprising an extracellular TGFβ1 binding domain of TGFβR1; an IL-21R transmembrane domain; and an IL-21R intracellular signaling domain.

[0102] In a specific embodiment, the cell comprises a first polynucleotide encoding a MAGEA4 TCR; and a second polynucleotide encoding a fusion polypeptide comprising: a TGFβR2 polypeptide comprising an extracellular TGFβ1 binding domain of TGFβR2, an IL-18R1 transmembrane domain; and an IL-18R1 intracellular signaling domain; a viral self-cleaving 2A peptide; and a TGFβR1 polypeptide comprising an extracellular TGFβ1 binding domain of TGFβR1; an IL-18RAP transmembrane domain and an IL-18RAP intracellular signaling domain.

[0103] In various embodiments, the cell comprises a first polynucleotide encoding a MAGEA4 TCR; and a second polynucleotide encoding a fusion polypeptide comprising: a TGFβR2 polypeptide comprising an extracellular TGFβ1 binding domain of TGFβR2, an IL-18RAP transmembrane domain; and an IL-18RAP intracellular signaling domain; a viral self-cleaving 2A peptide; and a TGFβR1 polypeptide comprising an extracellular TGFβ1 binding domain of TGFβR1; an IL-18R1 transmembrane domain, and an IL-18R1 intracellular signaling domain.

[0104] In various embodiments, the cell comprises a first polynucleotide encoding a MAGEA4 TCR; and a second polynucleotide encoding a fusion polypeptide comprising: a TGFβR2 polypeptide comprising an extracellular TGFβ1 binding domain of TGFβR2, an IL-1R1 transmembrane domain; and an IL-1R1 intracellular signaling domain; a viral self-cleaving 2A peptide; and a TGFβR1 polypeptide comprising an extracellular TGFβ1 binding domain of TGFβR1; an IL-1RAP transmembrane domain and an IL-1RAP intracellular signaling domain.

[0105] In another embodiment, the cell comprises a first polynucleotide encoding a MAGEA4 TCR; and a second polynucleotide encoding a fusion polypeptide comprising: a TGFβR2 polypeptide comprising the extracellular TGFβ1 binding domain of TGFβR2, an IL-1RAP transmembrane domain; and an IL-1RAP intracellular signaling domain; a viral self-cleaving 2A peptide; and a TGFβR1 polypeptide comprising the extracellular TGFβ1 binding domain of TGFβR1; an IL-1R1 transmembrane domain, and an IL-1R1 intracellular signaling domain.

[0106] In some embodiments, the cell comprises a first polynucleotide encoding a MAGEA4 TCR; and a second polynucleotide encoding a fusion polypeptide comprising: a TGFβR2 polypeptide comprising an extracellular TGFβ1 binding domain of TGFβR2, an IFNAR1 transmembrane domain; and an IFNAR1 intracellular signaling domain; a viral self-cleaving 2A peptide; and a TGFβR1 polypeptide comprising an extracellular TGFβ1 binding domain of TGFβR1; an IFNAR2 transmembrane domain, and an IFNAR2 intracellular signaling domain.

[0107] In some embodiments, the cell comprises a first polynucleotide encoding a MAGEA4 TCR; and a second polynucleotide encoding a fusion polypeptide comprising: a TGFβR2 polypeptide comprising an extracellular TGFβ1 binding domain of TGFβR2, an IFNAR2 transmembrane domain; and an IFNAR2 intracellular signaling domain; a viral self-cleaving 2A peptide; and a TGFβR1 polypeptide comprising an extracellular TGFβ1 binding domain of TGFβR1; an IFNAR1 transmembrane domain, and an IFNAR1 intracellular signaling domain.

[0108] In various embodiments, the cell comprises a first polynucleotide encoding a MAGEA4 TCR; and a second polynucleotide encoding a fusion polypeptide comprising: a TGFβR2 polypeptide comprising an extracellular TGFβ1 binding domain of TGFβR2, a TLR1 transmembrane domain; and a TLR1 intracellular signaling domain; a viral self-cleaving 2A peptide; and a TGFβR1 polypeptide comprising an extracellular TGFβ1 binding domain of TGFβR1; a TLR1 transmembrane domain, and a TLR1 intracellular signaling domain.

[0109] In various embodiments, the cell comprises a first polynucleotide encoding a MAGEA4 TCR; and a second polynucleotide encoding a fusion polypeptide comprising: a TGFβR2 polypeptide comprising an extracellular TGFβ1 binding domain of TGFβR2, a TLR2 transmembrane domain; and a TLR2 intracellular signaling domain; a viral self-cleaving 2A peptide; and a TGFβR1 polypeptide comprising an extracellular TGFβ1 binding domain of TGFβR1; a TLR2 transmembrane domain and a TLR2 intracellular signaling domain.

[0110] In another embodiment, the cell comprises a first polynucleotide encoding a MAGEA4 TCR; and a second polynucleotide encoding a fusion polypeptide comprising: a TGFβR2 polypeptide comprising the extracellular TGFβ1 binding domain of TGFβR2, a TLR3 transmembrane domain; and a TLR3 intracellular signaling domain; a viral self-cleaving 2A peptide; and a TGFβR1 polypeptide comprising the extracellular TGFβ1 binding domain of TGFβR1; a TLR3 transmembrane domain and a TLR3 intracellular signaling domain.

[0111] In certain embodiments, the cell comprises a first polynucleotide encoding a MAGEA4 TCR; and a second polynucleotide encoding a fusion polypeptide comprising: a TGFβR2 polypeptide comprising an extracellular TGFβ1 binding domain of TGFβR2, a TLR4 transmembrane domain; and a TLR4 intracellular signaling domain; a viral self-cleaving 2A peptide; and a TGFβR1 polypeptide comprising an extracellular TGFβ1 binding domain of TGFβR1; a TLR4 transmembrane domain and a TLR4 intracellular signaling domain.

[0112] In a specific embodiment, the cell comprises a first polynucleotide encoding a MAGEA4 TCR; and a second polynucleotide encoding a fusion polypeptide comprising: a TGFβR2 polypeptide comprising the extracellular TGFβ1 binding domain of TGFβR2, a TLR5 transmembrane domain; and a TLR5 intracellular signaling domain; a viral self-cleaving 2A peptide; and a TGFβR1 polypeptide comprising the extracellular TGFβ1 binding domain of TGFβR1; a TLR5 transmembrane domain and a TLR5 intracellular signaling domain.

[0113] In some embodiments, the cell comprises a first polynucleotide encoding a MAGEA4 TCR; and a second polynucleotide encoding a fusion polypeptide comprising: a TGFβR2 polypeptide comprising an extracellular TGFβ1 binding domain of TGFβR2, a TLR6 transmembrane domain; and a TLR6 intracellular signaling domain; a viral self-cleaving 2A peptide; and a TGFβR1 polypeptide comprising an extracellular TGFβ1 binding domain of TGFβR1; a TLR6 transmembrane domain, and a TLR6 intracellular signaling domain.

[0114] In additional embodiments, the cell comprises a first polynucleotide encoding a MAGEA4 TCR; and a second polynucleotide encoding a fusion polypeptide comprising: a TGFβR2 polypeptide comprising the extracellular TGFβ1 binding domain of TGFβR2, a TLR7 transmembrane domain; and a TLR7 intracellular signaling domain; a viral self-cleaving 2A peptide; and a TGFβR1 polypeptide comprising the extracellular TGFβ1 binding domain of TGFβR1; a TLR7 transmembrane domain and a TLR7 intracellular signaling domain.

[0115] In various embodiments, the cell comprises a first polynucleotide encoding a MAGEA4 TCR; and a second polynucleotide encoding a fusion polypeptide comprising: a TGFβR2 polypeptide comprising the extracellular TGFβ1 binding domain of TGFβR2, a TLR8 transmembrane domain; and a TLR8 intracellular signaling domain; a viral self-cleaving 2A peptide; and a TGFβR1 polypeptide comprising the extracellular TGFβ1 binding domain of TGFβR1; a TLR8 transmembrane domain; and a TLR8 intracellular signaling domain.

[0116] In various embodiments, the cell comprises a first polynucleotide encoding a MAGEA4 TCR; and a second polynucleotide encoding a fusion polypeptide comprising: a TGFβR2 polypeptide comprising the extracellular TGFβ1 binding domain of TGFβR2, a TLR9 transmembrane domain; and a TLR9 intracellular signaling domain; a viral self-cleaving 2A peptide; and a TGFβR1 polypeptide comprising the extracellular TGFβ1 binding domain of TGFβR1; a TLR9 transmembrane domain and a TLR9 intracellular signaling domain.

[0117] In various embodiments, the cell comprises a first polynucleotide encoding a MAGEA4 TCR; and a second polynucleotide encoding a fusion polypeptide comprising: a TGFβR2 polypeptide comprising an extracellular TGFβ1 binding domain of TGFβR2, a TLR10 transmembrane domain; and a TLR10 intracellular signaling domain; a viral self-cleaving 2A peptide; and a TGFβR1 polypeptide comprising an extracellular TGFβ1 binding domain of TGFβR1; a TLR10 transmembrane domain and a TLR10 intracellular signaling domain.

[0118] In a specific embodiment, the viral self-cleaving 2A polypeptide is selected from the group consisting of: foot-and-mouth disease virus (FMDV) (F2A) peptide, equine rhinitis virus A (ERAV) (E2A) peptide, β-tetrasomal virus of the genus Tatyanavirus (TaV) (T2A) peptide, porcine Teschovirus-1 (PTV-1) (P2A) peptide, Theiler virus 2A peptide and encephalomyocarditis virus 2A peptide.

[0119] In various embodiments, the fusion polypeptide comprises the amino acid sequence set forth in SEQ ID NO:5.

[0120] In additional embodiments, the MAGEA4 TCR binds to the peptide GVYDGREHTV presented by HLA-A*02:01 encoded molecules.

[0121] In some embodiments, the MAGEA4 TCR comprises: an alpha chain comprising the amino acid sequence set forth in SEQ ID NO:2 and a beta chain comprising the amino acid sequence set forth in SEQ ID NO:3.

[0122] In various embodiments, the MAGEA4 TCR comprises an alpha chain comprising the amino acid sequence set forth in SEQ ID NO:5 and a beta chain comprising the amino acid sequence set forth in SEQ ID NO:6.

[0123] In certain embodiments, the cells are hematopoietic cells.

[0124] In further embodiments, the cell is a T cell.

[0125] In certain embodiments, the cells are CD3+, CD4+ and / or CD8+ cells.

[0126] In various embodiments, the cells are immune effector cells.

[0127] In some embodiments, the cell is a cytotoxic T lymphocyte (CTL), a tumor infiltrating lymphocyte (TIL), or a helper T cell.

[0128] In certain embodiments, the cell is a natural killer (NK) cell or a natural killer T (NKT) cell.

[0129] In certain embodiments, the source of the cells is peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, or a tumor.

[0130] In various embodiments, the compositions comprise cells expressing a MAGEA4 TCR and a fusion polypeptide contemplated herein.

[0131] In additional embodiments, a pharmaceutical composition comprises a pharmaceutically acceptable carrier and cells expressing the MAGEA4 TCR and a fusion polypeptide contemplated herein.

[0132] In certain embodiments, a method of treating a subject in need thereof comprises administering to the subject an effective amount of a composition contemplated herein.

[0133] In various embodiments, a method of treating, preventing, or ameliorating at least one symptom of cancer, infectious diseases, autoimmune diseases, inflammatory diseases, and immunodeficiency, or conditions associated therewith, comprises administering to the subject an effective amount of a composition contemplated herein.

[0134] In certain embodiments, the method of treating a solid cancer comprises administering to the subject an effective amount of a composition contemplated herein.

[0135] In some embodiments, the solid cancer comprises liver cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, bladder cancer, brain cancer, sarcoma, head and neck cancer, bone cancer, thyroid cancer, kidney cancer, or skin cancer.

[0136] In certain embodiments, the solid cancer is pancreatic cancer, lung cancer, or breast cancer.

[0137] In certain embodiments, a method of treating a hematological malignancy comprises administering to the subject an effective amount of a composition contemplated herein.

[0138] In various embodiments, the hematological malignancy is a leukemia, lymphoma, or multiple myeloma. BRIEF DESCRIPTION OF THE DRAWINGS

[0139] Figure 1 Shown are STAT4 and SMAD2 / 3 phosphorylation in untransduced (UTD) T cells, T cells transduced with LVV encoding the MAGEA4 TCR, and T cells transduced with LVV encoding the MAGEA4 TCR and LVV encoding an IL-12-responsive chimeric TGFβ receptor (CTBR12) cultured for 20 minutes in the presence or absence of TGFβ1.

[0140] Figure 2 Figure 2 shows the presence or absence of TGFβ1 (10 ng / ml) when cultured alone or with A375 MAGEA4 at a 1:1 E:T ratio. + IFNγ secretion by UTD T cells, MAGEA4TCR T cells, and MAGEA4TCR / CTBR12 T cells after 24 h of tumor cell culture.

[0141] Figure 3 A375 MAGEA4 in NSG tumor xenograft mouse models treated with UTD T cells, MAGEA4 TCR T cells, or MAGEA4 TCR / CTBR12 T cells are shown. + Tumor cell volume.

[0142] Figure 4 Shown are STAT4 and SMAD2 / 3 phosphorylation in T cells transduced with LVV encoding a MAGEA4-paired enhanced TCR (eTCR) and in T cells transduced with LVV encoding a MAGEA4 eTCR and an IL-12-responsive chimeric TGFβ receptor (CTBR12) cultured for 20 min in the presence or absence of TGFβ1.

[0143] Figure 5 Shown are the results of culture alone, with TGFβ1, or with A375 MAGEA4 at a 1:1 E:T ratio in the presence or absence of TGFβ1 (10 ng / ml). + IFNγ secretion by UTD T cells, MAGEA4 eTCR T cells, and MAGEA4 eTCR / CTBR12 T cells after 24 h of tumor cell culture.

[0144] A brief description of sequence identifiers

[0145] SEQ ID NO: 1 shows the amino acid sequence of the MAGEA4 epitope.

[0146] SEQ ID NO: 2 shows the amino acid sequence of human MAGEA4 TCR α chain.

[0147] SEQ ID NO: 3 shows the amino acid sequence of human MAGEA4 TCR β chain.

[0148] SEQ ID NO: 4 shows the amino acid sequence of human MAGEA4 TCR fusion polypeptide.

[0149] SEQ ID NO: 5 shows the amino acid sequence of human MAGEA4 eTCR α chain.

[0150] SEQ ID NO: 6 shows the amino acid sequence of human MAGEA4 eTCR β chain.

[0151] SEQ ID NO: 7 shows the amino acid sequence of human MAGEA4 eTCR fusion polypeptide.

[0152] SEQ ID NO: 8 shows the amino acid sequence of the IL-12-responsive chimeric TGFβ receptor (CTBR12).

[0153] SEQ ID NOs: 9-19 show the amino acid sequences of various linkers.

[0154] SEQ ID NOs: 20-44 show the amino acid sequences of protease cleavage sites and self-cleaving polypeptide cleavage sites.

[0155] SEQ ID NO: 45 shows the nucleotide sequence of the Kozak sequence.

[0156] In the aforementioned sequences, if X is present, it refers to any amino acid or the absence of a certain amino acid. Specific implementation plan

[0157] A. Overview

[0158] T cell receptor (TCR) expressing T cells have demonstrated limited, if any, efficacy in solid tumor indications, in part due to the immunosuppressive solid tumor microenvironment (TME). Tumor cells and tumor-infiltrating lymphocytes overproduce immunosuppressive cytokines including TGFβ, which contribute to the immunosuppressive tumor microenvironment. TGFβ inhibits T cell function through multiple mechanisms. TGFβ is frequently associated with tumor metastasis and invasion, suppressing immune cell function and poor prognosis in cancer patients. TGFβ signaling in tumor-specific CTLs through TGFβR2 attenuates their function and frequency in tumors, and blocking CD8 + TGFβ signaling on T cells leads to more rapid tumor surveillance and the presence of more CTLs at tumor sites.To date, strategies to inhibit TGFβ in clinical settings have not yielded significant therapeutic benefit.

[0159] The present disclosure generally relates to immune effector cells expressing MAGEA4 TCR and polypeptides converting immunosuppressive TGFβ signals into immunostimulatory signals, and to cells expressing polypeptides. Without wishing to be bound by any particular theory, the polypeptides encompassed herein are chimeric TGFβ receptors (CTBRs), which comprise the TGFβ binding domains of TGFβR1 and TGFβR2, which, when connected to an immunostimulatory endodomain and co-expressed in immune effector cells, can be exposed to TGFβ and converted from immunosuppressive signals into immunostimulatory signals that stimulate the activity and function of immune effector cells. The co-expression of chimeric TGFβ receptor polypeptides in immune effector cells, for example, by restoring or increasing proinflammatory cytokine secretion, makes cells resistant to the immunosuppressive effects of TGFβ. In a specific preferred embodiment, MAGEA4 TCR is human MAGEA4 paired to enhance TCR (eTCR), and the chimeric TGFβ receptor is CTBR12.

[0160] In various embodiments, the present disclosure contemplates, in part, immune effector cells expressing a MAGEA4 TCR and a CTBR polypeptide that converts an immunosuppressive TGFβ signal into an immunostimulatory signal mediated via or through one or more intracellular domains of one or more immune receptors.

[0161] In various embodiments, the present disclosure contemplates, in part, immune effector cells expressing a MAGEA4 TCR and a CTBR polypeptide that converts an immunosuppressive TGFβ signal into an immunostimulatory signal mediated via or through one or more intracellular domains of one or more cytokine receptors.

[0162] In various embodiments, the present disclosure contemplates, in part, immune effector cells expressing a MAGEA4 TCR and a CTBR polypeptide that converts an immunosuppressive TGFβ signal into an immunostimulatory signal mediated via or through one or more intracellular domains of one or more interleukin receptors.

[0163] In various embodiments, the present disclosure contemplates, in part, immune effector cells expressing a MAGEA4 TCR and a CTBR polypeptide that converts an immunosuppressive TGFβ signal into an immunostimulatory signal mediated via or through one or more intracellular domains of one or more pattern recognition receptors.

[0164] In various embodiments, the present disclosure contemplates, in part, immune effector cells expressing a MAGEA4 TCR and a CTBR polypeptide that converts an immunosuppressive TGFβ signal into an immunostimulatory signal mediated via or through one or more intracellular domains of one or more toll-like receptors.

[0165] In certain embodiments, the present disclosure contemplates, in part, immune effector cells expressing a MAGEA4 TCR and a CTBR polypeptide comprising a TGFβR1 extracellular domain, a transmembrane domain, and one or more intracellular domains of one or more immune receptors that binds TGFβ; and a CTBR polypeptide comprising a TGFβR2 extracellular domain, a transmembrane domain, and one or more intracellular domains of one or more immune receptors that binds TGFβ. In one embodiment, the polypeptides are linked to each other by a polypeptide cleavage signal, such as a 2A polypeptide cleavage signal.

[0166] In certain embodiments, the present disclosure contemplates, in part, immune effector cells expressing a MAGEA4 TCR (e.g., SEQ ID NOs: 2-4), preferably a MAGEA4 paired-enhanced TCR (eTCR; e.g., SEQ ID NOs: 5-7), preferably a MAGEA4 paired-enhanced TCR that binds the MAGEA4 peptide GVYDGREHTV presented by an HLA-A*02:01 encoded molecule, and a fusion polypeptide encoding a chimeric TGFβ receptor (CTBR) comprising the TGFβR1 extracellular domain, a transmembrane domain, and one or more intracellular domains of one or more immune receptors that binds TGFβ; and a polypeptide comprising the TGFβR2 extracellular domain, a transmembrane domain, and one or more intracellular domains of one or more immune receptors that binds TGFβ.

[0167] In specific embodiments, the transmembrane domain and the intracellular signaling domain are isolated from an IL-12 receptor, an IL-7 receptor, an IL-15 receptor, an IL-21 receptor, an IL-2 receptor, an IL-1 receptor, an IL-18 receptor, an IL-36 receptor, a type I IFN receptor, a TLR1 receptor, a TLR2 receptor, a TLR3 receptor, a TLR4 receptor, a TLR5 receptor, a TLR6 receptor, a TLR7 receptor, a TLR8 receptor, a TLR9 receptor, or a TLR10 receptor.

[0168] In certain embodiments, the transmembrane domain and the intracellular signaling domain are isolated from IL-12Rβ2, IL-7Rα, IL-2Rγ, IL-2Rβ, IL-21R, IL-18R1, IL-18RAP, IL-1R1, IL-1RAP, IFNAR1, IFNAR2, IL-1RL2, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, or TLR10.

[0169] In a preferred embodiment, the fusion polypeptide is an IL-12-responsive CTBR (CTBR12; eg, SEQ ID NO: 8).

[0170] Techniques for 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 can generally be performed as described in various general and more specific references in microbiology, molecular biology, biochemistry, molecular genetics, cell biology, virology, and immunology that are cited and discussed throughout this specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (updated July 2008 by John Wiley and Sons); Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, GreenePub. Associates and Wiley-Interscience; Glover, DNA Cloning: A Practical Approach, Volume I & II (IRL Press, Oxford Univ. Press USA, 1985); Current Protocols in Immunology (Editors: 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 ed., 1985); Transcription and Translation (B. Hames & S. Higgins ed., 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 ed., 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, ed., 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 ed., (Blackwell Scientific Publications, Oxford, 1988); Current Protocols in Immunology (Q. E. Colligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, and W. Strober, eds., 1991); Annual Review of Immunology; and monographs in journals such as Advances in Immunology.

[0171] B. Definition

[0172] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. Although any methods and materials similar or equivalent to the methods and materials described herein can be used to practice or test specific embodiments, preferred embodiments of compositions, methods and materials are described herein. For the purposes of this disclosure, the following terms are defined below.

[0173] As used herein, the articles "a" and "an" refer to one or more than one (i.e., at least one or one or more) the grammatical object of the article. As an example, "an element" means one element or one or more elements.

[0174] The use of alternatives such as "or" should be understood to mean one, two, or any combination of the alternatives.

[0175] The term "and / or" should be understood to mean one or both of the alternatives.

[0176] 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 quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that is within the range of approximately ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0177] Throughout this specification, unless the context requires otherwise, the words "comprise / comprises / comprising" should be understood to imply the inclusion of a stated step or element or group of steps or elements, but not the exclusion of any other step or element or any other group of steps or elements. "Consisting of is intended to include and be limited to what follows the phrase "consisting of." Thus, the phrase "consisting of indicates that the listed elements are essential or required, and that no other elements can be present. "Consisting essentially of is intended to include any element listed after the phrase, and is limited to other elements that do not interfere with or affect the activity or action described in the disclosure regarding the listed elements. Thus, the phrase "consisting essentially of indicates that the listed elements are essential or required, and that no other elements are present that materially affect the activity or action of the listed elements.

