Engineered T cells

Abstract

The present disclosure provides improved compositions for adoptive T cell therapy to treat, prevent, or ameliorate at least one symptom of, or conditions associated with, cancer, infectious disease, autoimmune disease, inflammatory disease, and immune deficiency.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62 / 845,311, filed May 8, 2019, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing Statement The sequence listing for this application has been submitted in text format in lieu of paper and is incorporated herein by reference. The name of the text file containing the sequence listing is BLBD_122_01WO_ST25. The text file is 37 KB, was created on May 6, 2020, and is being submitted electronically via EFS-Web simultaneously with the filing of this application. [Background technology]

[0003] The present disclosure relates to adoptive cell therapy. More specifically, the present disclosure relates to improved signaling molecules, cells, and methods of using them. Summary of the Invention [Means for solving the problem]

[0004] The present disclosure relates generally, in part, to improved adoptive immunotherapy and, in particular embodiments, to immune effector cells and chimeric TGFβ receptors (CTBRs), compositions, and methods of using same, comprising an αβ TCR that binds to MAGEA4, preferably a human pairing-enhanced αβ TCR that binds to MAGEA4, preferably a polynucleotide encoding the MAGEA4 peptide GVYDGREHTV (SEQ ID NO: 1) presented by an HLA-A*02:01-encoded molecule.

[0005] In various embodiments, the cell comprises a first polynucleotide encoding an engineered αβTCR that binds to MAGEA4 (MAGEA4 TCR), and a second polynucleotide encoding a fusion polypeptide comprising a first polypeptide comprising the extracellular TGFβ1-binding domain, transmembrane domain, and immunoreceptor intracellular signaling domain of TGFβR2, and a second polypeptide comprising a polypeptide cleavage signal and the extracellular TGFβ1-binding domain, transmembrane domain, and immunoreceptor intracellular signaling domain of TGFβR1.

[0006] In various embodiments, the cell comprises a first polynucleotide encoding a human pairing-enhanced αβTCR that binds to MAGEA4 (MAGEA4 eTCR), and a second polynucleotide encoding a fusion polypeptide comprising a first polypeptide comprising the extracellular TGFβ1-binding domain, transmembrane domain, and immunoreceptor intracellular signaling domain of TGFβR2, and a second polypeptide comprising a polypeptide cleavage signal and the extracellular TGFβ1-binding domain, transmembrane domain, and immunoreceptor intracellular signaling domain of TGFβR1.

[0007] In certain embodiments, the immunoreceptor 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.

[0008] In certain embodiments, the immunoreceptor 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.

[0009] In certain embodiments, the immunoreceptor intracellular signaling domain of the first polypeptide is an IL-12Rβ2 intracellular signaling domain and the immunoreceptor intracellular signaling domain of the second polypeptide is an IL-12Rβ1 intracellular signaling domain.

[0010] In various embodiments, the transmembrane domain of the first polypeptide comprises an IL-12Rβ2 transmembrane domain.

[0011] In a further embodiment, the transmembrane domain of the second polypeptide comprises the IL-12Rβ1 transmembrane domain.

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

[0013] In various embodiments, the transmembrane domain of the first polypeptide comprises an IL-12Rβ1 transmembrane domain.

[0014] In certain embodiments, the transmembrane domain of the second polypeptide comprises the IL-12Rβ2 transmembrane domain.

[0015] In certain embodiments, the immunoreceptor intracellular signaling domain of the first polypeptide is an IL-7Rα intracellular signaling domain and the immunoreceptor intracellular signaling domain of the second polypeptide is an IL-2Rγ intracellular signaling domain.

[0016] In a further embodiment, the transmembrane domain of the first polypeptide comprises an IL-7Rα transmembrane domain.

[0017] In additional embodiments, the transmembrane domain of the second polypeptide comprises an IL-2Rγ transmembrane domain.

[0018] In various embodiments, the immunoreceptor intracellular signaling domain of the first polypeptide is an IL-2Rγ intracellular signaling domain and the immunoreceptor intracellular signaling domain of the second polypeptide is an IL-7Rα intracellular signaling domain.

[0019] In some embodiments, the transmembrane domain of the first polypeptide comprises an IL-2Rγ transmembrane domain.

[0020] In certain embodiments, the transmembrane domain of the second polypeptide comprises an IL-7Rα transmembrane domain.

[0021] In certain embodiments, the immunoreceptor intracellular signaling domain of the first polypeptide is an IL-2Rβ intracellular signaling domain and the immunoreceptor intracellular signaling domain of the second polypeptide is an IL-2Rγ intracellular signaling domain.

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

[0023] In a further embodiment, the transmembrane domain of the second polypeptide comprises an IL-2Rγ transmembrane domain.

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

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

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

[0027] In additional embodiments, the immunoreceptor intracellular signaling domain of the first polypeptide is an IL-21R intracellular signaling domain and the immunoreceptor intracellular signaling domain of the second polypeptide is an IL-2Rγ intracellular signaling domain.

[0028] In various embodiments, the transmembrane domain of the first polypeptide comprises an IL-21R transmembrane domain.

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

[0030] In a specific embodiment, the immunoreceptor intracellular signaling domain of the first polypeptide is an IL-2Rγ intracellular signaling domain and the immunoreceptor intracellular signaling domain of the second polypeptide is an IL-21R intracellular signaling domain.

[0031] In a further embodiment, the transmembrane domain of the first polypeptide comprises an IL-2Rγ transmembrane domain.

[0032] In some embodiments, the transmembrane domain of the second polypeptide comprises an IL-21R transmembrane domain.

[0033] In additional embodiments, the immunoreceptor intracellular signaling domain of the first polypeptide is an IL-18R1 intracellular signaling domain and the immunoreceptor intracellular signaling domain of the second polypeptide is an IL-18RAP intracellular signaling domain.

[0034] In various embodiments, the transmembrane domain of the first polypeptide comprises an IL-18R1 transmembrane domain.

[0035] In various embodiments, the transmembrane domain of the second polypeptide comprises an IL-18RAP transmembrane domain.

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

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

[0038] In a further embodiment, the transmembrane domain of the second polypeptide comprises an IL-18R1 transmembrane domain.

[0039] In certain embodiments, the immunoreceptor intracellular signaling domain of the first polypeptide is an IL-1R1 intracellular signaling domain and the immunoreceptor intracellular signaling domain of the second polypeptide is an IL-1RAP intracellular signaling domain.

[0040] In various embodiments, the transmembrane domain of the first polypeptide comprises an IL-1R1 transmembrane domain.

[0041] In some embodiments, the transmembrane domain of the second polypeptide comprises an IL-1RAP transmembrane domain.

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

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

[0044] In certain embodiments, the transmembrane domain of the second polypeptide comprises the IL-1R1 transmembrane domain.

[0045] In certain embodiments, the immunoreceptor intracellular signaling domain of the first polypeptide is an IL-1RAP intracellular signaling domain and the immunoreceptor intracellular signaling domain of the second polypeptide is an IL-1RL2 intracellular signaling domain.

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

[0047] In additional embodiments, the transmembrane domain of the second polypeptide comprises the IL-1RL2 transmembrane domain.

[0048] In a further embodiment, the immunoreceptor intracellular signaling domain of the first polypeptide is an IL-1RL2 intracellular signaling domain and the immunoreceptor intracellular signaling domain of the second polypeptide is an IL-1RAP intracellular signaling domain.

[0049] In various embodiments, the transmembrane domain of the first polypeptide comprises an IL-1RL2 transmembrane domain.

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

[0051] In additional embodiments, the immunoreceptor intracellular signaling domain of the first polypeptide is an IFNAR1 intracellular signaling domain and the immunoreceptor intracellular signaling domain of the second polypeptide is an IFNAR2 intracellular signaling domain.

[0052] In various embodiments, the transmembrane domain of the first polypeptide comprises an IFNAR1 transmembrane domain.

[0053] In certain embodiments, the transmembrane domain of the second polypeptide comprises an IFNAR2 transmembrane domain.

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

[0055] In some embodiments, the transmembrane domain of the first polypeptide comprises an IFNAR2 transmembrane domain.

[0056] In various embodiments, the transmembrane domain of the second polypeptide comprises an IFNAR1 transmembrane domain.

[0057] In further embodiments, the immunoreceptor intracellular signaling domain of the first polypeptide is a TLR1 intracellular signaling domain, and the immunoreceptor intracellular signaling domain of the second polypeptide is a TLR1 intracellular signaling domain.

[0058] In certain embodiments, the transmembrane domain of the first polypeptide comprises a TLR1 transmembrane domain.

[0059] In certain embodiments, the transmembrane domain of the second polypeptide comprises a TLR1 transmembrane domain.

[0060] In some embodiments, the immunoreceptor intracellular signaling domain of the first polypeptide is a TLR2 intracellular signaling domain, and the immunoreceptor intracellular signaling domain of the second polypeptide is a TLR2 intracellular signaling domain.

[0061] In various embodiments, the transmembrane domain of the first polypeptide comprises a TLR2 transmembrane domain.

[0062] In additional embodiments, the transmembrane domain of the second polypeptide comprises a TLR2 transmembrane domain.

[0063] In various embodiments, the immunoreceptor intracellular signaling domain of the first polypeptide is a TLR3 intracellular signaling domain, and the immunoreceptor intracellular signaling domain of the second polypeptide is a TLR3 intracellular signaling domain.

[0064] In certain embodiments, the transmembrane domain of the first polypeptide comprises a TLR3 transmembrane domain.

[0065] In certain embodiments, the transmembrane domain of the second polypeptide comprises a TLR3 transmembrane domain.

[0066] In further embodiments, the immunoreceptor intracellular signaling domain of the first polypeptide is a TLR4 intracellular signaling domain, and the immunoreceptor intracellular signaling domain of the second polypeptide is a TLR4 intracellular signaling domain.

[0067] In various embodiments, the transmembrane domain of the first polypeptide comprises a TLR4 transmembrane domain.

[0068] In some embodiments, the transmembrane domain of the second polypeptide comprises a TLR4 transmembrane domain.

[0069] In various embodiments, the immunoreceptor intracellular signaling domain of the first polypeptide is a TLR5 intracellular signaling domain, and the immunoreceptor intracellular signaling domain of the second polypeptide is a TLR5 intracellular signaling domain.

[0070] In certain embodiments, the transmembrane domain of the first polypeptide comprises a TLR5 transmembrane domain.

[0071] In additional embodiments, the transmembrane domain of the second polypeptide comprises a TLR5 transmembrane domain.

[0072] In various embodiments, the immunoreceptor intracellular signaling domain of the first polypeptide is a TLR6 intracellular signaling domain, and the immunoreceptor intracellular signaling domain of the second polypeptide is a TLR6 intracellular signaling domain.

[0073] In various embodiments, the transmembrane domain of the first polypeptide comprises a TLR6 transmembrane domain.

[0074] In certain embodiments, the transmembrane domain of the second polypeptide comprises a TLR6 transmembrane domain.

[0075] In certain embodiments, the immunoreceptor intracellular signaling domain of the first polypeptide is a TLR7 intracellular signaling domain, and the immunoreceptor intracellular signaling domain of the second polypeptide is a TLR7 intracellular signaling domain.

[0076] In various embodiments, the transmembrane domain of the first polypeptide comprises a TLR7 transmembrane domain.

[0077] In a further embodiment, the transmembrane domain of the second polypeptide comprises a TLR7 transmembrane domain.

[0078] In some embodiments, the immunoreceptor intracellular signaling domain of the first polypeptide is a TLR8 intracellular signaling domain, and the immunoreceptor intracellular signaling domain of the second polypeptide is a TLR8 intracellular signaling domain.

[0079] In additional embodiments, the transmembrane domain of the first polypeptide comprises a TLR8 transmembrane domain.

[0080] In various embodiments, the transmembrane domain of the second polypeptide comprises a TLR8 transmembrane domain.

[0081] In certain embodiments, the immunoreceptor intracellular signaling domain of the first polypeptide is a TLR9 intracellular signaling domain, and the immunoreceptor intracellular signaling domain of the second polypeptide is a TLR9 intracellular signaling domain.

[0082] In certain embodiments, the transmembrane domain of the first polypeptide comprises a TLR9 transmembrane domain.

[0083] In certain embodiments, the transmembrane domain of the second polypeptide comprises a TLR9 transmembrane domain.

[0084] In various embodiments, the immunoreceptor intracellular signaling domain of the first polypeptide is a TLR10 intracellular signaling domain and the immunoreceptor intracellular signaling domain of the second polypeptide is a TLR10 intracellular signaling domain.

[0085] In some embodiments, the transmembrane domain of the first polypeptide comprises a TLR10 transmembrane domain.

[0086] In certain embodiments, the transmembrane domain of the second polypeptide comprises a TLR10 transmembrane domain.

[0087] In certain embodiments, the polypeptide cleavage signal is a viral self-cleaving polypeptide.

[0088] In various embodiments, the polypeptide cleavage signal is a viral self-cleaving 2A polypeptide.

[0089] In certain embodiments, 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 A virus (ERAV) (E2A) peptide, Thosea asigna virus (TaV) (T2A) peptide, porcine teschovirus-1 (PTV-1) (P2A) peptide, tylovirus 2A peptide, and encephalomyocarditis virus 2A peptide.

[0090] In various embodiments, the fusion polypeptide comprises the amino acid sequence set forth in SEQ ID NO:5.

[0091] 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, the IL-12Rβ2 transmembrane domain, and the IL-12Rβ2 intracellular signaling domain, and a viral self-cleaving 2A peptide and a TGFβR1 polypeptide comprising the extracellular TGFβ1-binding domain of TGFβR1, the IL-12Rβ1 transmembrane domain, and the IL-12Rβ1 intracellular signaling domain.

[0092] 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 the extracellular TGFβ1-binding domain of TGFβR2, the IL-12Rβ1 transmembrane domain, and the IL-12Rβ1 intracellular signaling domain, and a viral self-cleaving 2A peptide and a TGFβR1 polypeptide comprising the extracellular TGFβ1-binding domain of TGFβR1, the IL-12Rβ2 transmembrane domain, and the IL-12Rβ2 intracellular signaling domain.

[0093] In one embodiment, the cell comprises a first polynucleotide encoding a MAGEA4 TCR, a TGFβR2 polypeptide comprising the extracellular TGFβ1-binding domain of TGFβR2, the IL-7Rα transmembrane domain, and the IL-7Rα intracellular signaling domain, and a TGFβR1 polypeptide comprising the viral self-cleaving 2A peptide and the extracellular TGFβ1-binding domain of TGFβR1, the IL-2Rγ transmembrane domain, and the IL-2Rγ intracellular signaling domain.

[0094] In some embodiments, the cell comprises a first polynucleotide encoding a MAGEA4 TCR, a TGFβR2 polypeptide comprising the extracellular TGFβ1-binding domain, the IL-2Rγ transmembrane domain, and the IL-2Rγ intracellular signaling domain of TGFβR2, and a second polynucleotide encoding a fusion polypeptide comprising a viral self-cleaving 2A peptide and a TGFβR1 polypeptide comprising the extracellular TGFβ1-binding domain, the IL-7Rα transmembrane domain, and the IL-7Rα intracellular signaling domain of TGFβR1.

[0095] In additional embodiments, the cell comprises a first polynucleotide encoding a MAGEA4 TCR, a TGFβR2 polypeptide comprising the extracellular TGFβ1-binding domain of TGFβR2, the IL-2Rβ transmembrane domain, and the IL-2Rβ intracellular signaling domain, and a second polynucleotide encoding a fusion polypeptide comprising a viral self-cleaving 2A peptide and a TGFβR1 polypeptide comprising the extracellular TGFβ1-binding domain of TGFβR1, the IL-2Rγ transmembrane domain, and the IL-2Rγ intracellular signaling domain.

[0096] In a further embodiment, the cell comprises a first polynucleotide encoding a MAGEA4 TCR, a TGFβR2 polypeptide comprising the extracellular TGFβ1-binding domain of TGFβR2, the IL-2Rγ transmembrane domain, and the IL-2Rγ intracellular signaling domain, and a second polynucleotide encoding a fusion polypeptide comprising 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.

[0097] In various embodiments, the cell comprises a first polynucleotide encoding a MAGEA4 TCR, a TGFβR2 polypeptide comprising the extracellular TGFβ1-binding domain of TGFβR2, the IL-21R transmembrane domain, and the IL-21R intracellular signaling domain, and a second polynucleotide encoding a fusion polypeptide comprising a viral self-cleaving 2A peptide and a TGFβR1 polypeptide comprising the extracellular TGFβ1-binding domain of TGFβR1, the IL-2Rγ transmembrane domain, and the IL-2Rγ intracellular signaling domain.

[0098] In one 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, the IL-2Rγ transmembrane domain, and the IL-2Rγ intracellular signaling domain, and a TGFβR1 polypeptide comprising the viral self-cleaving 2A peptide and the extracellular TGFβ1-binding domain of TGFβR1, the IL-21R transmembrane domain, and the IL-21R intracellular signaling domain.

[0099] In certain embodiments, the cell comprises a first polynucleotide encoding a MAGEA4 TCR, a TGFβR2 polypeptide comprising the extracellular TGFβ1-binding domain of TGFβR2, the IL-18R1 transmembrane domain, and the IL-18R1 intracellular signaling domain, and a second polynucleotide encoding a fusion polypeptide comprising a viral self-cleaving 2A peptide and a TGFβR1 polypeptide comprising the extracellular TGFβ1-binding domain of TGFβR1, the IL-18RAP transmembrane domain, and the IL-18RAP intracellular signaling domain.

[0100] In various embodiments, the cell comprises a first polynucleotide encoding a MAGEA4 TCR, a TGFβR2 polypeptide comprising the extracellular TGFβ1-binding domain of TGFβR2, the IL-18RAP transmembrane domain, and the IL-18RAP intracellular signaling domain, and a second polynucleotide encoding a fusion polypeptide comprising a viral self-cleaving 2A peptide and a TGFβR1 polypeptide comprising the extracellular TGFβ1-binding domain of TGFβR1, the IL-18R1 transmembrane domain, and the IL-18R1 intracellular signaling domain.

[0101] In various embodiments, the cell comprises a first polynucleotide encoding a MAGEA4 TCR, a TGFβR2 polypeptide comprising the extracellular TGFβ1-binding domain of TGFβR2, the IL-1R1 transmembrane domain, and the IL-1R1 intracellular signaling domain, and a second polynucleotide encoding a fusion polypeptide comprising a viral self-cleaving 2A peptide and a TGFβR1 polypeptide comprising the extracellular TGFβ1-binding domain of TGFβR1, the IL-1RAP transmembrane domain, and the IL-1RAP intracellular signaling domain.

[0102] In a further embodiment, the cell comprises a first polynucleotide encoding a MAGEA4 TCR, a TGFβR2 polypeptide comprising the extracellular TGFβ1-binding domain of TGFβR2, the IL-1RAP transmembrane domain, and the IL-1RAP intracellular signaling domain, and a TGFβR1 polypeptide comprising the viral self-cleaving 2A peptide and the extracellular TGFβ1-binding domain of TGFβR1, the IL-1R1 transmembrane domain, and the IL-1R1 intracellular signaling domain.

[0103] In some embodiments, the cell comprises a first polynucleotide encoding a MAGEA4 TCR, a TGFβR2 polypeptide comprising the extracellular TGFβ1-binding domain of TGFβR2, the IFNAR1 transmembrane domain, and the IFNAR1 intracellular signaling domain, and a second polynucleotide encoding a fusion polypeptide comprising a viral self-cleaving 2A peptide and a TGFβR1 polypeptide comprising the extracellular TGFβ1-binding domain of TGFβR1, the IFNAR2 transmembrane domain, and the IFNAR2 intracellular signaling domain.

[0104] In some embodiments, the cell comprises a first polynucleotide encoding a MAGEA4 TCR, a TGFβR2 polypeptide comprising the extracellular TGFβ1-binding domain of TGFβR2, the IFNAR2 transmembrane domain, and the IFNAR2 intracellular signaling domain, and a second polynucleotide encoding a fusion polypeptide comprising a viral self-cleaving 2A peptide and a TGFβR1 polypeptide comprising the extracellular TGFβ1-binding domain of TGFβR1, the IFNAR1 transmembrane domain, and the IFNAR1 intracellular signaling domain.

[0105] In various embodiments, the cell comprises a first polynucleotide encoding a MAGEA4 TCR, a TGFβR2 polypeptide comprising the extracellular TGFβ1-binding domain of TGFβR2, the TLR1 transmembrane domain, and the TLR1 intracellular signaling domain, and a second polynucleotide encoding a fusion polypeptide comprising a viral self-cleaving 2A peptide and a TGFβR1 polypeptide comprising the extracellular TGFβ1-binding domain of TGFβR1, the TLR1 transmembrane domain, and the TLR1 intracellular signaling domain.

[0106] In various embodiments, the cell comprises a first polynucleotide encoding a MAGEA4 TCR, a TGFβR2 polypeptide comprising the extracellular TGFβ1-binding domain of TGFβR2, the TLR2 transmembrane domain, and the TLR2 intracellular signaling domain, and a second polynucleotide encoding a fusion polypeptide comprising a viral self-cleaving 2A peptide and a TGFβR1 polypeptide comprising the extracellular TGFβ1-binding domain of TGFβR1, the TLR2 transmembrane domain, and the TLR2 intracellular signaling domain.

[0107] In a further embodiment, the cell comprises a first polynucleotide encoding a MAGEA4 TCR, a TGFβR2 polypeptide comprising the extracellular TGFβ1-binding domain of TGFβR2, the TLR3 transmembrane domain, and the TLR3 intracellular signaling domain, and a second polynucleotide encoding a fusion polypeptide comprising a viral self-cleaving 2A peptide and a TGFβR1 polypeptide comprising the extracellular TGFβ1-binding domain of TGFβR1, the TLR3 transmembrane domain, and the TLR3 intracellular signaling domain.

[0108] In one embodiment, the cell comprises a first polynucleotide encoding a MAGEA4 TCR, a TGFβR2 polypeptide comprising the extracellular TGFβ1-binding domain of TGFβR2, the TLR4 transmembrane domain, and the TLR4 intracellular signaling domain, and a second polynucleotide encoding a fusion polypeptide comprising a viral self-cleaving 2A peptide and a TGFβR1 polypeptide comprising the extracellular TGFβ1-binding domain of TGFβR1, the TLR4 transmembrane domain, and the TLR4 intracellular signaling domain.

[0109] In certain embodiments, the cell comprises a first polynucleotide encoding a MAGEA4 TCR, a TGFβR2 polypeptide comprising the extracellular TGFβ1-binding domain of TGFβR2, the TLR5 transmembrane domain, and the TLR5 intracellular signaling domain, and a second polynucleotide encoding a fusion polypeptide comprising a viral self-cleaving 2A peptide and a TGFβR1 polypeptide comprising the extracellular TGFβ1-binding domain of TGFβR1, the TLR5 transmembrane domain, and the TLR5 intracellular signaling domain.