[0178] Reference throughout this specification to "one embodiment," "a specific embodiment," "a related embodiment," "an embodiment," "an additional embodiment," or "another embodiment" or combinations thereof means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the aforementioned phrases throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. It should also be understood that a positive recitation of a feature in one embodiment serves as a basis for excluding that feature from a particular embodiment.

[0179] "Antigen (Ag)" refers to a compound, composition, or substance that can stimulate antibody production or a T cell response in an animal, including compositions injected or absorbed into an animal (e.g., compositions comprising a cancer-specific protein). Exemplary antigens include, but are not limited to, lipids, carbohydrates, polysaccharides, glycoproteins, peptides, or nucleic acids. Antigens react with products that elicit specific humoral or cellular immunity, including products induced by heterologous antigens such as the disclosed antigens.

[0180] A "target antigen" or "target antigen of interest" is an antigen to which a binding domain encompassed herein is designed to bind. In certain embodiments, the target antigen is selected from the group consisting of: alpha folate receptor, 5T4, αvβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, FRα, GD2, GD3, Glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-A1+NY-ESO-1, HLA-A2+NY-ESO-1, HLA-A3+NY-ESO-1, IL-11Rα, IL-13Rα2, Lambda, Lewis-Y, Kappa, Mesothelin, Muc1, Muc16, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, Survivin, STn, TAG72, TEM, VEGFR2 and WT-1. In a preferred embodiment, the target antigen is MAGEA4.

[0181] MAGE-A4 belongs to a group of so-called cancer / testis antigens. Cancer / testis antigens are expressed in various malignancies and germ cells, but not in other adult tissues. Therefore, MAGE-A4 is an interesting immunotherapy target. The human gene encoding MAGE-A4 is designated MAGEA4 (ENSG00000147381).

[0182] In one embodiment, the antigen is an MHC-peptide complex, such as a class I MHC-peptide complex or a class II MHC-peptide complex.

[0183] "Linkers" refer to amino acid residues added between polypeptide domains for proper spacing and conformation of the molecule.

[0184] Illustrative examples of linkers suitable for use in the specific embodiments contemplated herein include, but are not limited to, the following amino acid sequences: GGG; DGGGS (SEQ ID NO: 9); TGEKP (SEQ ID NO: 10) (see, e.g., Liu et al., PNAS 5525-5530 (1997)); GGRR (SEQ ID NO: 11) (Pomerantz et al., 1995, supra); (GGGGS) n , wherein n=1, 2, 3, 4 or 5 (SEQ ID NO: 12) (Kim et al., PNAS 93, 1156-1160 (1996.); EGKSSGSGSESKVD (SEQ ID NO: 13) (Chaudhary et al., 1990, Proc. Natl. Acad. Sci. USA 87: 1066-1070); KESGSVSSEQLAQFRSLD (SEQ ID NO: 14) (Bird et al., 1988, Science 242: 423-426), GGRRGGGS (SEQ ID NO: 15); LRQRDGERP (SEQ ID NO: 16); LRQKDGGGSERP (SEQ ID NO: 17); LRQKD(GGGS)2ERP (SEQ ID NO: 18). Alternatively, a computer program capable of modeling the DNA binding site and the peptide itself can be used (Desjarlais & Berg, PNAS 90:2256-2260 (1993), PNAS 91:11099-11103 (1994) or rationally design flexible linkers by phage display methods. In one embodiment, the linker comprises the following amino acid sequence: GSTSGSGKPGSGEGSTKG (SEQ ID NO: 19) (Cooper et al., Blood, 101 (4): 1637-1644 (2003)).

[0185] A "transmembrane domain" or "TM domain" is a domain that anchors a polypeptide to the plasma membrane of a cell. A TM domain can be derived from natural, synthetic, semisynthetic or recombinant sources.

[0186] "Intracellular signaling domain" refers to a portion of a protein that transduces an effector function signal and directs the cell to perform a specialized function. Although the entire intracellular signaling domain can generally be employed, in many cases it is not necessary to use the entire domain. To the extent that a truncated portion of an intracellular signaling domain is used, such a truncated portion can be used in place of the entire domain as long as it transduces an effector function signal. The term intracellular signaling domain is intended to include any truncated portion of an intracellular signaling domain that is sufficient to transduce an effector function signal.

[0187] The term "effector function" or "effector cell function" refers to the specialized functions of immune effector cells. Effector functions include, but are not limited to, activation, cytokine production, proliferation, and cytotoxic activity, including the release of cytotoxic factors, or other cellular responses initiated by antigen binding to receptors expressed on immune effector cells.

[0188] "Immune disorder" refers to a disease that causes a response from the immune system. In certain embodiments, the term "immune disorder" refers to cancer, an autoimmune disease, or an immunodeficiency. In one embodiment, immune disorders encompass infectious diseases.

[0189] As used herein, the term "cancer" generally refers to a class of diseases or conditions in which abnormal cells divide without control and may invade nearby tissues.

[0190] As used herein, the term "malignant" refers to a group of cancer cells that show one or more of uncontrolled growth (i.e., division beyond normal limits), invasion (i.e., invasion and destruction of adjacent tissues), and metastasis (i.e., spread to other locations in the body via the lymph or blood). As used herein, the term "metastasis" refers to the spread of cancer from one part of the body to another. Tumors formed by cells that have spread are called "metastatic tumors" or "metastasis". Metastatic tumors contain cells similar to those in the original (primary) tumor.

[0191] As used herein, the term "benign" or "non-malignant" refers to a tumor that can grow larger but does not spread to other parts of the body. Benign tumors are self-limited and usually do not invade or metastasize.

[0192] "Cancer cell" refers to a single cell of a cancerous growth or tissue. Cancer cells include solid cancers and liquid cancers. "Tumor" or "tumor cell" generally refers to a swelling or lesion formed by an abnormal growth of cells, which can be benign, pre-malignant, or malignant. Most cancers form tumors, but liquid cancers such as leukemias do not necessarily form tumors. For those cancers that form tumors, the terms cancer (cell) and tumor (cell) are used interchangeably. The amount of tumor in an individual is the "tumor burden," which can be measured as the number, volume, or weight of tumors.

[0193] The term "relapse" refers to the return of cancer diagnosis or signs and symptoms after a period of improvement or remission.

[0194] "Remission" is also called "clinical remission" and includes both partial remission and complete remission. In a partial remission, some but not all signs and symptoms of cancer have disappeared. In a complete remission, all signs and symptoms of cancer have disappeared, although the cancer may still be in the body.

[0195] "Refractory" means that the cancer is resistant or unresponsive to treatment with a particular therapeutic agent. A cancer can be refractory at the start of treatment (i.e., unresponsive to initial exposure to the therapeutic agent), or it can become refractory because it develops resistance to the therapeutic agent during the first treatment period or during subsequent treatment periods.

[0196] "Antigen negative" refers to cells that do not express the antigen or express negligible amounts of undetectable antigen. In one embodiment, the antigen negative cell does not bind to a receptor directed to the antigen. In one embodiment, the antigen negative cell does not substantially bind to a receptor directed to the antigen.

[0197] "Autoimmune disease" refers to a disease in which the body produces an immunogenic (i.e., immune system) response to a certain component of its own tissue. In other words, the immune system loses its ability to identify a certain tissue or system in the body as "self" and targets and attacks the tissue or system as if the tissue or system were foreign. Autoimmune diseases can be classified as diseases in which mainly one organ is affected (e.g., hemolytic anemia and anti-immune thyroiditis) and diseases in which the autoimmune disease process is diffused through many tissues (e.g., systemic lupus erythematosus). For example, multiple sclerosis is considered to be caused by T cell attacks on the sheaths of the nerve fibers surrounding the brain and spinal cord. This results in loss of coordination, weakness, and blurred vision. Autoimmune diseases are known in the art and include, for example, Hashimoto's thyroiditis, Grave's disease, lupus, multiple sclerosis, rheumatoid arthritis, hemolytic anemia, anti-immune thyroiditis, systemic lupus erythematosus, celiac disease, Crohn's disease, colitis, diabetes, scleroderma, psoriasis, and the like.

[0198] "Immunodeficiency" means a state of a patient whose immune system has been damaged by disease or the administration of chemicals. This condition leaves the system lacking the number and type of blood cells needed to defend against foreign substances. Immunodeficiency conditions or diseases are known in the art and include, for example, AIDS (Acquired Immune Deficiency Syndrome), SCID (Severe Combined Immunodeficiency Disease), Selective IgA Deficiency, Common Variable Immunodeficiency, X-linked agammaglobulinemia, chronic granulomatous disease, hyper-IgM syndrome, and diabetes.

[0199] "Infectious disease" refers to a disease that can be transmitted from person to person or from organism to organism and is caused by a microbial or viral agent (e.g., the common cold). Infectious diseases are known in the art and include, for example, hepatitis, sexually transmitted diseases (e.g., chlamydia, gonorrhea), tuberculosis, HIV / AIDS, diphtheria, hepatitis B, hepatitis C, cholera, and influenza.

[0200] As used herein, the terms "individual" and "subject" are often used interchangeably and refer to any animal that exhibits symptoms of cancer or other immune disorders that can be treated with the compositions and methods encompassed herein elsewhere. Suitable subjects (e.g., patients) include experimental animals (such as mice, rats, rabbits, or guinea pigs), farm animals, and domestic animals or pets (such as cats or dogs). Non-human primates and preferably human patients are included. Typical subjects include human patients who have, have been diagnosed as having, or are at risk of having cancer or another immune condition.

[0201] As used herein, the term "patient" refers to a subject who has been diagnosed with cancer or another immune disorder that can be treated with the compositions and methods disclosed elsewhere herein.

[0202] As used herein, "treatment" or "treating" encompasses any beneficial or desired effect on the symptoms or pathology of a disease or pathological condition and may even encompass a minimal reduction in one or more measurable markers of the disease or condition being treated. Optionally, treatment may involve a reduction in the disease or condition or a delay in the progression of the disease or condition, such as delaying tumor growth. "Treatment" does not necessarily indicate complete eradication or cure of the disease or condition or its associated symptoms.

[0203] As used herein, "prevent" and similar words such as "prevented" or "preventing" refer to actions that prevent, inhibit, or reduce the likelihood of the occurrence or recurrence of a disease or condition. Prevention 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 words also encompass reducing the intensity, effects, symptoms, and / or burden of a disease or condition prior to the onset or recurrence of the disease or condition.

[0204] As used herein, the phrase "alleviating at least one symptom of" refers to reducing one or more symptoms of the disease or condition being treated. In certain embodiments, the disease or condition being treated is cancer, wherein the one or more symptoms alleviated include, but are not limited to, weakness, fatigue, shortness of breath, easy bruising and bleeding, frequent infections, swollen lymph nodes, abdominal swelling or pain (due to enlarged abdominal organs), bone or joint pain, bone fractures, unintentional weight loss, loss of appetite, night sweats, persistent mild fever, and decreased urination (due to impaired kidney function).

[0205] "Enhancement" or "promotion" or "increase" or "amplification" generally refers to that the compositions encompassed herein can produce, induce or cause a greater physiological response (i.e., downstream effect) compared to the response caused by a vehicle or control molecule / composition. Measurable physiological responses can include an increase in T cell expansion, activation, persistence, cytokine secretion and / or an increase in cancer cell killing ability, as well as other aspects apparent from the understanding of the art and the description herein. An "increased" or "enhanced" amount is typically a "statistically significant" amount and can include 1.1 times, 1.2 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times or more (e.g., 500 times, 1000 times) of the response produced by a vehicle or control composition (including all integers and decimal points therebetween and above 1, such as 1.5, 1.6, 1.7, 1.8, etc.).

[0206] "Decrease" or "attenuate" or "become less" or "reduce" or "mitigate" generally refers to the ability of the compositions encompassed herein to produce, elicit or induce less of a response (i.e., a physiological response) than the response elicited by vehicle or a control molecule / composition. A "decreased" or "reduced" amount is generally a "statistically significant" amount and can include a decrease of 1.1-fold, 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold or more (e.g., 500-fold, 1000-fold) (including all integers and decimal points in between and above 1, such as 1.5, 1.6, 1.7, 1.8, etc.) compared to the response produced by vehicle, a control composition, or a response in a particular cell lineage (reference response).

[0207] "Maintain" or "maintain" or "no change" or "no substantial change" or "no substantial decrease" generally refers to the ability of the compositions contemplated herein to produce, elicit or cause a substantially similar or comparable physiological response (i.e., downstream effect) in a cell compared to the response elicited by vehicle, a control molecule / composition, or a response in a particular cell lineage. A comparable response is one that is not significantly different or measurably different from a reference response.

[0208] C.MAGEA4 T cell receptor

[0209] The MAGEA4 T cell receptor (TCR) recognizes peptide fragments of MAGEA4 when presented by major histocompatibility complex (MHC) molecules. There are two different types of MHC molecules, MHC I and MHC II, which deliver peptides from different cellular compartments to the cell surface. Engagement of the TCR with the antigen and MHC leads to the activation of immune effector cells through a series of biochemical events mediated by associated enzymes, co-receptors, and specific accessory molecules.

[0210] The TCR encompassed herein is a heterodimer complex comprising TCR alpha (TCR α) chains and TCR beta (TCR β) chains. Human TCR α locus is located on chromosome 14 (14q11.2). Mature TCR α chains comprise variable domains and constant (C) domains derived from the reorganization of variable (V) segments and connection (J) segments. Human TCR β locus is located on chromosome 7 (7q34). Mature TCR β chains comprise variable domains and one of two constant (C) domains derived from the reorganization of variable (V) segments, diversity (D) segments and connection (J) segments.

[0211] In certain embodiments, the TCR binds MAGEA4.

[0212] In certain embodiments, the TCR is a human TCR that binds MAGEA4.

[0213] In a preferred embodiment, the TCR is a human paired-enhancer TCR that binds MAGEA4.

[0214] The pairing-enhanced MAGEA4 TCRs encompassed herein are engineered to increase TCR stability, TCR expression, specific TCR pairing, and functional avidity.

[0215] In certain embodiments, the constant domains of the MAGEA4 TCRα and MAGEA4 TCRβ chains are engineered or modified to increase TCR stability, TCR expression, specific TCR pairing, and functional avidity.

[0216] To effectively enhance correct pairing of MAGEA4 TCR sequences and to avoid mispairing with endogenous TCR chains, the MAGEA4 pairing-enhanced TCRs contemplated herein comprise minimally murinized TCRα and TCRβ constant domains and also comprise hydrophobic amino acid substitutions in the TCRα transmembrane domain.

[0217] In a preferred embodiment, the MAGEA4 paired enhanced TCR (eTCR) comprises: a MAGEA4 TCR alpha chain comprising a constant domain comprising the most humanizing amino acid substitutions at positions 90, 91, 92 and 93, and hydrophobic amino acid substitutions at positions 115, 118 and 119 of the constant region; and a MAGEA4 TCR beta chain comprising a constant domain comprising the most humanizing amino acids at positions 18, 22, 133, 136 and 139.

[0218] In a preferred embodiment, the MAGEA4 eTCR comprises: a TCR alpha chain comprising a constant domain comprising the following most murine amino acid substitutions P90S, E91D, S92V, and S93P, and the following hydrophobic amino acid substitutions S115L, G118V, and F119L in the transmembrane domain of the constant region; and a TCR beta chain comprising a constant domain comprising the following most murine amino acid substitutions E18K, S22A, F133I, E / V136A, and Q139H.

[0219] In certain preferred embodiments, the MAGEA4 eTCR comprises a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 5 and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 6. In other certain preferred embodiments, the MAGEA4 eTCR is expressed as a fusion polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 7.

[0220] D. Chimeric TGFβ receptor (CTBR)

[0221] In certain embodiments, cells comprising a polynucleotide encoding a human MAGEA4 TCR or a human MAGEA4 paired enhancing TCR (eTCR), and a chimeric TGFβ receptor that transduces an immunostimulatory signal upon exposure to TGFβ, including but not limited to TGFβ1, are contemplated.

[0222] As used herein, the term "chimeric TGFβ receptor" refers to one or more non-naturally occurring polypeptides that convert TGFβ immunosuppressive signals from the tumor microenvironment into immunostimulatory signals in T cells, for example, stimulating immune effector cell activity and function, thereby increasing the production and / or secretion of proinflammatory cytokines. In certain embodiments, the term "chimeric TGFβ receptor" is used interchangeably with the term "CTBR."

[0223] In certain embodiments, a CTBR polypeptide comprises the extracellular TGFβ binding domain, transmembrane domain, and intracellular signaling domain of an immune receptor, including but not limited to cytokine receptors, interleukin receptors, pattern recognition receptors, and toll-like receptors, of TGFβR2; a polypeptide cleavage signal; and the extracellular TGFβ binding domain, transmembrane domain, and intracellular signaling domain of an immune receptor, including but not limited to cytokine receptors, interleukin receptors, pattern recognition receptors, and toll-like receptors, of TGFβR1.

[0224] In a specific embodiment, the CTBR is a fusion polypeptide comprising: a first polypeptide comprising the extracellular TGFβ binding domain, a transmembrane domain, an intracellular signaling domain of an immune receptor, including but not limited to cytokine receptors, interleukin receptors, pattern recognition receptors, and toll-like receptors, of TGFβR2; a polypeptide cleavage signal; and a second polypeptide comprising the extracellular TGFβ binding domain, a transmembrane domain, and an intracellular signaling domain of an immune receptor, including but not limited to cytokine receptors, interleukin receptors, pattern recognition receptors, and toll-like receptors, of TGFβR1.

[0225] In other specific embodiments, the CTBR is a polypeptide complex comprising: a polypeptide comprising the extracellular TGFβ binding domain, a transmembrane domain, and an intracellular signaling domain of an immune receptor, including but not limited to cytokine receptors, interleukin receptors, pattern recognition receptors, and toll-like receptors, of TGFβR2; and a polypeptide comprising the extracellular TGFβ binding domain, a transmembrane domain, and an intracellular signaling domain of an immune receptor, including but not limited to cytokine receptors, interleukin receptors, pattern recognition receptors, and toll-like receptors, of TGFβR1.

[0226] As used herein, the term "immunoreceptor" refers to a receptor expressed on the surface of an immune cell that modulates an immune response when bound to its cognate ligand. Suitable immunoreceptors for use in specific embodiments include, but are not limited to, cytokine receptors, interleukin receptors, pattern recognition receptors, and toll-like receptors, wherein signaling through the immunoreceptor stimulates an immune response.

[0227] Illustrative examples of immunoreceptor transmembrane and intracellular signaling domains that can be used in specific embodiments contemplated herein include, but are not limited to, transmembrane and intracellular signaling domains isolated from an IL-12 receptor, an IL-7 receptor, an IL-15 receptor, an IL-21 receptor, an IL-2 receptor, an IL-1 receptor, an IL-18 receptor, an IL-36 receptor, a type I IFN receptor, a TLR1 receptor, a TLR2 receptor, a TLR3 receptor, a TLR4 receptor, a TLR5 receptor, a TLR6 receptor, a TLR7 receptor, a TLR8 receptor, a TLR9 receptor, or a TLR10 receptor.

[0228] Additional illustrative examples of immunoreceptor transmembrane and intracellular signaling domains that can be used in specific embodiments contemplated herein include, but are not limited to, transmembrane and intracellular signaling domains isolated from IL-12Rβ2, IL-7Rα, IL-2Rγ, IL-2Rβ, IL-21R, IL-18R1, IL-18RAP, IL-1R1, IL-1RAP, IFNAR1, IFNAR2, IL-1RL2, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, or TLR10.

[0229] Illustrative examples of cytokine receptor transmembrane and intracellular signaling domains that can be used in the specific embodiments contemplated herein include, but are not limited to, transmembrane and intracellular signaling domains isolated from IL-12Rβ2, IL-7Rα, IL-2Rγ, IL-2Rβ, IL-21R, IL-18R1, IL-18RAP, IL-1R1, IL-1RAP, IFNAR1, IFNAR2, and IL-1RL2.

[0230] Illustrative examples of interleukin receptor transmembrane and intracellular signaling domains that can be used in the specific embodiments contemplated herein include, but are not limited to, transmembrane and intracellular signaling domains isolated from IL-12Rβ2, IL-7Rα, IL-2Rγ, IL-2Rβ, IL-21R, IL-18R1, IL-18RAP, IL-1R1, IL-1RAP, and IL-1RL2.

[0231] Illustrative examples of toll-like receptor transmembrane and intracellular signaling domains that can be used in specific embodiments contemplated herein include, but are not limited to, transmembrane and intracellular signaling domains isolated from TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, and TLR10.

[0232] 1. CTBR12 peptide

[0233] Interleukin-12 (IL-12) is a cytokine that promotes T cell function and activity, in part by increasing IFNγ expression, increasing T cell proliferation, and enhancing IL-12 signaling. IL-12 binds to both interleukin-12 receptor β1 (IL-12Rβ1, also known as CD212) and interleukin-12 receptor β2 (IL-12Rβ2).

[0234] IL-12 signaling through IL-12Rβ1 and IL-12Rβ2 leads to phosphorylation of STAT3, STAT4, and STAT5. Phosphorylated STAT3 / STAT4 translocates to the nucleus and binds to the IFNγ promoter to increase IFNγ expression. Phosphorylated STAT4 also recruits the proto-oncogene (c-Jun) to the IFNγ promoter to increase IFNγ expression and enhances IL-12 signaling by increasing the transcription of IL-12Rβ2. STAT5 phosphorylation increases T cell proliferation.

[0235] IL-12 signaling also increases interleukin 2 receptor α (IL-2R) expression by recruiting STAT4 and c-Jun to the IL-2R promoter, thereby enhancing T cell proliferation.

[0236] In various embodiments, one or more immune effector cells are modified by introducing one or more polynucleotides or vectors encoding one or more CTBR12 polypeptides, including immune effector cells expressing MAGEA4 TCR or MAGEA4 eTCR. In various embodiments, one or more immune effector cells are modified by introducing one or more polynucleotides or vectors encoding CTBR12 and MAGEA4 TCR or MAGEA4 eTCR.

[0237] In certain embodiments, CTBR12 converts the immunosuppressive TGFβ signal into an IL-12-mediated immunostimulatory signal. In certain embodiments, the CTBR12 contemplated herein comprises: the extracellular TGFβ1 binding domain, transmembrane domain, and IL-12Rβ1 intracellular signaling domain of TGFβR1; a polypeptide cleavage signal; and the extracellular TGFβ1 binding domain, transmembrane domain, and IL-12Rβ2 intracellular signaling domain of TGFβR2. In certain embodiments, the CTBR12 contemplated herein comprises: the extracellular TGFβ1 binding domain, transmembrane domain, and IL-12Rβ2 intracellular signaling domain of TGFβR1; a polypeptide cleavage signal; and the extracellular TGFβ1 binding domain, transmembrane domain, and IL-12Rβ1 intracellular signaling domain of TGFβR2.

[0238] In certain embodiments, the CTBR12 contemplated herein include a fusion polypeptide comprising: a first polypeptide comprising an extracellular TGFβ1 binding domain, a transmembrane domain, and an IL-12Rβ1 intracellular signaling domain of TGFβR1; a polypeptide cleavage signal; and a second polypeptide comprising an extracellular TGFβ1 binding domain, a transmembrane domain, and an IL-12Rβ2 intracellular signaling domain of TGFβR2. In certain embodiments, the CTBR12 contemplated herein include a fusion polypeptide comprising: a first polypeptide comprising an extracellular TGFβ1 binding domain, a transmembrane domain, and an IL-12Rβ2 intracellular signaling domain of TGFβR1; a polypeptide cleavage signal; and a second polypeptide comprising an extracellular TGFβ1 binding domain, a transmembrane domain, and an IL-12Rβ1 intracellular signaling domain of TGFβR2.

[0239] In certain embodiments, CTBR12 is a polypeptide complex comprising a first polypeptide comprising: a polypeptide comprising the extracellular TGFβ1 binding domain, a transmembrane domain, and an IL-12Rβ1 intracellular signaling domain of TGFβR1; and a polypeptide comprising the extracellular TGFβ1 binding domain, a transmembrane domain, and an IL-12Rβ2 intracellular signaling domain of TGFβR2. In certain embodiments, CTBR12 is a polypeptide complex comprising a first polypeptide comprising: a polypeptide comprising the extracellular TGFβ1 binding domain, a transmembrane domain, and an IL-12Rβ2 intracellular signaling domain of TGFβR1; and a polypeptide comprising the extracellular TGFβ1 binding domain, a transmembrane domain, and an IL-12Rβ1 intracellular signaling domain of TGFβR2.

[0240] In certain embodiments, the polypeptide comprises a transmembrane domain of TGFβR1 or TGFβR2. In certain embodiments, the polypeptide comprises a transmembrane domain of IL-12Rβ1 or IL-12Rβ2. In one embodiment, the polypeptide comprises an extracellular TGFβ1 binding domain of TGFβR1 and an IL-12Rβ1 transmembrane domain and an intracellular signaling domain. In one embodiment, the polypeptide comprises an extracellular TGFβ1 binding domain of TGFβR2 and an IL-12Rβ2 transmembrane domain and an intracellular signaling domain. In one embodiment, the polypeptide comprises an extracellular TGFβ1 binding domain of TGFβR1 and an IL-12Rβ2 transmembrane domain and an intracellular signaling domain. In one embodiment, the polypeptide comprises an extracellular TGFβ1 binding domain of TGFβR2 and an IL-12Rβ1 transmembrane domain and an intracellular signaling domain.

[0241] In a specific embodiment, the polypeptide cleavage signal is a viral self-cleaving polypeptide; more preferably, a viral self-cleaving 2A polypeptide; and more preferably a viral self-cleaving polypeptide selected from the group consisting of: foot-and-mouth disease virus (FMDV) (F2A) peptide, equine rhinitis virus A (ERAV) (E2A) peptide, cleaved beta-tetrasomal virus (TaV) (T2A) peptide, porcine teschovirus-1 (PTV-1) (P2A) peptide, Theiler virus 2A peptide, and encephalomyocarditis virus 2A peptide. In one embodiment, the polypeptide cleavage signal is a P2A or T2A viral self-cleaving polypeptide.

[0242] 2. CTBR7 peptide

[0243] Interleukin-7 (IL-7) is a cytokine that promotes T cell function and activity, in part by improving T cell precursor survival and proliferation. IL-7 binds to the interleukin-7 receptor alpha (IL-7Rα, also known as CD127) and the interleukin-2 receptor, common gamma chain (IL-2Rγ, also known as CD132 and γc). IL-7 signaling activates the JAK / STAT, PI-3K, and Src kinase pathways, leading to the transcription of anti-apoptotic genes and genes that promote T cell precursor proliferation.

[0244] In various embodiments, one or more immune effector cells are modified by introducing one or more polynucleotides or vectors encoding one or more CTBR7 polypeptides, including immune effector cells expressing MAGEA4 TCR or MAGEA4 eTCR. In various embodiments, one or more immune effector cells are modified by introducing one or more polynucleotides or vectors encoding CTBR7 and MAGEA4 TCR or MAGEA4 eTCR.

[0245] In certain embodiments, the chimeric TGFβ receptor converts the immunosuppressive TGFβ signal into an IL-7-mediated immunostimulatory signal. In certain embodiments, the CTBR7 contemplated herein comprises: the extracellular TGFβ1 binding domain, transmembrane domain, and IL-7Rα intracellular signaling domain of TGFβR1; a polypeptide cleavage signal; and the extracellular TGFβ1 binding domain, transmembrane domain, and IL-2Rγ intracellular signaling domain of TGFβR2. In certain embodiments, the CTBR7 contemplated herein comprises: the extracellular TGFβ1 binding domain, transmembrane domain, and IL-2Rγ intracellular signaling domain of TGFβR1; a polypeptide cleavage signal; and the extracellular TGFβ1 binding domain, transmembrane domain, and IL-7Rα intracellular signaling domain of TGFβR2.

[0246] In certain embodiments, the CTBR7 contemplated herein include fusion polypeptides comprising: a first polypeptide comprising the extracellular TGFβ1 binding domain, a transmembrane domain, and an IL-7Rα intracellular signaling domain of TGFβR1; a polypeptide cleavage signal; and a second polypeptide comprising the extracellular TGFβ1 binding domain, a transmembrane domain, and an IL-2Rγ intracellular signaling domain of TGFβR2. In certain embodiments, the CTBR7 contemplated herein include fusion polypeptides comprising: a first polypeptide comprising the extracellular TGFβ1 binding domain, a transmembrane domain, and an IL-2Rγ intracellular signaling domain of TGFβR1; a polypeptide cleavage signal; and a second polypeptide comprising the extracellular TGFβ1 binding domain, a transmembrane domain, and an IL-7Rα intracellular signaling domain of TGFβR2.

[0247] In certain embodiments, CTBR7 is a polypeptide complex comprising a first polypeptide comprising: a polypeptide comprising the extracellular TGFβ1 binding domain, a transmembrane domain, and an IL-7Rα intracellular signaling domain of TGFβR1; and a polypeptide comprising the extracellular TGFβ1 binding domain, a transmembrane domain, and an IL-2Rγ intracellular signaling domain of TGFβR2. In certain embodiments, CTBR7 is a polypeptide complex comprising a first polypeptide comprising: a polypeptide comprising the extracellular TGFβ1 binding domain, a transmembrane domain, and an IL-2Rγ intracellular signaling domain of TGFβR1; and a polypeptide comprising the extracellular TGFβ1 binding domain, a transmembrane domain, and an IL-7Rα intracellular signaling domain of TGFβR2.

[0248] In certain embodiments, the polypeptide comprises a transmembrane domain of TGFβR1 or TGFβR2. In certain embodiments, the polypeptide comprises a transmembrane domain of IL-7Rα or IL-2Rγ. In one embodiment, the polypeptide comprises an extracellular TGFβ1 binding domain of TGFβR1 and an IL-7Rα transmembrane domain and an intracellular signaling domain. In one embodiment, the polypeptide comprises an extracellular TGFβ1 binding domain of TGFβR2 and an IL-2Rγ transmembrane domain and an intracellular signaling domain. In one embodiment, the polypeptide comprises an extracellular TGFβ1 binding domain of TGFβR1 and an IL-2Rγ transmembrane domain and an intracellular signaling domain. In one embodiment, the polypeptide comprises an extracellular TGFβ1 binding domain of TGFβR2 and an IL-7Rα transmembrane domain and an intracellular signaling domain.

[0249] In a specific embodiment, the polypeptide cleavage signal is a viral self-cleaving polypeptide; more preferably, a viral self-cleaving 2A polypeptide; and more preferably a viral self-cleaving polypeptide selected from the group consisting of: foot-and-mouth disease virus (FMDV) (F2A) peptide, equine rhinitis virus A (ERAV) (E2A) peptide, cleaved beta-tetrasomal virus (TaV) (T2A) peptide, porcine teschovirus-1 (PTV-1) (P2A) peptide, Theiler virus 2A peptide, and encephalomyocarditis virus 2A peptide. In one embodiment, the polypeptide cleavage signal is a P2A or T2A viral self-cleaving polypeptide.

[0250] 3. CTBR15 peptide

[0251] Interleukin-15 (IL-15) is a cytokine that promotes T cell function and activity by partially improving the survival and proliferation of T cell precursors. IL-15 binds with high affinity to IL-15Rα (also known as CD215), which then associates with a complex containing IL-2Rβ (also known as IL-15Rβ and CD122) and IL-2Rγ (also known as CD132 and γc) expressed on the same cell (cis presentation) or on different cells (trans presentation). IL-15 signaling activates the JAK / STAT, PI-3K, and Src kinase pathways and leads to the transcription of anti-apoptotic genes and genes that promote the proliferation of T cell precursors.

[0252] In various embodiments, one or more immune effector cells, including immune effector cells expressing MAGEA4 TCR or MAGEA4 eTCR, are modified by introducing one or more polynucleotides or vectors encoding one or more CTBR15 polypeptides, and optionally a polynucleotide or vector encoding IL-15Rα. In various embodiments, one or more immune effector cells are modified by introducing one or more polynucleotides or vectors encoding CTBR15 and MAGEA4 TCR or MAGEA4 eTCR, and optionally a polynucleotide or vector encoding an IL-15Rα polypeptide.

[0253] In certain embodiments, the chimeric TGFβ receptor converts the immunosuppressive TGFβ signal into an IL-15-mediated immunostimulatory signal. In certain embodiments, the CTBR15 contemplated herein comprises: the extracellular TGFβ1 binding domain, transmembrane domain, and IL-2Rβ intracellular signaling domain of TGFβR1; a polypeptide cleavage signal; and the extracellular TGFβ1 binding domain, transmembrane domain, and IL-2Rγ intracellular signaling domain of TGFβR2. In certain embodiments, the CTBR15 contemplated herein comprises: the extracellular TGFβ1 binding domain, transmembrane domain, and IL-2Rγ intracellular signaling domain of TGFβR1; a polypeptide cleavage signal; and the extracellular TGFβ1 binding domain, transmembrane domain, and IL-2Rβ intracellular signaling domain of TGFβR2.

[0254] In certain embodiments, the CTBR15 contemplated herein include a fusion polypeptide comprising: a first polypeptide comprising the extracellular TGFβ1 binding domain, a transmembrane domain, and an IL-2Rβ intracellular signaling domain of TGFβR1; a polypeptide cleavage signal; and a second polypeptide comprising the extracellular TGFβ1 binding domain, a transmembrane domain, and an IL-2Rγ intracellular signaling domain of TGFβR2. In certain embodiments, the CTBR15 contemplated herein include a fusion polypeptide comprising: a first polypeptide comprising the extracellular TGFβ1 binding domain, a transmembrane domain, and an IL-2Rγ intracellular signaling domain of TGFβR1; a polypeptide cleavage signal; and a second polypeptide comprising the extracellular TGFβ1 binding domain, a transmembrane domain, and an IL-2Rβ intracellular signaling domain of TGFβR2.

[0255] In certain embodiments, CTBR15 is a polypeptide complex comprising a first polypeptide comprising: a polypeptide comprising the extracellular TGFβ1 binding domain, a transmembrane domain, and an IL-2Rβ intracellular signaling domain of TGFβR1; and a polypeptide comprising the extracellular TGFβ1 binding domain, a transmembrane domain, and an IL-2Rγ intracellular signaling domain of TGFβR2. In certain embodiments, CTBR15 is a polypeptide complex comprising a first polypeptide comprising: a polypeptide comprising the extracellular TGFβ1 binding domain, a transmembrane domain, and an IL-2Rγ intracellular signaling domain of TGFβR1; and a polypeptide comprising the extracellular TGFβ1 binding domain, a transmembrane domain, and an IL-2Rβ intracellular signaling domain of TGFβR2.

[0256] In certain embodiments, the polypeptide comprises a transmembrane domain of TGFβR1 or TGFβR2. In certain embodiments, the polypeptide comprises a transmembrane domain of IL-2Rβ or IL-2Rγ. In one embodiment, the polypeptide comprises an extracellular TGFβ1 binding domain of TGFβR1 and an IL-2Rβ transmembrane domain and an intracellular signaling domain. In one embodiment, the polypeptide comprises an extracellular TGFβ1 binding domain of TGFβR2 and an IL-2Rγ transmembrane domain and an intracellular signaling domain. In one embodiment, the polypeptide comprises an extracellular TGFβ1 binding domain of TGFβR1 and an IL-2Rγ transmembrane domain and an intracellular signaling domain. In one embodiment, the polypeptide comprises an extracellular TGFβ1 binding domain of TGFβR2 and an IL-2Rβ transmembrane domain and an intracellular signaling domain.

[0257] In a specific embodiment, the polypeptide cleavage signal is a viral self-cleaving polypeptide; more preferably, a viral self-cleaving 2A polypeptide; and more preferably a viral self-cleaving polypeptide selected from the group consisting of: foot-and-mouth disease virus (FMDV) (F2A) peptide, equine rhinitis virus A (ERAV) (E2A) peptide, cleaved beta-tetrasomal virus (TaV) (T2A) peptide, porcine teschovirus-1 (PTV-1) (P2A) peptide, Theiler virus 2A peptide, and encephalomyocarditis virus 2A peptide. In one embodiment, the polypeptide cleavage signal is a P2A or T2A viral self-cleaving polypeptide.

[0258] 4.CTBR21 peptide

[0259] Interleukin-21 (IL-21) is a cytokine that promotes T cell function and activity, in part by improving the survival and proliferation of T cell precursors. IL-21 binds to the interleukin-21 receptor (IL-21R, also known as CD360) and IL-2Rγ (also known as CD132 and γc). IL-21 signaling activates the JAK / STAT, PI-3K, and Src kinase pathways, leading to the transcription of anti-apoptotic genes and genes that promote T cell precursor proliferation.

[0260] In various embodiments, one or more immune effector cells are modified by introducing one or more polynucleotides or vectors encoding one or more CTBR21 polypeptides, including immune effector cells expressing MAGEA4 TCR or MAGEA4 eTCR. In various embodiments, one or more immune effector cells are modified by introducing one or more polynucleotides or vectors encoding CTBR21 and MAGEA4 TCR or MAGEA4 eTCR.

[0261] In certain embodiments, the chimeric TGFβ receptor converts the immunosuppressive TGFβ signal into an IL-21-mediated immunostimulatory signal. In certain embodiments, the CTBR21 encompassed herein comprises: the extracellular TGFβ1 binding domain, transmembrane domain, and IL-21R intracellular signaling domain of TGFβR1; a polypeptide cleavage signal; and the extracellular TGFβ1 binding domain, transmembrane domain, and IL-2Rγ intracellular signaling domain of TGFβR2. In certain embodiments, the CTBR21 encompassed herein comprises: the extracellular TGFβ1 binding domain, transmembrane domain, and IL-2Rγ intracellular signaling domain of TGFβR1; a polypeptide cleavage signal; and the extracellular TGFβ1 binding domain, transmembrane domain, and IL-21R intracellular signaling domain of TGFβR2.

[0262] In certain embodiments, the CTBR21 contemplated herein comprises a fusion polypeptide comprising: a first polypeptide comprising an extracellular TGFβ1 binding domain, a transmembrane domain, and an IL-21R intracellular signaling domain of TGFβR1; a polypeptide cleavage signal; and a second polypeptide comprising an extracellular TGFβ1 binding domain, a transmembrane domain, and an IL-2Rγ intracellular signaling domain of TGFβR2. In certain embodiments, the CTBR21 contemplated herein comprises a fusion polypeptide comprising: a first polypeptide comprising an extracellular TGFβ1 binding domain, a transmembrane domain, and an IL-2Rγ intracellular signaling domain of TGFβR1; a polypeptide cleavage signal; and a second polypeptide comprising an extracellular TGFβ1 binding domain, a transmembrane domain, and an IL-21R intracellular signaling domain of TGFβR2.

[0263] In certain embodiments, CTBR21 is a polypeptide complex comprising a first polypeptide comprising: a polypeptide comprising the extracellular TGFβ1 binding domain, a transmembrane domain, and an IL-21R intracellular signaling domain of TGFβR1; and a polypeptide comprising the extracellular TGFβ1 binding domain, a transmembrane domain, and an IL-2Rγ intracellular signaling domain of TGFβR2. In certain embodiments, CTBR21 is a polypeptide complex comprising a first polypeptide comprising: a polypeptide comprising the extracellular TGFβ1 binding domain, a transmembrane domain, and an IL-2Rγ intracellular signaling domain of TGFβR1; and a polypeptide comprising the extracellular TGFβ1 binding domain, a transmembrane domain, and an IL-21R intracellular signaling domain of TGFβR2.

[0264] In certain embodiments, the polypeptide comprises a transmembrane domain of TGFβR1 or TGFβR2. In certain embodiments, the polypeptide comprises a transmembrane domain of IL-21R or IL-2Rγ. In one embodiment, the polypeptide comprises an extracellular TGFβ1 binding domain of TGFβR1 and an IL-21R transmembrane domain and an intracellular signaling domain. In one embodiment, the polypeptide comprises an extracellular TGFβ1 binding domain of TGFβR2 and an IL-2Rγ transmembrane domain and an intracellular signaling domain. In one embodiment, the polypeptide comprises an extracellular TGFβ1 binding domain of TGFβR1 and an IL-2Rγ transmembrane domain and an intracellular signaling domain. In one embodiment, the polypeptide comprises an extracellular TGFβ1 binding domain of TGFβR2 and an IL-21R transmembrane domain and an intracellular signaling domain.

[0265] In a specific embodiment, the polypeptide cleavage signal is a viral self-cleaving polypeptide; more preferably, a viral self-cleaving 2A polypeptide; and more preferably a viral self-cleaving polypeptide selected from the group consisting of: foot-and-mouth disease virus (FMDV) (F2A) peptide, equine rhinitis virus A (ERAV) (E2A) peptide, cleaved beta-tetrasomal virus (TaV) (T2A) peptide, porcine teschovirus-1 (PTV-1) (P2A) peptide, Theiler virus 2A peptide, and encephalomyocarditis virus 2A peptide. In one embodiment, the polypeptide cleavage signal is a P2A or T2A viral self-cleaving polypeptide.

[0266] 5.CTBR18 peptide

[0267] Interleukin-18 (IL-18) is a cytokine that promotes T cell function and activity by, in part, increasing IFNγ expression, increasing T cell proliferation, and preventing activation-induced cell death (AICD). IL-18 binds to interleukin-18 receptor 1 (IL-18R1, also known as CD218a) and interleukin-18 receptor accessory protein (IL-18RAP, CD218b).

[0268] IL-18 signaling through IL-18R1 and IL-18RAP leads to activation of the MyD88 adaptor protein and phosphorylation of IRAK4. Phosphorylation of IRAK4 and subsequent phosphorylation of IRAK1 / 2 ultimately leads to activation of NF-κB and AP-1 transcription factors to increase IFNγ expression and enhance sensitivity to IL-12. The transcriptional program induced by IL-18 also increases T cell proliferation and protects against AICD.

[0269] In various embodiments, one or more immune effector cells, including immune effector cells expressing MAGEA4 TCR or MAGEA4 eTCR, are modified by introducing one or more polynucleotides or vectors encoding one or more CTBR18 polypeptides. In various embodiments, one or more immune effector cells are modified by introducing one or more polynucleotides or vectors encoding CTBR18 and MAGEA4 TCR or MAGEA4 eTCR.

[0270] In certain embodiments, the chimeric TGFβ receptor converts the immunosuppressive TGFβ signal into an IL-18-mediated immunostimulatory signal. In certain embodiments, the CTBR18 contemplated herein comprises: the extracellular TGFβ1 binding domain, transmembrane domain, and IL-18RAP intracellular signaling domain of TGFβR1; a polypeptide cleavage signal; and the extracellular TGFβ1 binding domain, transmembrane domain, and IL-18R1 intracellular signaling domain of TGFβR2. In certain embodiments, the CTBR18 contemplated herein comprises: the extracellular TGFβ1 binding domain, transmembrane domain, and IL-18R1 intracellular signaling domain of TGFβR1; a polypeptide cleavage signal; and the extracellular TGFβ1 binding domain, transmembrane domain, and IL-18RAP intracellular signaling domain of TGFβR2.

[0271] In certain embodiments, the CTBR18 contemplated herein include fusion polypeptides comprising: a first polypeptide comprising the extracellular TGFβ1 binding domain, a transmembrane domain, and an IL-18R1 intracellular signaling domain of TGFβR1; a polypeptide cleavage signal; and a second polypeptide comprising the extracellular TGFβ1 binding domain, a transmembrane domain, and an IL-18RAP intracellular signaling domain of TGFβR2. In certain embodiments, the CTBR18 contemplated herein include fusion polypeptides comprising: a first polypeptide comprising the extracellular TGFβ1 binding domain, a transmembrane domain, and an IL-18RAP intracellular signaling domain of TGFβR1; a polypeptide cleavage signal; and a second polypeptide comprising the extracellular TGFβ1 binding domain, a transmembrane domain, and an IL-18R1 intracellular signaling domain of TGFβR2.

[0272] In certain embodiments, CTBR18 is a polypeptide complex comprising a first polypeptide comprising: a polypeptide comprising the extracellular TGFβ1 binding domain, a transmembrane domain, and the intracellular signaling domain of IL-18RAP of TGFβR1; and a polypeptide comprising the extracellular TGFβ1 binding domain, a transmembrane domain, and the intracellular signaling domain of IL-18R1 of TGFβR2. In certain embodiments, CTBR18 is a polypeptide complex comprising a first polypeptide comprising: a polypeptide comprising the extracellular TGFβ1 binding domain, a transmembrane domain, and the intracellular signaling domain of IL-18R1 of TGFβR1; and a polypeptide comprising the extracellular TGFβ1 binding domain, a transmembrane domain, and the intracellular signaling domain of IL-18RAP of TGFβR2.

[0273] In certain embodiments, the polypeptide comprises a transmembrane domain of TGFβR1 or TGFβR2. In certain embodiments, the polypeptide comprises a transmembrane domain of IL-18R1 or IL-18RAP. In one embodiment, the polypeptide comprises an extracellular TGFβ1 binding domain of TGFβR1 and an IL-18RAP transmembrane domain and an intracellular signaling domain. In one embodiment, the polypeptide comprises an extracellular TGFβ1 binding domain of TGFβR2 and an IL-18R1 transmembrane domain and an intracellular signaling domain. In one embodiment, the polypeptide comprises an extracellular TGFβ1 binding domain of TGFβR1 and an IL-18R1 transmembrane domain and an intracellular signaling domain. In one embodiment, the polypeptide comprises an extracellular TGFβ1 binding domain of TGFβR2 and an IL-18RAP transmembrane domain and an intracellular signaling domain.