[0110] In some embodiments, the cell comprises a first polynucleotide encoding a MAGEA4 TCR, a TGFβR2 polypeptide comprising the extracellular TGFβ1-binding domain of TGFβR2, the TLR6 transmembrane domain, and the TLR6 intracellular signaling domain, and a second polynucleotide encoding a fusion polypeptide comprising a viral self-cleaving 2A peptide and a TGFβR1 polypeptide comprising the extracellular TGFβ1-binding domain of TGFβR1, the TLR6 transmembrane domain, and the TLR6 intracellular signaling domain.

[0111] In additional embodiments, the cell comprises a first polynucleotide encoding a MAGEA4 TCR, a TGFβR2 polypeptide comprising the extracellular TGFβ1-binding domain of TGFβR2, the TLR7 transmembrane domain, and the TLR7 intracellular signaling domain, and a second polynucleotide encoding a fusion polypeptide comprising a viral self-cleaving 2A peptide and a TGFβR1 polypeptide comprising the extracellular TGFβ1-binding domain of TGFβR1, the TLR7 transmembrane domain, and the TLR7 intracellular signaling domain.

[0112] In various embodiments, the cell comprises a first polynucleotide encoding a MAGEA4 TCR, a TGFβR2 polypeptide comprising the extracellular TGFβ1-binding domain of TGFβR2, the TLR8 transmembrane domain, and the TLR8 intracellular signaling domain, and a second polynucleotide encoding a fusion polypeptide comprising a viral self-cleaving 2A peptide and a TGFβR1 polypeptide comprising the extracellular TGFβ1-binding domain of TGFβR1, the TLR8 transmembrane domain, and the TLR8 intracellular signaling domain.

[0113] In various embodiments, the cell comprises a first polynucleotide encoding a MAGEA4 TCR, a TGFβR2 polypeptide comprising the extracellular TGFβ1-binding domain of TGFβR2, the TLR9 transmembrane domain, and the TLR9 intracellular signaling domain, and a second polynucleotide encoding a fusion polypeptide comprising a viral self-cleaving 2A peptide and a TGFβR1 polypeptide comprising the extracellular TGFβ1-binding domain of TGFβR1, the TLR9 transmembrane domain, and the TLR9 intracellular signaling domain.

[0114] In various embodiments, the cell comprises a first polynucleotide encoding a MAGEA4 TCR, a TGFβR2 polypeptide comprising the extracellular TGFβ1-binding domain of TGFβR2, the TLR10 transmembrane domain, and the TLR10 intracellular signaling domain, and a second polynucleotide encoding a fusion polypeptide comprising a viral self-cleaving 2A peptide and a TGFβR1 polypeptide comprising the extracellular TGFβ1-binding domain of TGFβR1, the TLR10 transmembrane domain, and the TLR10 intracellular signaling domain.

[0115] In certain embodiments, the viral self-cleaving 2A polypeptide is selected from the group consisting of: a foot-and-mouth disease virus (FMDV) (F2A) peptide, an equine rhinitis A virus (ERAV) (E2A) peptide, a Thosea asigna virus (TaV) (T2A) peptide, a porcine teschovirus-1 (PTV-1) (P2A) peptide, a tylovirus 2A peptide, and an encephalomyocarditis virus 2A peptide.

[0116] In various embodiments, the fusion polypeptide comprises the amino acid sequence set forth in SEQ ID NO:5.

[0117] In a further embodiment, the MAGEA4 TCR binds to the peptide GVYDGREHTV presented by HLA-A*02:01 encoded molecules.

[0118] In some embodiments, the MAGEA4 TCR is 2 and an alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 3 and a beta chain comprising the amino acid sequence set forth in

[0119] In various embodiments, the MAGEA4 TCR is 5 and an alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 6 and a beta chain comprising the amino acid sequence set forth in

[0120] In certain embodiments, the cells are hematopoietic cells.

[0121] In additional embodiments, the cell is a T cell.

[0122] In certain embodiments, the cells are CD3+, CD4+, and / or CD8+ cells.

[0123] In various embodiments, the cell is an immune effector cell.

[0124] In some embodiments, the cell is a cytotoxic T lymphocyte (CTL), a tumor-infiltrating lymphocyte (TIL), or a helper T cell.

[0125] In certain embodiments, the cells are natural killer (NK) cells or natural killer T (NKT) cells.

[0126] 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.

[0127] In various embodiments, the composition comprises a cell expressing a MAGEA4 TCR and a fusion polypeptide as contemplated herein.

[0128] In a further embodiment, a pharmaceutical composition comprises a pharmaceutically acceptable carrier, cells expressing a MAGEA4 TCR, and a fusion polypeptide as contemplated herein.

[0129] 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.

[0130] In various embodiments, a method for treating, preventing, or alleviating at least one symptom of cancer, infectious disease, autoimmune disease, inflammatory disease, and immune deficiency, or a condition related thereto, comprises administering to a subject an effective amount of a composition contemplated herein.

[0131] In certain embodiments, the method of treating a solid tumor comprises administering to a subject an effective amount of a composition contemplated herein.

[0132] 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.

[0133] In certain embodiments, the solid cancer is pancreatic cancer, lung cancer, or breast cancer.

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

[0135] In various embodiments, the hematological malignancy is leukemia, lymphoma, or multiple myeloma. [Brief explanation of the drawings]

[0136] [Figure 1] Figure 1 shows the phosphorylation of STAT4 and SMAD2 / 3 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 an IL-12-responsive chimeric TGFβ receptor (CTBR12), cultured for 20 min in the presence or absence of TGFβ1. [Figure 2] Figure 2 shows the amount of IFNγ secreted by UTD T cells, MAGEA4 TCR T cells, and MAGEA4 TCR / CTBR12 T cells cultured alone or with A375 MAGEA4+ tumor cells at an E:T ratio of 1:1 for 24 hours in the presence or absence of TGFβ1 (10 ng / ml). [Figure 3] Figure 3 shows A375 MAGEA4+ tumor cell volume in an NSG tumor xenograft mouse model treated with UTD T cells, MAGEA4 TCR T cells, or MAGEA4 TCR / CTBR12 T cells. [Figure 4] Figure 4 shows the phosphorylation of STAT4 and SMAD2 / 3 in T cells transduced with LVV encoding the MAGEA4 pairing-enhanced TCR (eTCR) and in T cells transduced with LVV encoding the MAGEA4 eTCR and an IL-12-responsive chimeric TGFβ receptor (CTBR12) cultured for 20 min in the presence or absence of TGFβ1. [Figure 5]Figure 5 shows the amount of IFNγ secreted by UTD T cells, MAGEA4 eTCR T cells, and MAGEA4 eTCR / CTBR12 T cells cultured alone, with TGFβ1, or with A375 MAGEA4+ tumor cells at an E:T ratio of 1:1 for 24 hours in the presence or absence of TGFβ1 (10 ng / ml).

[0137] A brief explanation of sequence numbers SEQ ID NO: 1 sets forth the amino acid sequence of the MAGEA4 epitope. SEQ ID NO: 2 sets forth the amino acid sequence of the human MAGEA4 TCR alpha chain. SEQ ID NO: 3 sets forth the amino acid sequence of the human MAGEA4 TCR β chain. SEQ ID NO: 4 sets forth the amino acid sequence of the human MAGEA4 TCR fusion polypeptide. SEQ ID NO: 5 sets forth the amino acid sequence of the human MAGEA4 eTCR alpha chain. SEQ ID NO: 6 sets forth the amino acid sequence of the human MAGEA4 eTCR β chain. SEQ ID NO: 7 sets forth the amino acid sequence of the human MAGEA4 eTCR fusion polypeptide. SEQ ID NO: 8 sets forth the amino acid sequence of an IL-12-responsive chimeric TGFβ receptor (CTBR12). SEQ ID NOs: 9 to 19 are set to the amino acid sequences of various linkers. SEQ ID NOs: 20 to 44 are set as the amino acid sequences of the protease cleavage site and the self-cleaving polypeptide cleavage site. SEQ ID NO: 45 sets forth the nucleotide sequence of the Kozak sequence. In the above sequences, X, if present, refers to any amino acid or the absence of an amino acid. DETAILED DESCRIPTION OF THE INVENTION

[0138] A. Overview T cell receptor (TCR) expressing T cells have shown limited, if any, efficacy in solid tumor indications, due in part to the immunosuppressive solid tumor microenvironment (TME). Excessive production of immunosuppressive cytokines, including TGFβ, by tumor cells and tumor-infiltrating lymphocytes contributes to the immunosuppressive tumor microenvironment. TGFβ inhibits T cell function through various mechanisms. TGFβ is frequently associated with tumor metastasis and infiltration, inhibited immune cell function, and poor prognosis in cancer patients. TGFβ signaling via TGFβR2 in tumor-specific CTLs attenuates their function and frequency in tumors, leading to the suppression of CD8 T cell proliferation by monoclonal antibodies. + Blocking TGFβ signaling on T cells leads to more rapid tumor surveillance and the presence of more CTLs at the tumor site. To date, strategies to inhibit TGFβ in clinical settings have not provided significant therapeutic benefit.

[0139] The present disclosure generally relates to immune effector cells expressing a MAGEA4 TCR, a polypeptide that converts immunosuppressive TGFβ signals into immunostimulatory signals, and cells expressing the polypeptide. Without intending to be bound by any particular theory, the polypeptide contemplated herein is a chimeric TGFβ receptor (CTBR) comprising the TGFβ-binding domains of TGFβR1 and TGFβR2 linked to an immunostimulatory endodomain, which, when co-expressed in immune effector cells, can convert TGFβ exposure from an immunosuppressive signal into an immunostimulatory signal that stimulates immune effector cell activity and function. Co-expression of the chimeric TGFβ receptor polypeptide in immune effector cells renders the cells resistant to the immunosuppressive effects of TGFβ, for example, by restoring or increasing proinflammatory cytokine secretion. In a particularly preferred embodiment, the MAGEA4 TCR is a human MAGEA4 pairing-enhanced TCR (eTCR), and the chimeric TGFβ receptor is CTBR12.

[0140] In various embodiments, the present disclosure contemplates immune effector cells expressing MAGEA4 TCR and CTBR polypeptides that, in part, convert immunosuppressive TGFβ signals into immunostimulatory signals mediated by or through one or more intracellular domains of one or more immune receptors.

[0141] In various embodiments, the present disclosure contemplates immune effector cells expressing MAGEA4 TCR and CTBR polypeptides that, in part, convert immunosuppressive TGFβ signals into immunostimulatory signals mediated by or through one or more intracellular domains of one or more cytokine receptors.

[0142] In various embodiments, the present disclosure contemplates immune effector cells expressing MAGEA4 TCR and CTBR polypeptides that, in part, convert immunosuppressive TGFβ signals into immunostimulatory signals mediated by or through one or more intracellular domains of one or more interleukin receptors.

[0143] In various embodiments, the present disclosure contemplates immune effector cells expressing MAGEA4 TCR and CTBR polypeptides that, in part, convert immunosuppressive TGFβ signals into immunostimulatory signals mediated by or through one or more intracellular domains of one or more pattern recognition receptors.

[0144] In various embodiments, the present disclosure contemplates immune effector cells expressing MAGEA4 TCR and CTBR polypeptides that, in part, convert immunosuppressive TGFβ signals into immunostimulatory signals mediated by or through one or more intracellular domains of one or more toll-like receptors.

[0145] In certain embodiments, the present disclosure contemplates, in part, an immune effector cell expressing a MAGEA4 TCR, wherein the CTBR polypeptide comprises a TGFβR1 extracellular domain that binds to TGFβ, a transmembrane domain, and one or more intracellular domains of one or more immune receptors, and the CTBR polypeptide comprises a TGFβR2 extracellular domain that binds to TGFβ, a transmembrane domain, and one or more intracellular domains of one or more immune receptors. In one embodiment, the polypeptides are linked to each other by a polypeptide cleavage signal, e.g., a 2A polypeptide cleavage signal.

[0146] 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 pairing-enhanced TCR (eTCR, e.g., SEQ ID NOs: 5-7), preferably a MAGEA4 pairing-enhanced TCR that binds to the MAGEA4 peptide GVYDGREHTV presented by an HLA-A*02:01-encoded molecule, and fusion polypeptides encoding a polypeptide comprising a chimeric TGFβ receptor (CTBR) comprising a TGFβR1 extracellular domain that binds to TGFβ, a transmembrane domain, and one or more intracellular domains of one or more immune receptors, and a TGFβR2 extracellular domain that binds to TGFβ, a transmembrane domain, and one or more intracellular domains of one or more immune receptors.

[0147] In certain 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.

[0148] 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.

[0149] In a preferred embodiment, the fusion polypeptide is an IL-12-responsive CTBR (CTBR12, eg, SEQ ID NO: 8).

[0150] Recombinant (i.e., engineered) DNA, peptide and oligonucleotide synthesis, immunoassays, tissue culture, transformation (e.g., electroporation, lipofection), enzymatic reactions, purification and related techniques and procedures may generally be performed as described in various general and more specific references in microbiology, molecular biology, biochemistry, molecular genetics, cell biology, virology and immunology, cited and discussed throughout this specification. For example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2008); Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Glover, DNA Cloning: A Practical Approach, vol.I & II(IRL Press,Oxford Univ.Press USA,1985);Current Protocols in Immunology(Edited by:John E.Coligan,Ada M.Kruisbeek,David H.Margulies,Ethan M.Shevach,Warren Strober 2001 John Wiley & Sons,NY,NY);Real-Time PCR:Current Technology and Applications,Edited by Julie Logan,Kirstin Edwards and Nick Saunders,2009,Caister Academic Press,Norfolk,UK;Anand,Techniques for the Analysis of Complex Genomes,(Academic Press,New York,1992);Guthrie and Fink,Guide to Yeast Genetics and Molecular Biology(Academic Press,New York,1991);Oligonucleotide Synthesis(N.Gait,Ed.,1984);Nucleic Acid The Hybridization(B.Hames & S.Higgins,Eds.,1985);Transcription and Translation(B.Hames & S.Higgins,Eds.,1984);Animal Cell Culture(R.Freshney,Ed.,1986);Perbal,A Practical Guide to Molecular Cloning(1984);Next-Generation Genome Sequencing(Janitz,2008 Wiley-VCH);PCR Protocols(Methods in Molecular Biology)(Park,Ed.,3rd Edition,2010 Humana Press);Immobilized Cells And Enzymes(IRL Press,1986);the treatise,Methods In Enzymology(Academic Press,Inc.,N.Y.);Gene Transfer Vectors For Mammalian Cells(J.H.Miller and M.P.Calos eds., 1987, Cold Spring Harbor Laboratory); Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D.M. Weir and C.C. Blackwell, eds., 1986); Roitt, Essential Immunology, 6th Edition, (Blackwell Scientific Publications, Oxford, 1988); Current Protocols in Immunology (Q.E. Coligigan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach and W. Strober, eds., 1991); Annual Review of Immunology; and research articles in journals such as, for example, Advances in Immunology.

[0151] B. Definition Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used to practice or test particular embodiments, preferred compositions, methods and materials embodiments are disclosed herein. For purposes of this disclosure, the following terms are defined below.

[0152] The articles "a," "an," and "the" are used herein to refer to one or to more than one (i.e., to at least one, or to one or more) of the grammatical object of the article. By way of example, "an element" means one element or one or more elements.

[0153] The use of the alternative (eg, "or") should be understood to mean either one, both, or any combination of the alternatives.

[0154] The term "and / or" should be understood to mean either one or both of the alternatives.

[0155] As used herein, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by up to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% relative to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the term "about" or "approximately" refers to a range of ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length relative to the reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0156] Throughout this specification, unless otherwise required, the terms "comprise" and "comprising" should be understood to imply the inclusion of the recited step or element or group of steps or elements, but not the exclusion of any other step or element or group of steps or elements. "Consisting of" means including, but limited to, what follows the words "consisting of." Thus, the words "consisting of" indicate that the listed elements are necessary or mandatory, and that no other elements may be present. "Consisting essentially of" means including any elements listed after the words, and any elements limited to other elements that do not interfere with or contribute to the activity or function specified in this disclosure for the listed elements. Thus, the words "consisting essentially of" indicate that the listed elements are necessary or mandatory, but that there are no other elements that materially affect the activity or function of the listed elements.

[0157] References throughout this specification to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "an embodiment," "an additional embodiment," or "a further embodiment," or combinations thereof, mean that the particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment. Thus, the appearances of such phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. Furthermore, it should be understood that the affirmative recitation of a feature in one embodiment serves as grounds for the exclusion of that feature in certain embodiments.

[0158] "Antigen (Ag)" refers to a compound, composition, or substance that can stimulate antibody production or a T-cell response in an animal, including compositions (e.g., compositions containing a cancer-specific protein) that are injected into or absorbed into an animal. Examples of antigens include, but are not limited to, lipids, carbohydrates, polysaccharides, glycoproteins, peptides, or nucleic acids. Antigens react with the products of specific humoral or cellular immunity, including those induced by heterologous antigens, such as the antigens disclosed herein.

[0159] A "target antigen" or "target antigen of interest" is an antigen to which a binding domain contemplated 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, and 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, Mucl, Mucl6, 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.

[0160] MAGE-A4 belongs to a group of so-called cancer / testis antigens. Cancer / testis antigens are expressed in various malignant tumors and germ cells, but not in other adult tissues. Therefore, MAGE-A4 is an interesting immunotherapy target antigen. The human gene encoding MAGE-A4 is designated MAGEA4 (ENSG00000147381).

[0161] In one embodiment, the antigen is an MHC-peptide complex, eg, a class I MHC-peptide complex or a class II MHC-peptide complex.

[0162] "Linker" refers to multiple amino acid residues between various polypeptide domains, added for proper spacing and conformation of the molecule.

[0163] Exemplary linkers suitable for use in certain 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) (e.g., Liu et al., PNAS 5525-5530 (1997)); GGRR (SEQ ID NO: 11) (Pomerantz et al. 1995, supra); (GGGGS) n, where 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); KESGSVSSSEQLAQFRSLD (SEQ ID NO: 14) (Bird et al., 1988, Science 242:423-426), GGRRGGGS (SEQ ID NO: 15); LQRDGERP (SEQ ID NO: 16); LRQKDGGGSERP (SEQ ID NO: 17); LRQKD(GGGS)2ERP (SEQ ID NO: 18). Alternatively, flexible linkers can be modeled using a computer program (Desjarlais & Berg, 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). Alternatively, flexible linkers can be modeled using a computer program that can model both the DNA binding site and the peptide itself (Desjarlais & Berg, PNAS 90:2256-2260 (1993), PNAS 91:11099-11103 (1994), or rationally designed by phage display. 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)).

[0164] A "transmembrane domain" or "TM domain" is a domain that anchors a polypeptide to a cell membrane. TM domains may be derived from either natural, synthetic, semi-synthetic, or recombinant sources.

[0165] "Intracellular signaling domain" refers to a protein portion that transmits an effector function signal, instructing the cell to perform a specialized function. While the entire intracellular signaling domain can usually be employed, it is often 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, so long as it transduces an effector function signal. The term intracellular signaling domain is meant to include any truncated portion of the intracellular signaling domain sufficient to transduce an effector function signal.

[0166] 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 release of cytotoxic factors, or other cellular responses triggered by antigen binding to receptors expressed on immune effector cells.

[0167] "Immune disorder" refers to a disease that provokes 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, an immune disorder includes an infectious disease.

[0168] As used herein, the term "cancer" generally relates to certain diseases or conditions in which abnormal cells divide uncontrollably and can invade nearby tissues.

[0169] As used herein, the term "malignant" refers to a cancer in which tumor cells exhibit one or more of the following: uncontrolled growth (i.e., division beyond normal limits), invasion (i.e., invasion and destruction of adjacent tissues), and metastasis (i.e., spread to other parts of the body via the lymph or blood). As used herein, the term "metastasizing" refers to the spread of cancer from one part of the body to another. A tumor formed by the spread cells is called a "metastatic tumor" or "metastatic cancer." A metastatic tumor contains cells similar to those of the original tumor (primary tumor).

[0170] As used herein, the terms "benign" or "non-malignant" refer to tumors that can grow large but do not spread to other parts of the body. Benign tumors are self-limited and often do not invade or metastasize.

[0171] "Cancer cell" refers to an individual cell of a cancerous growth or cancerous tissue. Cancer cells include both solid and liquid cancers. "Tumor" or "tumor cell" generally refers to an expansion due to abnormal cell proliferation or a lesion formed by abnormal cell proliferation, which may be benign, precancerous, or malignant. Most cancers form tumors, but liquid cancers, such as leukemia, do not necessarily form tumors. For 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.

[0172] The term "recurrence" refers to the diagnosis of a return of cancer, or the diagnosis of signs and symptoms of a return of cancer, after a period of improvement or remission.

[0173] "Remission," also called "clinical remission," includes both partial and complete remission. In a partial remission, some, but not all, signs and symptoms of cancer disappear. In a complete remission, all signs and symptoms of cancer disappear, although cancer may still be present in the body.

[0174] "Refractory" refers to a cancer that is resistant or unresponsive to therapy with a particular therapeutic agent. A cancer may be refractory from the start of treatment (i.e., unresponsive to initial exposure to the therapeutic agent) or may become refractory as a result of developing resistance to the therapeutic agent either during the initial treatment period or during subsequent treatment periods.

[0175] "Antigen negative" refers to cells that do not express an antigen or that express an antigen in such small amounts that it cannot be detected. In one embodiment, antigen negative cells do not bind to the target receptor for the antigen. In one embodiment, antigen negative cells do not substantially bind to the target receptor for the antigen.

[0176] An "autoimmune disease" refers to a condition in which the body mounts an immunogenic (i.e., immune system) response against some of the body's own tissue components. In other words, the immune system loses the ability to recognize some of the body's tissues or systems as "self" and attacks them as foreign. Autoimmune diseases can be classified as those that primarily affect one organ (e.g., hemolytic anemia and autoimmune thyroiditis) or those in which the autoimmune disease process spreads to many tissues (e.g., systemic lupus erythematosus). Multiple sclerosis, for example, is thought to arise when T cells attack the sheaths surrounding nerve fibers in the brain and spinal cord. This results in loss of coordination, weakness, and vision problems. Autoimmune diseases are known in the art and include, for example, Hashimoto's thyroiditis, Graves' disease, lupus erythematosus, multiple sclerosis, rheumatoid arthritis, hemolytic anemia, anti-immune thyroiditis, systemic lupus erythematosus, celiac disease, Crohn's disease, colitis, diabetes, scleroderma, psoriasis, and the like.

[0177] "Immunodeficiency" refers to a condition in a patient in which the immune system is impaired by disease or chemical exposure. This condition results in the immune system being deficient in the number and types of blood cells needed to defend against foreign agents. Immunodeficiency conditions or diseases are known in the art and include, for example, AIDS (acquired immunodeficiency syndrome), SCID (severe combined immunodeficiency), selective IgA deficiency, common variable immunodeficiency, X-linked agammaglobulinemia, chronic granulomatous disease, hyper-IgM syndrome, and diabetes.

[0178] "Infectious disease" refers to a disease that can be transmitted from person to person or from organism to organism and is caused by a microorganism or virus (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.

[0179] 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 otherwise contemplated herein. Suitable subjects (e.g., patients) include experimental animals (e.g., mice, rats, rabbits, or guinea pigs), farm animals, domestic animals, or pets (e.g., cats or dogs). Non-human primates, preferably human patients, are also included. Typical subjects include human patients who have, are at risk of, or have been diagnosed with cancer or another immune disorder.

[0180] As used herein, the term "patient" refers to a subject diagnosed with cancer or another immune disorder that can be treated with the compositions and methods otherwise disclosed herein.

[0181] As used herein, "treatment" or "treating" includes any beneficial or desired effect on the symptoms or pathology of a disease or condition, and may include even a minimal reduction in one or more measurable markers of the disease or condition being treated. Treatment may optionally include a reduction in the disease or condition, or a delay in the progression of the disease or condition, e.g., a delay in tumor growth. "Treatment" does not necessarily indicate a complete elimination or cure of the disease or condition, or its associated symptoms.

[0182] As used herein, "prevent" and similar terms, such as "prevented" and "preventing," refer to an approach aimed at preventing, inhibiting, or reducing the likelihood of occurrence or recurrence of a disease or condition. It also refers to delaying the onset or recurrence of a disease or condition, or delaying the onset or recurrence of symptoms of a disease or condition. As used herein, "prevention" and similar terms also include reducing the intensity, effects, symptoms, and / or burden of a disease or condition before the onset or recurrence of the disease or condition.

[0183] As used herein, "amelioration of at least one symptom of" refers to a decrease in one or more symptoms of the disease or condition for which the subject is being treated. In certain embodiments, the disease or condition for which treatment is being treated is cancer, in which case the one or more symptoms that are improved include, but are not limited to, weakness, fatigue, shortness of breath, easy bruising and bleeding, frequent infections, enlarged lymph nodes, abdominal distension or pain (caused by distended abdominal organs), bone or joint pain, broken bones, unexpected weight loss, loss of appetite, night sweats, persistent low-grade fever, and decreased urination (caused by impaired kidney function).

[0184] "Enhance," "promote," "increase," or "expand" generally refer to the ability of a composition contemplated herein to produce, elicit, or generate a greater physiological response (i.e., downstream effect) compared to the response induced by a vehicle or control molecule / composition. Measurable physiological responses include, among others, increased T cell expansion, activation, persistence, cytokine secretion, and / or cancer cell killing capacity, as will be apparent from an understanding of the art and the present disclosure. An "increased" or "enhanced" amount is typically a "statistically significant" amount and may include an increase of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, or more (e.g., 500-fold, 1000-fold) (including all integers and decimal points between and above 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) over the response induced by a vehicle or control composition.

[0185] "Decrease," or "lower," or "reduce," or "diminish," or "attenuate" generally refers to the ability of a composition contemplated herein to produce, elicit, or generate a physiological response (i.e., a downstream effect) that is smaller than the response induced by a vehicle or control molecule / composition. The amount of "reduction" or "decreased" is typically a "statistically significant" amount and can include a decrease of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, or more (e.g., 500-fold, 1000-fold) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) of the response induced by a vehicle, a control composition, or a response in a particular cell line.

[0186] "Maintain," or "preserve," or "maintain," or "no change," or "no substantial change," or "no substantial decrease" generally refer to the ability of a composition contemplated herein to produce, elicit, or produce a substantially similar or equivalent physiological response (i.e., downstream effect) in a cell compared to the response produced by a vehicle, a control molecule / composition, or the response in a particular cell line. An equivalent response is one that is not substantially different, or not measurably different, from the reference response.

[0187] C.MAGEA4 T cell receptor The MAGEA4 T cell receptor (TCR) recognizes peptide fragments of MAGEA4 when presented by major histocompatibility complex (MHC) molecules. There are two distinct classes of MHC molecules, MHC I and MHC II, which deliver peptides to the cell surface from different cellular compartments. Engagement of the TCR with antigen and MHC leads to the activation of immune effector cells through a series of biochemical events mediated by associated enzymes, co-receptors, and specialized accessory molecules.

[0188] The TCR contemplated herein is a heterodimeric complex comprising a TCR alpha (TCRα) chain and a TCR beta (TCRβ) chain. The human TCRα locus is located on chromosome 14 (14q11.2). The mature TCRα chain comprises a variable domain derived from recombination of a variable (V) segment and a joining (J) segment, and a constant (C) domain. The human TCRβ locus is located on chromosome 7 (7q34). The mature TCRβ chain comprises a variable domain derived from recombination of a variable (V) segment, a diversity (D) segment, and a joining (J) segment, and one of two constant (C) domains.

[0189] In certain embodiments, the TCR binds to MAGEA4.

[0190] In certain embodiments, the TCR is a human TCR that binds to MAGEA4.

[0191] In a preferred embodiment, the TCR is a human pairing-enhanced TCR that binds to MAGEA4.

[0192] The pairing-enhanced MAGEA4 TCRs contemplated herein are engineered to increase TCR stability, TCR expression, specific TCR pairing, and functional avidity.

[0193] In certain embodiments, the constant domains of the MAGEA4 TCR alpha and MAGEA4 TCR beta chains are engineered or modified to enhance TCR stability, TCR expression, specific TCR pairing, and functional avidity.

[0194] To efficiently enhance correct pairing of the MAGEA4 TCR sequence and avoid mispairing with endogenous TCR chains, the MAGEA4 pairing-enhanced TCRs contemplated herein contain minimally murine TCRα and TCRβ constant domains and further contain hydrophobic amino acid substitutions in the TCRα transmembrane domain.

[0195] In a preferred embodiment, the MAGEA4 pairing-enhanced TCR (eTCR) comprises a MAGEA4 TCR α chain comprising a constant domain comprising minimally murine amino acid substitutions at positions 90, 91, 92, and 93 and hydrophobic amino acid substitutions at positions 115, 118, and 119 in the constant region, and a MAGEA4 TCR β chain comprising a constant domain comprising minimally murine amino acid substitutions at positions 18, 22, 133, 136, and 139.

[0196] In a preferred embodiment, the MAGEA4 eTCR comprises a TCR α chain comprising a constant domain with the following minimally murine amino acid substitutions: P90S, E91D, S92V, and S93P, and the following hydrophobic amino acid substitutions in the transmembrane domain of the constant region: S115L, G118V, and F119L, and a TCR β chain comprising a constant domain with the following minimally murine amino acid substitutions: E18K, S22A, F133I, E / V136A, and Q139H.

[0197] In certain 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.

[0198] D. Chimeric TGFβ receptor (CTBR) In certain embodiments, cells are contemplated that comprise a polynucleotide encoding a human MAGEA4 TCR or a human MAGEA4 pairing-enhanced TCR (eTCR) and a chimeric TGFβ receptor that transmits an immunostimulatory signal upon exposure to TGFβ, including but not limited to TGFβ1.

[0199] 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, e.g., stimulating the activity and function of immune effector cells and increasing the production and / or secretion of proinflammatory cytokines. In certain embodiments, the term "chimeric TGFβ receptor" is used interchangeably with the term "CTBR."

[0200] In certain embodiments, the CTBR polypeptide comprises the extracellular TGFβ binding domain of TGFβR2, a transmembrane domain, an intracellular signaling domain of an immune receptor, including, but not limited to, a cytokine receptor, an interleukin receptor, a pattern recognition receptor, and a toll-like receptor; a polypeptide cleavage signal; and the extracellular TGFβ binding domain of TGFβR1, a transmembrane domain, an intracellular signaling domain of an immune receptor, including, but not limited to, a cytokine receptor, an interleukin receptor, a pattern recognition receptor, and a toll-like receptor.

[0201] In certain embodiments, the CTBR is a fusion polypeptide comprising: a first polypeptide comprising the extracellular TGFβ binding domain of TGFβR2, a transmembrane domain, an intracellular signaling domain of an immunoreceptor, including, but not limited to, a cytokine receptor, an interleukin receptor, a pattern recognition receptor, and a toll-like receptor; a polypeptide cleavage signal; and a second polypeptide comprising the extracellular TGFβ binding domain of TGFβR1, a transmembrane domain, an intracellular signaling domain of an immunoreceptor, including, but not limited to, a cytokine receptor, an interleukin receptor, a pattern recognition receptor, and a toll-like receptor.

[0202] In other specific embodiments, the CTBR is a complex of polypeptides comprising a polypeptide comprising the extracellular TGFβ binding domain of TGFβR2, a transmembrane domain, an intracellular signaling domain of an immune receptor, including but not limited to, a cytokine receptor, an interleukin receptor, a pattern recognition receptor, and a toll-like receptor, and a polypeptide comprising the extracellular TGFβ binding domain of TGFβR1, a transmembrane domain, an intracellular signaling domain of an immune receptor, including but not limited to, a cytokine receptor, an interleukin receptor, a pattern recognition receptor, and a toll-like receptor.

[0203] As used herein, the term "immunoreceptor" refers to a receptor expressed on the surface of an immune cell that regulates an immune response upon binding its cognate ligand. Immunoreceptors suitable for use in certain embodiments include, but are not limited to, cytokine receptors, interleukin receptors, pattern recognition receptors, and toll-like receptors, and signaling through the immunoreceptor stimulates an immune response.

[0204] Illustrative examples of immune receptor transmembrane domains and intracellular signaling domains that may be used in certain embodiments contemplated herein include, but are not limited to, transmembrane domains and intracellular signaling domains isolated from IL-12 receptor, IL-7 receptor, IL-15 receptor, IL-21 receptor, IL-2 receptor, IL-1 receptor, IL-18 receptor, IL-36 receptor, type I IFN receptor, TLR1 receptor, TLR2 receptor, TLR3 receptor, TLR4 receptor, TLR5 receptor, TLR6 receptor, TLR7 receptor, TLR8 receptor, TLR9 receptor, or TLR10 receptor.

[0205] Further examples of immune receptor transmembrane domains and intracellular signaling domains that may be used in certain embodiments contemplated herein include, but are not limited to, transmembrane domains 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.

[0206] Examples of cytokine receptor transmembrane domains and intracellular signaling domains that may be used in certain embodiments contemplated herein include, but are not limited to, transmembrane domains 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.

[0207] Examples of interleukin receptor transmembrane domains and intracellular signaling domains that may be used in certain embodiments contemplated herein include, but are not limited to, transmembrane domains 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.

[0208] Examples of toll-like receptor transmembrane domains and intracellular signaling domains that may be used in certain embodiments contemplated herein include, but are not limited to, transmembrane domains and intracellular signaling domains isolated from TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, and TLR10.

[0209] 1. CTBR12 polypeptide 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 interleukin-12 receptor, beta 1 (IL-12Rβ1, also known as CD212) and interleukin-12 receptor, beta 2 (IL-12Rβ2).

[0210] IL-12 signaling via IL-12Rβ1 and IL-12Rβ2 leads to the phosphorylation of STAT3, STAT4, and STAT5. Phosphorylated STAT3 / STAT4 translocates to the nucleus and binds to the IFNγ promoter, increasing IFNγ expression. Phosphorylated STAT4 also recruits the Jun oncogene (c-Jun) to the IFNγ promoter, increasing IFNγ expression and enhancing IL-12 signaling by increasing IL-12Rβ2 transcription. Phosphorylation of STAT5 increases T cell proliferation.

[0211] IL-12 signaling also increases the expression of interleukin 2 receptor, alpha (IL-2R) by recruiting STAT4 and c-Jun to the promoter of IL-2R, thereby promoting T cell proliferation.

[0212] In various embodiments, one or more immune effector cells, including immune effector cells expressing a MAGEA4 TCR or a MAGEA4 eTCR, are modified by introducing one or more polynucleotides or vectors encoding one or more CTBR12 polypeptides. In various embodiments, the one or more immune effector cells are modified by introducing one or more polynucleotides or vectors encoding CTBR12 and a MAGEA4 TCR or a MAGEA4 eTCR.

[0213] In certain embodiments, CTBR12 converts immunosuppressive TGFβ signals into immunostimulatory signals via IL-12. In certain embodiments, 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, 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.

[0214] In certain embodiments, CTBR12 contemplated herein comprises a fusion polypeptide comprising a first polypeptide comprising the extracellular TGFβ1-binding domain, transmembrane domain, and IL-12Rβ1 intracellular signaling domain of TGFβR1; a polypeptide cleavage signal; and a second polypeptide comprising the extracellular TGFβ1-binding domain, transmembrane domain, and IL-12Rβ2 intracellular signaling domain of TGFβR2. In certain embodiments, CTBR12 contemplated herein comprises a fusion polypeptide comprising a first polypeptide comprising the extracellular TGFβ1-binding domain, transmembrane domain, and IL-12Rβ2 intracellular signaling domain of TGFβR1; a polypeptide cleavage signal; and a second polypeptide comprising the extracellular TGFβ1-binding domain, transmembrane domain, and IL-12Rβ1 intracellular signaling domain of TGFβR2.

[0215] In certain embodiments, CTBR12 is a polypeptide complex comprising a first polypeptide comprising a polypeptide comprising the extracellular TGFβ1-binding domain, transmembrane domain, and IL-12Rβ1 intracellular signaling domain of TGFβR1, and a polypeptide comprising the extracellular TGFβ1-binding domain, transmembrane domain, and 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, transmembrane domain, and IL-12Rβ2 intracellular signaling domain of TGFβR1, and a polypeptide comprising the extracellular TGFβ1-binding domain, transmembrane domain, and IL-12Rβ1 intracellular signaling domain of TGFβR2.

[0216] 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, an IL-12Rβ1 transmembrane domain, and an intracellular signaling domain of TGFβR1. In one embodiment, the polypeptide comprises an extracellular TGFβ1-binding domain, an IL-12Rβ2 transmembrane domain, and an intracellular signaling domain of TGFβR2. In one embodiment, the polypeptide comprises an extracellular TGFβ1-binding domain, an IL-12Rβ2 transmembrane domain, and an intracellular signaling domain of TGFβR1. In one embodiment, the polypeptide comprises an extracellular TGFβ1-binding domain, an IL-12Rβ2 transmembrane domain, and an intracellular signaling domain of TGFβR2. In one embodiment, the polypeptide comprises an extracellular TGFβ1-binding domain, an IL-12Rβ1 transmembrane domain, and an intracellular signaling domain of TGFβR2.

[0217] In certain embodiments, the polypeptide cleavage signal is a viral self-cleaving polypeptide, more preferably a viral self-cleaving 2A polypeptide, more preferably a viral self-cleaving polypeptide selected from the group consisting of: foot-and-mouth disease virus (FMDV) (F2A) peptide, equine rhinitis A virus (ERAV) (E2A) peptide, Thosea asigna virus (TaV) (T2A) peptide, porcine teschovirus-1 (PTV-1) (P2A) peptide, tylovirus 2A peptide, and encephalomyocarditis virus 2A peptide. In one embodiment, the polypeptide cleavage signal is a P2A or T2A viral self-cleaving polypeptide.

[0218] 2. CTBR7 polypeptide Interleukin-7 (IL-7) is a cytokine that promotes T cell function and activity, in part by improving the survival and proliferation of T cell precursors. IL-7 binds to interleukin-7 receptor alpha (IL-7Rα, also known as CD127) and 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 antiapoptotic genes and genes that promote proliferation of T cell precursors.

[0219] In various embodiments, one or more immune effector cells, including immune effector cells expressing a MAGEA4 TCR or a MAGEA4 eTCR, are modified by introducing one or more polynucleotides or vectors encoding one or more CTBR7 polypeptides. In various embodiments, the one or more immune effector cells are modified by introducing one or more polynucleotides or vectors encoding CTBR7 and a MAGEA4 TCR or a MAGEA4 eTCR.

[0220] In certain embodiments, the chimeric TGFβ receptor converts immunosuppressive TGFβ signals into immunostimulatory signals via IL-7. 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.

[0221] In certain embodiments, CTBR7 contemplated herein comprises a fusion polypeptide comprising a first polypeptide comprising the extracellular TGFβ1-binding domain, transmembrane domain, and IL-7Rα intracellular signaling domain of TGFβR1; a polypeptide cleavage signal; and a second polypeptide comprising the extracellular TGFβ1-binding domain, transmembrane domain, and IL-2Rγ intracellular signaling domain of TGFβR2. In certain embodiments, CTBR7 contemplated herein comprises a fusion polypeptide comprising a first polypeptide comprising the extracellular TGFβ1-binding domain, transmembrane domain, and IL-2Rγ intracellular signaling domain of TGFβR1; a polypeptide cleavage signal; and a second polypeptide comprising the extracellular TGFβ1-binding domain, transmembrane domain, and IL-7Rα intracellular signaling domain of TGFβR2.

[0222] In certain embodiments, CTBR7 is a polypeptide complex comprising a first polypeptide comprising a polypeptide comprising the extracellular TGFβ1-binding domain, transmembrane domain, and IL-7Rα intracellular signaling domain of TGFβR1, and a polypeptide comprising the extracellular TGFβ1-binding domain, transmembrane domain, and 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, transmembrane domain, and IL-2Rγ intracellular signaling domain of TGFβR1, and a polypeptide comprising the extracellular TGFβ1-binding domain, transmembrane domain, and IL-7Rα intracellular signaling domain of TGFβR2.

[0223] 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, an IL-7Rα transmembrane domain, and an intracellular signaling domain of TGFβR1. In one embodiment, the polypeptide comprises an extracellular TGFβ1-binding domain, an IL-2Rγ transmembrane domain, and an intracellular signaling domain of TGFβR2. In one embodiment, the polypeptide comprises an extracellular TGFβ1-binding domain, an IL-2Rγ transmembrane domain, and an intracellular signaling domain of TGFβR1. In one embodiment, the polypeptide comprises an extracellular TGFβ1-binding domain, an IL-7Rα transmembrane domain, and an intracellular signaling domain of TGFβR2.

[0224] In certain embodiments, the polypeptide cleavage signal is a viral self-cleaving polypeptide, more preferably a viral self-cleaving 2A polypeptide, more preferably a viral self-cleaving polypeptide selected from the group consisting of: foot-and-mouth disease virus (FMDV) (F2A) peptide, equine rhinitis A virus (ERAV) (E2A) peptide, Thosea asigna virus (TaV) (T2A) peptide, porcine teschovirus-1 (PTV-1) (P2A) peptide, tylovirus 2A peptide, and encephalomyocarditis virus 2A peptide. In one embodiment, the polypeptide cleavage signal is a P2A or T2A viral self-cleaving polypeptide.

[0225] 3. CTBR15 polypeptide Interleukin-15 (IL-15) is a cytokine that promotes T cell function and activity, in part by 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 either 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, leading to the transcription of anti-apoptotic genes and genes that promote T cell precursor proliferation.

[0226] In various embodiments, one or more immune effector cells, including immune effector cells expressing a MAGEA4 TCR or a 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, the one or more immune effector cells are modified by introducing one or more polynucleotides or vectors encoding CTBR15 and a MAGEA4 TCR or a MAGEA4 eTCR, and optionally, a polynucleotide or vector encoding an IL-15Rα polypeptide.

[0227] In certain embodiments, the chimeric TGFβ receptor converts an immunosuppressive TGFβ signal into an immunostimulatory signal via IL-15. 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.

[0228] In certain embodiments, a CTBR15 contemplated herein comprises a fusion polypeptide comprising a first polypeptide comprising the extracellular TGFβ1-binding domain, transmembrane domain, and IL-2Rβ intracellular signaling domain of TGFβR1; a polypeptide cleavage signal; and a second polypeptide comprising the extracellular TGFβ1-binding domain, transmembrane domain, and IL-2Rγ intracellular signaling domain of TGFβR2. In certain embodiments, a CTBR15 contemplated herein comprises a fusion polypeptide comprising a first polypeptide comprising the extracellular TGFβ1-binding domain, transmembrane domain, and IL-2Rγ intracellular signaling domain of TGFβR1; a polypeptide cleavage signal; and a second polypeptide comprising the extracellular TGFβ1-binding domain, transmembrane domain, and IL-2Rβ intracellular signaling domain of TGFβR2.

[0229] In certain embodiments, CTBR15 is a polypeptide complex comprising a first polypeptide comprising a polypeptide comprising the extracellular TGFβ1-binding domain, transmembrane domain, and IL-2Rβ intracellular signaling domain of TGFβR1, and a polypeptide comprising the extracellular TGFβ1-binding domain, transmembrane domain, and 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, transmembrane domain, and IL-2Rγ intracellular signaling domain of TGFβR1, and a polypeptide comprising the extracellular TGFβ1-binding domain, transmembrane domain, and IL-2Rβ intracellular signaling domain of TGFβR2.

[0230] 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, an IL-2Rβ transmembrane domain, and an intracellular signaling domain of TGFβR1. In one embodiment, the polypeptide comprises an extracellular TGFβ1-binding domain, an IL-2Rγ transmembrane domain, and an intracellular signaling domain of TGFβR2. In one embodiment, the polypeptide comprises an extracellular TGFβ1-binding domain, an IL-2Rγ transmembrane domain, and an intracellular signaling domain of TGFβR1. In one embodiment, the polypeptide comprises an extracellular TGFβ1-binding domain, an IL-2Rγ transmembrane domain, and an intracellular signaling domain of TGFβR2.

[0231] In certain embodiments, the polypeptide cleavage signal is a viral self-cleaving polypeptide, more preferably a viral self-cleaving 2A polypeptide, more preferably a viral self-cleaving polypeptide selected from the group consisting of: foot-and-mouth disease virus (FMDV) (F2A) peptide, equine rhinitis A virus (ERAV) (E2A) peptide, Thosea asigna virus (TaV) (T2A) peptide, porcine teschovirus-1 (PTV-1) (P2A) peptide, tylovirus 2A peptide, and encephalomyocarditis virus 2A peptide. In one embodiment, the polypeptide cleavage signal is a P2A or T2A viral self-cleaving polypeptide.

[0232] 4. CTBR21 polypeptide 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 antiapoptotic genes and genes that promote the proliferation of T cell precursors.

[0233] In various embodiments, one or more immune effector cells, including immune effector cells expressing a MAGEA4 TCR or a MAGEA4 eTCR, are modified by introducing one or more polynucleotides or vectors encoding one or more CTBR21 polypeptides. In various embodiments, the one or more immune effector cells are modified by introducing one or more polynucleotides or vectors encoding CTBR21 and a MAGEA4 TCR or a MAGEA4 eTCR.

[0234] In certain embodiments, the chimeric TGFβ receptor converts an immunosuppressive TGFβ signal into an immunostimulatory signal via IL-21. In certain embodiments, the CTBR21 contemplated 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 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-21R intracellular signaling domain of TGFβR2.