[0274] In a specific embodiment, the polypeptide cleavage signal is a viral self-cleaving polypeptide; more preferably, a viral self-cleaving 2A polypeptide; and more preferably a viral self-cleaving polypeptide selected from the group consisting of: foot-and-mouth disease virus (FMDV) (F2A) peptide, equine rhinitis virus A (ERAV) (E2A) peptide, cleaved beta-tetrasomal virus (TaV) (T2A) peptide, porcine teschovirus-1 (PTV-1) (P2A) peptide, Theiler virus 2A peptide, and encephalomyocarditis virus 2A peptide. In one embodiment, the polypeptide cleavage signal is a P2A or T2A viral self-cleaving polypeptide.

[0275] 6.CTBR1 polypeptide

[0276] Interleukin-1 (IL-1) is a cytokine that promotes T cell function and activity by, in part, increasing IFNγ expression, enhancing T cell proliferation, and enhancing protection against activation-induced cell death (AICD). IL-1 binds to interleukin-1 receptor 1 (IL-1R1, also known as CD121a) and interleukin-1 receptor accessory protein (IL-1RAP).

[0277] IL-1 signaling through IL-1R1 and IL-1RAP leads to activation of the MyD88 adaptor protein and phosphorylation of IRAK4. Phosphorylation of IRAK4 and subsequent phosphorylation of IRAK1 / 2 ultimately leads to activation of NF-κB and AP-1 transcription factors to increase IFNγ expression and enhance sensitivity to IL-12. The transcriptional program induced by IL-1 also increases T cell proliferation and protects against AICD.

[0278] In various embodiments, one or more immune effector cells are modified by introducing one or more polynucleotides or vectors encoding one or more CTBR1 polypeptides, including immune effector cells expressing MAGEA4 TCR or MAGEA4 eTCR. In various embodiments, one or more immune effector cells are modified by introducing one or more polynucleotides or vectors encoding CTBR1 and MAGEA4 TCR or MAGEA4 eTCR.

[0279] In certain embodiments, the chimeric TGFβ receptor converts the immunosuppressive TGFβ signal into an IL-1-mediated immunostimulatory signal. In certain embodiments, the CTBR1 contemplated herein comprises: the extracellular TGFβ1 binding domain, transmembrane domain, and IL-1RAP intracellular signaling domain of TGFβR1; a polypeptide cleavage signal; and the extracellular TGFβ1 binding domain, transmembrane domain, and IL-1R1 intracellular signaling domain of TGFβR2. In certain embodiments, the CTBR1 contemplated herein comprises: the extracellular TGFβ1 binding domain, transmembrane domain, and IL-1R1 intracellular signaling domain of TGFβR1; a polypeptide cleavage signal; and the extracellular TGFβ1 binding domain, transmembrane domain, and IL-1RAP intracellular signaling domain of TGFβR2.

[0280] In certain embodiments, the CTBR1 contemplated herein include fusion polypeptides comprising: a first polypeptide comprising the extracellular TGFβ1 binding domain, a transmembrane domain, and an IL-1R1 intracellular signaling domain of TGFβR1; a polypeptide cleavage signal; and a second polypeptide comprising the extracellular TGFβ1 binding domain, a transmembrane domain, and an IL-1RAP intracellular signaling domain of TGFβR2. In certain embodiments, the CTBR1 contemplated herein include fusion polypeptides comprising: a first polypeptide comprising the extracellular TGFβ1 binding domain, a transmembrane domain, and an IL-1RAP intracellular signaling domain of TGFβR1; a polypeptide cleavage signal; and a second polypeptide comprising the extracellular TGFβ1 binding domain, a transmembrane domain, and an IL-1R1 intracellular signaling domain of TGFβR2.

[0281] In certain embodiments, CTBR1 is a polypeptide complex comprising a first polypeptide comprising: a polypeptide comprising the extracellular TGFβ1 binding domain, a transmembrane domain, and an IL-1RAP intracellular signaling domain of TGFβR1; and a polypeptide comprising the extracellular TGFβ1 binding domain, a transmembrane domain, and an IL-1R1 intracellular signaling domain of TGFβR2. In certain embodiments, CTBR1 is a polypeptide complex comprising a first polypeptide comprising: a polypeptide comprising the extracellular TGFβ1 binding domain, a transmembrane domain, and an IL-1R1 intracellular signaling domain of TGFβR1; and a polypeptide comprising the extracellular TGFβ1 binding domain, a transmembrane domain, and an IL-1RAP intracellular signaling domain of TGFβR2.

[0282] In certain embodiments, the polypeptide comprises a transmembrane domain of TGFβR1 or TGFβR2. In certain embodiments, the polypeptide comprises a transmembrane domain of IL-1R1 or IL-1RAP. In one embodiment, the polypeptide comprises an extracellular TGFβ1 binding domain of TGFβR1 and an IL-1RAP transmembrane domain and an intracellular signaling domain. In one embodiment, the polypeptide comprises an extracellular TGFβ1 binding domain of TGFβR2 and an IL-1R1 transmembrane domain and an intracellular signaling domain. In one embodiment, the polypeptide comprises an extracellular TGFβ1 binding domain of TGFβR1 and an IL-1R1 transmembrane domain and an intracellular signaling domain. In one embodiment, the polypeptide comprises an extracellular TGFβ1 binding domain of TGFβR2 and an IL-1RAP transmembrane domain and an intracellular signaling domain.

[0283] In a specific embodiment, the polypeptide cleavage signal is a viral self-cleaving polypeptide; more preferably, a viral self-cleaving 2A polypeptide; and more preferably a viral self-cleaving polypeptide selected from the group consisting of: foot-and-mouth disease virus (FMDV) (F2A) peptide, equine rhinitis virus A (ERAV) (E2A) peptide, cleaved beta-tetrasomal virus (TaV) (T2A) peptide, porcine teschovirus-1 (PTV-1) (P2A) peptide, Theiler virus 2A peptide, and encephalomyocarditis virus 2A peptide. In one embodiment, the polypeptide cleavage signal is a P2A or T2A viral self-cleaving polypeptide.

[0284] 7.CTBR.TLR peptide

[0285] Toll-like receptors (TLR1 to TLR10) are pattern recognition receptors that detect invading pathogens and activate innate and adaptive immune responses. Activation of TLRs by various ligands leads to the induction of proinflammatory transcriptional programs and the expression of multiple inflammatory cytokines.

[0286] TLR signaling occurs through homodimerization of the TLR signaling domain, leading to activation of the MyD88 adaptor protein and phosphorylation of IRAK4. Phosphorylation of IRAK4 and subsequent phosphorylation of IRAK1 / 2 ultimately leads to activation of NF-κB and AP-1 transcription factors, increasing inflammatory cytokine production and inducing proliferation. TLR activation can also lead to activation of IRF3 and IRF7 transcription factors.

[0287] In various embodiments, one or more immune effector cells are modified by introducing one or more polynucleotides or vectors encoding one or more CTBR.TLR polypeptides, including immune effector cells expressing MAGEA4 TCR or MAGEA4 eTCR. In various embodiments, one or more immune effector cells are modified by introducing one or more polynucleotides or vectors encoding CTBR.TLR and MAGEA4 TCR or MAGEA4 eTCR.

[0288] In certain embodiments, the chimeric TGFβ receptor converts the immunosuppressive TGFβ signal into a TLR-mediated immunostimulatory signal. In certain embodiments, the CTBR.TLRs encompassed herein include: the extracellular TGFβ1 binding domain, transmembrane domain, and TLR intracellular signaling domain of TGFβR1; a polypeptide cleavage signal; and the extracellular TGFβ1 binding domain, transmembrane domain, and the same TLR signaling domain of TGFβR2.

[0289] In certain embodiments, the CTBR.TLRs encompassed herein include fusion polypeptides comprising: a first polypeptide comprising the extracellular TGFβ1 binding domain, a transmembrane domain, and a TLR intracellular signaling domain of TGFβR1; a polypeptide cleavage signal; and a second polypeptide comprising the extracellular TGFβ1 binding domain, a transmembrane domain, and the same TLR signaling domain of TGFβR2.

[0290] In a specific embodiment, the CTBR.TLR is a polypeptide complex comprising a first polypeptide comprising: a polypeptide comprising the extracellular TGFβ1 binding domain, a transmembrane domain, and a TLR intracellular signaling domain of TGFβR1; and a polypeptide comprising the extracellular TGFβ1 binding domain, a transmembrane domain, and the same TLR intracellular signaling domain of TGFβR2.

[0291] In certain embodiments, the polypeptide comprises a transmembrane domain of TGFβR1 or TGFβR2. In certain embodiments, the polypeptide comprises a transmembrane domain of a TLR. In one embodiment, the polypeptide comprises an extracellular TGFβ1 binding domain of TGFβR1 and a TLR transmembrane domain and an intracellular signaling domain. In one embodiment, the polypeptide comprises an extracellular TGFβ1 binding domain of TGFβR2 and a TLR transmembrane domain and an intracellular signaling domain.

[0292] In a specific embodiment, the polypeptide cleavage signal is a viral self-cleaving polypeptide; more preferably, a viral self-cleaving 2A polypeptide; and more preferably a viral self-cleaving polypeptide selected from the group consisting of: foot-and-mouth disease virus (FMDV) (F2A) peptide, equine rhinitis virus A (ERAV) (E2A) peptide, cleaved beta-tetrasomal virus (TaV) (T2A) peptide, porcine teschovirus-1 (PTV-1) (P2A) peptide, Theiler virus 2A peptide, and encephalomyocarditis virus 2A peptide. In one embodiment, the polypeptide cleavage signal is a P2A or T2A viral self-cleaving polypeptide.

[0293] E. Peptide

[0294] Various polypeptides are contemplated herein, including but not limited to MAGEA4 TCR, MAGEA4 eTCR, CTBR, fusion proteins comprising the foregoing polypeptides, and fragments thereof. Unless otherwise indicated, "polypeptide," "peptide," and "protein" are used interchangeably and are conventionally defined as amino acid sequences. In one embodiment, "polypeptide" includes fusion polypeptides and other variants. Polypeptides can be prepared using any of a variety of well-known recombinant and / or synthetic techniques. Polypeptides are not limited to a specific length; for example, they can comprise the full-length protein sequence, fragments of the full-length protein, or fusion proteins, and can include post-translational modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, and the like, as well as other naturally occurring and non-naturally occurring modifications known in the art.

[0295] As used herein, "isolated peptide" or "isolated polypeptide" and the like refer to a peptide or polypeptide molecule that is separated and / or purified in vitro from the cellular environment and from association with other components of the cell, i.e., the peptide or polypeptide molecule is not significantly associated with substances in the body.

[0296] Polypeptides include "polypeptide variants." A polypeptide variant may differ from a naturally occurring polypeptide by one or more substitutions, deletions, additions, and / or insertions. Such variants may be naturally occurring or may be generated synthetically, for example, by modifying one or more of the above-described polypeptide sequences. For example, in certain embodiments, it may be desirable to improve the binding affinity and / or other biological properties of a polypeptide by introducing one or more substitutions, deletions, additions, and / or insertions into the polypeptide. In specific embodiments, the polypeptide comprises a polypeptide 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 reference sequences contemplated herein, typically wherein the variant maintains at least one biological activity of the reference sequence.

[0297] Polypeptide variants include biologically active "polypeptide fragments". Illustrative examples of biologically active polypeptide fragments include DNA binding domains, nuclease domains, and the like. 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 comprise an amino acid chain that is at least 5 to about 1700 amino acids long. It will be appreciated that in certain embodiments, a 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, 1500, 1600, 1700 or more amino acids in length.

[0298] In certain embodiments, the polypeptides described herein may comprise one or more amino acids designated as "X." "X," if present in an amino acid SEQ ID NO, refers to any one or more amino acids. In certain embodiments, a SEQ ID NO representing a fusion protein includes a sequence of consecutive X residues that cumulatively represent any amino acid sequence.

[0299] As described above, polypeptides can be altered in various ways, including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of a reference polypeptide can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel (1985, Proc. Natl. Acad. Sci. USA. 82: 488-492), Kunkel et al., (1987, Methods in Enzymol, 154: 367-382), U.S. Patent No. 4,873,192, Watson, JD et al. (Molecular Biology of the Gene, Fourth Edition, Benjamin / Cummings, Menlo Park, Calif., 1987), and references cited therein. Guidance regarding appropriate amino acid substitutions that do not affect the biological activity of the protein of interest can be found in the model of Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC).

[0300] In certain embodiments, polypeptide variants comprises one or more conservative replacements." conservative replacement " is that amino acid is replaced by another amino acid with similar characteristics, makes the technical staff of peptide chemistry field can expect the secondary structure of polypeptide and the replacement that hydrophilicity is constant basically.Modification can be carried out at the structure of the polynucleotide and polypeptide contained in the specific embodiment and still obtained the variant or the derivative polypeptide with desired characteristics being encoded functional molecule.When wishing to change the amino acid sequence of polypeptide to produce the variant polypeptide of equivalence or even improvement, those skilled in the art for example can change one or more codons of coding DNA sequence, for example according to Table 1.

[0301] Table 1 - Amino acid codons

[0302]

[0303] Guidance on determining which amino acid residues can be substituted, inserted or deleted without eliminating biological activity can be found using computer programs well known in the art such as DNASTAR, DNA Strider, Geneious, MacVector or Vector NTI software. Preferably, the amino acid changes in the protein variants disclosed herein are conservative amino acid changes, i.e., replacements of similarly charged or uncharged amino acids. Conservative amino acid changes involve replacement of one of a group of amino acids associated with a side chain. Naturally occurring amino acids are generally divided into four groups: acidic amino acids (aspartic acid, glutamic acid), basic amino acids (lysine, arginine, histidine), non-polar amino acids (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) and uncharged polar amino acids (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan and tyrosine are sometimes collectively classified as aromatic amino acids. In peptides or proteins, suitable conservative amino acid substitutions are known to those skilled in the art and can generally be made without changing the biological activity of the resulting molecule. Those skilled in the art will recognize that, generally speaking, single amino acid substitutions in non-essential regions of a polypeptide will not substantially alter biological activity (see, e.g., Watson et al. Molecular Biology of the Gene, 4th ed., 1987, The Benjamin / Cummings Pub. Co., p. 224).

[0304] In one embodiment, where expression of two or more polypeptides is desired, the polynucleotide sequences encoding the two or more polypeptides may be separated by an IRES sequence, as disclosed elsewhere herein.

[0305] Polypeptides encompassed in certain embodiments include fusion polypeptides. In certain embodiments, fusion polypeptides and polynucleotides encoding fusion polypeptides are provided. Fusion polypeptides and fusion proteins refer to polypeptides having at least two, three, four, five, six, seven, eight, nine, or ten polypeptide segments.

[0306] In another embodiment, two or more polypeptides may be expressed as a fusion protein comprising one or more self-cleaving polypeptide sequences as disclosed elsewhere herein.

[0307] Fusion polypeptides may comprise one or more polypeptide domains or segments, including but not limited to signal peptides, cell penetrating peptide domains (CPP), DNA binding domains, nuclease domains, etc., epitope tags (e.g., maltose binding protein ("MBP"), glutathione S-transferase (GST), HIS6, MYC, FLAG, V5, VSV-G, and HA), polypeptide linkers, and polypeptide cleavage signals. Fusion polypeptides are typically C-terminally linked to the N-terminus, but they can also be C-terminally linked to the C-terminus, N-terminally linked to the N-terminus, or N-terminally linked to the C-terminus. In certain embodiments, the polypeptides of the fusion protein can follow any order. Fusion polypeptides or fusion proteins can also include conservatively modified variants, polymorphic variants, alleles, mutants, subsequences, and interspecies homologs, as long as the desired activity of the fusion polypeptide is preserved. Fusion polypeptides can be made by chemical synthesis or by chemical linkage between the two parts, or can generally be prepared using other standard techniques. The linked DNA sequences that constitute the fusion polypeptide are operably linked to suitable transcriptional or translational control elements as disclosed elsewhere herein.

[0308] The fusion polypeptide may optionally comprise a linker that can be used to connect one or more polypeptides or one or more domains within a polypeptide. Peptide linker sequences can be used to separate any two or more polypeptide groups by a distance sufficient to ensure that each polypeptide folds into its appropriate secondary and tertiary structures, thereby allowing the polypeptide domains to perform their desired functions. Such peptide linker sequences are incorporated into the fusion polypeptide using standard techniques in the art. Suitable peptide linker sequences can be selected based on the following factors: (1) their ability to present a flexible extended conformation; (2) their inability to present a secondary structure that can interact with functional epitopes on the first and second polypeptides; and (3) the lack of hydrophobic or charged residues that may react with functional epitopes on the polypeptides. Preferred peptide linker sequences contain Gly, Asn, and Ser residues. Other nearly neutral amino acids, such as Thr and Ala, may also be used in the linker sequence. Amino acid sequences that can be usefully used as linkers include Maratea et al., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258-8262, 1986; U.S. Patent No. 4,935,233 and U.S. Patent No. 4,751,180. When a particular fusion polypeptide segment contains a non-essential N-terminal amino acid region that can be used to separate functional domains and prevent spatial interference, a linker sequence is not required. Preferred linkers are typically flexible amino acid subsequences synthesized as part of a recombinant fusion protein. The linker polypeptide can be between 1 and 200 amino acids in length, between 1 and 100 amino acids in length, or between 1 and 50 amino acids in length, including all integer values therebetween.

[0309] Exemplary polypeptide cleavage signals include polypeptide cleavage recognition sites, such as protease cleavage sites, nuclease cleavage sites (eg, 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).

[0310] Suitable protease cleavage sites and self-cleaving peptides are known to the skilled person (see, for example, Ryan et al., 1997. J. Gener. Virol. 78, 699-722; Scymczak et al. (2004) Nature Biotech. 5, 589-594). Exemplary 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 PI (P35) protease, byovirus NIa protease, protease encoded by byovirus RNA-2, aphthovirus L protease, enterovirus 2A protease, rhinovirus 2A protease, picorna 3C protease, comovirus 24K protease, nepovirus 24K protease, rice tungro sphericalvirus (RTSV) 3C-like protease, parsnip yellow fleck virus (PYVF) 3C-like protease, heparin, thrombin, factor Xa, and enterokinase. In one embodiment, tobacco etch virus (TEV) protease cleavage sites are preferred due to their higher cleavage stringency, such as EXXYXQ(G / S) (SEQ ID NO:20), such as ENLYFQG (SEQ ID NO:21) and ENLYFQS (SEQ ID NO:22), where X represents any amino acid (TEV cleavage occurs between Q and G or Q and S).

[0311] In certain embodiments, the self-cleaving polypeptide site comprises a 2A or 2A-like site, sequence, or domain (Donnelly et al., 2001. J. Gen. Virol. 82: 1027-1041). In specific embodiments, the viral 2A peptide is an aphthous 2A peptide, a potato virus 2A peptide, or a cardiovirus 2A peptide.

[0312] In one embodiment, the viral 2A peptide is selected from the group consisting of: foot-and-mouth disease virus (FMDV) (F2A) peptide, equine rhinitis virus A (ERAV) (E2A) peptide, beta-tetrasomal virus of the genus Tatyanavirus (TaV) (T2A) peptide, porcine Teschovirus-1 (PTV-1) (P2A) peptide, Theiler virus 2A peptide, and encephalomyocarditis virus 2A peptide.

[0313] Illustrative examples of 2A sites are provided in Table 2.

[0314] Table 2:

[0315] SEQ ID NO:23 GSGATNFSLLKQAGDVEENPGP SEQ ID NO:24 ATNFSLLKQAGDVEENPGP SEQ ID NO:25 LLKQAGDVEENPGP SEQ ID NO:26 GSGEGRGSLLTCGDVEENPGP SEQ ID NO:27 EGRGSLLTCGDVEENPGP SEQ ID NO:28 LLTCGDVEENPGP SEQ ID NO:29 GSGQCTNYALLKLAGDVESNPGP SEQ ID NO:30 QCTNYALLKLAGDVESNPGP SEQ ID NO:31 LLKLAGDVESNPGP SEQ ID NO:32 GSGVKQTLNFDLLKLAGDVESNPGP SEQ ID NO:33 VKQTLNFDLLKLAGDVESNPGP SEQ ID NO:34 LLKLAGDVESNPGP SEQ ID NO:35 LLNFDLLKLAGDVESNPGP SEQ ID NO:36 TLNFDLLKLAGDVESNPGP SEQ ID NO:37 LLKLAGDVESNPGP SEQ ID NO:38 NFDLLKLAGDVESNPGP SEQ ID NO:39 QLLNFDLLKLAGDVESNPGP SEQ ID NO:40 APVKQTLNFDLLKLAGDVESNPGP SEQ ID NO:41 VTELLYRMKRAETYCPRPLLAIHPTEARHKQKIVAPVKQT SEQ ID NO:42 LNFDLLKLAGDVESNPGP SEQ ID NO:43 LLAIHPTEARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP SEQ ID NO:44 EARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP

[0316] In preferred embodiments, the polypeptide comprises a MAGEA4 TCR, a MAGEA4 eTCR, or one or more CTBR polypeptides.

[0317] F. Polynucleotides

[0318] In certain embodiments, polynucleotides encoding MAGEA4 TCRs, CTBRs, engineered TCRs, fusion proteins comprising the aforementioned polypeptides, and fragments thereof are provided. As used herein, the terms "polynucleotide" or "nucleic acid" refer to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and DNA / RNA hybrids. Polynucleotides can be single-stranded or double-stranded and recombinant, synthetic, or isolated. Polynucleotides include, but are not limited to, pre-messenger RNA (pre-mRNA), messenger RNA (mRNA), synthetic RNA, synthetic mRNA, genomic DNA (gDNA), PCR amplified DNA, complementary DNA (cDNA), synthetic DNA, or recombinant DNA. A polynucleotide is 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, as well as all intermediate lengths of nucleotides, i.e., polymeric forms of ribonucleotides or deoxyribonucleotides or modified forms of either type of nucleotide. It will be readily understood that in this context, "intermediate lengths" means 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, the 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.

[0319] In certain embodiments, the polynucleotide may undergo codon optimization. As used herein, the term "codon optimized" refers to replacing codons in a polynucleotide encoding a polypeptide so as to increase the expression, stability, and / or activity of the polypeptide. Factors that affect codon optimization include, but are not limited to, one or more of the following: (i) variations in codon preference between two or more organisms or genes or synthetically constructed preference tables; (ii) variations in the degree of codon preference within an organism, gene, or gene set; (iii) systematic variations in codons, including environmental variations; (iv) variations in codons derived from decoding tRNAs; (v) variations in codons derived from GC% in one position of a triplet; (vi) variations in the degree of similarity to a reference sequence, such as a naturally occurring sequence; (vii) variations in the codon frequency cutoff; (viii) structural properties of mRNA transcribed from a DNA sequence; (ix) prior knowledge of the function of the DNA sequence upon which the codon substitution set was designed; (x) systematic variations in the codon set for each amino acid; and / or (xi) isolated removal of pseudo-translation start sites.