[0235] In certain embodiments, CTBR21 contemplated herein comprises a fusion polypeptide comprising a first polypeptide comprising the extracellular TGFβ1-binding domain, transmembrane domain, and IL-21R intracellular signaling domain of TGFβR1; a polypeptide cleavage signal; and a second polypeptide comprising the extracellular TGFβ1-binding domain, transmembrane domain, and IL-2Rγ intracellular signaling domain of TGFβR2. In certain embodiments, CTBR21 contemplated herein comprises a fusion polypeptide comprising a first polypeptide comprising the extracellular TGFβ1-binding domain, transmembrane domain, and IL-2Rγ intracellular signaling domain of TGFβR1; a polypeptide cleavage signal; and a second polypeptide comprising the extracellular TGFβ1-binding domain, transmembrane domain, and IL-2Rγ intracellular signaling domain of TGFβR2.

[0236] In certain embodiments, CTBR21 is a polypeptide complex comprising a first polypeptide comprising a polypeptide comprising the extracellular TGFβ1-binding domain, transmembrane domain, and IL-21R intracellular signaling domain of TGFβR1, and a polypeptide comprising the extracellular TGFβ1-binding domain, transmembrane domain, and 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, transmembrane domain, and IL-2Rγ intracellular signaling domain of TGFβR1, and a polypeptide comprising the extracellular TGFβ1-binding domain, transmembrane domain, and IL-21R intracellular signaling domain of TGFβR2.

[0237] 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, an IL-21R transmembrane domain, and an intracellular signaling domain. In one embodiment, the polypeptide comprises an extracellular TGFβ1-binding domain of TGFβR2, an IL-2Rγ transmembrane domain, and an intracellular signaling domain. In one embodiment, the polypeptide comprises an extracellular TGFβ1-binding domain of TGFβR1, an IL-2Rγ transmembrane domain, and an intracellular signaling domain. In one embodiment, the polypeptide comprises an extracellular TGFβ1-binding domain of TGFβR2, an IL-21R transmembrane domain, and an intracellular signaling domain.

[0238] In certain embodiments, the polypeptide cleavage signal is a viral self-cleaving polypeptide, more preferably a viral self-cleaving 2A polypeptide, more preferably a viral self-cleaving polypeptide selected from the group consisting of: foot-and-mouth disease virus (FMDV) (F2A) peptide, equine rhinitis A virus (ERAV) (E2A) peptide, Thosea asigna virus (TaV) (T2A) peptide, porcine teschovirus-1 (PTV-1) (P2A) peptide, tylovirus 2A peptide, and encephalomyocarditis virus 2A peptide. In one embodiment, the polypeptide cleavage signal is a P2A or T2A viral self-cleaving polypeptide.

[0239] 5. CTBR18 polypeptide Interleukin-18 (IL-18) is a cytokine that promotes T cell function and activity, in part, by increasing IFNγ expression, increasing T cell proliferation, and protecting against 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).

[0240] IL-18 signaling via IL-18R1 and IL-18RAP leads to activation via the MyD88 adaptor protein and phosphorylation of IRAK4. Phosphorylation of IRAK4 and subsequent phosphorylation of IRAK1 / 2 ultimately leads to activation of NF-kappaB and AP-1 transcription factors, increasing IFNγ expression and enhancing sensitivity to IL-12. IL-18-induced transcriptional programs also increase T cell proliferation and protect against AICD.

[0241] In various embodiments, one or more immune effector cells, including immune effector cells expressing a MAGEA4 TCR or a MAGEA4 eTCR, are modified by introducing one or more polynucleotides or vectors encoding one or more CTBR18 polypeptides. In various embodiments, the one or more immune effector cells are modified by introducing one or more polynucleotides or vectors encoding CTBR18 and a MAGEA4 TCR or a MAGEA4 eTCR.

[0242] In certain embodiments, the chimeric TGFβ receptor converts an immunosuppressive TGFβ signal into an immunostimulatory signal via IL-18. 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.

[0243] In certain embodiments, CTBR18 contemplated herein comprises a fusion polypeptide comprising a first polypeptide comprising the extracellular TGFβ1-binding domain, transmembrane domain, and IL-18R1 intracellular signaling domain of TGFβR1; a polypeptide cleavage signal; and a second polypeptide comprising the extracellular TGFβ1-binding domain, transmembrane domain, and IL-18RAP intracellular signaling domain of TGFβR2. In certain embodiments, CTBR18 contemplated herein comprises a fusion polypeptide comprising a first polypeptide comprising the extracellular TGFβ1-binding domain, transmembrane domain, and IL-18RAP intracellular signaling domain of TGFβR1; a polypeptide cleavage signal; and a second polypeptide comprising the extracellular TGFβ1-binding domain, transmembrane domain, and IL-18RAP intracellular signaling domain of TGFβR2.

[0244] In certain embodiments, CTBR18 is a polypeptide complex comprising a first polypeptide comprising a polypeptide comprising the extracellular TGFβ1-binding domain, transmembrane domain, and IL-18RAP intracellular signaling domain of TGFβR1, and a polypeptide comprising the extracellular TGFβ1-binding domain, transmembrane domain, and IL-18R1 intracellular signaling domain of TGFβR2. In certain embodiments, CTBR18 is a polypeptide complex comprising a first polypeptide comprising a polypeptide comprising the extracellular TGFβ1-binding domain, transmembrane domain, and IL-18R1 intracellular signaling domain of TGFβR1, and a polypeptide comprising the extracellular TGFβ1-binding domain, transmembrane domain, and IL-18R1 intracellular signaling domain of TGFβR2.

[0245] 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, an IL-18RAP transmembrane domain, and an intracellular signaling domain. In one embodiment, the polypeptide comprises an extracellular TGFβ1-binding domain of TGFβR2, an IL-18R1 transmembrane domain, and an intracellular signaling domain. In one embodiment, the polypeptide comprises an extracellular TGFβ1-binding domain of TGFβR1, an IL-18R1 transmembrane domain, and an intracellular signaling domain. In one embodiment, the polypeptide comprises an extracellular TGFβ1-binding domain of TGFβR2, an IL-18RAP transmembrane domain, and an intracellular signaling domain.

[0246] In certain embodiments, the polypeptide cleavage signal is a viral self-cleaving polypeptide, more preferably a viral self-cleaving 2A polypeptide, more preferably a viral self-cleaving polypeptide selected from the group consisting of: foot-and-mouth disease virus (FMDV) (F2A) peptide, equine rhinitis A virus (ERAV) (E2A) peptide, Thosea asigna virus (TaV) (T2A) peptide, porcine teschovirus-1 (PTV-1) (P2A) peptide, tylovirus 2A peptide, and encephalomyocarditis virus 2A peptide. In one embodiment, the polypeptide cleavage signal is a P2A or T2A viral self-cleaving polypeptide.

[0247] 6. CTBR1 polypeptide Interleukin-1 (IL-1) is a cytokine that promotes T cell function and activity, in part by increasing IFNγ expression, increasing T cell proliferation, and enhancing protection from 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).

[0248] IL-1 signaling through IL-1R1 and IL-1RAP leads to activation via the MyD88 adaptor protein and phosphorylation of IRAK4. Phosphorylation of IRAK4 and subsequent phosphorylation of IRAK1 / 2 ultimately leads to activation of NF-kappaB and AP-1 transcription factors, increasing IFNγ expression and enhancing sensitivity to IL-12. IL-1-induced transcriptional programs also increase T cell proliferation and protect against AICD.

[0249] In various embodiments, one or more immune effector cells, including immune effector cells expressing a MAGEA4 TCR or a MAGEA4 eTCR, are modified by introducing one or more polynucleotides or vectors encoding one or more CTBR1 polypeptides. In various embodiments, the one or more immune effector cells are modified by introducing one or more polynucleotides or vectors encoding CTBR1 and a MAGEA4 TCR or a MAGEA4 eTCR.

[0250] In certain embodiments, the chimeric TGFβ receptor converts immunosuppressive TGFβ signals into immunostimulatory signals via IL-1. 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.

[0251] In certain embodiments, the CTBR1 contemplated herein comprises a fusion polypeptide comprising a first polypeptide comprising the extracellular TGFβ1-binding domain, transmembrane domain, and IL-1R1 intracellular signaling domain of TGFβR1; a polypeptide cleavage signal; and a second polypeptide comprising the extracellular TGFβ1-binding domain, transmembrane domain, and IL-1RAP intracellular signaling domain of TGFβR2. In certain embodiments, the CTBR1 contemplated herein comprises a fusion polypeptide comprising a first polypeptide comprising the extracellular TGFβ1-binding domain, transmembrane domain, and IL-1RAP intracellular signaling domain of TGFβR1; a polypeptide cleavage signal; and a second polypeptide comprising the extracellular TGFβ1-binding domain, transmembrane domain, and IL-1R1 intracellular signaling domain of TGFβR2.

[0252] In certain embodiments, CTBR1 is a polypeptide complex comprising a first polypeptide comprising a polypeptide comprising the extracellular TGFβ1-binding domain, transmembrane domain, and IL-1RAP intracellular signaling domain of TGFβR1, and a polypeptide comprising the extracellular TGFβ1-binding domain, transmembrane domain, and 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, transmembrane domain, and IL-1R1 intracellular signaling domain of TGFβR1, and a polypeptide comprising the extracellular TGFβ1-binding domain, transmembrane domain, and IL-1RAP intracellular signaling domain of TGFβR2.

[0253] 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, an IL-1RAP transmembrane domain, and an intracellular signaling domain. In one embodiment, the polypeptide comprises an extracellular TGFβ1-binding domain of TGFβR2, an IL-1R1 transmembrane domain, and an intracellular signaling domain. In one embodiment, the polypeptide comprises an extracellular TGFβ1-binding domain of TGFβR1, an IL-1R1 transmembrane domain, and an intracellular signaling domain. In one embodiment, the polypeptide comprises an extracellular TGFβ1-binding domain of TGFβR2, an IL-1RAP transmembrane domain, and an intracellular signaling domain.

[0254] In certain embodiments, the polypeptide cleavage signal is a viral self-cleaving polypeptide, more preferably a viral self-cleaving 2A polypeptide, more preferably a viral self-cleaving polypeptide selected from the group consisting of: foot-and-mouth disease virus (FMDV) (F2A) peptide, equine rhinitis A virus (ERAV) (E2A) peptide, Thosea asigna virus (TaV) (T2A) peptide, porcine teschovirus-1 (PTV-1) (P2A) peptide, tylovirus 2A peptide, and encephalomyocarditis virus 2A peptide. In one embodiment, the polypeptide cleavage signal is a P2A or T2A viral self-cleaving polypeptide.

[0255] 7. CTBR.TLR polypeptide Toll-like receptors (TLR1-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 pro-inflammatory transcriptional programs and the expression of multiple inflammatory cytokines.

[0256] TLR signaling occurs via homodimerization of the TLR signaling domain, leading to activation via the MyD88 adaptor protein and IRAK4 phosphorylation. Phosphorylation of IRAK4 and subsequent phosphorylation of IRAK1 / 2 ultimately leads to activation of NF-kappaB and AP-1 transcription factors, increasing the production of proinflammatory cytokines and inducing proliferation. TLR activation can also lead to activation of IRF3 and IRF7 transcription factors.

[0257] In various embodiments, one or more immune effector cells, including immune effector cells expressing a MAGEA4 TCR or a MAGEA4 eTCR, are modified by introducing one or more polynucleotides or vectors encoding one or more CTBR.TLR polypeptides. In various embodiments, the one or more immune effector cells are modified by introducing one or more polynucleotides or vectors encoding a CTBR.TLR and a MAGEA4 TCR or a MAGEA4 eTCR.

[0258] In certain embodiments, the chimeric TGFβ receptor converts immunosuppressive TGFβ signals into immunostimulatory signals via TLRs. In certain embodiments, the CTBR.TLR contemplated herein comprises 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 identical TLR intracellular signaling domain of TGFβR2.

[0259] In certain embodiments, the CTBR.TLR contemplated herein comprises a fusion polypeptide comprising a first polypeptide comprising the extracellular TGFβ1-binding domain, transmembrane domain, and TLR intracellular signaling domain of TGFβR1; a polypeptide cleavage signal; and a second polypeptide comprising the extracellular TGFβ1-binding domain, transmembrane domain, and the same TLR intracellular signaling domain of TGFβR2.

[0260] In certain embodiments, CTBR.TLR is a complex of polypeptides comprising a first polypeptide comprising a polypeptide comprising the extracellular TGFβ1-binding domain, transmembrane domain, and TLR intracellular signaling domain of TGFβR1, and a polypeptide comprising the extracellular TGFβ1-binding domain, transmembrane domain, and the same TLR intracellular signaling domain of TGFβR2.

[0261] 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, a TLR transmembrane domain, and an intracellular signaling domain. In one embodiment, the polypeptide comprises an extracellular TGFβ1-binding domain of TGFβR2, a TLR transmembrane domain, and an intracellular signaling domain.

[0262] In certain embodiments, the polypeptide cleavage signal is a viral self-cleaving polypeptide, more preferably a viral self-cleaving 2A polypeptide, more preferably a viral self-cleaving polypeptide selected from the group consisting of: foot-and-mouth disease virus (FMDV) (F2A) peptide, equine rhinitis A virus (ERAV) (E2A) peptide, Thosea asigna virus (TaV) (T2A) peptide, porcine teschovirus-1 (PTV-1) (P2A) peptide, tylovirus 2A peptide, and encephalomyocarditis virus 2A peptide. In one embodiment, the polypeptide cleavage signal is a P2A or T2A viral self-cleaving polypeptide.

[0263] E. Polypeptides Various polypeptides are contemplated herein, including, but not limited to, MAGEA4 TCR, MAGEA4 eTCR, CTBR, and fusion proteins comprising the aforementioned polypeptides and fragments thereof. The terms "polypeptide," "peptide," and "protein" are used interchangeably and follow their conventional meanings unless otherwise specified, i.e., as amino acid sequences. In one embodiment, "polypeptide" includes fusion polypeptides and other variants. Polypeptides can be prepared using any of a variety of known recombinant and / or synthetic techniques. Polypeptides are not limited to a particular length; for example, they can include full-length protein sequences, fragments of full-length proteins, or fusion proteins, and can include post-translational modifications of polypeptides, such as glycosylation, acetylation, phosphorylation, and other modifications, both naturally occurring and non-naturally occurring, known in the art.

[0264] As used herein, "isolated peptide" or "isolated polypeptide" or the like refers to the in vitro isolation and / or purification of a peptide or polypeptide molecule from its cellular environment and from association with other components of a cell, i.e., not substantially associated with in vivo substances.

[0265] Polypeptides include "polypeptide variants." Polypeptide variants may differ from naturally occurring polypeptides by one or more substitutions, deletions, additions, and / or insertions. Such variants may be natural or synthetically produced, 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 certain embodiments, polypeptides include those having at least about 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 86%, 97%, 98%, or 99% amino acid identity to any of the reference sequences contemplated herein, and typically such variants retain at least one biological activity of the reference sequence.

[0266] Polypeptide variants include biologically active "polypeptide fragments." 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 contain an amino acid chain of at least 5 to about 1700 amino acids in length. In certain embodiments, the fragment comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, or more amino acids in length.

[0267] In certain embodiments, the polypeptides described herein may contain one or more amino acids designated "X." When present in an amino acid SEQ ID NO:, "X" refers to any one or more amino acids. In certain embodiments, a SEQ ID NO representing a fusion protein contains a sequence of consecutive X residues that cumulatively represent any amino acid sequence.

[0268] As mentioned above, polypeptides may be modified in various ways, including amino acid substitution, deletion, truncation and insertion. Methods for such manipulation are generally known in the art. For example, amino acid sequence variants of reference polypeptides can be created by mutations in DNA. Methods for mutagenesis and nucleotide sequence modification are known in the art. For example, see 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, J.D. et al., (Molecular Biology of the Gene, Fourth Edition, Benjamin / Cummings, Menlo Park, Calif., 1987), and the references cited therein. Guidance regarding appropriate amino acid substitutions that do not affect the biological activity of the protein of interest can be found in the model Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC).

[0269] In some embodiments, a polypeptide variant contains one or more conservative substitutions. A "conservative substitution" is one in which an amino acid is replaced with another amino acid with similar properties, and one skilled in the art of peptide chemistry would predict that such a substitution would not substantially alter the secondary structure and hydrophilic properties of the polypeptide. In certain embodiments, modifications may be made to the contemplated polynucleotide and polypeptide structures while still obtaining functional molecules encoding variant or derivative polypeptides with desired characteristics. If it is desired to alter the amino acid sequence of a polypeptide to generate an equivalent or improved variant polypeptide, one skilled in the art can change one or more codons in the encoding DNA sequence, for example, according to Table 1. [Table 1]

[0270] Guidance for determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological activity can be found using computer programs known in the art, such as DNASTAR, DNA Strider, Geneious, MacVector, or Vector NTI software. Preferably, the amino acid changes in the protein variants disclosed herein are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes include substitutions of members of a family of amino acids that are related by their side chains. Natural amino acids are generally divided into four families: acidic amino acids (aspartic acid, glutamic acid), basic amino acids (lysine, arginine, histidine), nonpolar amino acids (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar amino acids (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes collectively classified as aromatic amino acids. In peptides or proteins, suitable conservative substitutions of amino acids are known to those skilled in the art and can generally be made without altering the biological activity of the resulting molecule. Those skilled in the art recognize that single amino acid substitutions in non-essential regions of a polypeptide generally do not significantly alter biological activity (see, for example, Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p. 224).

[0271] In one embodiment, when expression of more than one polypeptide is desired, the polynucleotide sequences encoding them may be separated by an IRES sequence, as otherwise disclosed herein.

[0272] In certain embodiments, contemplated polypeptides 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 2, 3, 4, 5, 6, 7, 8, 9, or 10 polypeptide segments.

[0273] In another embodiment, two or more polypeptides may be expressed as a fusion protein containing one or more self-cleaving polypeptide sequences, as disclosed elsewhere herein.

[0274] Fusion polypeptides may contain one or more polypeptide domains or segments, including, but not limited to, a signal peptide, a cell-penetrating peptide domain (CPP), a DNA-binding domain, a nuclease domain, etc., an epitope tag (e.g., maltose-binding protein ("MBP"), glutathione S-transferase (GST), HIS6, MYC, FLAG, V5, VSV-G, and HA), a polypeptide linker, and a polypeptide cleavage signal. Fusion polypeptides are typically joined C-terminally to N-terminally, but they may also be joined C-terminally to C-terminally, N-terminally to N-terminally, or N-terminally to C-terminally. In certain embodiments, the polypeptides of the fusion protein may be in any order. Fusion polypeptides or fusion proteins may include conservatively modified variants, polymorphic variants, alleles, mutants, subsequences, and interspecies homologs, provided that the desired activity of the fusion polypeptide is preserved. Fusion polypeptides may be made by chemical synthesis or by chemical conjugation between two moieties, or may generally be prepared using other standard methods. The ligated DNA sequence comprising the fusion polypeptide is operably linked to suitable transcriptional or translational control elements as disclosed elsewhere herein.

[0275] Fusion polypeptides may optionally contain a linker that can be used to link one or more polypeptides or domains within the polypeptide. A peptide linker sequence can be utilized to separate any two or more polypeptide components by a distance sufficient to ensure that each polypeptide folds into its appropriate secondary and tertiary structure so that the polypeptide domains can perform their desired functions. Such peptide linker sequences are incorporated into fusion polypeptides using standard techniques in the art. Suitable peptide linker sequences can be selected based on the following factors: (1) the ability to accommodate a flexible, extended conformation, (2) the ability to accommodate a secondary structure that can interact with functional epitopes on the first and second polypeptides, and (3) the absence of hydrophobic or charged residues that can react with the polypeptide functional epitopes. Preferred peptide linker sequences contain Gly, Asn, and Ser residues. Other near-neutral amino acids, such as Thr and Ala, can also be used in linker sequences. Amino acid sequences that can be usefully employed as linkers include those described in Maratea et al., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258-8262, 1986; U.S. Pat. Nos. 4,935,233 and 4,751,180. Linker sequences are not required if a particular fusion polypeptide segment contains a non-essential N-terminal amino acid region that can be used to separate functional domains and prevent steric interference. Preferred linkers are typically flexible amino acid subsequences synthesized as part of the recombinant fusion protein. Linker polypeptides can be 1 to 200 amino acids in length, 1 to 100 amino acids in length, or 1 to 50 amino acids in length, including all integer values between those values.

[0276] Examples of polypeptide cleavage signals include polypeptide cleavage recognition sites, such as protease cleavage sites, nuclease cleavage sites (e.g., rare restriction enzyme recognition sites, self-cleaving ribozyme recognition sites), and self-cleaving viral oligopeptides (see deFelipe and Ryan, 2004. Traffic, 5(8);616-26).

[0277] Suitable protease cleavage sites and self-cleaving peptides are known to those skilled in the art (see, for example, Ryan et al., 1997. J. Gener. Virol. 78, 699-722; Scymczak et al. (2004) Nature Biotech. 5, 589-594). Examples of protease cleavage sites include, but are not limited to, cleavage sites for potyvirus NIa protease (e.g., tobacco etch virus protease), potyvirus HC protease, potyvirus P1 (P35) protease, byovirus NIa protease, biovirus RNA-2-encoded protease, aphthovirus L protease, enterovirus 2A protease, rhinovirus 2A protease, picorna 3C protease, comovirus 24K protease, nepovirus 24K protease, RTSV (Waika virus) 3C-like protease, PYVF (parsnip yellow mottle virus) 3C-like protease, heparin, thrombin, factor Xa, and enterokinase. Due to their high cleavage stringency, in one embodiment TEV (tobacco etch virus) protease cleavage sites, e.g., EXXYXQ(G / S) (SEQ ID NO: 20), such as ENLYFQG (SEQ ID NO: 21) and ENLYFQS (SEQ ID NO: 22), are preferred, where X represents any amino acid (TEV cleavage occurs between Q and G or between Q and S).

[0278] 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 certain embodiments, the viral 2A peptide is an aphthovirus 2A peptide, a potyvirus 2A peptide, or a cardiovirus 2A peptide.

[0279] In one embodiment, the viral 2A peptide is selected from the group consisting of: a foot-and-mouth disease virus (FMDV) (F2A) peptide, an equine rhinitis A virus (ERAV) (E2A) peptide, a Thosea asigna virus (TaV) (T2A) peptide, a porcine teschovirus-1 (PTV-1) (P2A) peptide, a tylovirus 2A peptide, and an encephalomyocarditis virus 2A peptide.

[0280] Examples of 2A sites are provided in Table 2. [Table 2]

[0281] In preferred embodiments, the polypeptide comprises a MAGEA4 TCR, a MAGEA4 eTCR, or one or more CTBR polypeptides.