[0320] As used herein, the term "nucleotide" refers to a heterocyclic nitrogenous base in an N-glycosidic linkage to a phosphorylated sugar. Nucleotides are understood to include natural bases, as well as a variety of modified bases recognized in the art. Such bases are generally located at the 1' position of the nucleotide sugar moiety. Nucleotides generally comprise a base, a sugar, and a phosphate group. In ribonucleic acid (RNA), the sugar is ribose, and in deoxyribonucleic acid (DNA), the sugar is deoxyribose, i.e., a sugar without the hydroxyl group present in ribose. Exemplary natural nitrogenous bases include purines, i.e., adenosine (A) and guanidine (G); and pyrimidines, i.e., cytidine (C) and thymidine (T) (or uracil (U) in the case of RNA). The C-1 atom of deoxyribose is bonded to the N-1 of pyrimidine or the N-9 of purine. Nucleotides are typically monophosphates, diphosphates, or triphosphates. Nucleotides may be unmodified or modified at the sugar, phosphate and / or base moieties (also interchangeably referred to as nucleotide analogs, nucleotide derivatives, modified nucleotides, non-natural nucleotides, and non-standard nucleotides; see, e.g., WO 92 / 07065 and WO 93 / 15187). Examples of modified nucleic acid bases are summarized in Limbach et al. (1994, Nucleic Acids Res. 22, 2183-2196).

[0321] Nucleotide can also be considered as the phosphate ester of nucleoside, and esterification occurs on the hydroxyl of C-5 that is connected to sugar.As used herein, term " nucleoside " refers to the heterocyclic nitrogenous base in the N-glycosidic linkage with sugar.Nucleoside is generally recognized in the art to include natural base, and also includes well-known modified base.Such base is generally located at the 1 ' position of nucleoside sugar part.Nucleoside generally comprises base and glycosyl.Nucleoside can be unmodified or experience modification (also can be interchangeably referred to as nucleoside analog, nucleoside derivative, modified nucleoside, non-natural nucleoside or non-standard nucleoside) at sugar and / or base part.In addition, as mentioned above, the example of modified nucleic acid base is summarized in Limbach et al. (1994, Nucleic Acids Res.22,2183-2196).

[0322] In various exemplary embodiments, polynucleotides encompassed herein include, but are not limited to, polynucleotides encoding MAGEA4 TCR, MAGEA4 eTCR, one or more CTBR polypeptides, fusion polypeptides, and expression vectors, viral vectors, and transfer plasmids comprising the polynucleotides encompassed herein.

[0323] As used herein, the terms "polynucleotide variant" and "variant" and the like refer to polynucleotides that exhibit substantial sequence identity to a reference polynucleotide sequence or that hybridize to a reference sequence under stringent conditions as defined below. These terms also encompass polynucleotides that differ from a reference polynucleotide by the addition, deletion, substitution, or modification of at least one nucleotide. Thus, the terms "polynucleotide variant" and "variant" include polynucleotides in which one or more nucleotides are added or deleted or modified, or replaced with a different nucleotide. In this regard, it is well understood in the art that certain changes, including mutations, additions, deletions, and substitutions, may be made to a reference polynucleotide such that the altered polynucleotide retains the biological function or activity of the reference polynucleotide.

[0324] In one embodiment, the polynucleotide comprises a nucleotide sequence that hybridizes to a target nucleic acid sequence under stringent conditions. Hybridization under "stringent conditions" describes a hybridization scheme in which nucleotide sequences that are at least 60% identical to each other remain hybridized. Generally, the stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) of the specific sequence at a given ionic strength and pH. The Tm is the temperature (under a given ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequence is generally present in excess at the Tm, 50% of the probes are occupied at equilibrium.

[0325] As used herein, the description "sequence identity" or, for example, "a sequence that is 50% identical to..." refers to the degree to which the sequences are identical on a nucleotide-by-nucleotide basis or on an amino acid-by-amino acid basis within the comparison window. Thus, the "percentage of sequence identity" can be calculated by comparing two optimally aligned sequences within the comparison window, determining the number of positions at which identical nucleic acid bases (e.g., A, T, C, G, I) or identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) appear in the two sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to obtain 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 wherein the polypeptide variant maintains at least one biological activity of the reference polypeptide.

[0326] 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" is at least 12, but often 15 to 18 and usually at least 25 monomer units in length, including nucleotides and amino acid residues. Because two polynucleotides can each contain (1) a sequence that is similar between the two polynucleotides (i.e., only a portion of the complete polynucleotide sequence); and (2) a sequence that is different between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing the sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of at least 6, typically about 50 to about 100, more typically about 100 to about 150 contiguous positions, in which one sequence is compared after optimally aligning it with a reference sequence having the same number of contiguous positions. In order to carry out optimal comparison to two sequences, the comparison window can include additions or deletions of about 20% or less (i.e., gaps) compared to the reference sequence (not including added or deleted sequences). The optimal sequence alignment for comparing the comparison window can be performed by computer-implemented algorithms (GAP, BESTFIT, FASTA, and TFASTA, Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by an inspection and optimal comparison (i.e., producing the highest homology percentage within the comparison window) generated by any of the various selected methods. Reference can also be made to the BLAST program family disclosed in, for example, 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, Unit 19.3 of Chapter 15.

[0327] As used herein, an "isolated polynucleotide" refers to a polynucleotide that has been purified from its naturally occurring sequences on either side, e.g., a DNA fragment that has been removed from the sequences that normally surround it. An "isolated polynucleotide" also refers to complementary DNA (cDNA), recombinant DNA, or other polynucleotides that do not exist in nature and have been made by the hand of man.

[0328] In various embodiments, the polynucleotides include mRNAs encoding the polypeptides contemplated herein. In certain embodiments, the mRNA comprises a cap, one or more nucleotides, and a poly(A) tail.

[0329] Terms describing the orientation of polynucleotides include: 5' (usually the end of the polynucleotide with a free phosphate group) and 3' (usually the end of the polynucleotide with a free hydroxyl group (OH)). Polynucleotide sequences can be annotated in a 5' to 3' orientation or a 3' to 5' orientation. For DNA and mRNA, the 5' to 3' strand is designated as the "sense" strand, "positive" strand, or "coding" strand because its sequence is consistent with the sequence of the pre-messenger (pre-mRNA) [although it is uracil (U) in RNA and thymine (T) in DNA]. For DNA and mRNA, the complementary 3' to 5' strand, which is the strand transcribed by RNA polymerase, is designated as the "template" strand, "antisense" strand, "minus" strand, or "non-coding" strand. As used herein, the term "reverse orientation" refers to a 5' to 3' sequence written in a 3' to 5' orientation or a 3' to 5' sequence written in a 5' to 3' orientation.

[0330] The terms "complementary" and "complementarity" refer to polynucleotides (i.e., nucleotide sequences) that are related by the base pairing rules. For example, the complementary strand of the DNA sequence 5'AGTCATG 3' is 3'TCAGTA C5'. The latter sequence is usually written as a reverse complement sequence, with the 5' end on the left and the 3' end on the right, i.e., 5'CATGACT 3'. A sequence that is identical to its reverse complement sequence is called a palindrome. Complementarity can be "partial," in which only the bases of some nucleic acids match according to the base pairing rules. Alternatively, there can be "complete" or "overall" complementarity between nucleic acids.

[0331] In addition, it will be appreciated by those skilled in the art that due to the degeneracy of the genetic code, there are many nucleotide sequences encoding polypeptides or their variant fragments, as described herein. In these polynucleotides, some have minimum homology to the nucleotide sequence of any natural gene. Nevertheless, in specific embodiments, polynucleotides that vary due to codon usage differences are particularly encompassed, for example, polynucleotides that are optimized for human and / or primate codon selection. In specific embodiments, polynucleotides are codons optimized for expression and / or stability. In addition, the alleles of the genes comprising the polynucleotide sequences provided herein can also be used. Alleles are endogenous genes that change as a result of one or more sudden changes of nucleotides, such as deletions, additions, and / or substitutions.

[0332] As used herein, the term "nucleic acid cassette" or "expression cassette" refers to a gene sequence in a vector that can express RNA and subsequently express a polypeptide. In one embodiment, the nucleic acid cassette contains a target gene, such as a target polynucleotide. In another embodiment, the nucleic acid cassette contains one or more expression control sequences, such as a promoter, an enhancer, a polyadenylation sequence, and a target gene, such as a target polynucleotide. The vector may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleic acid cassettes. The nucleic acid cassette is oriented sequentially by position within the vector so that the nucleic acid in the cassette can be transcribed into RNA and, when necessary, translated into a protein or polypeptide, undergoes appropriate post-translational modifications required for activity in the transformed cell, and is translocated to the appropriate compartment by targeting the appropriate intracellular compartment or secreting into the extracellular compartment to obtain biological activity. Preferably, the 3' and 5' ends of the cassette are suitable for rapid insertion into the vector, for example, they have restriction endonuclease sites at each end. In a preferred embodiment, the nucleic acid cassette contains a therapeutic gene sequence for treating, preventing, or ameliorating a genetic disorder. The cassette can be removed as a single unit and inserted into a plasmid or viral vector.

[0333] Polynucleotides include a polynucleotide of interest. As used herein, the term "polynucleotide of interest" refers to a polynucleotide encoding a polypeptide or fusion polypeptide, or a polynucleotide used as a template to transcribe an inhibitory polynucleotide, as contemplated herein.

[0334] As disclosed elsewhere herein or as known in the art, regardless of the length of the coding sequence itself, the polynucleotides encompassed herein can be combined with other DNA sequences, such as promoters and / or enhancers, untranslated regions (UTRs), signal sequences, Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosome entry sites (IRES), recombinase recognition sites (e.g., LoxP sites, FRT sites, and Att sites), stop codons, transcription termination signals, and polynucleotides encoding self-cleaving polypeptides, epitope tags, so that the overall length of the polynucleotides can vary significantly. Thus, it is contemplated that polynucleotide fragments of virtually any length can be employed, with the overall length preferably being limited by ease of preparation and use in the intended recombinant DNA protocol.

[0335] Polynucleotides can be prepared, manipulated, expressed and / or delivered using any of a variety of well-established techniques known and available in the art. In order to express the desired polypeptide, the nucleotide sequence encoding the polypeptide can be inserted into an appropriate vector.

[0336] Illustrative examples of vectors include, but are not limited to, plasmids, autonomously replicating sequences, and transposable elements, such as SleepingBeauty, PiggyBac.

[0337] Additional illustrative 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.

[0338] Illustrative examples of viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40).

[0339] Illustrative examples of expression vectors include, but are not limited to, the pClneo vector (Promega) for expression in mammalian cells; the pLenti4 / V5-DEST vector (Promega) for lentivirus-mediated gene transfer and expression in mammalian cells; TM 、pLenti6 / V5-DEST TM and pLenti6.2 / V5-GW / lacZ (Invitrogen). In certain embodiments, the coding sequence of the polypeptides disclosed herein can be linked to such expression vectors to express the polypeptides in mammalian cells.

[0340] In a specific embodiment, carrier is an episomal vector or maintains an extrachromosomal carrier. As used herein, term "episomal" refers to a carrier that can replicate and is not integrated into the host's chromosomal DNA and can not gradually lose from the host cell of division, which also means that the carrier replicates extrachromosomally or additionally.

[0341] "Expression control sequences," "control elements," or "regulatory sequences" present in an expression vector are those untranslated regions of the vector (origin of replication, selection cassette, promoter, enhancer, translation initiation signal (Shine Dalgarno sequence or Kozak sequence), introns, polyadenylation sequence, 5' and 3' untranslated regions) that interact with host cell proteins to effect transcription and translation. These elements can vary in their strength and specificity. Depending on the vector system and host utilized, a variety of suitable transcription and translation elements can be used, including ubiquitous promoters and inducible promoters.

[0342] In a specific embodiment, polynucleotide comprises a vector, and the vector includes but is not limited to expression vectors and viral vectors. The vector may include one or more exogenous, endogenous or heterologous control sequences, such as promoters and / or enhancers. "Endogenous control sequence" is a sequence naturally connected to a given gene in the genome. "Exogenous control sequence" is a sequence that is placed in juxtaposition with a gene so that the transcription of this gene is guided by the connected enhancer / promoter by genetic manipulation (i.e., molecular biology techniques). "Heterologous control sequence" is an exogenous sequence from a different species than the cell manipulated by the gene. "Synthetic" control sequence may include one or more endogenous and / or exogenous sequences and / or elements of a sequence that provides optimal promoter and / or enhancer activity for a particular therapy as determined in vitro or in a computer.

[0343] As used herein, the term "promoter" refers to a recognition site for a polynucleotide (DNA or RNA) to which RNA polymerase binds. RNA polymerase initiates and transcribes a polynucleotide operably linked to the promoter. In a specific embodiment, a promoter that functions in mammalian cells includes an AT-rich region approximately 25 to 30 bases upstream of the start transcription site and / or another sequence found 70 to 80 bases upstream of the start of transcription, i.e., a CNCAAT region in which N can be any nucleotide.

[0344] The term "enhancer" refers to a segment of DNA that contains sequences that can provide enhanced transcription and, in some cases, can function independently of its orientation relative to another control sequence. An enhancer can function synergistically or additively with a promoter and / or other enhancer elements. The term "promoter / enhancer" refers to a segment of DNA that contains sequences that can provide both promoter and enhancer functions.

[0345] The term "operably linked" refers to a juxtaposition in which the described components are in a relationship permitting them to function in their intended manner. In one embodiment, the term refers to a functional linkage between a nucleic acid expression control sequence (e.g., a promoter and / or enhancer) and a second polynucleotide sequence, e.g., a polynucleotide of interest, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.

[0346] As used herein, the term "constitutive expression control sequence" refers to a promoter, enhancer, or promoter / enhancer that continuously or continually permits transcription of an operably linked sequence. A constitutive expression control sequence can be a "ubiquitous" promoter, enhancer, or promoter / enhancer that permits expression in a wide variety of cell and tissue types, or a "cell-specific," "cell type-specific," "cell lineage-specific," or "tissue-specific" promoter, enhancer, or promoter / enhancer that permits expression in a restricted variety of cell and tissue types, respectively.

[0347] Exemplary ubiquitous expression control sequences suitable for use in particular embodiments include, but are not limited to, cytomegalovirus (CMV) immediate early promoter, 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, H5, P7.5 promoter and P11 promoter from vaccinia virus, elongation factor 1-alpha (EF1a) promoter, early growth response 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 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 β-actin (CAG) promoter, β-actin promoter and myeloproliferative sarcoma virus enhancer negative control region deleted and d1587rev primer binding site replaced (MND) U3 promoter (Haas et al., Journal of Virology. 2003; 77(17): 9439-9450).

[0348] In one embodiment, the vector includes the MNDU3 promoter.

[0349] In one embodiment, the vector comprises an EF1a promoter comprising the first intron of the human EF1a gene.

[0350] In one embodiment, the vector includes the EF1a promoter lacking the first intron of the human EF1a gene.

[0351] In certain embodiments, it may be desirable to use cell-, cell type-, cell lineage-, or tissue-specific expression control sequences to achieve cell type-specific, lineage-specific, or tissue-specific expression of a desired polynucleotide sequence (e.g., to express a particular nucleic acid encoding a polypeptide only in a subset of a cell type, cell lineage, or tissue, or during a particular stage of development).

[0352] In certain embodiments, it may be desirable to express the polynucleotide from a T cell-specific promoter.

[0353] 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 specific physiological, biological or disease state, etc. This definition is not intended to exclude cell type or tissue specific expression. Certain embodiments provide conditional expression of a polynucleotide of interest, for example, by controlling expression by subjecting a cell, tissue, organism, etc., to a treatment or condition that results in increased or decreased expression of the polynucleotide or a polynucleotide encoded by the polynucleotide of interest.

[0354] Illustrative examples of inducible promoters / systems include, but are not limited to, steroid-inducible promoters such as promoters of genes encoding glucocorticoid 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-regulatable system (Sirin et al., 2003, Gene, 323:67), cumate-inducible gene switches (WO 2002 / 088346), tetracycline-dependent regulatory systems, etc. Inducing agents include, but are not limited to, glucocorticoids, estrogens, mifepristone (RU486), metals, interferons, small molecules, cumate, tetracycline, doxycycline, and variants thereof.

[0355] As used herein, "internal ribosome entry site" or "IRES" refers to an element that facilitates direct entry of internal ribosomes into a cistron (protein coding region) into a start codon such as ATG, thereby leading to cap-independent translation of the gene. See, for example, Jackson et al., 1990. Trends Biochem Sci 15(12):477-83) and Jackson and Kaminski. 1995. RNA 1(10):985-1000. Examples of IRES commonly used by those skilled in the art include those described in U.S. Patent No. 6,692,736. Other examples of "IRES" known in the art include, but are not limited to, IRES obtainable from picornaviruses (Jackson et al., 1990) and IRES obtainable from viral or cellular mRNA sources, such as immunoglobulin heavy chain binding protein (BiP), vascular endothelial growth factor (VEGF) (Huez et al., 1998. Mol. Cell. Biol. 18(11):6178-6190), fibroblast growth factor 2 (FGF-2) and insulin-like growth factor (IGFII), translation initiation factor eIF4G and yeast transcription factors TFIID and HAP4, Ephelomencycarditis virus (EMCV) (Duke et al., 1992. J. Virol 66(3):1602-9) and VEGF IRES commercially available from Novagen (Huez et al., 1998. Mol Cell Biol 18(11):6178-90). IRESs have been reported in viral genomes of species from the families Picornaviridae, Dicistroviridae, and Flaviviridae, as well as in HCV, Friend murine leukemia virus (FrMLV), and Moloney murine leukemia virus (MoMLV).

[0356] In one embodiment, the IRES used in the polynucleotides contemplated herein is the EMCV IRES.

[0357] In certain embodiments, the polynucleotide comprises a polynucleotide having a consensus Kozak sequence and encoding a desired polypeptide. As used herein, the term "Kozak sequence" refers to a short nucleotide sequence that greatly promotes the initial binding of mRNA to the small subunit of the ribosome and increases translation. The consensus Kozak sequence is (GCC)RCCATGG (SEQ ID NO: 45), 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).

[0358] Elements that guide efficient termination and polyadenylation of heterologous nucleic acid transcripts will increase the expression of heterologous genes. Transcription termination signals are generally found downstream of the polyadenylation signal. In specific embodiments, the vector comprises a polyadenylation sequence at the 3' end of the polynucleotide encoding the polypeptide to be expressed. As used herein, the term "polyA site" or "polyA sequence" refers to a DNA sequence that guides the termination and polyadenylation of nascent RNA transcripts caused by RNA polymerase II. The polyadenylation sequence can promote mRNA stability by adding a polyadenylic acid tail at the 3' end of the coding sequence, thereby promoting increased translation efficiency. Cleavage and polyadenylation are guided by the polyA sequence in the RNA. The core polyA sequence of mammalian mRNA precursors (pre-mRNA) has two recognition elements flanking the cleavage-polyadenylation site. Typically, the nearly invariant AAUAAA hexamer is located 20-50 nucleotides upstream of the more variable element rich in U or GU residues. Cleavage of the nascent transcript occurs between these two elements and is coupled to add up to 250 adenosines to the 5' cleavage product. In certain embodiments, the core poly(A) sequence is an ideal poly(A) sequence (e.g., AATAAA, ATTAAA, AGTAAA). In certain embodiments, the poly(A) sequence is the SV40 poly(A) sequence, the bovine growth hormone poly(A) sequence (BGHpA), the rabbit β-globin poly(A) sequence (rβgpA), or another suitable heterologous or endogenous poly(A) sequence known in the art.

[0359] In some embodiments, the polynucleotide or cell containing the polynucleotide utilizes a suicide gene, including an inducible suicide gene for reducing the risk of direct toxicity and / or uncontrolled proliferation. In a specific embodiment, the suicide gene does not confers immunity to the host containing the polynucleotide or cell. An example of a suicide gene that can be used is caspase-9 or caspase-8 or cytosine deaminase. Specific chemical inducers of dimerization (CID) can be used to activate caspase-9.

[0360] In certain embodiments, one or more polynucleotides encoding MAGEA4 TCRα chain and TCRβ chain (including eTCR chain) and / or one or more CTBR polypeptides are introduced into cells (e.g., immune effector cells) via non-viral or viral vectors. 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 linked to a vector nucleic acid molecule, for example, inserted into a vector nucleic acid molecule. The vector may include a sequence that directs autonomous replication in the cell, or may include a sequence sufficient to allow integration into the host cell DNA. In certain embodiments, a non-viral vector is used to deliver one or more polynucleotides encompassed herein into T cells.

[0361] Illustrative examples of non-viral vectors include, but are not limited to, mRNA, plasmids (eg, DNA plasmids or RNA plasmids), transposons, cosmids, and bacterial artificial chromosomes.

[0362] Exemplary methods of non-viral delivery of polynucleotides encompassed in particular embodiments include, but are not limited to, electroporation, sonoporation, lipofection, microinjection, gene guns, virions, liposomes, immunoliposomes, nanoparticles, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, DEAE-dextran mediated transfer, gene guns, and heat shock.

[0363] Illustrative examples of polynucleotide delivery systems suitable for use in the specific embodiments contemplated herein include, but are not limited to, those provided by Amaxa Biosystems, Maxcyte, Inc., BTX Molecular Delivery Systems, and Copernicus Therapeutics Inc. Lipofectamine transfection reagents are commercially available (e.g., Transfectam TM and Lipofectin TM Cationic and neutral lipids suitable for efficient receptor-recognition lipofection of polynucleotides have been described in the literature. See, for example, Liu et al. (2003) Gene Therapy. 10:180–187; and Balazs et al. (2011) Journal of Drug Delivery. 2011:1-12. Antibody-targeted, bacterial-derived, inanimate nanocell-based delivery is also contemplated in certain embodiments.

[0364] In various embodiments, the polynucleotide is an mRNA that is introduced into a cell for transient expression of a desired polypeptide. As used herein, "transient" refers to expression of a non-integrated transgene over a period of hours, days, or weeks, wherein the period of expression is less than the period of expression of the polynucleotide if integrated into the genome or contained within a stable plasmid replicon in the cell.

[0365] In certain embodiments, viral vectors are used to deliver one or more polynucleotides contemplated herein into T cells.

[0366] Illustrative examples of viral vector systems suitable for use in specific embodiments contemplated herein include, but are not limited to, adeno-associated virus (AAV), retrovirus (including lentivirus), herpes simplex virus, adenovirus, and vaccinia virus vectors.