[0282] F. Polynucleotides In certain embodiments, polynucleotides are provided that encode MAGEA4 TCR, CTBR, engineered TCR, fusion proteins comprising the aforementioned polypeptides and fragments thereof. As used herein, the term "polynucleotide" or "nucleic acid" refers to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and DNA / RNA hybrids. Polynucleotides may be single-stranded or double-stranded, and may be recombinant, synthetic, or isolated. Polynucleotides include, but are not limited to, pre-messenger RNA (pre-mRNA), messenger RNA (mRNA), synthetic RNA, synthetic mRNA, genomic DNA (gDNA), PCR-amplified DNA, complementary DNA (cDNA), synthetic DNA, or recombinant DNA. Polynucleotide refers to a polymeric form of nucleotides having a length of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 1000, at least 5000, at least 10000, or at least 15000 or more nucleotides, including ribonucleotides or deoxyribonucleotides, or modified forms of either type of nucleotide, as well as all intermediate lengths. In this context, "intermediate length" will be readily understood to mean any length between the recited values, such as 6, 7, 8, 9, etc., 101, 102, 103, etc., 151, 152, 153, etc., 201, 202, 203, etc. In certain embodiments, a polynucleotide or variant has at least or about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a reference sequence.

[0283] In certain embodiments, polynucleotides may be codon-optimized. As used herein, the term "codon optimization" refers to substituting codons in a polynucleotide encoding a polypeptide to increase the expression, stability, and / or activity of the polypeptide. Factors that influence codon optimization include, but are not limited to, one or more of: (i) variation in codon bias between two or more organisms or genes, or synthetically constructed bias tables; (ii) variation in the degree of codon bias within an organism, gene, or set of genes; (iii) systematic variation of codons with context; (iv) variation of codons with their decoding tRNAs; (v) variation of codons with GC % either overall or at any single position in triplicates; (vi) variation in the degree of similarity to a reference sequence, e.g., a naturally occurring sequence; (vii) variation in codon frequency cutoff; (viii) structural properties of mRNA transcribed from a DNA sequence; (ix) prior knowledge of the function of the DNA sequence on which the design of the codon substitution set is based; (x) systematic variation of the codon set for each amino acid; and / or (xi) isolated removal of spurious translation start sites.

[0284] As used herein, the term "nucleotide" refers to a heterocyclic nitrogenous base in N-glycosidic linkage with a phosphorylated sugar. Nucleotides are understood to include natural bases and a wide variety of modified bases recognized in the art. Such bases are typically located at the 1' position of the nucleotide sugar moiety. Nucleotides generally contain a base, sugar, and a phosphate group. In ribonucleic acid (RNA), the sugar is ribose, and in deoxyribonucleic acid (DNA), the sugar is deoxyribose. That is, deoxyribose is a sugar lacking the hydroxyl group present in ribose. Examples of natural nitrogenous bases include the purines adenosine (A) and guanidine (G) and the pyrimidines cytidine (C) and thymidine (T) (or, in the case of RNA, uracil (U)). The C-1 atom of deoxyribose is linked to the N-1 atom of a pyrimidine or the N-9 atom of a purine. Nucleotides are typically mono-, di-, or triphosphates. Nucleotides can be unmodified or modified at the sugar, phosphate, and / or base moieties (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 nucleobases are summarized in Limbach et al. (1994, Nucleic Acids Res. 22, 2183-2196).

[0285] Nucleotides can also be considered as phosphate esters of nucleosides, with esterification occurring at the hydroxyl group attached to C-5 of the sugar. As used herein, the term "nucleoside" refers to a heterocyclic nitrogenous base in N-glycosidic linkage with a sugar. Nucleosides are recognized in the art to include natural bases and also known modified bases. Such bases are generally located at the 1' position of the nucleoside sugar moiety. Nucleosides generally contain a base and a sugar group. Nucleosides can be unmodified or modified in the sugar and / or base moieties (interchangeably referred to as nucleoside analogs, nucleoside derivatives, modified nucleosides, non-natural nucleosides, or non-standard nucleosides). As mentioned above, examples of modified nucleobases are summarized in Limbach et al. (1994, Nucleic Acids Res. 22, 2183-2196).

[0286] In various embodiments, polynucleotides contemplated herein include, but are not limited to, polynucleotides encoding MAGEA4 TCR, MAGEA4 eTCR, one or more CTBR polypeptides, fusion polypeptides, as well as expression vectors, viral vectors, and transfer plasmids comprising polynucleotides contemplated herein.

[0287] As used herein, terms such as "polynucleotide variant" and "variant" refer to a polynucleotide that exhibits substantial sequence identity with a reference polynucleotide sequence, or hybridizes with a reference sequence under stringent conditions as defined herein.These terms also include polynucleotides that are distinguished from a reference polynucleotide by the addition, deletion, substitution or modification of at least one nucleotide.Therefore, the terms "polynucleotide variant" and "variant" include polynucleotides in which one or more nucleotides are added, deleted, or modified, or replaced with another nucleotide.In this regard, it is well known in the art that certain changes, including mutations, additions, deletions and substitutions, can be made to a reference polynucleotide, thereby allowing the modified polynucleotide to retain the biological function or activity of the reference polynucleotide.

[0288] 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 protocol in which nucleotide sequences that are at least 60% identical to each other remain hybridized. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) of a specific sequence at a defined ionic strength and pH. Tm is the temperature (under defined 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. The target sequence is generally present in excess, so that at Tm, 50% of the probes are occupied at equilibrium.

[0289] The "sequence identity" listed, or for example, "50% identical sequences" as used herein, refers to the degree to which sequences are identical on a nucleotide-to-nucleotide basis or an amino acid-to-amino acid basis over a comparison window.Therefore, "percentage of sequence identity" can be calculated by: comparing two optimally aligned sequences over a comparison window; determining the number of positions where the same nucleic acid base (e.g., A, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) exists in both sequences; calculating the number of matching positions; dividing the number of matching positions by the total number of positions in the comparison window (i.e., window size); and multiplying the result by 100 to calculate the percentage of sequence identity. Included are nucleotides and polypeptides having at least about 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 86%, 97%, 98%, or 99% sequence identity to any of the reference sequences described herein; typically, the polypeptide variant retains at least one biological activity of the reference polypeptide.

[0290] Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include "reference sequence," "comparison window," "sequence identity," "percentage of sequence identity," and "substantial identity." A "reference sequence" comprises nucleotides and amino acid residues that are at least 12 monomeric units in length, often 15-18 monomeric units, and often at least 25 monomeric units in length. Two polynucleotides may each contain (1) similar sequences between the two polynucleotides (i.e., only a portion of the complete polynucleotide sequence) and (2) divergent sequences between the two polynucleotides. Sequence comparison between two (or more) polynucleotides is typically performed by comparing the sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of at least six contiguous positions, usually about 50 to about 100, more commonly about 100 to about 150, and a sequence is compared to the reference sequence over the same number of contiguous positions after the two sequences are optimally aligned. The comparison window may contain no more than about 20% additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. Optimal alignment of the sequences for aligning the comparison window can be performed by computerized implementation of algorithms (GAP, BESTFIT, FASTA, and TFASTA) in Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive, Madison, WI, USA, or by inspection and best alignment (i.e., resulting in the highest homology across the comparison window) generated by any of the various methods selected. See, for example, the BLAST family of programs disclosed by Altschul et al., 1997, Nucl. Acids Res. 25:3389.A detailed discussion of sequence analysis can be found in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons Inc, 1994-1998, Chapter 15, Unit 19.3.

[0291] As used herein, an "isolated polynucleotide" refers to a polynucleotide that has been purified from sequences that naturally flank it, e.g., a DNA fragment that has been removed from sequences that normally flank it. "Isolated polynucleotide" also refers to complementary DNA (cDNA), recombinant DNA, or other polynucleotides that are not found in nature and are made by the hand of man.

[0292] In various embodiments, the polynucleotide comprises an mRNA encoding a polypeptide contemplated herein, hi some embodiments, the mRNA comprises a cap, one or more nucleotides, and a poly(A) tail.

[0293] Terms describing the orientation of a polynucleotide include 5' (usually the end of a polynucleotide having a free phosphate group) and 3' (usually the end of a polynucleotide having a free hydroxyl (OH) group). Polynucleotide sequences can be annotated in the 5' to 3' direction or the 3' to 5' direction. For DNA and mRNA, the 5' to 3' strand is designated the "sense," "plus," or "coding" strand because its sequence is identical to that of the pre-messenger (pre-mRNA) [except for uracil (U) in RNA instead of thymine (T) in DNA]. For DNA and mRNA, the complementary 3' to 5' strand, which is the strand transcribed by RNA polymerase, is designated the "template," "antisense," "minus," or "non-coding" strand. As used herein, the term "reverse" refers to a 5' to 3' sequence written in the 3' to 5' direction or a 3' to 5' sequence written in the 5' to 3' direction.

[0294] The terms "complementary" and "complementarity" refer to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, the complementary strand of the DNA sequence 5'AGTCTATTG 3' is 3'TCAGTAC 5'. The latter sequence is often written as a reverse complement, 5'ATGGACT 3', with the 5' end to the left and the 3' end to the right. A sequence equivalent to its reverse complement is said to be a palindromic sequence. Complementarity can be "partial," in which only a portion of the nucleic acid bases match according to the base-pairing rules. Alternatively, there can be "complete" or "total" complementarity between nucleic acids.

[0295] Furthermore, those skilled in the art will recognize that, as a result of the degeneracy of the genetic code, there are numerous nucleotide sequences that encode the polypeptides described herein or variant fragments thereof. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any native gene. However, polynucleotides that vary due to differences in codon usage are contemplated, and in certain embodiments, polynucleotides optimized for, for example, human and / or primate codon preferences are contemplated. In certain embodiments, polynucleotides are codon-optimized for expression and / or stability. Furthermore, alleles of genes comprising the polynucleotide sequences provided herein may also be used. Alleles are endogenous genes that are altered as a result of one or more mutations, such as deletions, additions, and / or substitutions of nucleotides.

[0296] As used herein, the term "nucleic acid cassette" or "expression cassette" refers to a gene sequence in a vector capable of expressing RNA and subsequently expressing a polypeptide. In one embodiment, the nucleic acid cassette contains a gene of interest, e.g., a polynucleotide of interest. In another embodiment, the nucleic acid cassette contains one or more expression control sequences, e.g., a promoter, an enhancer, a poly(A) sequence, and a gene of interest, e.g., a polynucleotide of interest. A vector may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleic acid cassettes. The nucleic acid cassettes are positionally and sequentially oriented within the vector, allowing the nucleic acid in the cassette to be transcribed into RNA, translated into a protein or polypeptide if necessary, subjected to appropriate post-translational modifications required for activity in transformed cells, targeted to an appropriate intracellular compartment for translocation to a compartment suitable for biological activity, or secreted into an extracellular compartment. Preferably, the cassette has its 3' and 5' ends adapted for insertion into a vector, e.g., it has restriction endonuclease sites at each end. In a preferred embodiment, the nucleic acid cassette contains the sequence of a therapeutic gene used to treat, prevent, or ameliorate a genetic disorder. The cassette can be extracted and inserted as a single unit into a plasmid or viral vector.

[0297] Polynucleotides include polynucleotides of interest. As used herein, the term "polynucleotide of interest" refers to a polynucleotide that encodes a polypeptide or fusion polypeptide, or serves as a template for transcription of an inhibitory polynucleotide, as contemplated herein.

[0298] Polynucleotides contemplated herein, regardless of the length of the coding sequence itself, may be combined with other DNA sequences disclosed elsewhere herein or known in the art, such as promoters and / or enhancers, untranslated regions (UTRs), signal sequences, Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosome entry sites (IRES), recombinase recognition sites (e.g., LoxP, FRT, and Att sites), stop codons, transcription termination signals, and polynucleotides encoding self-cleaving polypeptides, epitope tags, etc., and as a result, their overall length may vary significantly. It is therefore contemplated that polynucleotide fragments of almost any length may be employed, with the overall length preferably being limited by ease of preparation and use in the intended recombinant DNA protocol.

[0299] Polynucleotides may be prepared, manipulated, expressed, and / or delivered using any of a variety of established techniques known and available in the art. To express a desired polypeptide, a nucleotide sequence encoding the polypeptide may be inserted into an appropriate vector.

[0300] Examples of vectors include, but are not limited to, plasmids, autonomously replicating sequences, and transposable elements, such as Sleeping Beauty and PiggyBac.

[0301] Additional examples of vectors include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal viruses.

[0302] Examples of viruses useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, and papovaviruses (e.g., SV40).

[0303] Exemplary expression vectors include, but are not limited to, pClneo vector (Promega) for expression in mammalian cells, pLenti4 / V5-DEST™, pLenti6 / V5-DEST™, and pLenti6.2 / V5-GW / lacZ (Invitrogen) for lentiviral-mediated gene transfer and expression in mammalian cells. In certain embodiments, the coding sequences of the polypeptides disclosed herein can be ligated into such expression vectors for expression of the polypeptides in mammalian cells.

[0304] In certain embodiments, the vector is an episomal vector, or a vector that is maintained extrachromosomally. As used herein, the term "episomal" refers to a vector that can replicate without being integrated into the chromosomal DNA of a host, and is not gradually reduced with the division of the host cell, which also means that the vector replicates extrachromosomally or episomally.

[0305] "Expression control sequences," "control elements," or "regulatory sequences" present in an expression vector are the untranslated regions of the vector—origins of replication, selection cassettes, promoters, enhancers, translation initiation signals (Shine Dalgarno or Kozak sequences), introns, polyadenylation sequences, and 5' and 3' untranslated regions—all of which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including ubiquitous and inducible promoters, may be used.

[0306] In certain embodiments, the polynucleotide comprises a vector, including, but not limited to, an expression vector and a viral vector. The vector may contain one or more exogenous, endogenous, or heterologous regulatory sequences, such as promoters and / or enhancers. An "endogenous regulatory sequence" is a sequence naturally linked to a given gene in the genome. An "exogenous regulatory sequence" is a sequence placed in juxtaposition to a gene by genetic engineering (i.e., molecular biological techniques), such that transcription of the gene is induced by the linked enhancer / promoter. A "heterologous regulatory sequence" is an exogenous sequence derived from a species different from the cell being genetically engineered. A "synthetic" regulatory sequence may contain one or more endogenous and / or exogenous sequence elements, and / or sequence elements determined in vitro or in silico, to provide optimal promoter and / or enhancer activity for a particular therapy.

[0307] As used herein, the term "promoter" refers to a recognition site in a polynucleotide (DNA or RNA) to which an RNA polymerase binds. The RNA polymerase initiates transcription of a polynucleotide operably linked to the promoter. In certain embodiments, promoters that operate in mammalian cells contain an AT-rich region located approximately 25-30 bases upstream from the site where transcription is initiated, and / or a separate sequence, a CNCAAT region, located 70-80 bases upstream from the transcription start site, where N can be any nucleotide.

[0308] The term "enhancer" refers to a DNA segment containing a sequence that can provide enhanced transcription, and in some cases can function regardless of orientation relative to another regulatory sequence. Enhancers can function cooperatively or additively with promoter elements and / or other enhancer elements. The term "promoter / enhancer" refers to a DNA segment containing a sequence that can provide both promoter and enhancer functions.

[0309] The term "operably linked" refers to a juxtaposition wherein the described components are in a relationship permitting them to function in their intended manner. In one embodiment, the term refers to the functional linkage between a nucleic acid expression control sequence (e.g., a promoter and / or enhancer) and a second polynucleotide sequence, e.g., a polynucleotide of interest, where the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.

[0310] As used herein, the term "structural expression control sequence" refers to a promoter, enhancer, or promoter / enhancer that permits continuous or sequential transcription of an operably linked sequence. A structural expression control sequence may be a "ubiquitous" promoter, enhancer, or promoter / enhancer that permits expression in a variety of cell and tissue types, or it may be a "cell-specific," "cell type-specific," "cell line-specific," or "tissue-specific" promoter, enhancer, or promoter / enhancer that permits expression in restricted cell and tissue types, respectively.

[0311] Examples of ubiquitous expression control sequences suitable for use in certain embodiments include, but are not limited to, the cytomegalovirus (CMV) immediate early promoter, the viral Simian Virus 40 (SV40) (e.g., early or late), Moloney Murine Leukemia Virus (MoMLV) LTR promoter, Rous Sarcoma Virus (RSV) LTR, herpes simplex virus (HSV) (thymidine kinase) promoter, the H5, P7.5, and P11 promoters from vaccinia virus, elongation factor 1 alpha (EF1a) promoter, early growth response protein 1 (EGR1), ferritin H (FerH), ferritin L (FerL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70 kDa protein 5 (HSPA5), heat shock protein 90 kDa, and the like. beta, member 1 (HSP90B1), heat shock protein 70kDa (HSP70), beta-kinesin (β-KIN), human ROSA 26 locus (Irions et al., Nature Biotechnology 25, 1477-1482 (2007)), ubiquitin C promoter (UBC), phosphoglycerate kinase-1 (PGK) promoter, cytomegalovirus enhancer / chicken beta-actin (CAG) promoter, beta-actin promoter and myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer binding site substitution (MND) U3 promoter (Haas et al. Journal of Virology. 2003;77(17):9439-9450).

[0312] In one embodiment, the vector contains the MNDU3 promoter.

[0313] In one embodiment, the vector contains the EF1a promoter including the first intron of the human EF1a gene.

[0314] In one embodiment, the vector contains the EF1a promoter lacking the first intron of the human EF1a gene.

[0315] In certain embodiments, it may be desirable to use cell-, cell type-, cell line-, or tissue-specific expression control sequences to achieve cell-type-, cell line-, or tissue-specific expression of a desired polynucleotide sequence (e.g., expressing a nucleic acid encoding a particular polypeptide only in a subset of cell types, cell lines, or tissues, or only at a particular stage of development).

[0316] In certain embodiments, it may be desirable to express the polynucleotide from a T cell specific promoter.

[0317] As used herein, "conditional expression" can refer to any type of conditional expression, including, but not limited to, inducible expression, repressible expression, and expression in cells or tissues that are in a particular physiological, biological, or disease state. This definition is not intended to exclude cell-type or tissue-specific expression. Certain embodiments provide for conditional expression of a polynucleotide of interest, e.g., expression controlled by exposing a cell, tissue, or organism to a treatment or condition that results in expression of the polynucleotide or that increases or decreases expression of a polynucleotide encoded by the polynucleotide of interest.

[0318] Examples of inducible promoters / systems include, but are not limited to, steroid-inducible promoters, such as promoters of genes encoding glucocorticoid receptors or estrogen receptors (inducible by treatment with the corresponding hormone), metallothionein promoters (inducible by treatment with various heavy metals), MX-1 promoters (inducible by interferon), the "GeneSwitch" mifepristone-regulated system (Sirin et al., 2003, Gene, 323:67), cumate-inducible gene switches (WO 2002 / 088346), tetracycline-dependent regulatory systems, etc. Inducers include, but are not limited to, glucocorticoids, estrogen, mifepristone (RU486), metals, interferons, small molecules, cumate, tetracycline, doxycycline, and variants thereof.

[0319] As used herein, "internal ribosome entry site" or "IRES" refers to a factor that promotes direct entry of an internal ribosome into an initiation codon, such as ATG, of a cistron (protein-coding region), resulting in cap-independent gene translation. See, 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 employed by those skilled in the art include those described in U.S. Pat. No. 6,692,736. Further 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), the translation initiation factor eIF4G, and the yeast transcription factors TFIID and HAP4, the encephalomyocarditis virus (EMCV) IRES available from Novagen (Duke et al., 1992. J. Virol 66(3):1602-9), and the VEGF IRES (Huez et al., 1998. Mol Cell Biol 18(11):6178-90). IRESes have also been reported in viral genomes of species from the Picornaviridae, Dicistroviridae, and Flaviviridae families, as well as in HCV, Friend murine leukemia virus (FrMLV), and Moloney murine leukemia virus (MoMLV).

[0320] In one embodiment, the IRES used in the polynucleotides contemplated herein is the EMCV IRES.

[0321] 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 significantly promotes the initial binding of mRNA to the small ribosomal subunit, increasing 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).

[0322] Factors that induce efficient termination and polyadenylation of heterologous nucleic acid transcripts increase heterologous gene expression. Transcription termination signals are generally located downstream of polyadenylation signals. In certain embodiments, vectors contain a polyadenylation sequence 3' to the polynucleotide encoding the polypeptide to be expressed. As used herein, the terms "polyA site" or "polyA sequence" refer to a DNA sequence that induces both termination and polyadenylation of the nascent RNA transcript by RNA polymerase II. Polyadenylation sequences can promote mRNA stability by adding a polyA tail to the 3' end of the coding sequence, thus contributing to improved translation efficiency. Cleavage and polyadenylation are directed by poly(A) sequences in the RNA. The core poly(A) sequence of mammalian pre-mRNAs has two recognition elements flanking the cleavage polyadenylation site. Typically, a nearly invariant AAUAAA hexamer is present 20-50 nucleotides upstream of a more variable element rich in U or GU residues. Cleavage of the initial transcript occurs between these two elements, adding up to 250 adenosines to the 5' cleavage product. In certain embodiments, the core poly(A) sequence is a poly(A) sequence of choice (e.g., AATAAA, ATTAAA, AGTAAA). In certain embodiments, the poly(A) sequence is SV40 poly(A), bovine growth hormone poly(A) sequence (BGHpA), rabbit β-globin poly(A) sequence (rβgpA), or another suitable heterologous or endogenous poly(A) sequence known in the art.

[0323] In some embodiments, the polynucleotide or cells harboring the polynucleotide utilize a suicide gene, including an inducible suicide gene, to reduce the risk of direct toxicity and / or uncontrolled amplification. In certain embodiments, the suicide gene is not immunogenic to the host or cells harboring the polynucleotide. Examples of suicide genes that can be used are caspase-9, caspase-8, or cytosine deaminase. Caspase-9 can be activated using a specific chemical inducer of dimerization (CID).

[0324] In certain embodiments, one or more polynucleotides encoding the eTCR chain, and / or the MAGEA4 TCR α chain and TCR β chain comprising one or more CTBR polypeptides are introduced into a cell (e.g., an immune effector cell) by a non-viral or viral vector. The term "vector" is used herein to refer to a nucleic acid molecule capable of transmitting or transporting another nucleic acid molecule. The transmitted nucleic acid is generally inserted, for example, into a vector nucleic acid molecule. The vector may contain a sequence that directs autonomous replication within the cell or may contain a sequence sufficient to allow integration into host cell DNA. In certain embodiments, a non-viral vector is used to deliver one or more polynucleotides contemplated herein to a T cell.

[0325] Examples of non-viral vectors include, but are not limited to, mRNA, plasmids (eg, DNA or RNA plasmids), transposons, cosmids, and bacterial artificial chromosomes.