[0367] In a specific embodiment, a polycistronic polynucleotide encoding a MAGEA4 TCR (SEQ ID NO: 4) comprising a TCR α chain (SEQ ID NO: 2) and a TCR β chain (SEQ ID NO: 3) and a polycistronic polynucleotide encoding a CTBR (SEQ ID NO: 8) are introduced into the cell using a non-viral or viral vector. In a specific embodiment, a polycistronic polynucleotide encoding a fusion protein encoding a MAGEA4 eTCR (SEQ ID NO: 7) comprising an eTCR α chain (SEQ ID NO: 5) and an eTCR β chain (SEQ ID NO: 6) and a polycistronic polynucleotide encoding a CTBR (SEQ ID NO: 8) are introduced into the cell using a non-viral or viral vector.

[0368] In certain embodiments, a polycistronic polynucleotide encoding a MAGEA4 TCR (SEQ ID NO: 4) comprising a TCR alpha chain (SEQ ID NO: 2) and a TCR beta chain (SEQ ID NO: 3) and a CTBR (SEQ ID NO: 8) is introduced into the cell using a non-viral or viral vector. In certain embodiments, a polycistronic polynucleotide encoding a fusion protein encoding a MAGEA4 eTCR (SEQ ID NO: 7) comprising an eTCR alpha chain (SEQ ID NO: 5) and an eTCR beta chain (SEQ ID NO: 6) and a CTBR (SEQ ID NO: 8) is introduced into the cell using a non-viral or viral vector.

[0369] G. Genetically modified cells

[0370] In various embodiments, cells are modified to express a MAGEA4 TCR or a MAGEA4eTCR and a CTBR for use in treating cancer. Cells can be non-genetically modified to express the polypeptides encompassed herein, or in certain preferred embodiments, cells can be genetically modified to express the polypeptides encompassed herein. As used herein, the terms "genetically engineered" or "genetically modified" refer to the addition of additional genetic material in the form of DNA or RNA to the total genetic material of a cell. In certain embodiments, the terms "genetically modified cell," "modified cell," and "redirected cell" are used interchangeably.

[0371] In certain embodiments, the MAGEA4 TCR and one or more CTBR polypeptides encompassed herein are introduced and expressed in immune effector cells to increase resistance of the cells to immunosuppressive signals in the TME mediated by TGFβ. In certain embodiments, the MAGEA4 eTCR and one or more CTBR polypeptides are introduced and expressed in immune effector cells.

[0372] "Immune effector cells" are any cells of the immune system that have one or more effector functions (e.g., cytotoxic cell killing activity, cytokine secretion, induction of ADCC and / or CDC). Exemplary immune effector cells encompassed herein are T lymphocytes, particularly cytotoxic T cells (CTLs; CD8+ T cells), TILs, and helper T cells (HTLs; CD4+ T cells. In one embodiment, immune effector cells include natural killer (NK) cells. In one embodiment, immune effector cells include natural killer T (NKT) cells. Immune effector cells can be autologous / self ("self") or non-autologous ("non-self," e.g., allogeneic, isogenic, or xenogeneic).

[0373] As used herein, "autologous" refers to cells from the same subject. As used herein, "allogeneic" refers to cells from the same species that are genetically different from the comparison cells. As used herein, "isogenic" refers to cells from a different subject that are genetically identical to the comparison cells. As used herein, "xenogeneic" refers to cells from a different species than the comparison cells. In preferred embodiments, the cells are autologous.

[0374] Exemplary immune effector cells suitable for introduction of the CTBR polypeptides contemplated herein include T lymphocytes. The terms "T cell" or "T lymphocyte" are art-recognized and are intended to encompass thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. T cells can be T helper (Th) cells, such as T helper 1 (Th1) or T helper 2 (Th2) cells. T cells can be helper T cells (HTL; CD4+ T cells)CD4 + cells, cytotoxic T cells (CTL; CD8 + T cells), CD4 + CD8 + T cells, CD4 - CD8 - T cells or any other T cell subset. Other exemplary T cell populations suitable for use in certain embodiments include naive T cells and memory T cells.

[0375] As will be appreciated by those skilled in the art, other cells may also be used as immune effector cells with MAGEA4TCR or MAGEA4 eTCR and one or more CTBR polypeptides as contemplated herein. In particular, immune effector cells also include NK cells, NKT cells, neutrophils and macrophages. Immune effector cells also include progenitor cells of effector cells, wherein such progenitor cells can be induced to differentiate into immune effector cells in vivo or in vitro. Therefore, in specific embodiments, immune effector cells include progenitor cells of immune effector cells, such as hematopoietic stem cells (HSCs) contained in CD34+ cell populations derived from umbilical cord blood, bone marrow or mobilized peripheral blood, which differentiate into mature immune effector cells after administration in a subject, or which can induce the HSCs to differentiate into mature immune effector cells in vitro.

[0376] As used herein, immune effector cells that have been genetically engineered to contain specific chimeric receptors may be referred to as "antigen-specific redirected immune effector cells."

[0377] 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) that typically acts as a cell-cell adhesion factor and participates in the entry of T cells into lymph nodes. The CD34+ cell population contains hematopoietic stem cells (HSCs), which differentiate and contribute to all hematopoietic lineages when administered to a patient, including cells of the T cell, NK cell, NKT cell, neutrophil, and monocyte / macrophage lineages.

[0378] In a specific embodiment, a method for preparing immune effector cells expressing MAGEA4 TCR or MAGEA4eTCR and chimeric TGFβ receptor polypeptides as encompassed herein is provided. In one embodiment, the method includes transfection or transduction of immune effector cells separated from an individual so that the immune effector cells express MAGEA4 TCR or MAGEA4 eTCR and one or more chimeric TGFβ receptor polypeptides as encompassed herein. In one embodiment, the method includes transfection or transduction of immune effector cells separated from an individual so that the immune effector cells express MAGEA4 TCR or MAGEA4eTCR and one or more chimeric TGFβ receptor polypeptides and MAGEA4 TCR or MAGEA4 eTCR encompassed herein. In certain embodiments, immune effector cells are separated from an individual and genetically modified without further manipulation in vitro. Such cells can then be directly administered to an individual again. In another embodiment, immune effector cells are first activated and stimulated to proliferate in vitro before genetic modification. In this regard, immune effector cells can be cultured before and / or after genetic modification.

[0379] In certain embodiments, prior to the in vitro manipulation or genetic modification of the immune effector cells described herein, the source of cells is obtained from a subject. In certain embodiments, the modified immune effector cells comprise T cells.

[0380] T cells can be obtained from a variety of sources, including but not limited to peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, any number of techniques known to those skilled in the art, such as sedimentation (e.g., FICOLL) can be used. TM T cells are obtained by isolation) from a unit of blood collected from a subject.

[0381] In other embodiments, isolated or purified T cell populations are used.In some embodiments, following isolation of PBMCs, cytotoxic and helper T lymphocytes may be sorted into naive, memory, and effector T cell subsets before or after activation, expansion, and / or genetic modification.

[0382] In one embodiment, the isolated or purified T cell population expresses one or more of the following markers, including but not limited to: CD3 + 、CD4 + 、CD8 + or a combination thereof.

[0383] In certain embodiments, T cells are isolated from an individual and first activated and stimulated to proliferate in vitro before being modified to express a chimeric TGF receptor polypeptide.

[0384] To achieve a sufficient therapeutic dose of a T cell composition, the T cells are typically subjected to one or more rounds of stimulation, activation, and / or expansion. T cells may generally be activated and expanded using methods such as those described in, for example, 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; and 6,867,041, each of which is incorporated herein by reference in its entirety. In specific embodiments, T cells are activated and expanded for about 6 hours, about 12 hours, about 18 hours, or about 24 hours prior to introduction of the vector or polynucleotide encoding the MAGEA4 TCR or MAGEA4 eTCR and chimeric TGFβ receptor polypeptide.

[0385] In one embodiment, the T cells are activated while being modified.

[0386] In various embodiments, a method for producing immune effector cells includes activating a cell population including T cells and expanding the T cell population. T cell activation can be achieved by providing a primary stimulation signal through a T cell TCR / CD3 complex and providing a secondary costimulatory signal through an auxiliary molecule (e.g., CD28).

[0387] The TCR / CD3 complex can be stimulated by contacting the T cell with an appropriate CD3 binding agent (e.g., a CD3 ligand or an anti-CD3 monoclonal antibody). Illustrative examples of CD3 antibodies include, but are not limited to, OKT3, G19-4, BC3, and 64.1.

[0388] In addition to the primary stimulation signal provided by the TCR / CD3 complex, the induction of T cell responses also requires a second costimulatory signal. In a specific embodiment, a CD28 binding agent can be used to provide a costimulatory signal. Illustrative examples of CD28 binding agents include, but are not limited to, natural CD 28 ligands, for example, natural ligands of CD28 (e.g., members of the B7 protein family, such as B7-1 (CD80) and B7-2 (CD86); and anti-CD28 monoclonal antibodies or fragments thereof capable of cross-linking CD28 molecules, for example, monoclonal antibodies 9.3, B-T3, XR-CD28, KOLT-2, 15E8, 248.23.2, and EX5.3D10.

[0389] In one embodiment, the molecule that provides the primary stimulatory signal, eg, the molecule that provides stimulation via the TCR / CD3 complex, and the co-stimulatory molecule are coupled to the same surface.

[0390] In certain embodiments, the binding agent that provides the stimulatory and co-stimulatory signals is localized on the surface of the cell. This can be achieved by transfecting or transducing the cell with a nucleic acid encoding the binding agent in a form suitable for expression of the binding agent on the cell surface, or alternatively coupling the binding agent to the cell surface.

[0391] In another embodiment, molecules that provide the primary stimulatory signal, such as molecules that provide stimulation through the TCR / CD3 complex, and co-stimulatory molecules are displayed on antigen presenting cells.

[0392] In one embodiment, the molecule that provides the primary stimulation signal, eg, the molecule that provides stimulation through the TCR / CD3 complex, and the co-stimulatory molecule are provided on separate surfaces.

[0393] In certain embodiments, one of the binding agents that provides the stimulatory signal and the co-stimulatory signal is soluble (provided in solution) and the other or additional binding agents are provided on one or more surfaces.

[0394] In certain embodiments, both the binding agents providing the stimulatory signal and the co-stimulatory signal are provided in soluble form (provided in solution).

[0395] In various embodiments, the methods contemplated herein for preparing T cells comprise activating T cells using anti-CD3 and anti-CD28 antibodies.

[0396] In one embodiment, the T cells activated by the methods encompassed herein are expanded and further include cultivating a cell population comprising the T cells for several hours (about 3 hours) to about 7 days to about 28 days or any hourly integer value therebetween. In another embodiment, the T cell composition can be cultivated for 14 days. In a specific embodiment, the T cells are cultivated for about 21 days. In another embodiment, the T cell composition is cultivated for about 2 to 3 days. It can also be expected that several stimulation / activation / amplification cycles are required so that the culture time of the T cells can be 60 days or longer.

[0397] In certain embodiments, conditions suitable for T cell culture comprise an appropriate culture medium (e.g., minimal essential medium or RPMI medium 1640 or X-vivo 15 (Lonza)) and one or more factors necessary for proliferation and viability, including, but not limited to, serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, IL-21, GM-CSF, IL-10, IL-12, IL-15, TGFβ, and TNF-α, or any other additives suitable for cell growth known to those of skill in the art.

[0398] Additional illustrative examples of cell culture media include, but are not limited to, RPMI 1640, Clicks, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15 and X-Vivo 20, Optimizer, supplemented with amino acids, sodium pyruvate, and vitamins, serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones and / or one or more cytokines in an amount sufficient to allow T cell growth and expansion.

[0399] Antibiotics (e.g., penicillin and streptomycin) are included only during experimental culture and not during culture of cells to be injected into a subject. Target cells are maintained under conditions necessary to support growth, such as an appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air plus 5% CO 2 ).

[0400] In specific embodiments, PBMCs or isolated T cells are contacted with stimulatory and co-stimulatory agents, such as anti-CD3 and anti-CD28 antibodies, typically attached to beads or other surfaces, in culture medium with appropriate cytokines, such as IL-2, IL-7, and / or IL-15.

[0401] In other embodiments, artificial APCs (aAPCs) are prepared by engineered K562, U937, 721.221, T2 and C1R cells to guide the stable expression and secretion of various costimulatory molecules and cytokines. In a specific embodiment, K32 or U32 aAPCs are used to guide one or more antibody-based stimulatory molecules to be displayed on the surface of AAPC cells. T cell populations can be expanded by aAPCs expressing various costimulatory molecules, including but not limited to CD137L (4-1BBL), CD134L (OX40L) and / or CD80 or CD86. Finally, aAPCs provide an efficient platform for expanding genetically modified T cells and maintaining CD28 expression on CD8 T cells. The aAPCs provided in WO 03 / 057171 and US2003 / 0147869 are incorporated herein by reference in their entirety.

[0402] In certain embodiments, one or more polynucleotides encoding a MAGEA4 TCR or MAGEA4 eTCR and a chimeric TGFβ receptor are introduced into a T cell population. In certain embodiments, a polynucleotide encoding a chimeric TGFβ receptor is introduced into a T cell population expressing a MAGEA4 TCR or MAGEA4 eTCR. In certain embodiments, a polynucleotide encoding a MAGEA4 TCR or MAGEA4 eTCR is introduced into a T cell population expressing a chimeric TGFβ receptor. In certain embodiments, a polynucleotide encoding a MAGEA4 TCR or MAGEA4 eTCR and a chimeric TGFβ receptor are introduced into a T cell population. In certain embodiments, a polynucleotide encoding a MAGEA4 TCR or MAGEA4 eTCR and a chimeric TGFβ receptor are introduced into a T cell population simultaneously. Polynucleotides can be introduced into T cells by microinjection, transfection, lipofection, heat shock, electroporation, transduction, gene gun, microinjection, DEAE-dextran-mediated transfer, and the like.

[0403] In a preferred embodiment, the polynucleotide is introduced into T cells by viral transduction.

[0404] Suitable for introducing polynucleotides into immune effector cells or CD34 + Illustrative examples of viral vector systems in cells include, but are not limited to, adeno-associated virus (AAV), retrovirus, herpes simplex virus, adenovirus, vaccinia virus vectors for gene transfer.

[0405] In one embodiment, the polynucleotide is introduced into T cells by AAV transduction.

[0406] In one embodiment, the polynucleotide is introduced into T cells by retroviral transduction.

[0407] In one embodiment, the polynucleotide is introduced into T cells by lentiviral transduction.

[0408] In one embodiment, the polynucleotide is introduced into T cells by adenoviral transduction.

[0409] In one embodiment, the polynucleotide is introduced into T cells by herpes simplex virus transduction.

[0410] In one embodiment, the polynucleotide is introduced into T cells by vaccinia viral transduction.

[0411] H. Compositions and Formulations

[0412] Compositions encompassed herein can include one or more MAGEA4 TCR polypeptides, MAGEA4 eTCR polypeptides, CTBR polypeptides, polynucleotides, vectors comprising the same, genetically modified immune effector cells, and the like, as contemplated herein. Compositions include, but are not limited to, pharmaceutical compositions. In preferred embodiments, the compositions comprise one or more cells modified to express MAGEA4 TCR and CTBR. In preferred embodiments, the compositions comprise one or more cells modified to express MAGEA4 eTCR and CTBR12 polypeptides.

[0413] A "pharmaceutical composition" refers to a composition formulated in a pharmaceutically acceptable or physiologically acceptable solution that is administered to cells or animals, alone or in combination with one or more other therapeutic modalities. It should also be understood that the composition can also be administered in combination with other agents, such as cytokines, growth factors, hormones, small molecules, chemotherapeutic agents, prodrugs, drugs, antibodies, or other various pharmaceutically active agents, if desired. There is virtually no limitation on the other components that may be included in the composition, provided that the additional agents do not adversely affect the ability of the composition to deliver the intended therapy. In a preferred embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, diluent, or excipient and one or more cells that have been modified to express the MAGEA4 TCR and CTBR, preferably the MAGEA4 eTCR and CTBR12 polypeptide.

[0414] The phrase "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0415] As used herein, "pharmaceutically acceptable carrier, diluent or excipient" includes, but is not limited to, isotonic saline; Ringer's solution; ethanol; phosphate buffered saline; and any other compatible substance employed in pharmaceutical formulations.

[0416] In certain embodiments, the composition comprises an amount of immune effector cells expressing MAGEA4 TCR and CTBR, preferably MAGEA4eTCR and CTBR12 polypeptides. As used herein, the term "amount" refers to an "effective amount" or "effective amount" of genetically modified therapeutic cells, such as T cells, to achieve beneficial or desired prophylactic or therapeutic results, including clinical results.

[0417] A "prophylactically effective amount" refers to an amount of genetically modified therapeutic cells effective to achieve the desired prophylactic result. Typically, but not necessarily, a prophylactic amount is less than a therapeutically effective amount because a prophylactic dose is administered to a subject prior to or at an early stage of disease.

[0418] A "therapeutically effective amount" of genetically modified therapeutic cells can vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the stem and progenitor cells to elicit a desired response in the individual. A therapeutically effective amount is also a dose in which any toxic or deleterious effects of the virus or transduced therapeutic cells are outweighed by the therapeutically beneficial effects. The term "therapeutically effective amount" includes an amount that is effective to "treat" a subject (e.g., a patient). When a therapeutic amount is indicated, the precise amount of the composition to be administered can be determined by a physician taking into account individual differences in age, weight, tumor size, extent of infection or metastasis, and the condition of the patient (subject).

[0419] Generally speaking, the pharmaceutical composition comprising the T cells described herein can be taken as 10 6 to 10 13 cells / kg body weight, preferably 10 8 to 10 13 The number of cells will depend on the desired end use of the composition, as will the type of cells contained therein. For the uses provided herein, the volume of cells is typically one liter or less, and can be 500 ml or less, or even 250 ml or 100 ml or less. Thus, the desired cell density is typically greater than 10 6 cells / ml and usually greater than 10 7 cells / ml, usually 10 8 cells / ml or greater. Clinically relevant numbers of immune cells can be assigned to cumulatively equal to or greater than 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 or 10 13 The compositions can be administered multiple times at doses within these ranges. The cells can be allogeneic, syngeneic, xenogeneic, or autologous to the patient being treated.

[0420] The composition is preferably formulated for parenteral administration, eg, intravascular (intravenous or intraarterial), intraperitoneal, or intramuscular administration.

[0421] Liquid pharmaceutical compositions, whether they are solutions, suspensions or other similar forms, may include one or more of the following: a sterile diluent, such as water for injection; a saline solution, preferably normal saline; Ringer's solution or isotonic sodium chloride. Parenteral formulations may be filled in ampoules, disposable syringes or multiple-dose vials made of glass or plastic. Injectable pharmaceutical compositions are preferably sterile.

[0422] In one embodiment, the T cell compositions encompassed herein are formulated in a pharmaceutically acceptable cell culture medium. Such compositions are suitable for administration to human subjects. In a specific embodiment, the pharmaceutically acceptable cell culture medium is a serum-free medium.

[0423] Serum-free culture media have several advantages over serum-containing culture media, including simplified and better-defined compositions, reduced levels of contaminants, elimination of possible infectious agent sources, and reduced costs. In various embodiments, serum-free culture media are animal-free and can optionally be protein-free. Optionally, the culture media can contain biopharmaceutically acceptable recombinant proteins. "Animal-free" culture media refers to culture media whose compositions are derived from non-animal sources. Recombinant proteins replace natural animal proteins in animal-free culture media, and nutrients are obtained from synthetic, plant, or microbial sources. In contrast, "protein-free" culture media are defined as being substantially protein-free.

[0424] Illustrative examples of serum-free culture media used in certain embodiments include, but are not limited to, QBSF-60 (Quality Biological, Inc.), StemPro-34 (Life Technologies), and X-VIVO 10.

[0425] In a preferred embodiment, the compositions comprising the immune effector cells contemplated herein are formulated in a solution comprising PlasmaLyte A.

[0426] In another preferred embodiment, the composition comprising the immune effector cells encompassed herein is formulated in a solution comprising a cryopreservation medium. For example, cryopreservation medium with a cryopreservation agent can be used to maintain high cell viability results after thawing. Illustrative examples of cryopreservation medium used in specific embodiments include, but are not limited to, CryoStor CS10, CryoStor CS5, and CryoStor CS2.

[0427] In a more preferred embodiment, the composition comprising the immune effector cells contemplated herein is formulated in a solution comprising 50:50 PlasmaLyte A:CryoStor CS10.

[0428] In certain embodiments, the composition comprises an effective amount of genome-edited immune effector cells modified to express MAGEA4 TCR and CTBR, preferably MAGEA4 eTCR and CTBR12 polypeptides, alone or in combination with one or more therapeutic agents. Thus, the immune effector cell composition can be administered alone or in combination with other known cancer treatments, such as radiotherapy, chemotherapy, transplantation, immunotherapy, hormone therapy, photodynamic therapy, and the like. The composition can also be administered in combination with antibiotics. Such therapeutic agents are acceptable in the art as standard treatments for specific disease states such as specific cancers as described herein. Exemplary therapeutic agents encompassed in certain embodiments include cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory agents, chemotherapeutic agents, radiotherapeutic agents, therapeutic antibodies, or other active agents and adjuvants.

[0429] In certain embodiments, compositions comprising genome-edited immune effector cells modified to express MAGEA4 TCR and CTBR, preferably MAGEA4 eTCR and CTBR12 polypeptides, can be administered in conjunction with any number of chemotherapeutic agents.

[0430] In certain embodiments, a composition comprising an immune effector modified to express a MAGEA4 TCR and CTBR, preferably a MAGEA4 eTCR and CTBR12 polypeptide, is administered with a therapeutic antibody. Illustrative examples of therapeutic antibodies suitable for combination with the modified T cells contemplated in particular embodiments include, but are not limited to, atezolizumab, avelumab, bavituximab, bevacizumab (avastin), bivatuzumab, blinatumomab, conatumumab, crizotinib, daratumumab, duligotumab, dacetuzumab, dalotuzumab, durvalumab, elotuzumab (HuL uc63), gemtuzumab, ibritumomab, indatuximab, inotuzumab, ipilimumab, lorvotuzumab, lucatumumab, milatuzumab, moxetumomab, nivolumab, ocaratuzumab, ofatumumab, pembrolizumab, rituximab, cetuximab, siltuximab, teprotumumab, and ublituximab.

[0431] In certain embodiments, the formulation of pharmaceutically acceptable carrier solutions is well known to those skilled in the art, as is the development of suitable dosing and treatment regimens for the use of the specific compositions described herein in a variety of treatment regimens, including, for example, enteral and parenteral, such as intravascular, intravenous, intraarterial, intraosseous, intraventricular, intracerebral, intracranial, intraspinal, intrathecal, and intramedullary administration and formulation. It will be understood by those skilled in the art that certain embodiments contemplated herein may include other formulations, such as those well known in the pharmaceutical arts and described, for example, in Remington: The Science and Practice of Pharmacy, Volumes I and II, 22nd Edition, ed.: Loyd V. Allen Jr. Philadelphia, PA: Pharmaceutical Press; 2012, each of which is incorporated herein by reference in its entirety.