[0326] Non-viral methods of polynucleotide delivery contemplated in certain embodiments include, but are not limited to, electroporation, sonoporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, nanoparticles, polycation or lipid:nucleic acid complexes, naked DNA, artificial virions, DEAE-dextran mediated transfer, gene guns, and heat shock.

[0327] Examples of polynucleotide delivery systems suitable for use in certain contemplated embodiments include, but are not limited to, systems provided by Amaxa Biosystems, Maxcyte, Inc., BTX Molecular Delivery Systems, and Copernicus Therapeutics Inc. Lipofection reagents are commercially available (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids suitable for efficient receptor-recognition lipofection of polynucleotides have been described in the literature. See, for example, Liu et al. (2003) Gene Therapy. 10:180-187; and Balazs et al. (2011) Journal of Drug Delivery. 2011:1-12. Antibody-targeted delivery, bacteria-guided delivery, and non-biological nanocell-based delivery are also contemplated in certain embodiments.

[0328] In various embodiments, the polynucleotide is the mRNA that is introduced into cells to transiently express desired polypeptide.As used herein, " transient " refers to the expression of non-integrated transgene for a period of hours, days or weeks, and the expression period is shorter than the expression period of polynucleotide when it is integrated into the genome of cells or contained in a stable plasmid replicon.

[0329] In certain embodiments, viral vectors are used to deliver one or more polynucleotides contemplated herein to T cells.

[0330] Examples of viral vector systems suitable for use in certain embodiments contemplated herein include, but are not limited to, adeno-associated virus (AAV), retrovirus (including lentivirus), herpes simplex virus, adenovirus, and vaccinia virus vectors.

[0331] 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 polycistronic polynucleotide encoding CTBR (SEQ ID NO: 8) are introduced into cells using a non-viral or viral vector. In certain embodiments, a polycistronic polynucleotide encoding a fusion protein encoding a MAGEA4 TCR (SEQ ID NO: 7) comprising a TCR alpha chain (SEQ ID NO: 5) and a TCR beta chain (SEQ ID NO: 6), and a polycistronic polynucleotide encoding CTBR (SEQ ID NO: 8) are introduced into cells using a non-viral or viral vector.

[0332] 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) are introduced into cells using a non-viral or viral vector. In certain embodiments, a polycistronic polynucleotide encoding a fusion protein encoding a MAGEA4 TCR (SEQ ID NO: 7) comprising a TCR alpha chain (SEQ ID NO: 5) and a TCR beta chain (SEQ ID NO: 6), and a CTBR (SEQ ID NO: 8) are introduced into cells using a non-viral or viral vector.

[0333] G. Genetically Modified Cells In various embodiments, cells are modified to express MAGEA4 TCR or MAGEA4 eTCR and CTBR for use in treating cancer. Cells may be non-genetically modified to express the polypeptides contemplated herein, or in certain preferred embodiments, cells may be genetically modified to express the polypeptides contemplated 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 in a cell. The terms "genetically modified cells," "modified cells," and "redirected cells" are used interchangeably in certain embodiments.

[0334] In certain embodiments, the MAGEA4 TCR and one or more CTBR polypeptides contemplated herein are introduced into and expressed in immune effector cells to improve the cells' resistance to immunosuppressive signals in the TME mediated by TGFβ, hi certain embodiments, the MAGEA4 eTCR and one or more CTBR polypeptides are introduced into and expressed in immune effector cells.

[0335] An "immune effector cell" is any cell of the immune system that has one or more effector functions (e.g., cytotoxic cell-killing activity, secretion of cytokines, induction of ADCC and / or CDC). Exemplary immune effector cells contemplated 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 comprise natural killer (NK) cells. In one embodiment, immune effector cells comprise natural killer T (NKT) cells. Immune effector cells can be autologous / autogeneic ("self") or non-autologous ("non-self", e.g., allogeneic, syngeneic, or xenogeneic).

[0336] As used herein, "autologous" refers to cells derived from the same subject.As used herein, "allogeneic" refers to cells of the same species that are genetically different from the cells being compared.As used herein, "syngeneic" refers to cells of a different subject that are genetically identical to the cells being compared.As used herein, "xenogeneic" refers to cells of a different species than the cells being compared.In a preferred embodiment, the cells are autologous.

[0337] Examples of immune effector cells suitable for the introduction of the CTBR polypeptide contemplated herein include T lymphocytes. The term "T cell" or "T lymphocyte" is art-recognized and is intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. T cells may be T helper (Th) cells, such as T helper 1 (Th1) cells or T helper 2 (Th2) cells. T cells may be helper T cells (HT1; CD4 + T cell) CD4 + T cells, cytotoxic T cells (CTL:CD8 + T cells), CD4 + CD8 + T cells, CD4 - CD8 - The T cells may be T cells, or any other subset of T cells. Other examples of T cell populations suitable for use in certain embodiments include naive T cells and memory T cells.

[0338] As will be appreciated by those skilled in the art, other cells can also be used as immune effector cells having a MAGEA4 TCR 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 precursors of effector cells, where such precursor cells may be induced to differentiate into immune effector cells in vivo or in vitro. Thus, in certain embodiments, immune effector cells include precursors of immune effector cells, such as hematopoietic stem cells (HSCs) contained within the CD34+ population of cells derived from, for example, umbilical cord blood, bone marrow, or mobilized peripheral blood, which differentiate into mature immune effector cells when administered to a subject or may be induced to differentiate into mature immune effector cells in vitro.

[0339] As used herein, immune effector cells that have been genetically engineered to contain a particular chimeric receptor may be referred to as "antigen-specific redirected immune effector cells."

[0340] As used herein, the term "CD34+ cells" refers to cells that express CD34 protein on their cell surface. As used herein, "CD34" refers to a cell surface glycoprotein (e.g., sialomucin protein) that often acts as a cell-cell adhesion factor and is involved in the entry of T cells into lymph nodes. CD34+ cell populations contain hematopoietic stem cells (HSCs), which, when administered to patients, differentiate and give rise to all hematopoietic lineages, including T cells, NK cells, NKT cells, neutrophils, and cells of the monocyte / macrophage lineage.

[0341] In certain embodiments, methods are provided for generating immune effector cells expressing a MAGEA4 TCR or MAGEA4 eTCR and a chimeric TGFβ receptor polypeptide contemplated herein. In one embodiment, the method comprises transfecting or transducing immune effector cells isolated from an individual such that the immune effector cells express a MAGEA4 TCR or MAGEA4 eTCR and one or more chimeric TGFβ receptor polypeptides contemplated herein. In one embodiment, the method comprises transfecting or transducing immune effector cells isolated from an individual such that the immune effector cells express a MAGEA4 TCR or MAGEA4 eTCR and one or more chimeric TGFβ receptor polypeptides and multiple MAGEA4 TCRs or MAGEA4 eTCRs contemplated herein. In certain embodiments, the immune effector cells are isolated from an individual and genetically modified without further in vitro manipulation. Such cells may then be directly readministered to the individual. In a further embodiment, the immune effector cells are first activated in vitro and stimulated to proliferate before being genetically modified. In this regard, the immune effector cells may be cultured before and / or after being genetically modified.

[0342] In certain embodiments, a source of cells is obtained from a subject prior to in vitro manipulation or genetic modification of the immune effector cells described herein, hi certain embodiments, the engineered immune effector cells comprise T cells.

[0343] T cells can be obtained from many sources, including, but not limited to, 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, and tumors. In one embodiment, T cells can be obtained from a unit of blood drawn from a subject using any number of techniques known to those skilled in the art, such as sedimentation, for example, FICOLL™ separation.

[0344] In other embodiments, isolated or purified T cell populations are used. In some embodiments, after isolation of PBMCs, both cytotoxic and helper T lymphocytes may be sorted into naive, memory, and effector T cell subpopulations before or after activation, expansion, and / or genetic modification.

[0345] In one embodiment, the isolated or purified T cell population includes, but is not limited to, CD3 + , CD4 + , CD8 + or express one or more of the markers, including a combination thereof.

[0346] In one embodiment, T cells are isolated from an individual and first activated and stimulated to expand in vitro and then modified to express a chimeric TGFβ receptor polypeptide.

[0347] To obtain a sufficient therapeutic dose of a T cell composition, T cells often undergo one or more rounds of stimulation, activation, and / or expansion. T cells can generally be activated and expanded using methods described, for example, in U.S. Patent Nos. 6,352,694, 6,534,055, 6,905,680, 6,692,964, 5,858,358, 6,887,466, 6,905,681, 7,144,575, 7,067,318, 7,172,869, 7,232,566, 7,175,843, 5,883,223, 6,905,874, 6,797,514, and 6,867,041. These patents are incorporated by reference in their entireties. In certain 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 the chimeric TGFβ receptor polypeptide.

[0348] In one embodiment, the T cells are simultaneously activated when they are modified.

[0349] In various embodiments, methods for generating immune effector cells include activating a cell population comprising T cells and expanding the T cell population. T cell activation can be achieved by providing a primary stimulatory signal via the TCR / CD3 complex of the T cells and a secondary costimulatory signal via an accessory molecule such as CD28.

[0350] The TCR / CD3 complex may be stimulated by contacting the T cell with a suitable CD3-binding agent, such as a CD3 ligand or an anti-CD3 monoclonal antibody, including, but not limited to, OKT3, G19-4, BC3, and 64.1.

[0351] In addition to the primary stimulatory signal provided via the TCR / CD3 complex, induction of a T cell response requires a secondary costimulatory signal. In certain embodiments, a CD28 binding agent may be used to provide the costimulatory signal. Exemplary CD28 binding agents include, but are not limited to, natural CD28 ligands, such as natural ligands for CD28 (e.g., members of the B7 family of proteins, e.g., B7-1 (CD80) and B7-2 (CD86)); and anti-CD28 monoclonal antibodies, or fragments thereof capable of cross-linking the CD28 molecule, such as monoclonal antibodies 9.3, B-T3, XR-CD28, KOLT-2, 15E8, 248.23.2, and EX5.3D10.

[0352] In one embodiment, the molecule that provides the primary stimulatory signal, eg, a molecule that provides stimulation via the TCR / CD3 complex, and the costimulatory molecule are linked to the same surface.

[0353] In certain embodiments, the binding agents that provide the stimulatory and costimulatory signals are localized on the cell surface, which can be achieved by transfecting or transducing the cell with a nucleic acid encoding the binding agent in a form suitable for expression on the cell surface, or by linking the binding agent to the cell surface.

[0354] In another embodiment, a molecule that provides a primary stimulatory signal, eg, a molecule that provides stimulation via the TCR / CD3 complex, and a costimulatory molecule are presented on an antigen-presenting cell.

[0355] In one embodiment, the molecule that provides the primary stimulatory signal, eg, a molecule that provides stimulation via the TCR / CD3 complex, and the costimulatory molecule are displayed on separate surfaces.

[0356] In certain embodiments, one of the binding agents providing the stimulatory and costimulatory signals is soluble (provided in solution) and the other agent is provided on one or more surfaces.

[0357] In certain embodiments, the binding agents that provide the stimulatory signal and the costimulatory signal are both provided in soluble form (provided in solution).

[0358] In various embodiments, the methods of generating T cells contemplated herein include activating T cells with an anti-CD3 antibody and an anti-CD28 antibody.

[0359] In one embodiment, expansion of T cells activated by the methods contemplated herein further comprises culturing the population of cells comprising the T cells for a period of time ranging from several hours (about 3 hours) to about 7 days to about 28 days, or any integer number of hours therebetween. In another embodiment, the T cell composition may be cultured for 14 days. In a specific embodiment, the T cells are cultured for about 21 days. In another embodiment, the T cell composition is cultured for about 2-3 days. Multiple cycles of stimulation / activation / expansion may be desirable, potentially extending the T cell culture period to 60 days or longer.

[0360] In certain embodiments, conditions suitable for T cell culture include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or X-vivo 15 (Lonza)) and one or more factors necessary for growth and activity, 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 supplement suitable for cell growth known to one of skill in the art.

[0361] Further exemplary 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, and either serum-free or supplemented with an appropriate amount of serum (or plasma) or a combination of predetermined hormones and / or cytokines in amounts sufficient for T cell growth and expansion.

[0362] Antibiotics, such as penicillin and streptomycin, are included only in the experimental cultures and not in the cell cultures intended for injection into subjects. 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% CO2).

[0363] In certain embodiments, PBMCs, or isolated T cells, are contacted with stimulatory and costimulatory agents, such as anti-CD3 and anti-CD28 antibodies, typically bound to beads or other surfaces, in culture medium containing appropriate cytokines, such as IL-2, IL-7, and / or IL-15.

[0364] In other embodiments, artificial APCs (aAPCs) are generated by engineering K562, U937, 721.221, T2, and C1R cells to induce stable expression and secretion of various costimulatory molecules and cytokines. In specific embodiments, K32 or U32 aAPCs are used to induce the presentation of one or more antibody-based stimulatory molecules on the aAPC cell surface. T cell populations may be expanded with 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 the expansion of genetically modified T cells and the maintenance of CD28 expression on CD8 T cells. The aAPCs presented in WO03 / 057171 and US2003 / 0147869 are incorporated herein by reference in their entireties.

[0365] 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 is introduced into a T cell population. In certain embodiments, a polynucleotide encoding a MAGEA4 TCR or MAGEA4 eTCR and a polynucleotide encoding a chimeric TGFβ receptor are introduced into a T cell population simultaneously. Polynucleotides may be introduced into T cells by microinjection, transfection, lipofection, heat shock, electroporation, transduction, gene gun, microinjection, DEAE-dextran mediated transfer, and the like.

[0366] In a preferred embodiment, the polynucleotide is introduced into the T cell by viral transduction.

[0367] The polynucleotides are then transduced into immune effector cells or CD34 + Illustrative examples of viral vector systems suitable for introducing into cells include, but are not limited to, adeno-associated virus (AAV), retrovirus, herpes simplex virus, adenovirus, and vaccinia virus vectors for gene transfer.

[0368] In one embodiment, the polynucleotide is introduced into the T cell by AAV transduction.

[0369] In one embodiment, the polynucleotide is introduced into the T cell by retroviral transduction.

[0370] In one embodiment, the polynucleotide is introduced into the T cells by lentiviral transduction.

[0371] In one embodiment, the polynucleotide is introduced into the T cell by adenoviral transduction.

[0372] In one embodiment, the polynucleotide is introduced into the T cell by herpes simplex virus transduction.

[0373] In one embodiment, the polynucleotide is introduced into the T cell by vaccinia virus transduction.

[0374] H. Compositions and Formulations Compositions contemplated herein may comprise one or more MAGEA4 TCR polypeptides, MAGEA4 eTCR polypeptides, CTBR polypeptides, polynucleotides, vectors comprising same, genetically modified immune effector cells, etc. contemplated herein. Compositions include, but are not limited to, pharmaceutical compositions. In a preferred embodiment, the composition comprises one or more cells modified to express MAGEA4 TCR and CTBR. In a preferred embodiment, the composition comprises one or more cells modified to express MAGEA4 eTCR and CTBR12 polypeptides.

[0375] A "pharmaceutical composition" refers to a composition formulated in a pharmaceutically or physiologically acceptable solution for administration to a cell or animal, alone or in combination with one or more other therapeutic modalities. It should also be understood that, if desired, the composition may be administered in conjunction with other agents as well, such as cytokines, growth factors, hormones, small molecules, chemotherapeutic agents, prodrugs, drugs, antibodies, or various other pharmaceutically active agents. There is virtually no limit to the other components that may be included in the composition, provided that the added agents do not adversely affect the ability of the composition to deliver its intended therapy. In a preferred embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, diluent, or excipient and one or more cells modified to express MAGEA4 TCR and CTBR, preferably MAGEA4 eTCR and CTBR12 polypeptides.

[0376] As used herein, the phrase "pharmaceutically acceptable" is employed to refer to those compounds, substances, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0377] As used herein, "pharmaceutically acceptable carrier, diluent, or excipient" includes, but is not limited to, isotonic saline, Ringer's solution, ethyl alcohol, phosphate buffer, and any other compatible substance used in pharmaceutical formulations.

[0378] In certain embodiments, the composition comprises a quantity of immune effector cells expressing MAGEA4 TCR and CTBR, preferably MAGEA4 eTCR and CTBR12 polypeptides. As used herein, the term "amount" refers to an "effective amount" or "effective quantity" of genetically modified therapeutic cells, such as T cells, to achieve a beneficial or desired prophylactic or therapeutic result, including a clinical result.

[0379] A "prophylactically effective amount" refers to an amount of genetically modified therapeutic cells effective to achieve a desired prophylactic result. Typically, but not necessarily, a prophylactically effective amount is less than a therapeutically effective amount, since a prophylactic dose is used in subjects prior to or at an early stage of disease.

[0380] A "therapeutically effective amount" of genetically modified therapeutic cells can vary depending on factors such as the individual's condition, age, sex, and weight, as well as the ability of the stem and progenitor cells to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or harmful effects of the virus or transduced therapeutic cells are outweighed by the therapeutically beneficial effects. The term "therapeutically effective amount" includes an amount effective to "treat" a subject (e.g., a patient). When a therapeutic amount is indicated, the exact amount of the composition to be administered can be determined by a physician, taking into account individual differences in the patient's (subject's) age, weight, tumor size, extent of infection or metastasis, and condition.

[0381] Generally, pharmaceutical compositions comprising the T cells described herein are administered in a dose of 10 6 ~10 13 cells / kg body weight, preferably 10 8 ~10 13It can be said that a dose of cells / kg body weight can be administered, including all integer values within the range. The number of cells depends on the intended end use of the composition and the type of cells contained in the composition. For the uses presented herein, the cells are generally in a volume of liter or less, and can be 500 ml or less, or even 250 ml or 100 ml or less. Therefore, the desired cell density is often 10 6 cells / ml, typically greater than 10 7 cells / ml, typically >10 8 Clinically relevant immune cell counts may be divided into multiple infusions, cumulatively exceeding 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 or 10 13 The compositions may be administered multiple times at doses within these ranges. The cells may be allogeneic, syngeneic, xenogeneic, or autologous to the patient undergoing therapy.

[0382] The compositions are preferably formulated for parenteral administration, eg, intravascular (intravenous or intraarterial), intraperitoneal, or intramuscular administration.

[0383] Liquid pharmaceutical compositions, whether in solution, suspension, or other similar form, may contain one or more sterile diluents such as water for injection, saline, preferably saline, Ringer's solution, or isotonic saline. Parenteral preparations may be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic. Pharmaceutical compositions for injection are preferably sterile.

[0384] In one embodiment, the T cell compositions contemplated herein are formulated in a pharmaceutically acceptable cell culture medium. Such compositions are suitable for administration to human subjects. In certain embodiments, the pharmaceutically acceptable cell culture medium is a serum-free medium.

[0385] Serum-free media have several advantages over serum-containing media, including a simpler and more transparent composition, reduced contaminant load, elimination of potential sources of infectious agents, and reduced cost. In various embodiments, serum-free media are animal-free and optionally protein-free. Optionally, the media may contain biopharmaceutical-acceptable recombinant proteins. "Animal-free" media refers to media whose components are derived from non-animal sources. Recombinant proteins replace natural animal proteins in animal-free media, and the nutrients are obtained from synthetic, plant, or microbial sources. "Protein-free" media, in contrast, are defined as being substantially protein-free.

[0386] Examples of serum-free media for use in certain compositions include, but are not limited to, QBSF-60 (Quality Biological, Inc.), StemPro-34 (Life Technologies), and X-VIVO 10.

[0387] In a preferred embodiment, compositions comprising immune effector cells contemplated herein are formulated in a solution comprising PlasmaLyte A.

[0388] In another preferred embodiment, the compositions contemplated herein containing immune effector cells are formulated in a solution containing a cryopreservation medium. For example, a cryopreservation medium containing a cryopreservative may be used to maintain high cell viability after thawing. Examples of cryopreservation media used in certain compositions include, but are not limited to, CryoStor CS10, CryoStor CS5, and CryoStor CS2.

[0389] In a more preferred embodiment, compositions comprising immune effector cells contemplated herein are formulated in a solution comprising 50:50 PlasmaLyte A to CryoStor CS10.

[0390] In certain embodiments, the composition comprises an effective amount of genome-edited immune effector cells engineered 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 may be administered alone or in combination with other known cancer treatments, such as radiation therapy, chemotherapy, transplantation, immunotherapy, hormonal therapy, or photodynamic therapy. The composition may also be administered in combination with antibiotics. Such therapeutic agents may be art-recognized as standard treatments for certain disease states described herein, such as certain cancers. Examples of contemplated therapeutic agents include, in certain embodiments, cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory agents, chemotherapeutic agents, radiation therapy, therapeutic antibodies, or other active adjunctive agents.

[0391] In certain embodiments, the composition comprising genome-edited immune effector cells modified to express MAGEA4 TCR and CTBR, preferably MAGEA4 eTCR and CTBR12 polypeptides, may be administered in conjunction with any number of chemotherapeutic agents.

[0392] In certain embodiments, compositions comprising immune effectors engineered to express a MAGEA4 TCR and CTBR, preferably a MAGEA4 eTCR and CTBR12 polypeptide, are administered in conjunction with a therapeutic antibody. Examples of therapeutic antibodies suitable for use in combination with engineered T cells contemplated in certain embodiments include, but are not limited to, atezolizumab, avelumab, bavituximab, bevacizumab (Avastin), bivatuzumab, blinatumomab, conatumumab, crizotinib, daratumumab, durigotumab, dacetuzumab, dalotuzumab, and durba. These include ramucirumab, elotuzumab (HuLuc63), gemtuzumab, ibritumomab, indatuximab, inotuzumab, ipilimumab, lorvotuzumab, lucatumumab, milatuzumab, moxetumomab, nivolumab, ocaratuzumab, ofatumumab, pembrolizumab, rituximab, siltuximab, teprotumumab, and ublituximab.

[0393] In certain embodiments, the formulation of pharmaceutically acceptable carrier solutions is known in the art, as is the development of appropriate dosing and treatment regimens for use of the particular compositions described herein in a variety of treatment regimens, including, for example, enteral and parenteral, e.g., intravascular, intravenous, intraarterial, intraosseous, intraventricular, intracerebral, intracranial, intrathecal, intrathecal, and intramedullary administration and formulation. Those of skill in the art will appreciate that certain embodiments contemplated herein are known, for example, in the pharmaceutical arts, and can be found in, for example, Remington: The Science and Practice of Pharmacy, volume I and volume II. 22 nd It will be understood that the present invention may include other formulations such as those described in "Therapeutic Agents for the Treatment of Acute Acetaminophen," 1999 Edition. Edited by Loyd V. Allen Jr. Philadelphia, PA: Pharmaceutical Press; 2012, which is incorporated herein by reference in its entirety.

[0394] I. Treatment method Immune effector cells comprising MAGEA4 TCR T cells or MAGEA4 eTCR T cells containing CTBR as contemplated herein provide improved methods of adoptive immunotherapy for use in the prevention, treatment, and amelioration of cancer, or for preventing, treating, or ameliorating at least one symptom associated with cancer.