[0432] I. Treatment Methods

[0433] Immune effector cells comprising the CTBRs contemplated herein, including MAGEA4 TCR T cells or MAGEA4 eTCRT cells, provide improved methods for adoptive immunotherapy for preventing, treating, and ameliorating cancer, or for preventing, treating, or ameliorating at least one symptom associated with cancer.

[0434] Immune effector cells comprising the MAGEA4 TCR or MAGEA4 eTCR and CTBR encompassed herein provide improved pharmaceutical products for preventing, treating, or ameliorating at least one symptom of cancer, GVHD, infectious diseases, autoimmune diseases, inflammatory diseases, or immunodeficiency. As used herein, the term "pharmaceutical product" refers to modified cells produced using the compositions and methods encompassed herein. In certain embodiments, the pharmaceutical product comprises genetically modified immune effector cells, T cells modified to express MAGEA4 TCR or MAGEA4 eTCR, further modified to express CTBR polypeptides. In addition, the modified T cells encompassed in certain embodiments provide safer and more effective adoptive cell therapy because they are resistant to T cell exhaustion and exhibit increased durability and persistence in the tumor microenvironment, which can lead to sustained therapy.

[0435] In certain embodiments, an effective amount of modified immune effector cells or T cells comprising or expressing MAGEA4 TCR or MAGEA4eTCR and CTBR is administered to a subject to prevent, treat, or ameliorate at least one symptom of cancer, GVHD, infectious disease, autoimmune disease, inflammatory disease, or immunodeficiency.

[0436] In certain embodiments, a method for preventing, treating, or ameliorating at least one symptom of cancer comprises administering to a subject an effective amount of modified immune effector cells or T cells comprising or expressing CTBR and MAGEA4 TCR or MAGEA4eTCR. The genetically modified cells are more durable and long-lasting pharmaceutical products because the cells are more resistant to immunosuppressive signals from the tumor microenvironment by converting immunosuppressive TGFβ signals into immunostimulatory signals.

[0437] In certain embodiments, the modified immune effector cells contemplated herein are used to treat solid tumors or cancers.

[0438] In certain embodiments, the modified immune effector cells encompassed herein are used to treat solid tumors or cancers, including but not limited to adrenal cancer, adrenocortical cancer, anal cancer, appendix cancer, astrocytoma, atypical teratoid / rhabdoid tumor, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain / CNS cancer, breast cancer, bronchial tumors, cardiac tumors, cervical cancer, bile duct cancer, chondrosarcoma, chordoma, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma in situ (DCIS), and chondrosarcoma. ) Endometrial cancer, ependymoma, esophageal cancer, nasal glioma, Ewing's sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, eye cancer, fallopian tube cancer, fibrosarcoma, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid, 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, lip cancer, fat meat tumors, liver cancer, lung cancer, non-small cell lung cancer, lung carcinoid, malignant mesothelioma, medullary carcinoma, medulloblastoma, meningioma, melanoma, Merkel cell carcinoma, midline cancer, oral cancer, mucosal sarcoma, myelodysplastic syndrome, myeloproliferative neoplasms, nasal cavity and sinus cancer, nasopharyngeal cancer, neuroblastoma, oligodendroglioma, oral cancer, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, islet cell tumor, papillary carcinoma, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer , pheochromocytoma, pinealoma, pituitary tumor, pleuropulmonary blastoma, primary peritoneal cancer, prostate cancer, rectal cancer, retinoblastoma, renal cell carcinoma, renal pelvis and ureter cancer, rhabdomyosarcoma, salivary gland cancer, sebaceous gland cancer, skin cancer, soft tissue sarcoma, squamous cell carcinoma, small cell lung cancer, small intestine cancer, stomach cancer, sweat gland cancer, synovioma, testicular cancer, pharyngeal cancer, thymus cancer, thyroid cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vascular cancer, vulvar cancer and Wilms tumor.

[0439] In certain embodiments, the modified immune effector cells contemplated herein are used to treat solid tumors or cancers, including but not limited to liver cancer, pancreatic cancer, lung cancer, breast cancer, bladder cancer, brain cancer, bone cancer, thyroid cancer, kidney cancer, or skin cancer.

[0440] In certain embodiments, the modified immune effector cells contemplated herein are used to treat various cancers, including but not limited to pancreatic cancer, bladder cancer, and lung cancer.

[0441] In certain embodiments, the modified immune effector cells contemplated herein are used to treat liquid or hematological cancers.

[0442] In certain embodiments, the modified immune effector cells contemplated herein are used to treat B-cell malignancies, including but not limited to leukemias, lymphomas, and multiple myeloma.

[0443] In certain embodiments, the modified immune effector cells encompassed herein are used to treat liquid cancers, including but not limited to leukemias, lymphomas, and multiple myeloma: 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 myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML) and polycythemia vera, Hodgkin lymphoma, nodular lymphocyte-predominant Hodgkin lymphoma, Burkitt lymphoma, lymphoma), small lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, mantle cell lymphoma, marginal zone lymphoma, mycosis fungoides, anaplastic large cell lymphoma, Sézary syndrome, precursor T-lymphoblastic lymphoma, multiple myeloma, overt multiple myeloma, smoldering multiple myeloma, plasma cell leukemia, nonsecretory myeloma, IgD myeloma, osteosclerotic myeloma, solitary plasmacytoma of bone, and extramedullary plasmacytoma.

[0444] Preferred cells for use in the methods contemplated herein include autologous / self ("self") cells, preferably hematopoietic cells, more preferably T cells, and more preferably immune effector cells.

[0445] In certain embodiments, methods are provided comprising administering a therapeutically effective amount of modified immune effector cells encompassed herein, or compositions comprising the same, alone or in combination with one or more therapeutic agents, to a patient in need thereof. In certain embodiments, the cells are used to treat patients at risk for cancer, GVHD, infectious diseases, autoimmune diseases, inflammatory diseases, or immunodeficiency. Thus, certain embodiments include treating or preventing cancer, infectious diseases, autoimmune diseases, inflammatory diseases, or immunodeficiency, or ameliorating at least one symptom thereof, comprising administering a therapeutically effective amount of genome-edited cells encompassed herein to a subject in need thereof.

[0446] In one embodiment, a method of treating cancer, GVHD, infectious disease, autoimmune disease, inflammatory disease, or immunodeficiency in a subject in need thereof comprises administering an effective amount, e.g., a therapeutically effective amount, of a composition comprising modified immune effector cells as contemplated herein. The amount and frequency of administration will be determined by factors such as the patient's condition and the type and severity of the patient's disease, although appropriate dosages can be determined through clinical trials.

[0447] In one illustrative embodiment, the effective amount of modified immune effector cells provided to a subject is at least 2×10 6 cells / kg, at least 3×10 6 cells / kg, at least 4×10 6 cells / kg, at least 5×10 6 cells / kg, at least 6×10 6 cells / kg, at least 7×10 6 cells / kg, at least 8×10 6 cells / kg, at least 9×10 6 cells / kg, or at least 10×10 6 cells / kg or more, including all intermediate doses of cells.

[0448] In another illustrative embodiment, the effective amount of modified immune effector cells provided to a subject is about 2×10 6 cells / kg, about 3×10 6 cells / kg, about 4×10 6 cells / kg, about 5×10 6 cells / kg, about 6×10 6 cells / kg, about 7×10 6 cells / kg, about 8×10 6 cells / kg, about 9×10 6 cells / kg, or approximately 10×10 6cells / kg or more, including all intermediate doses of cells.

[0449] In another illustrative embodiment, the effective amount of modified immune effector cells provided to a subject is about 2×10 6 cells / kg to about 10×10 6 cells / kg, about 3×10 6 cells / kg to about 10×10 6 cells / kg, about 4×10 6 cells / kg to about 10×10 6 cells / kg, about 5×10 6 cells / kg to about 10×10 6 cells / kg, 2×10 6 cells / kg to about 6×10 6 cells / kg, 2×10 6 cells / kg to about 7×10 6 cells / kg, 2×10 6 cells / kg to about 8×10 6 cells / kg, 3×10 6 cells / kg to about 6×10 6 cells / kg, 3×10 6 cells / kg to about 7×10 6 cells / kg, 3×10 6 cells / kg to about 8×10 6 cells / kg, 4×10 6 cells / kg to about 6×10 6 cells / kg, 4×10 6 cells / kg to about 7×10 6 cells / kg, 4×10 6 cells / kg to about 8×10 6 cells / kg, 5×10 6 cells / kg to about 6×10 6 cells / kg, 5×10 6 cells / kg to about 7×10 6 cells / kg, 5×10 6 cells / kg to about 8×10 6 cells / kg or 6×10 6 cells / kg to about 8×10 6 cells / kg, including all intermediate doses.

[0450] Those of ordinary skill in the art will recognize that multiple administrations of the compositions encompassed in a particular embodiment may be required to achieve the desired therapy. For example, the composition can be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times or more over a span 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.

[0451] In certain embodiments, it may be desirable to administer activated T cells to a subject, and subsequently redraw blood (or perform apheresis), activate T cells therefrom, and re-infuse these activated and amplified T cells to the patient. This process can be performed multiple times every few weeks. In certain embodiments, 10cc to 400cc of blood can be drawn to activate T cells. In certain embodiments, 20cc, 30cc, 40cc, 50cc, 60cc, 70cc, 80cc, 90cc, 100cc, 150cc, 200cc, 250cc, 300cc, 350cc, or 400cc or more of blood are drawn to activate T cells. Without being bound by theory, using this multiple blood draw / multiple re-infusion protocol can be used to select certain T cell populations.

[0452] In one embodiment, a method for treating a subject diagnosed with cancer comprises: removing immune effector cells from the subject; modifying the immune effector cells by introducing one or more vectors encoding MAGEA4 TCR or MAGEA4eTCR and a chimeric TGFβ receptor and generating a population of modified immune effector cells; and administering the modified immune effector cell population to the same subject. In a preferred embodiment, the immune effector cells comprise T cells.

[0453] Methods for administering the cell compositions encompassed in certain embodiments include any method that effectively results in the reintroduction of modified immune effector cells ex vivo or modified progenitors of immune effector cells that differentiate into mature immune effector cells when introduced into a subject. One method includes ex vivo modification of peripheral blood T cells by introducing one or more vectors encoding MAGEA4 TCR or MAGEA4 eTCR and a chimeric TGFβ receptor and returning the transduced cells to the subject.

[0454] 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.

[0455] Although the foregoing embodiments have been described in considerable detail by way of illustration and example for purposes of clarity of understanding, those skilled in the art will readily appreciate, based on the teachings contained herein, that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. The following examples are provided for illustration only and are not intended to be limiting. Those skilled in the art will readily recognize a variety of non-critical parameters that may be changed or modified to produce substantially similar results.

[0456] Example

[0457] Example 1

[0458] T cells expressing the TGFβ signal transducer MAGEA4 TCR transduce IL-12 signals and secrete IL-12 in response to MAGEA4 and TGF Β 1 increased IFNγ

[0459] Peripheral blood mononuclear cells (PBMCs) from healthy donors were activated with soluble anti-CD3 (50 ng / ml) and anti-CD28 (50 ng / ml) and transduced (MOI = 20) with lentiviral vectors (LVV) expressing (i) MAGEA4 TCR (e.g., SEQ ID NO: 4) or (ii) MAGEA4 TCR and IL-12-responsive chimeric TGFβ signal transducer (CTBR12) encoded on separate vectors, e.g., SEQ ID NOs: 4 and 8. After 10 days of culture in IL2-containing medium, cell products were harvested for in vitro analysis and cryopreserved.

[0460] CTBR12 signaling

[0461] IL-12 signaling involves receptor dimerization and activation of STAT4 via phosphorylation. STAT4 phosphorylation in response to TGFβ was assessed. Smad2 / 3 phosphorylation was also assessed to verify that CTBR12 blocked natural TGFβ signaling. MAGEA4 TCRT cells and MAGEA4 TCR / CTBR12 T cells were kept in serum-free medium overnight and then exposed to TGFβ1 (10 ng / ml) for 20 minutes. The cells were fixed, permeabilized, and stained with anti-phospho-Smad2 / 3 (pS465 / 467) and phospho-STAT4 (pY693). CTBR12 blocked the phosphorylation of Smad2 / 3 and activated STAT4 in T cells expressing MAGEA4 TCR ( Figure 1 , far right). These data indicate that CTBR12 can block native TGFβ signaling and transduce IL-12 signaling when co-expressed with the MAGEA4 TCR.

[0462] MAGEA4 TCR signaling

[0463] Functional TCRs secrete IFNγ in response to antigens, and secretion can be enhanced by IL-12 signaling. Untransduced (UTD) T cells, MAGEA4 TCR T cells, and MAGEA4 TCR / CTBR12 T cells were co-cultured with A375 MAGEA4 TCRs at a 1:1 E:T ratio in the presence or absence of TGFβ1 (10 ng / ml). + Tumor cells were co-cultured for 24 hours. After 24 hours, the amount of IFNγ secreted into the culture medium was measured. MAGEA4 TCR / CTBR12 T cells produced significantly greater amounts of IFNγ in the presence of TGFβ1 compared to all other treatments or control conditions. Figure 2 These data demonstrate that CTBR12 expression in MAGEA4 TCR T cells protects against TGFβ immunosuppression in vitro and promotes enhanced effector function.

[0464] Example 2

[0465] CTBR12 expression enhances the efficacy of MAGEA4 TCR T cells in vivo

[0466] A xenograft NOD.Cg-Prkdcscid IL2rgtm1Wjl / SzJ (NSG) mouse model was used to evaluate whether CTBR12 expression enhances the in vivo efficacy of MAGEA4 TCR T cells. NSG mice were subcutaneously implanted with A375 MAGEA4 + Tumor cells. Tumor volume was measured twice a week by caliper and calculated using the formula tumor volume = length × width × height × 0.52. When tumors reached an average volume of 50 mm3, mice were injected intravenously with 0.625 × 10 6 UTD T cells, 0.625×10 6 GVY tetramer-positive MAGEA4 TCR T cells or 0.625 × 10 6 GVY tetramer-positive MAGEA4 TCR / CTBR12 T cells. MAGEA4TCR / CTBR12 T cells significantly controlled tumor volume better than MAGEA4 TCR T cells or UTD control T cells. Figure 3 These data show that CTBR12 expression enhances the in vivo efficacy of MAGEA4 TCR T cells.

[0467] Example 3

[0468] T cells expressing the enhanced MAGEA4 TCR of the TGFβ signal transducer transduce IL-12 signals and secrete

[0469] Increased IFNγ in response to MAGEA4 and TGFβ1

[0470] Peripheral blood mononuclear cells (PBMCs) from healthy donors were activated with soluble anti-CD3 (50 ng / ml) and anti-CD28 (50 ng / ml) and transduced (MOI = 20) with lentiviral vectors (LVVs) expressing (i) MAGEA4 paired enhanced TCR (eTCR), e.g., SEQ ID NO: 7 or (ii) MAGEA4 eTCR and CTBR12 receptor (e.g., SEQ ID NO: 7 and 8) encoded on the same vector. After 10 days of culture in IL2-containing medium, cell products were harvested for in vitro analysis and cryopreserved.

[0471] CTBR12 signaling

[0472] IL-12 signaling involves receptor dimerization and activation of STAT4 via phosphorylation. STAT4 phosphorylation in response to TGFβ was assessed. Smad2 / 3 phosphorylation was also assessed to verify that CTBR12 blocked natural TGFβ signaling. MAGEA4 eTCRT cells and MAGEA4 eTCR / CTBR12 T cells were kept in serum-free medium overnight and then exposed to TGFβ1 (10 ng / ml) for 20 minutes. The cells were fixed, permeabilized, and stained with anti-phospho-Smad2 / 3 (pS465 / 467) and phospho-STAT4 (pY693). CTBR12 blocked the phosphorylation of Smad2 / 3 and activated STAT4 in T cells expressing MAGEA4 eTCR ( Figure 4 , far right). These data indicate that CTBR12 can block native TGFβ signaling and transduce IL-12 signaling when co-expressed with the MAGEA4 eTCR.

[0473] MAGEA4 TCR signaling

[0474] Functional TCRs secrete IFNγ in response to antigens, and secretion can be enhanced by IL-12 signaling. Untransduced (UTD) T cells, MAGEA4 eTCR T cells, and MAGEA4 eTCR / CTBR12 T cells were co-cultured with A375 MAGEA4 in the presence or absence of TGFβ1 (10 ng / ml) at a 1:1 E:T ratio. +Tumor cells were co-cultured for 24 hours. After 24 hours, the amount of IFNγ secreted into the culture medium was measured. TGFβ1 treatment inhibited IFNγ secretion by MAGEA4 eTCRT cells and enhanced IFNγ secretion by MAGEA4 eTCR / CTBR12 T cells. Figure 5 These data demonstrate that CTBR12 expression in MAGEA4 eTCRT cells protects against TGFβ immunosuppression in vitro and promotes enhanced effector function.

[0475] 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 encompass all possible embodiments, along with the full scope of equivalents to which such claims are entitled. Therefore, the claims are not limited by this disclosure. Sequence Listing <110> bluebird bio, Inc. Boyerinas, Benjamin Mann, Jasdeep Christian Ellinger Daniel Sommermeyer <120> Engineered T cells <130> BLUE-122.PC <150> US 62 / 845,311 <151> 2019-05-08 <160> 45 <170> PatentIn version 3.5 <210> 1 <211> 10 <212> PRT <213> Homo sapiens <400> 1 Gly Val Tyr Asp Gly Arg Glu His Thr Val 1 5 10 <210> 2 <211> 277 <212> PRT <213> Homo sapiens <400> 2 Met Ser Leu Ser Ser Leu Leu Lys Val Val Thr Ala Ser Leu Trp Leu 1 5 10 15 Gly Pro Gly Ile Ala Gln Lys Ile Thr Gln Thr Gln Pro Gly Met Phe 20 25 30 Val Gln Glu Lys Glu Ala Val Thr Leu Asp Cys Thr Tyr Asp Thr Ser 35 40 45 Asp Pro Ser Tyr Gly Leu Phe Trp Tyr Lys Gln Pro Ser Ser Gly Glu 50 55 60 Met Ile Phe Leu Ile Tyr Gln Gly Ser Tyr Asp Gln Gln Asn Ala Thr 65 70 75 80 Glu Gly Arg Tyr Ser Leu Asn Phe Gln Lys Ala Arg Lys Ser Ala Asn 85 90 95 Leu Val Ile Ser Ala Ser Gln Leu Gly Asp Ser Ala Met Tyr Phe Cys 100 105 110 Ala Met Ser Gly Gly Tyr Thr Gly Gly Phe Lys Thr Ile Phe Gly Ala 115 120 125 Gly Thr Arg Leu Phe Val Lys Ala Asn Ile Gln Asn Pro Asp Pro Ala 130 135 140 Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu 145 150 155 160 Phe Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser 165 170 175 Asp Val Tyr Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp 180 185 190 Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala 195 200 205 Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe 210 215 220 Pro Ser Pro Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe 225 230 235 240 Glu Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe 245 250 255 Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu 260 265 270 Arg Leu Trp Ser Ser 275 <210> 3 <211> 310 <212> PRT <213> Homo sapiens <400> 3 Met Gly Phe Arg Leu Leu Cys Cys Val Ala Phe Cys Leu Leu Gly Ala 1 5 10 15 Gly Pro Val Asp Ser Gly Val Thr Gln Thr Pro Lys His Leu Ile Thr 20 25 30 Ala Thr Gly Gln Arg Val Thr Leu Arg Cys Ser Pro Arg Ser Gly Asp 35 40 45 Leu Ser Val Tyr Trp Tyr Gln Gln Ser Leu Asp Gln Gly Leu Gln Phe 50 55 60 Leu Ile Gln Tyr Tyr Asn Gly Glu Glu Arg Ala Lys Gly Asn Ile Leu 65 70 75 80 Glu Arg Phe Ser Ala Gln Gln Phe Pro Asp Leu His Ser Glu Leu Asn 85 90 95 Leu Ser Ser Leu Glu Leu Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser 100 105 110 Ser Gly Gly Asp Gly Asp Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu 115 120 125 Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val 130 135 140 Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu 145 150 155 160 Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp 165 170 175 Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln 180 185 190 Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser 195 200 205 Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His 210 215 220 Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp 225 230 235 240 Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala 245 250 255 Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly 260 265 270 Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr 275 280 285 Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys 290 295 300 Arg Lys Asp Ser Arg Gly 305 310 <210> 4 <211> 613 <212> PRT <213> Artificial sequence <220> <223> Laboratory-made fusion proteins <400> 4 Met Gly Phe Arg Leu Leu Cys Cys Val Ala Phe Cys Leu Leu Gly Ala 1 5 10 15 Gly Pro Val Asp Ser Gly Val Thr Gln Thr Pro Lys His Leu Ile Thr 20 25 30 Ala Thr Gly Gln Arg Val Thr Leu Arg Cys Ser Pro Arg Ser Gly Asp 35 40 45 Leu Ser Val Tyr Trp Tyr Gln Gln Ser Leu Asp Gln Gly Leu Gln Phe 50 55 60 Leu Ile Gln Tyr Tyr Asn Gly Glu Glu Arg Ala Lys Gly Asn Ile Leu 65 70 75 80 Glu Arg Phe Ser Ala Gln Gln Phe Pro Asp Leu His Ser Glu Leu Asn 85 90 95 Leu Ser Ser Leu Glu Leu Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser 100 105 110 Ser Gly Gly Asp Gly Asp Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu 115 120 125 Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val 130 135 140 Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu 145 150 155 160 Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp 165 170 175 Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln 180 185 190 Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser 195 200 205 Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His 210 215 220 Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp 225 230 235 240 Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala 245 250 255 Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly 260 265 270 Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr 275 280 285 Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys 290 295 300 Arg Lys Asp Ser Arg Gly Arg Ala Lys Arg Gly Ser Gly Ala Thr Asn 305 310 315 320 Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro 325 330 335 Met Ser Leu Ser Ser Leu Leu Lys Val Val Thr Ala Ser Leu Trp Leu 340 345 350 Gly Pro Gly Ile Ala Gln Lys Ile Thr Gln Thr Gln Pro Gly Met Phe 355 360 365 Val Gln Glu Lys Glu Ala Val Thr Leu Asp Cys Thr Tyr Asp Thr Ser 370 375 380 Asp Pro Ser Tyr Gly Leu Phe Trp Tyr Lys Gln Pro Ser Ser Gly Glu 385 390 395 400 Met Ile Phe Leu Ile Tyr Gln Gly Ser Tyr Asp Gln Gln Asn Ala Thr 405 410 415 Glu Gly Arg Tyr Ser Leu Asn Phe Gln Lys Ala Arg Lys Ser Ala Asn 420 425 430 Leu Val Ile Ser Ala Ser Gln Leu Gly Asp Ser Ala Met Tyr Phe Cys 435 440 445 Ala Met Ser Gly Gly Tyr Thr Gly Gly Phe Lys Thr Ile Phe Gly Ala 450 455 460 Gly Thr Arg Leu Phe Val Lys Ala Asn Ile Gln Asn Pro Asp Pro Ala 465 470 475 480 Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu 485 490 495 Phe Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser 500 505 510 Asp Val Tyr Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp 515 520 525 Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala 530 535 540 Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe 545 550 555 560 Pro Ser Pro Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe 565 570 575 Glu Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe 580 585 590 Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu 595 600 605 Arg Leu Trp Ser Ser 610 <210> 5 <211> 277 <212> PRT <213> Artificial Sequence <220> <223> Laboratory-made - Synthetic construct <400> 5 Met Ser Leu Ser Ser Leu Leu Lys Val Val Thr Ala Ser Leu Trp Leu 1 5 10 15 Gly Pro Gly Ile Ala Gln Lys Ile Thr Gln Thr Gln Pro Gly Met Phe 20 25 30 Val Gln Glu Lys Glu Ala Val Thr Leu Asp Cys Thr Tyr Asp Thr Ser 35 40 45 Asp Pro Ser Tyr Gly Leu Phe Trp Tyr Lys Gln Pro Ser Ser Gly Glu 50 55 60 Met Ile Phe Leu Ile Tyr Gln Gly Ser Tyr Asp Gln Gln Asn Ala Thr 65 70 75 80 Glu Gly Arg Tyr Ser Leu Asn Phe Gln Lys Ala Arg Lys Ser Ala Asn 85 90 95 Leu Val Ile Ser Ala Ser Gln Leu Gly Asp Ser Ala Met Tyr Phe Cys 100 105 110 Ala Met Ser Gly Gly Tyr Thr Gly Gly Phe Lys Thr Ile Phe Gly Ala 115 120 125 Gly Thr Arg Leu Phe Val Lys Ala Asn Ile Gln Asn Pro Asp Pro Ala 130 135 140 Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu 145 150 155 160 Phe Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser 165 170 175 Asp Val Tyr Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp 180 185 190 Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala 195 200 205 Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe 210 215 220 Pro Ser Ser Asp Val Pro Cys Asp Val Lys Leu Val Glu Lys Ser Phe 225 230 235 240 Glu Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu Leu Val Ile Val Leu 245 250 255 Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu 260 265 270 Arg Leu Trp Ser Ser 275 <210> 6 <211> 310 <212> PRT <213> Artificial sequence <220> <223> Laboratory-made - synthetic constructs <400> 6 Met Gly Phe Arg Leu Leu Cys Cys Val Ala Phe Cys Leu Leu Gly Ala 1 5 10 15 Gly Pro Val Asp Ser Gly Val Thr Gln Thr Pro Lys His Leu Ile Thr 20 25 30 Ala Thr Gly Gln Arg Val Thr Leu Arg Cys Ser Pro Arg Ser Gly Asp 35 40 45 Leu Ser Val Tyr Trp Tyr Gln Gln Ser Leu Asp Gln Gly Leu Gln Phe 50 55 60 Leu Ile Gln Tyr Tyr Asn Gly Glu Glu Arg Ala Lys Gly Asn Ile Leu 65 70 75 80 Glu Arg Phe Ser Ala Gln Gln Phe Pro Asp Leu His Ser Glu Leu Asn 85 90 95 Leu Ser Ser Leu Glu Leu Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser 100 105 110 Ser Gly Gly Asp Gly Asp Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu 115 120 125 Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val 130 135 140 Phe Glu Pro Ser Lys Ala Glu Ile Ala His Thr Gln Lys Ala Thr Leu 145 150 155 160 Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp 165 170 175 Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln 180 185 190 Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser 195 200 205 Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His 210 215 220 Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp 225 230 235 240 Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala 245 250 255 Trp Gly Arg Ala Asp Cys Gly Ile Thr Ser Ala Ser Tyr His Gln Gly 260 265 270 Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr 275 280 285 Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys 290 295 300 Arg Lys Asp Ser Arg Gly 305 310 <210> 7 <211> 613 <212> PRT <213> Artificial Sequence <220> <223> Laboratory-made - Fusion Protein <400> 7 Met Gly Phe Arg Leu Leu Cys Cys Val Ala Phe Cys Leu Leu Gly Ala 1 5 10 15 Gly Pro Val Asp Ser Gly Val Thr Gln Thr Pro Lys His Leu Ile Thr 20 25 30 Ala Thr Gly Gln Arg Val Thr Leu Arg Cys Ser Pro Arg Ser Gly Asp 35 40 45 Leu Ser Val Tyr Trp Tyr Gln Gln Ser Leu Asp Gln Gly Leu Gln Phe 50 55 60 Leu Ile Gln Tyr Tyr Asn Gly Glu Glu Arg Ala Lys Gly Asn Ile Leu 65 70 75 80 Glu Arg Phe Ser Ala Gln Gln Phe Pro Asp Leu His Ser Glu Leu Asn 85 90 95 Leu Ser Ser Leu Glu Leu Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser 100 105 110 Ser Gly Gly Asp Gly Asp Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu 115 120 125 Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val 130 135 140 Phe Glu Pro Ser Lys Ala Glu Ile Ala His Thr Gln Lys Ala Thr Leu 145 150 155 160 Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp 165 170 175 Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln 180 185 190 Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser 195 200 205 Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His 210 215 220 Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp 225 230 235 240 Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala 245 250 255 Trp Gly Arg Ala Asp Cys Gly Ile Thr Ser Ala Ser Tyr His Gln Gly 260 265 270 Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr 275 280 285 Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys 290 295 300 Arg Lys Asp Ser Arg Gly Arg Ala Lys Arg Gly Ser Gly Ala Thr Asn 305 310 315 320 Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro 325 330 335 Met Ser Leu Ser Ser Leu Leu Lys Val Val Thr Ala Ser Leu Trp Leu 340 345 350 Gly Pro Gly Ile Ala Gln Lys Ile Thr Gln Thr Gln Pro Gly Met Phe 355 360 365 Val Gln Glu Lys Glu Ala Val Thr Leu Asp Cys Thr Tyr Asp Thr Ser 370 375 380 Asp Pro Ser Tyr Gly Leu Phe Trp Tyr Lys Gln Pro Ser Ser Gly Glu 385 390 395 400 Met Ile Phe Leu Ile Tyr Gln Gly Ser Tyr Asp Gln Gln Asn Ala Thr 405 410 415 Glu Gly Arg Tyr Ser Leu Asn Phe Gln Lys Ala Arg Lys Ser Ala Asn 420 425 430 Leu Val Ile Ser Ala Ser Gln Leu Gly Asp Ser Ala Met Tyr Phe Cys 435 440 445 Ala Met Ser Gly Gly Tyr Thr Gly Gly Phe Lys Thr Ile Phe Gly Ala 450 455 460 Gly Thr Arg Leu Phe Val Lys Ala Asn Ile Gln Asn Pro Asp Pro Ala 465 470 475 480 Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu 485 490 495 Phe Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser 500 505 510 Asp Val Tyr Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp 515 520 525 Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala 530 535 540 Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe 545 550 555 560 Pro Ser Ser Asp Val Pro Cys Asp Val Lys Leu Val Glu Lys Ser Phe 565 570 575 Glu Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu Leu Val Ile Val Leu 580 585 590 Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu 595 600 605 Arg Leu Trp Ser Ser 610 <210> 8 <211> 671 <212> PRT <213> Artificial sequence <220> <223> Laboratory-made - synthetic constructs <400> 8 Met Gly Arg Gly Leu Leu Arg Gly Leu Trp Pro Leu His Ile Val Leu 1 5 10 15 Trp Thr Arg Ile Ala Ser Thr Ile Pro Pro His Val Gln Lys Ser Val 20 25 30 Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro 35 40 45 Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln 50 55 60 Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro 65 70 75 80 Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr 85 90 95 Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile 100 105 110 Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys 115 120 125 Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn 130 135 140 Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Leu 145 150 155 160 Leu Leu Val Ile Phe Gln Trp Met Ala Phe Val Ala Pro Ser Ile Cys 165 170 175 Ile Ala Ile Ile Met Val Gly Ile Phe Ser Thr His Tyr Phe Gln Gln 180 185 190 Lys Val Phe Val Leu Leu Ala Ala Leu Arg Pro Gln Trp Cys Ser Arg 195 200 205 Glu Ile Pro Asp Pro Ala Asn Ser Thr Cys Ala Lys Lys Tyr Pro Ile 210 215 220 Ala Glu Glu Lys Thr Gln Leu Pro Leu Asp Arg Leu Leu Ile Asp Trp 225 230 235 240 Pro Thr Pro Glu Asp Pro Glu Pro Leu Val Ile Ser Glu Val Leu His 245 250 255 Gln Val Thr Pro Val Phe Arg His Pro Pro Cys Ser Asn Trp Pro Gln 260 265 270 Arg Glu Lys Gly Ile Gln Gly His Gln Ala Ser Glu Lys Asp Met Met 275 280 285 His Ser Ala Ser Ser Pro Pro Pro Pro Arg Ala Leu Gln Ala Glu Ser 290 295 300 Arg Gln Leu Val Asp Leu Tyr Lys Val Leu Glu Ser Arg Gly Ser Asp 305 310 315 320 Pro Lys Pro Glu Asn Pro Ala Cys Pro Trp Thr Val Leu Pro Ala Gly 325 330 335 Asp Leu Pro Thr His Asp Gly Tyr Leu Pro Ser Asn Ile Asp Asp Leu 340 345 350 Pro Ser His Glu Ala Pro Leu Ala Asp Ser Leu Glu Glu Leu Glu Pro 355 360 365 Gln His Ile Ser Leu Ser Val Phe Pro Ser Ser Ser Leu His Pro Leu 370 375 380 Thr Phe Ser Cys Gly Asp Lys Leu Thr Leu Asp Gln Leu Lys Met Arg 385 390 395 400 Cys Asp Ser Leu Met Leu Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu 405 410 415 Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Glu Ala Ala 420 425 430 Val Ala Ala Pro Arg Pro Arg Leo Leo Leo Leo Val Leo Ala Ala Ala 435 440 445 Ala Ala Ala Ala Ala Ala Leu Leu Pro Gly Ala Thr Ala Leu Gln Cys 450 455 460 Phe Cys His Leu Cys Thr Lys Asp Asn Phe Thr Cys Val Thr Asp Gly 465 470 475 480 Leu Cys Phe Val Ser Val Thr Glu Thr Thr Asp Lys Val Ile His Asn 485 490 495 Ser Met Cys Ile Ala Glu Ile Asp Leu Ile Pro Arg Asp Arg Pro Phe 500 505 510 Val Cys Ala Pro Ser Ser Lys Thr Gly Ser Val Thr Thr Thr Tyr Cys 515 520 525 Cys Asn Gln Asp His Cys Asn Lys Ile Glu Leu Pro Thr Thr Val Lys 530 535 540 Ser Ser Pro Gly Leu Gly Pro Val Glu Leu Trp Leu Ile Phe Phe Ala 545 550 555 560 Ser Leu Gly Ser Phe Leu Ser Ile Leu Leu Val Gly Val Leu Gly Tyr 565 570 575 Leu Gly Leu Asn Arg Ala Ala Arg His Leu Cys Pro Pro Leu Pro Thr 580 585 590 Pro Cys Ala Ser Ser Ala Ile Glu Phe Pro Gly Gly Lys Glu Thr Trp 595 600 605 Gln Trp Ile Asn Pro Val Asp Phe Gln Glu Glu Ala Ser Leu Gln Glu 610 615 620 Ala Leu Val Val Glu Met Ser Trp Asp Lys Gly Glu Arg Thr Glu Pro 625 630 635 640 Leu Glu Lys Thr Glu Leu Pro Glu Gly Ala Pro Glu Leu Ala Leu Asp 645 650 655 Thr Glu Leu Ser Leu Glu Asp Gly Asp Arg Cys Lys Ala Lys Met 660 665 670 <210> 9 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Exemplary linker sequences <400> 9 Asp Gly Gly Gly Ser 1 5 <210> 10 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Exemplary linker sequences <400> 10 Thr Gly Glu Lys Pro 1 5 <210> 11 <211> 4 <212> PRT <213> Artificial sequence <220> <223> Exemplary linker sequences <400> 11 Gly Gly Arg Arg 1 <210> 12 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Exemplary linker sequences <400> 12 Gly Gly Gly Gly Ser 1 5 <210> 13 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Exemplary linker sequences <400> 13 Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Val Asp 1 5 10 <210> 14 <211> 18 <212> PRT <213> Artificial sequence <220> <223> Exemplary linker sequences <400> 14 Lys Glu Ser Gly Ser Val Ser Ser Glu Gln Leu Ala Gln Phe Arg Ser 1 5 10 15 Leu Asp <210> 15 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Exemplary linker sequences <400> 15 Gly Gly Arg Arg Gly Gly Gly Ser 1 5 <210> 16 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Exemplary linker sequences <400> 16 Leu Arg Gln Arg Asp Gly Glu Arg Pro 1 5 <210> 17 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Exemplary linker sequences <400> 17 Leu Arg Gln Lys Asp Gly Gly Gly Ser Glu Arg Pro 1 5 10 <210> 18 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Exemplary linker sequences <400> 18 Leu Arg Gln Lys Asp Gly Gly Gly Ser Gly Gly Gly Ser Glu Arg Pro 1 5 10 15 <210> 19 <211> 18 <212> PRT <213> Artificial sequence <220> <223> Exemplary linker sequences <400> 19 Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr 1 5 10 15 Lys Gly <210> 20 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Cleavage sequence of TEV protease <220> <221> misc_feature <222> (2)..(3) <223> Xaa is any amino acid <220> <221> misc_feature <222> (5)..(5) <223> Xaa is any amino acid <220> <221> MISC_FEATURE <222> (7)..(7) <223> Xaa = Gly or Ser <400> 20 Glu Xaa Xaa Tyr Xaa Gln Xaa 1 5 <210> twenty one <211> 7 <212> PRT <213> Artificial sequence <220> <223> Cleavage sequence of TEV protease <400> twenty one Glu Asn Leu Tyr Phe Gln Gly 1 5 <210> twenty two <211> 7 <212> PRT <213> Artificial sequence <220> <223> Cleavage sequence of TEV protease <400> twenty two Glu Asn Leu Tyr Phe Gln Ser 1 5 <210> twenty three <211> twenty two <212> PRT <213> Artificial sequence <220> <223> Self-cleaving peptide containing 2A site <400> twenty three Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val 1 5 10 15 Glu Glu Asn Pro Gly Pro 20 <210> twenty four <211> 19 <212> PRT <213> Artificial sequence <220> <223> Self-cleaving peptide containing 2A site <400> twenty four Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn 1 5 10 15 Pro Gly Pro <210> 25 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Self-cleaving peptide containing 2A site <400> 25 Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro 1 5 10 <210> 26 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> Self-cleaving peptide containing 2A site <400> 26 Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu 1 5 10 15 Glu Asn Pro Gly Pro 20 <210> 27 <211> 18 <212> PRT <213> Artificial sequence <220> <223> Self-cleaving peptide containing 2A site <400> 27 Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro 1 5 10 15 Gly Pro <210> 28 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Self-cleaving peptide containing 2A site <400> 28 Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro 1 5 10 <210> 29 <211> twenty three <212> PRT <213> Artificial sequence <220> <223> Self-cleaving peptide containing 2A site <400> 29 Gly Ser Gly Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp 1 5 10 15 Val Glu Ser Asn Pro Gly Pro 20 <210> 30 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Self-cleaving peptide containing 2A site <400> 30 Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp Val Glu Ser 1 5 10 15 Asn Pro Gly Pro 20 <210> 31 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Self-cleaving peptide containing 2A site <400> 31 Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly Pro 1 5 10 <210> 32 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Self-cleaving peptide containing 2A site <400> 32 Gly Ser Gly Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala 1 5 10 15 Gly Asp Val Glu Ser Asn Pro Gly Pro 20 25 <210> 33 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> Self-cleaving peptide containing 2A site <400> 33 Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val 1 5 10 15 Glu Ser Asn Pro Gly Pro 20 <210> 34 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Self-cleaving peptide containing 2A site <400> 34 Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly Pro 1 5 10 <210> 35 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Self-cleaving peptide containing 2A site <400> 35 Leu Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn 1 5 10 15 Pro Gly Pro <210> 36 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Self-cleaving peptide containing 2A site <400> 36 Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn 1 5 10 15 Pro Gly Pro <210> 37 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Self-cleaving peptide containing 2A site <400> 37 Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly Pro 1 5 10 <210> 38 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Self-cleaving peptide containing 2A site <400> 38 Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly 1 5 10 15 Pro <210> 39 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Self-cleaving peptide containing 2A site <400> 39 Gln Leu Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser 1 5 10 15 Asn Pro Gly Pro 20 <210> 40 <211> twenty four <212> PRT <213> Artificial sequence <220> <223> Self-cleaving peptide containing 2A site <400> 40 Ala Pro Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly 1 5 10 15 Asp Val Glu Ser Asn Pro Gly Pro 20 <210> 41 <211> 40 <212> PRT <213> Artificial sequence <220> <223> Self-cleaving peptide containing 2A site <400> 41 Val Thr Glu Leu Leu Tyr Arg Met Lys Arg Ala Glu Thr Tyr Cys Pro 1 5 10 15 Arg Pro Leu Leu Ala Ile His Pro Thr Glu Ala Arg His Lys Gln Lys 20 25 30 Ile Val Ala Pro Val Lys Gln Thr 35 40 <210> 42 <211> 18 <212> PRT <213> Artificial sequence <220> <223> Self-cleaving peptide containing 2A site <400> 42 Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro 1 5 10 15 Gly Pro <210> 43 <211> 40 <212> PRT <213> Artificial sequence <220> <223> Self-cleaving peptide containing 2A site <400> 43 Leu Leu Ala Ile His Pro Thr Glu Ala Arg His Lys Gln Lys Ile Val 1 5 10 15 Ala Pro Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly 20 25 30 Asp Val Glu Ser Asn Pro Gly Pro 35 40 <210> 44 <211> 33 <212> PRT <213> Artificial sequence <220> <223> Self-cleaving peptide containing 2A site <400> 44 Glu Ala Arg His Lys Gln Lys Ile Val Ala Pro Val Lys Gln Thr Leu 1 5 10 15 Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly 20 25 30 Pro <210> 45 <211> 10 <212> DNA <213> Artificial sequence <220> <223> consensus Kozak sequence <400> 45 gccrccatgg 10

Claims

1. A cell comprising a first polynucleotide encoding a MAGEA4 TCR; and one or more polynucleotides encoding a chimeric TGFβ receptor, wherein the MAGEA4 TCR comprises: an α chain consisting of the amino acid sequence shown in SEQ ID NO: 2 and a β chain consisting of the amino acid sequence shown in SEQ ID NO: 3, or an α chain consisting of the amino acid sequence shown in SEQ ID NO: 5 and a β chain consisting of the amino acid sequence shown in SEQ ID NO: 6, and wherein the chimeric TGFβ receptor comprises (a) a TGFβR2 polypeptide comprising: (i) the extracellular TGFβ1-binding domain of TGFβR2; (ii) IL-12Rβ2 transmembrane domain; as well as (iii) IL-12Rβ2 intracellular signaling domain; and (b) a TGFβR1 polypeptide comprising: (i) the extracellular TGFβ1-binding domain of TGFβR1; (ii) IL-12Rβ1 transmembrane domain; as well as (iii) IL-12Rβ1 intracellular signaling domain.

2. The cell of claim 1, wherein the chimeric TGFβ receptor comprises a polypeptide cleavage signal between (a) and (b).

3. The cell of claim 2, wherein the polypeptide cleavage signal is a viral self-cleavage 2A peptide.

4. The cell of claim 3, wherein the viral self-cleaving 2A polypeptide is selected from the group consisting of: foot-and-mouth disease virus (F2A) peptide, equine rhinitis A virus (E2A) peptide, Tetrasomyces leucovorus beta-tetrasomy virus (T2A) peptide, porcine Teschovirus-1 (P2A) peptide, Theiler virus 2A peptide, and encephalomyocarditis virus 2A peptide.

5. The cell of any one of claims 2-4, wherein the chimeric TGFβ receptor consists of the amino acid sequence set forth in SEQ ID NO:

8.

6. The cell of any one of claims 1-4, wherein the MAGEA4 TCR binds to the peptide shown in SEQ ID NO: 1 presented by an HLA-A*02:01 encoded molecule.

7. The cell of any one of claims 1-4, wherein the MAGEA4 TCR comprises: The α chain consists of the amino acid sequence set forth in SEQ ID NO:2, and the β chain consists of the amino acid sequence set forth in SEQ ID NO:

3.

8. The cell of claim 7, wherein the MAGEA4 TCR consists of the amino acid sequence of SEQ ID NO:

4.

9. The cell of any one of claims 1-4, wherein the MAGEA4 TCR comprises: The α chain consists of the amino acid sequence set forth in SEQ ID NO:5 and the β chain consists of the amino acid sequence set forth in SEQ ID NO:

6.

10. The cell of claim 9, wherein the MAGEA4 TCR consists of the amino acid sequence of SEQ ID NO:

7.

11. A cell comprising a first polynucleotide encoding a MAGEA4 TCR; and a second polynucleotide encoding a chimeric TGFβ receptor, wherein the MAGEA4 TCR consists of the amino acid sequence of SEQ ID NO: 4 and the chimeric TGFβ receptor consists of the amino acid sequence of SEQ ID NO:

8.

12. A cell comprising a first polynucleotide encoding a MAGEA4 TCR; and a second polynucleotide encoding a chimeric TGFβ receptor, wherein the MAGEA4 TCR consists of the amino acid sequence of SEQ ID NO: 7 and the chimeric TGFβ receptor consists of the amino acid sequence of SEQ ID NO:

8.

13. The cell of any one of claims 1, 11 and 12, wherein the cell is a hematopoietic cell.

14. The cell of any one of claims 1, 11, and 12, wherein the cell is a T cell.

15. The cell of any one of claims 1, 11 and 12, wherein the cell is a CD3 + 、CD4 + and / or CD8 + cell.

16. The cell of any one of claims 1, 11, and 12, wherein the cell is an immune effector cell.

17. The cell of any one of claims 1, 11 and 12, wherein the cell is a cytotoxic T lymphocyte (CTL), a tumor infiltrating lymphocyte (TIL) or a helper T cell.

18. The cell of any one of claims 1, 11, and 12, wherein the cell is a natural killer (NK) cell or a natural killer T (NKT) cell.

19. The cell of any one of claims 1, 11, and 12, wherein the source of the cell is peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, or a tumor.

20. A composition comprising the cell of any one of claims 1-19.

21. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the cell of any one of claims 1-19.

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