[0395] The immune effector cells comprising a MAGEA4 TCR or MAGEA4 eTCR and a CTBR contemplated herein provide improved medicaments for use in the prevention, treatment, or amelioration of at least one symptom of cancer, GVHD, an infectious disease, an autoimmune disease, an inflammatory disease, or an immunodeficiency. As used herein, the term "medicament" refers to modified cells produced using the compositions and methods contemplated herein. In certain embodiments, the medicament comprises genetically modified immune effector cells, T cells modified to express a MAGEA4 TCR or a MAGEA4 eTCR, and T cells further modified to express a CTBR polypeptide. Furthermore, the modified T cells contemplated in certain embodiments exhibit improved durability and persistence in the tumor microenvironment, which can resist T cell exhaustion and result in sustained therapy, thereby providing safer and more effective adoptive cell therapy.

[0396] In certain embodiments, an effective amount of engineered immune effector cells or T cells comprising or expressing a MAGEA4 TCR or MAGEA4 eTCR and a CTBR is administered to a subject to prevent, treat, or ameliorate at least one symptom of cancer, GVHD, an infectious disease, an autoimmune disease, an inflammatory disease, or an immunodeficiency disorder.

[0397] 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 engineered immune effector cells or T cells comprising or expressing CTBR and MAGEA4 TCR or MAGEA4 eTCR. By converting immunosuppressive TGFβ signals into immunostimulatory signals, the genetically engineered cells are more resistant to immunosuppressive signals from the tumor microenvironment and are therefore more durable and long-lasting therapeutic agents.

[0398] In certain embodiments, the engineered immune effector cells contemplated herein are used to treat solid tumors or cancers.

[0399] In certain embodiments, the engineered immune effector cells contemplated herein are selected from, but are not limited to, adrenal carcinoma, adrenocortical carcinoma, anal cancer, appendiceal cancer, astrocytoma, atypical teratoid tumor / atypical rhabdomyoid tumor, basal cell carcinoma, bile duct carcinoma, bladder cancer, bone cancer, brain / CNS cancer, breast cancer, bronchial tumor, cardiac tumor, cervical cancer, bile duct carcinoma, chondrosarcoma, chordoma, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma in situ (DCIS), endometrial cancer, epithelial carcinoma, esophageal cancer, nasal neuroblastoma, Ewing's sarcoma, cranial Extragonadal germ cell tumor, extragonadal germ cell tumor, eye cancer, fallopian tube cancer, fibrous tissue sarcoma, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, glioma, glioblastoma, head and neck cancer, hemangioblastoma, hepatocellular carcinoma, hypopharyngeal cancer, intraocular melanoma, Kaposi's sarcoma, kidney cancer, laryngeal cancer, leiomyosarcoma, tongue cancer, liposarcoma, liver cancer, lung cancer, non-small cell lung cancer, embryonal carcinoid tumor, malignant mesothelioma, medullary carcinoma, medulloblastoma, meningioma, melanoma, Merkel cell carcinoma, midline carcinoma, oral cancer Used to treat solid tumors or cancers, including uterine cancer, myxosarcoma, myelodysplastic syndrome, myeloproliferative neoplasms, cancer of the nasal cavity and paranasal sinuses, nasopharyngeal carcinoma, neuroblastoma, oligodendroglioma, oral cancer (oral cavity cancer), oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, islet cell tumor, papillary carcinoma, paraganglioma, parathyroid carcinoma, penile cancer, pharyngeal carcinoma, pheochromocytoma, pinealoma, pituitary tumor, pleuropulmonary blastoma, primary pleurima, prostate cancer, rectal cancer, retinoblastoma, renal cell carcinoma, cancer of the renal pelvis and ureter, rhabdomyosarcoma, salivary gland cancer, sebaceous gland carcinoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, small cell lung cancer, small intestine cancer, gastric cancer, sweat gland carcinoma, synovial tumor, testicular cancer, pharyngeal cancer, thymic cancer, thyroid cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vascular cancer, vulvar cancer, and Wilms' tumor

[0400] In certain embodiments, the engineered 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.

[0401] In certain embodiments, the engineered immune effector cells contemplated herein are used to treat various cancers, including, but not limited to, pancreatic, bladder, and lung cancer.

[0402] In certain embodiments, the engineered immune effector cells contemplated herein are used to treat liquid or hematological cancers.

[0403] In certain embodiments, the engineered immune effector cells contemplated herein are used to treat B-cell malignancies, including, but not limited to, leukemia, lymphoma, and multiple myeloma.

[0404] In certain embodiments, the engineered immune effector cells contemplated herein are directed against, but are 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 myelogenous leukemia (CML), chronic myelomonocytic leukemia (CMML), and polycythemia vera, Hodgkin's lymphoma, nodular lymphocyte-predominant Hodgkin's lymphoma, It is used to treat liquid cancers including Burkitt's lymphoma, small lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, mantle cell lymphoma, marginal zone lymphoma, mycosis fungoides, anaplastic large cell lymphoma, Sézary syndrome, precursor T-lymphoblastic lymphoma, multiple myeloma, overt multiple myeloma, smoldering multiple myeloma, plasma cell leukemia, non-secretory myeloma, IgD myeloma, osteosclerotic myeloma, solitary bone plasmacytoma, and extramedullary plasmacytoma.

[0405] Preferred cells for use in the methods contemplated herein include self cells, preferably hematopoietic cells, more preferably T cells, and more preferably immune effector cells.

[0406] In certain embodiments, methods are provided that include administering a therapeutically effective amount of the engineered immune effector cells contemplated herein or a composition comprising the same to a patient in need thereof, alone or in combination with one or more therapeutic agents. In certain embodiments, the cells are used to treat a patient at risk of developing cancer, GVHD, an infectious disease, an autoimmune disease, an inflammatory disease, or an immune deficiency. Accordingly, certain embodiments include treating or preventing or ameliorating at least one symptom of cancer, an infectious disease, an autoimmune disease, an inflammatory disease, or an immune deficiency, comprising administering to a subject in need thereof a therapeutically effective amount of the genome-edited cells contemplated herein.

[0407] In one embodiment, a method for treating cancer, GVHD, an infectious disease, an autoimmune disease, an inflammatory disease, or an immune deficiency in a subject in need thereof comprises administering an effective amount, e.g., a therapeutically effective amount, of a composition comprising an engineered immune effector cell as contemplated herein. The amount and frequency of administration will be determined by factors such as the condition of the patient and the type and severity of the patient's disease, although appropriate dosages may be determined by clinical trials.

[0408] In one exemplary embodiment, the effective amount of engineered immune effector cells provided to a subject is at least 2 x 10 6 Cells / kg, at least 3x10 6 cells / kg, at least 4x10 6 Cells / kg, at least 5x10 6 cells / kg, at least 6x10 6 Cells / kg, at least 7x10 6 Cells / kg, at least 8x10 6 Cells / kg, at least 9x10 6 cells / kg, or at least 10x10 6 cells / kg, or more, including all cell doses in between.

[0409] In another exemplary embodiment, the effective amount of engineered immune effector cells provided to a subject is about 2x10 6 cells / kg, approximately 3x10 6cells / kg, approximately 4x10 6 cells / kg, approximately 5x10 6 cells / kg, approximately 6x10 6 cells / kg, approximately 7x10 6 cells / kg, approximately 8x10 6 cells / kg, approximately 9x10 6 cells / kg, or approximately 10x10 6 cells / kg, or more, including all cell doses in between.

[0410] In another exemplary embodiment, the effective amount of engineered immune effector cells provided to a subject is about 2x10 6 cells / kg ~ approx. 10x10 6 cells / kg, approximately 3x10 6 cells / kg ~ approx. 10x10 6 cells / kg, approximately 4x10 6 cells / kg ~ approx. 10x10 6 cells / kg, approximately 5x10 6 cells / kg ~ approx. 10x10 6 cells / kg, 2x10 6 cells / kg ~ approx. 6x10 6 cells / kg, 2x10 6 cells / kg ~ approx. 7x10 6 cells / kg, 2x10 6 cells / kg ~ approx. 8x10 6 cells / kg, 3x10 6 cells / kg ~ approx. 6x10 6 cells / kg, 3x10 6 cells / kg ~ approx. 7x10 6 cells / kg, 3x10 6 cells / kg ~ approx. 8x10 6 cells / kg, 4x10 6 cells / kg ~ approx. 6x10 6 cells / kg, 4x10 6 cells / kg ~ approx. 7x10 6 cells / kg, 4x10 6 cells / kg ~ approx. 8x10 6 cells / kg, 5x10 6 cells / kg ~ approx. 6x10 6 cells / kg, 5x10 6 cells / kg ~ approx. 7x10 6 cells / kg, 5x106 cells / kg ~ approx. 8x10 6 cells / kg, or 6x10 6 cells / kg ~ approx. 8x10 6 cells / kg and includes all cell doses in between.

[0411] Those skilled in the art will recognize that multiple administrations of contemplated compositions in certain embodiments may be required to achieve the desired therapeutic effect, for example, the compositions may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times over a period of 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 5 years, 10 years or more.

[0412] In some embodiments, it may be desirable to administer activated T cells to a subject, then withdraw blood from the subject (or perform apheresis), activate the T cells derived therefrom, and reinfuse these activated and expanded T cells back into the patient. This process may be performed multiple times, every few weeks. In some embodiments, T cells may be activated from a blood draw of 10 cc to 400 cc. In some embodiments, T cells are activated from a blood draw of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, 100 cc, 150 cc, 200 cc, 250 cc, 300 cc, 350 cc, or 400 cc or more. Without being bound by theory, the use of multiple blood draws / multiple reinfusion protocols may aid in the selection of certain populations of T cells.

[0413] In one embodiment, a method of treating a subject diagnosed with cancer comprises harvesting immune effector cells from the subject, modifying the immune effector cells by introducing one or more vectors encoding a MAGEA4 TCR or MAGEA4 eTCR and a chimeric TGFβ receptor, generating a population of modified immune effector cells, and administering the population of modified immune effector cells to the subject. In a preferred embodiment, the immune effector cells comprise T cells.

[0414] In certain embodiments, contemplated methods of administering cell compositions include any method effective to result in the reintroduction of modified immune effector cells ex vivo, or any method effective to result in the reintroduction of modified precursors of immune effector cells that differentiate into mature immune effector cells upon introduction into a subject. One method involves modifying peripheral blood T cells ex vivo by introducing one or more vectors encoding a MAGEA4 TCR or MAGEA4 eTCR and a chimeric TGFβ receptor, and returning the transduced cells to the subject.

[0415] 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.

[0416] Although the foregoing embodiments have been described in detail in the figures and examples for purposes of clarity and understanding, it will be readily apparent to those skilled in the art in light of the teachings contemplated herein that certain changes and modifications can be made without departing from the spirit or scope of the appended claims. The following examples are provided for illustrative purposes only and not for purposes of limitation. Those skilled in the art will readily recognize a variety of non-critical parameters that can be changed or modified to yield essentially similar results. [Example]

[0417] Example 1 MAGEA4 TCR T cells expressing the TGFβ signal converter transduce IL-12 signals and secrete increased IFNγ in response to MAGEA4 and TGFβ1. Peripheral blood mononuclear cells (PBMCs) from healthy donors were activated with soluble anti-CD3 (50 ng / ml) and anti-CD28 (50 ng / ml) to express (i) MAGEA4 TCR (e.g., SEQ ID NO: 4or (ii) a MAGEA4 TCR and an IL-12-responsive chimeric TGFβ signal converter (CTBR12) encoded on separate vectors, e.g., SEQ ID NO: 4 and 8 The cells were transduced with a lentiviral vector (LVV) expressing IL-2 at an MOI of 20. After 10 days of culture in IL2-containing medium, the cell products were harvested and cryopreserved for in vitro analysis.

[0418] CTBR12 signaling 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 confirm that CTBR12 blocks native TGFβ signaling. MAGEA4 TCR T cells and MAGEA4 TCR / CTBR12 T cells were rested overnight in serum-free medium and then exposed to TGFβ1 (10 ng / ml) for 20 minutes. Cells were fixed, permeabilized, and stained with anti-phospho-Smad2 / 3 (pS465 / 467) and anti-phospho-STAT4 (pY693). CTBR12 blocked Smad2 / 3 phosphorylation and activated STAT4 in T cells expressing MAGEA4 TCR (Figure 1, far right panel). These data indicate that CTBR12, when coexpressed with MAGEA4 TCR, can block native TGFβ signaling and transduce IL-12 signaling.

[0419] MAGEA4 TCR signaling Functional TCRs secrete IFNγ in response to antigen, and this secretion can be enhanced by IL-12 signaling. Untransduced (UTD) T cells, MAGEA4 TCR T cells, and MAGEA4 TCR / CTBR12 T cells were transduced with A375 MAGEA4 TCR in the presence or absence of TGFβ1 (10 ng / ml). +Tumor cells were co-cultured with the tumor cells at an E:T ratio of 1:1 for 24 hours. After 24 hours, the amount of IFNγ secreted into the medium was quantified. MAGEA4 TCR / CTBR12 T cells produced significantly greater amounts of IFNγ in the presence of TGFβ1 compared to all other treatment or control conditions (Figure 2). These data demonstrate that CTBR12 expression in MAGEA4 TCR T cells protects against TGFβ immunosuppression and promotes enhanced effector function in vitro.

[0420] Example 2 CTBR12 Expression Enhances MAGEA4 TCR T Cell Efficacy In Vivo We used the NOD.Cg-Prkdcscid IL2rgtm1Wjl / SzJ (NSG) xenograft mouse model to evaluate whether CTBR12 expression enhances MAGEA4 TCR T cell efficacy in vivo. A375 MAGEA4 + Tumor cells were implanted subcutaneously into NSG mice. Tumor volumes were measured twice weekly with a caliper and calculated using the formula: tumor volume = length x width x height x 0.52. When tumors reached a mean volume of 50 mm, 0.625 x 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 were injected intravenously into mice. MAGEA4 TCR / CTBR12 T cells controlled tumor volume significantly better than MAGEA4 TCR T cells or UTD control T cells (Figure 3). These data demonstrate that CTBR12 expression enhances the in vivo efficacy of MAGEA4 TCR T cells.

[0421] Example 3 Enhanced MAGEA4 TCR T cells expressing the TGFβ signal converter transduce IL-12 signals and secrete increased IFNγ in response to MAGEA4 and TGFβ1. Peripheral blood mononuclear cells (PBMCs) from healthy donors were activated with soluble anti-CD3 (50 ng / ml) and anti-CD28 (50 ng / ml) to express (i) a MAGEA4-paired enhanced TCR (eTCR), e.g., SEQ ID NO: 7 or (ii) a MAGEA4 eTCR and a CTBR12 receptor encoded in the same vector (e.g., SEQ ID NO: 7 and 8 The cells were transduced with a lentiviral vector (LVV) expressing the IL-2-containing gene (MOI = 20). After 10 days of culture in IL2-containing medium, the cell products were harvested and cryopreserved for in vitro analysis.

[0422] CTBR12 signaling 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 confirm that CTBR12 blocks native TGFβ signaling. MAGEA4 eTCR T cells and MAGEA4 eTCR / CTBR12 T cells were quiescent overnight in serum-free medium and then exposed to TGFβ1 (10 ng / ml) for 20 minutes. Cells were fixed, permeabilized, and stained with anti-phospho-Smad2 / 3 (pS465 / 467) and anti-phospho-STAT4 (pY693). CTBR12 blocked Smad2 / 3 phosphorylation and activated STAT4 in T cells expressing MAGEA4 eTCR (Figure 4, far right panel). These data indicate that CTBR12, when coexpressed with MAGEA4 eTCR, can block native TGFβ signaling and transduce IL-12 signaling.

[0423] MAGEA4 TCR signaling Functional TCRs secrete IFNγ in response to antigen, and this secretion can be enhanced by IL-12 signaling. Untransduced (UTD) T cells, MAGEA4 eTCR T cells, and MAGEA4 eTCR / CTBR12 T cells were transduced with A375 MAGEA4 in the presence or absence of TGFβ1 (10 ng / ml). +MAGEA4 eTCR T cells were co-cultured with tumor cells at an E:T ratio of 1:1 for 24 hours. After 24 hours, the amount of IFNγ secreted into the medium was quantified. TGFβ1 treatment suppressed IFNγ secretion by MAGEA4 eTCR T cells and enhanced IFNγ secretion by MAGEA4 eTCR / CTBR12 T cells (Figure 5). These data demonstrate that CTBR12 expression in MAGEA4 eTCR T cells protects them from TGFβ immunosuppression and promotes enhanced effector function in vitro.

[0424] In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the present disclosure. In certain embodiments, for example, the following items are provided: (Item 1) a cell comprising a first polynucleotide encoding a MAGEA4 TCR; (a) a first polypeptide, (i) the extracellular TGFβ1-binding domain of TGFβR2; (ii) a transmembrane domain, and (iii) a first polypeptide comprising an immunoreceptor intracellular signaling domain; and (b) a polypeptide cleavage signal; and (c) a second polypeptide, (i) the extracellular TGFβ1-binding domain of TGFβR1; (ii) a transmembrane domain, and (iii) a second polypeptide comprising an immunoreceptor intracellular signaling domain; and a second polynucleotide encoding a fusion polypeptide comprising: (Item 2) 2. The cell of item 1, wherein the immunoreceptor 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. (Item 3) 3. The cell of item 1 or item 2, wherein 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. (Item 4) 2. The cell of item 1, wherein the immunoreceptor intracellular signaling domain of the first polypeptide is an IL-12Rβ2 intracellular signaling domain and the immunoreceptor intracellular signaling domain of the second polypeptide is an IL-12Rβ1 intracellular signaling domain. (Item 5) 5. The cell of item 4, wherein the transmembrane domain of the first polypeptide comprises an IL-12Rβ2 transmembrane domain. (Item 6) 6. The cell of claim 4 or 5, wherein the transmembrane domain of the second polypeptide comprises an IL-12Rβ1 transmembrane domain. (Item 7) 2. The cell of item 1, wherein the immunoreceptor intracellular signaling domain of the first polypeptide is an IL-12Rβ1 intracellular signaling domain and the immunoreceptor intracellular signaling domain of the second polypeptide is an IL-12Rβ2 intracellular signaling domain. (Item 8) 8. The cell of item 7, wherein the transmembrane domain of the first polypeptide comprises an IL-12Rβ1 transmembrane domain. (Item 9) 9. The cell of claim 7 or 8, wherein the transmembrane domain of the second polypeptide comprises an IL-12Rβ2 transmembrane domain. (Item 10) 2. The cell of item 1, wherein the immunoreceptor intracellular signaling domain of the first polypeptide is an IL-7Rα intracellular signaling domain and the immunoreceptor intracellular signaling domain of the second polypeptide is an IL-2Rγ intracellular signaling domain. (Item 11) 11. The cell of item 10, wherein the transmembrane domain of the first polypeptide comprises an IL-7Rα transmembrane domain. (Item 12) 12. The cell of claim 10 or 11, wherein the transmembrane domain of the second polypeptide comprises an IL-2Rγ transmembrane domain. (Item 13) 2. The cell of item 1, wherein the immunoreceptor intracellular signaling domain of the first polypeptide is an IL-2Rγ intracellular signaling domain and the immunoreceptor intracellular signaling domain of the second polypeptide is an IL-7Rα intracellular signaling domain. (Item 14) 14. The cell of item 13, wherein the transmembrane domain of the first polypeptide comprises an IL-2Rγ transmembrane domain. (Item 15) 15. The cell of claim 13 or 14, wherein the transmembrane domain of the second polypeptide comprises an IL-7Rα transmembrane domain. (Item 16) 2. The cell of item 1, wherein the immunoreceptor intracellular signaling domain of the first polypeptide is an IL-2Rβ intracellular signaling domain and the immunoreceptor intracellular signaling domain of the second polypeptide is an IL-2Rγ intracellular signaling domain. (Item 17) 17. The cell of item 16, wherein the transmembrane domain of the first polypeptide comprises an IL-2Rβ transmembrane domain. (Item 18) 18. The cell of claim 16 or 17, wherein the transmembrane domain of the second polypeptide comprises an IL-2Rγ transmembrane domain. (Item 19) 2. The cell of item 1, wherein the immunoreceptor intracellular signaling domain of the first polypeptide is an IL-2Rγ intracellular signaling domain and the immunoreceptor intracellular signaling domain of the second polypeptide is an IL-2Rβ intracellular signaling domain. (Item 20) 20. The cell of item 19, wherein the transmembrane domain of the first polypeptide comprises an IL-2Rγ transmembrane domain. (Item 21) 21. The cell of claim 19 or 20, wherein the transmembrane domain of the second polypeptide comprises an IL-2Rβ transmembrane domain. (Item 22) 2. The cell of item 1, wherein the immunoreceptor intracellular signaling domain of the first polypeptide is an IL-21R intracellular signaling domain and the immunoreceptor intracellular signaling domain of the second polypeptide is an IL-2Rγ intracellular signaling domain. (Item 23) 23. The cell of item 22, wherein the transmembrane domain of the first polypeptide comprises an IL-21R transmembrane domain. (Item 24) 24. The cell of claim 22 or 23, wherein the transmembrane domain of the second polypeptide comprises an IL-2Rγ transmembrane domain. (Item 25) 2. The cell of item 1, wherein the immunoreceptor intracellular signaling domain of the first polypeptide is an IL-2Rγ intracellular signaling domain and the immunoreceptor intracellular signaling domain of the second polypeptide is an IL-21R intracellular signaling domain. (Item 26) 26. The cell of item 25, wherein the transmembrane domain of the first polypeptide comprises an IL-2Rγ transmembrane domain. (Item 27) 27. The cell of claim 25 or 26, wherein the transmembrane domain of the second polypeptide comprises an IL-21R transmembrane domain. (Item 28) 2. The cell of item 1, wherein the immunoreceptor intracellular signaling domain of the first polypeptide is an IL-18R1 intracellular signaling domain and the immunoreceptor intracellular signaling domain of the second polypeptide is an IL-18RAP intracellular signaling domain. (Item 29) 29. The cell of item 28, wherein the transmembrane domain of the first polypeptide comprises an IL-18R1 transmembrane domain. (Item 30) 30. The cell of claim 28 or 29, wherein the transmembrane domain of the second polypeptide comprises an IL-18RAP transmembrane domain. (Item 31) 2. The cell of item 1, wherein the immunoreceptor intracellular signaling domain of the first polypeptide is an IL-18RAP intracellular signaling domain and the immunoreceptor intracellular signaling domain of the second polypeptide is an IL-18R1 intracellular signaling domain. (Item 32) 32. The cell of claim 31, wherein the transmembrane domain of the first polypeptide comprises an IL-18RAP transmembrane domain. (Item 33) 33. The cell of claim 31 or 32, wherein the transmembrane domain of the second polypeptide comprises an IL-18R1 transmembrane domain. (Item 34) 2. The cell of item 1, wherein the immunoreceptor intracellular signaling domain of the first polypeptide is an IL-1R1 intracellular signaling domain and the immunoreceptor intracellular signaling domain of the second polypeptide is an IL-1RAP intracellular signaling domain. (Item 35) 35. The cell of item 34, wherein the transmembrane domain of the first polypeptide comprises an IL-1R1 transmembrane domain. (Item 36) 36. The cell of claim 34 or 35, wherein the transmembrane domain of the second polypeptide comprises an IL-1RAP transmembrane domain. (Item 37) 2. The cell of item 1, wherein the immunoreceptor intracellular signaling domain of the first polypeptide is an IL-1RAP intracellular signaling domain and the immunoreceptor intracellular signaling domain of the second polypeptide is an IL-1R1 intracellular signaling domain. (Item 38) 38. The cell of item 37, wherein the transmembrane domain of the first polypeptide comprises an IL-1RAP transmembrane domain. (Item 39) 39. The cell of claim 37 or 38, wherein the transmembrane domain of the second polypeptide comprises an IL-1R1 transmembrane domain. (Item 40) 2. The cell of item 1, wherein the immunoreceptor intracellular signaling domain of the first polypeptide is an IL-1RAP intracellular signaling domain and the immunoreceptor intracellular signaling domain of the second polypeptide is an IL-1RL2 intracellular signaling domain. (Item 41) 23. The cell of item 22, wherein the transmembrane domain of the first polypeptide comprises an IL-1RAP transmembrane domain. (Item 42) 42. The fusion polypeptide of claim 40 or 41, wherein the transmembrane domain of the second polypeptide comprises an IL-1RL2 transmembrane domain. (Item 43) 2. The cell of item 1, wherein the immunoreceptor intracellular signaling domain of the first polypeptide is an IL-1RL2 intracellular signaling domain and the immunoreceptor intracellular signaling domain of the second polypeptide is an IL-1RAP intracellular signaling domain. (Item 44) 44. The cell of item 43, wherein the transmembrane domain of the first polypeptide comprises an IL-1RL2 transmembrane domain. (Item 45) 45. The cell of claim 43 or 44, wherein the transmembrane domain of the second polypeptide comprises an IL-1RAP transmembrane domain. (Item 46) 2. The cell of item 1, wherein the immunoreceptor intracellular signaling domain of the first polypeptide is an IFNAR1 intracellular signaling domain and the immunoreceptor intracellular signaling domain of the second polypeptide is an IFNAR2 intracellular signaling domain. (Item 47) 47. The cell of item 46, wherein the transmembrane domain of the first polypeptide comprises an IFNAR1 transmembrane domain. (Item 48) 48. The cell of claim 46 or 47, wherein the transmembrane domain of the second polypeptide comprises an IFNAR2 transmembrane domain. (Item 49) 2. The cell of item 1, wherein the immunoreceptor intracellular signaling domain of the first polypeptide is an IFNAR2 intracellular signaling domain and the immunoreceptor intracellular signaling domain of the second polypeptide is an IFNAR1 intracellular signaling domain. (Item 50) 50. The cell of item 49, wherein the transmembrane domain of the first polypeptide comprises an IFNAR2 transmembrane domain. (Item 51) 51. The cell of claim 49 or 50, wherein the transmembrane domain of the second polypeptide comprises an IFNAR1 transmembrane domain. (Item 52) 2. The cell of item 1, wherein the immunoreceptor intracellular signaling domain of the first polypeptide is a TLR1 intracellular signaling domain, and the immunoreceptor intracellular signaling domain of the second polypeptide is a TLR1 intracellular signaling domain. (Item 53) 53. The cell of item 52, wherein the transmembrane domain of the first polypeptide comprises a TLR1 transmembrane domain. (Item 54) 54. The cell of claim 52 or 53, wherein the transmembrane domain of the second polypeptide comprises a TLR1 transmembrane domain. (Item 55) 2. The cell of item 1, wherein the immunoreceptor intracellular signaling domain of the first polypeptide is a TLR2 intracellular signaling domain, and the immunoreceptor intracellular signaling domain of the second polypeptide is a TLR2 intracellular signaling domain. (Item 56) 56. The cell of item 55, wherein the transmembrane domain of the first polypeptide comprises a TLR2 transmembrane domain. (Item 57) 57. The cell of claim 55 or 56, wherein the transmembrane domain of the second polypeptide comprises a TLR2 transmembrane domain. (Item 58) 2. The cell of item 1, wherein the immunoreceptor intracellular signaling domain of the first polypeptide is a TLR3 intracellular signaling domain, and the immunoreceptor intracellular signaling domain of the second polypeptide is a TLR3 intracellular signaling domain. (Item 59) 59. The cell of item 58, wherein the transmembrane domain of the first polypeptide comprises a TLR3 transmembrane domain. (Item 60) 60. The cell of claim 58 or 59, wherein the transmembrane domain of the second polypeptide comprises a TLR3 transmembrane domain. (Item 61) 2. The cell of item 1, wherein the immunoreceptor intracellular signaling domain of the first polypeptide is a TLR4 intracellular signaling domain, and the immunoreceptor intracellular signaling domain of the second polypeptide is a TLR4 intracellular signaling domain. (Item 62) 62. The cell of item 61, wherein the transmembrane domain of the first polypeptide comprises a TLR4 transmembrane domain. (Item 63) 63. The cell of claim 61 or 62, wherein the transmembrane domain of the second polypeptide comprises a TLR4 transmembrane domain. (Item 64) 2. The cell of item 1, wherein the immunoreceptor intracellular signaling domain of the first polypeptide is a TLR5 intracellular signaling domain, and the immunoreceptor intracellular signaling domain of the second polypeptide is a TLR5 intracellular signaling domain. (Item 65) 65. The cell of item 64, wherein the transmembrane domain of the first polypeptide comprises a TLR5 transmembrane domain. (Item 66) 66. The cell of claim 64 or 65, wherein the transmembrane domain of the second polypeptide comprises a TLR5 transmembrane domain. (Item 67) 2. The cell of item 1, wherein the immunoreceptor intracellular signaling domain of the first polypeptide is a TLR6 intracellular signaling domain, and the immunoreceptor intracellular signaling domain of the second polypeptide is a TLR6 intracellular signaling domain. (Item 68) 68. The cell of item 67, wherein the transmembrane domain of the first polypeptide comprises a TLR6 transmembrane domain. (Item 69) 69. The cell of claim 67 or 68, wherein the transmembrane domain of the second polypeptide comprises a TLR6 transmembrane domain. (Item 70) 2. The cell of item 1, wherein the immunoreceptor intracellular signaling domain of the first polypeptide is a TLR7 intracellular signaling domain, and the immunoreceptor intracellular signaling domain of the second polypeptide is a TLR7 intracellular signaling domain. (Item 71) 71. The cell of item 70, wherein the transmembrane domain of the first polypeptide comprises a TLR7 transmembrane domain. (Item 72) 72. The cell of claim 70 or 71, wherein the transmembrane domain of the second polypeptide comprises a TLR7 transmembrane domain. (Item 73) 2. The cell of item 1, wherein the immunoreceptor intracellular signaling domain of the first polypeptide is a TLR8 intracellular signaling domain, and the immunoreceptor intracellular signaling domain of the second polypeptide is a TLR8 intracellular signaling domain. (Item 74) 74. The cell of item 73, wherein the transmembrane domain of the first polypeptide comprises a TLR8 transmembrane domain. (Item 75) 75. The cell of claim 73 or 74, wherein the transmembrane domain of the second polypeptide comprises a TLR8 transmembrane domain. (Item 76) 2. The cell of item 1, wherein the immunoreceptor intracellular signaling domain of the first polypeptide is a TLR9 intracellular signaling domain, and the immunoreceptor intracellular signaling domain of the second polypeptide is a TLR9 intracellular signaling domain. (Item 77) 77. The cell of item 76, wherein the transmembrane domain of the first polypeptide comprises a TLR9 transmembrane domain. (Item 78) 78. The cell of claim 76 or 77, wherein the transmembrane domain of the second polypeptide comprises a TLR9 transmembrane domain. (Item 79) 2. The cell of item 1, wherein the immunoreceptor intracellular signaling domain of the first polypeptide is a TLR10 intracellular signaling domain, and the immunoreceptor intracellular signaling domain of the second polypeptide is a TLR10 intracellular signaling domain. (Item 80) 80. The cell of item 79, wherein the transmembrane domain of the first polypeptide comprises a TLR10 transmembrane domain. (Item 81) 81. The cell of claim 79 or 80, wherein the transmembrane domain of the second polypeptide comprises a TLR10 transmembrane domain. (Item 82) 82. The cell of any one of items 1 to 81, wherein the polypeptide cleavage signal is a viral self-cleaving polypeptide. (Item 83) 83. The cell of any one of items 1 to 82, wherein the polypeptide cleavage signal is a viral self-cleaving 2A polypeptide. (Item 84) 84. The cell of any one of items 1 to 83, wherein 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 A virus (ERAV) (E2A) peptide, Thosea asigna virus (TaV) (T2A) peptide, porcine teschovirus-1 (PTV-1) (P2A) peptide, tylovirus 2A peptide, and encephalomyocarditis virus 2A peptide. (Item 85) 10. The cell according to any one of items 1 to 9, wherein the fusion polypeptide comprises the amino acid sequence set forth in SEQ ID NO:5. (Item 86) a cell comprising a first polynucleotide encoding a MAGEA4 TCR; (a) a TGFβR2 polypeptide, (i) the extracellular TGFβ1-binding domain of TGFβR2; (ii) the IL-12Rβ2 transmembrane domain, and (iii) a TGFβR2 polypeptide comprising an IL-12Rβ2 intracellular signaling domain; and (b) viral self-cleaving 2A peptide; (c) a TGFβR1 polypeptide, (i) the extracellular TGFβ1-binding domain of TGFβR1; (ii) the IL-12Rβ1 transmembrane domain, and (iii) a cell comprising a TGFβR1 polypeptide comprising an IL-12Rβ1 intracellular signaling domain; and a second polynucleotide encoding a fusion polypeptide comprising the IL-12Rβ1 intracellular signaling domain. (Item 87) a cell comprising a first polynucleotide encoding a MAGEA4 TCR; (a) a TGFβR2 polypeptide, (i) the extracellular TGFβ1-binding domain of TGFβR2; (ii) the IL-12Rβ1 transmembrane domain, and (iii) a TGFβR2 polypeptide comprising an IL-12Rβ1 intracellular signaling domain; and (b) viral self-cleaving 2A peptide; (c) a TGFβR1 polypeptide, (i) the extracellular TGFβ1-binding domain of TGFβR1; (ii) the IL-12Rβ2 transmembrane domain, and (iii) a cell comprising a TGFβR1 polypeptide comprising an IL-12Rβ2 intracellular signaling domain; and a second polynucleotide encoding a fusion polypeptide comprising the TGFβR1 polypeptide. (Item 88) a cell comprising a first polynucleotide encoding a MAGEA4 TCR; (a) a TGFβR2 polypeptide, (i) the extracellular TGFβ1-binding domain of TGFβR2; (ii) the IL-7Rα transmembrane domain, and (iii) a TGFβR2 polypeptide comprising an IL-7Rα intracellular signaling domain; and (b) viral self-cleaving 2A peptide; (c) a TGFβR1 polypeptide, (i) the extracellular TGFβ1-binding domain of TGFβR1; (ii) the IL-2Rγ transmembrane domain, and (iii) a cell comprising a TGFβR1 polypeptide comprising an IL-2Rγ intracellular signaling domain; and a second polynucleotide encoding a fusion polypeptide comprising the TGFβR1 polypeptide. (Item 89) a cell comprising a first polynucleotide encoding a MAGEA4 TCR; (a) a TGFβR2 polypeptide, (i) the extracellular TGFβ1-binding domain of TGFβR2; (ii) the IL-2Rγ transmembrane domain, and (iii) a TGFβR2 polypeptide comprising an IL-2Rγ intracellular signaling domain; and (b) viral self-cleaving 2A peptide; (c) a TGFβR1 polypeptide, (i) the extracellular TGFβ1-binding domain of TGFβR1; (ii) the IL-7Rα transmembrane domain, and (iii) a cell comprising a TGFβR1 polypeptide comprising an IL-7Rα intracellular signaling domain; and a second polynucleotide encoding a fusion polypeptide comprising the IL-7Rα intracellular signaling domain. (Item 90) a cell comprising a first polynucleotide encoding a MAGEA4 TCR; (a) a TGFβR2 polypeptide, (i) the extracellular TGFβ1-binding domain of TGFβR2; (ii) the IL-2Rβ transmembrane domain, and (iii) a TGFβR2 polypeptide comprising an IL-2Rβ intracellular signaling domain; and (b) viral self-cleaving 2A peptide; (c) a TGFβR1 polypeptide, (i) the extracellular TGFβ1-binding domain of TGFβR1; (ii) the IL-2Rγ transmembrane domain, and (iii) a cell comprising a TGFβR1 polypeptide comprising an IL-2Rγ intracellular signaling domain; and a second polynucleotide encoding a fusion polypeptide comprising the TGFβR1 polypeptide. (Item 91) a cell comprising a first polynucleotide encoding a MAGEA4 TCR; (a) a TGFβR2 polypeptide, (i) the extracellular TGFβ1-binding domain of TGFβR2; (ii) the IL-2Rγ transmembrane domain, and (iii) a TGFβR2 polypeptide comprising an IL-2Rγ intracellular signaling domain; and (b) viral self-cleaving 2A peptide; (c) a TGFβR1 polypeptide, (i) the extracellular TGFβ1-binding domain of TGFβR1; (ii) the IL-2Rβ transmembrane domain, and (iii) a cell comprising a TGFβR1 polypeptide comprising an IL-2Rβ intracellular signaling domain; and a second polynucleotide encoding a fusion polypeptide comprising the TGFβR1 polypeptide. (Item 92) a cell comprising a first polynucleotide encoding a MAGEA4 TCR; (a) a TGFβR2 polypeptide, (i) the extracellular TGFβ1-binding domain of TGFβR2; (ii) the IL-21R transmembrane domain, and (iii) a TGFβR2 polypeptide comprising an IL-21R intracellular signaling domain; and (b) viral self-cleaving 2A peptide; (c) a TGFβR1 polypeptide, (i) the extracellular TGFβ1-binding domain of TGFβR1; (ii) the IL-2Rγ transmembrane domain, and (iii) a cell comprising a TGFβR1 polypeptide comprising an IL-2Rγ intracellular signaling domain; and a second polynucleotide encoding a fusion polypeptide comprising the TGFβR1 polypeptide. (Item 93) a cell comprising a first polynucleotide encoding a MAGEA4 TCR; (a) a TGFβR2 polypeptide, (i) the extracellular TGFβ1-binding domain of TGFβR2; (ii) the IL-2Rγ transmembrane domain, and (iii) a TGFβR2 polypeptide comprising an IL-2Rγ intracellular signaling domain; and (b) viral self-cleaving 2A peptide; (c) a TGFβR1 polypeptide, (i) the extracellular TGFβ1-binding domain of TGFβR1; (ii) the IL-21R transmembrane domain, and (iii) a cell comprising a TGFβR1 polypeptide comprising an IL-21R intracellular signaling domain; and a second polynucleotide encoding a fusion polypeptide comprising the TGFβR1 polypeptide. (Item 94) a cell comprising a first polynucleotide encoding a MAGEA4 TCR; (a) a TGFβR2 polypeptide, (i) the extracellular TGFβ1-binding domain of TGFβR2; (ii) the IL-18R1 transmembrane domain, and (iii) a TGFβR2 polypeptide comprising an IL-18R1 intracellular signaling domain; and (b) viral self-cleaving 2A peptide; (c) a TGFβR1 polypeptide, (i) the extracellular TGFβ1-binding domain of TGFβR1; (ii) the IL-18RAP transmembrane domain, and (iii) A cell comprising a TGFβR1 polypeptide comprising an IL-18RAP intracellular signaling domain, and a second polynucleotide encoding a fusion polypeptide comprising the TGFβR1 polypeptide. (Item 95) a cell comprising a first polynucleotide encoding a MAGEA4 TCR; (a) a TGFβR2 polypeptide, (i) the extracellular TGFβ1-binding domain of TGFβR2; (ii) the IL-18RAP transmembrane domain, and (iii) a TGFβR2 polypeptide comprising an IL-18RAP intracellular signaling domain; and (b) viral self-cleaving 2A peptide; (c) a TGFβR1 polypeptide, (i) the extracellular TGFβ1-binding domain of TGFβR1; (ii) the IL-18R1 transmembrane domain, and (iii) a cell comprising a TGFβR1 polypeptide comprising an IL-18R1 intracellular signaling domain; and a second polynucleotide encoding a fusion polypeptide comprising the TGFβR1 polypeptide. (Item 96) a cell comprising a first polynucleotide encoding a MAGEA4 TCR; (a) a TGFβR2 polypeptide, (i) the extracellular TGFβ1-binding domain of TGFβR2; (ii) the IL-1R1 transmembrane domain, and (iii) a TGFβR2 polypeptide comprising an IL-1R1 intracellular signaling domain; and (b) viral self-cleaving 2A peptide; (c) a TGFβR1 polypeptide, (i) the extracellular TGFβ1-binding domain of TGFβR1; (ii) the IL-1RAP transmembrane domain, and (iii) A cell comprising a TGFβR1 polypeptide comprising an IL-1RAP intracellular signaling domain, and a second polynucleotide encoding a fusion polypeptide comprising the TGFβR1 polypeptide. (Item 97) a cell comprising a first polynucleotide encoding a MAGEA4 TCR; (a) a TGFβR2 polypeptide, (i) the extracellular TGFβ1-binding domain of TGFβR2; (ii) the IL-1RAP transmembrane domain, and (iii) a TGFβR2 polypeptide comprising an IL-1RAP intracellular signaling domain; and (b) viral self-cleaving 2A peptide; (c) a TGFβR1 polypeptide, (i) the extracellular TGFβ1-binding domain of TGFβR1; (ii) the IL-1R1 transmembrane domain, and (iii) a cell comprising a TGFβR1 polypeptide comprising an IL-1R1 intracellular signaling domain; and a second polynucleotide encoding a fusion polypeptide comprising the TGFβR1 polypeptide. (Item 98) a cell comprising a first polynucleotide encoding a MAGEA4 TCR; (a) a TGFβR2 polypeptide, (i) the extracellular TGFβ1-binding domain of TGFβR2; (ii) the IFNAR1 transmembrane domain, and (iii) a TGFβR2 polypeptide comprising an IFNAR1 intracellular signaling domain; and (b) viral self-cleaving 2A peptide; (c) a TGFβR1 polypeptide, (i) the extracellular TGFβ1-binding domain of TGFβR1; (ii) the IFNAR2 transmembrane domain, and (iii) a cell comprising a TGFβR1 polypeptide comprising an IFNAR2 intracellular signaling domain, and a second polynucleotide encoding a fusion polypeptide comprising the IFNAR2 intracellular signaling domain. (Item 99) a cell comprising a first polynucleotide encoding a MAGEA4 TCR; (a) a TGFβR2 polypeptide, (i) the extracellular TGFβ1-binding domain of TGFβR2; (ii) the IFNAR2 transmembrane domain, and (iii) a TGFβR2 polypeptide comprising an IFNAR2 intracellular signaling domain; and (b) viral self-cleaving 2A peptide; (c) a TGFβR1 polypeptide, (i) the extracellular TGFβ1-binding domain of TGFβR1; (ii) the IFNAR1 transmembrane domain, and (iii) A cell comprising a TGFβR1 polypeptide comprising an IFNAR1 intracellular signaling domain, and a second polynucleotide encoding a fusion polypeptide comprising the IFNAR1 intracellular signaling domain. (Item 100) a cell comprising a first polynucleotide encoding a MAGEA4 TCR; (a) a TGFβR2 polypeptide, (i) the extracellular TGFβ1-binding domain of TGFβR2; (ii) the TLR1 transmembrane domain, and (iii) a TGFβR2 polypeptide comprising a TLR1 intracellular signaling domain; and (b) viral self-cleaving 2A peptide; (c) a TGFβR1 polypeptide, (i) the extracellular TGFβ1-binding domain of TGFβR1; (ii) the TLR1 transmembrane domain, and (iii) a cell comprising a TGFβR1 polypeptide comprising a TLR1 intracellular signaling domain; and a second polynucleotide encoding a fusion polypeptide comprising the TGFβR1 polypeptide. (Item 101) a cell comprising a first polynucleotide encoding a MAGEA4 TCR; (a) a TGFβR2 polypeptide, (i) the extracellular TGFβ1-binding domain of TGFβR2; (ii) the TLR2 transmembrane domain, and (iii) a TGFβR2 polypeptide comprising a TLR2 intracellular signaling domain; (b) viral self-cleaving 2A peptide; (c) a TGFβR1 polypeptide, (i) the extracellular TGFβ1-binding domain of TGFβR1; (ii) the TLR2 transmembrane domain, and (iii) a cell comprising a TGFβR1 polypeptide comprising a TLR2 intracellular signaling domain; and a second polynucleotide encoding a fusion polypeptide comprising the TGFβR1 polypeptide. (Item 102) a cell comprising a first polynucleotide encoding a MAGEA4 TCR; (a) a TGFβR2 polypeptide, (i) the extracellular TGFβ1-binding domain of TGFβR2; (ii) the TLR3 transmembrane domain, and (iii) a TGFβR2 polypeptide comprising a TLR3 intracellular signaling domain; and (b) viral self-cleaving 2A peptide; (c) a TGFβR1 polypeptide, (i) the extracellular TGFβ1-binding domain of TGFβR1; (ii) the TLR3 transmembrane domain, and (iii) A cell comprising a TGFβR1 polypeptide comprising a TLR3 intracellular signaling domain, and a second polynucleotide encoding a fusion polypeptide comprising the TGFβR1 polypeptide. (Item 103) a cell comprising a first polynucleotide encoding a MAGEA4 TCR; (a) a TGFβR2 polypeptide, (i) the extracellular TGFβ1-binding domain of TGFβR2; (ii) the TLR4 transmembrane domain, and (iii) a TGFβR2 polypeptide comprising a TLR4 intracellular signaling domain; and (b) viral self-cleaving 2A peptide; (c) a TGFβR1 polypeptide, (i) the extracellular TGFβ1-binding domain of TGFβR1; (ii) the TLR4 transmembrane domain, and (iii) A cell comprising a TGFβR1 polypeptide comprising a TLR4 intracellular signaling domain, and a second polynucleotide encoding a fusion polypeptide comprising the TGFβR1 polypeptide. (Item 104) a cell comprising a first polynucleotide encoding a MAGEA4 TCR; (a) a TGFβR2 polypeptide, (i) the extracellular TGFβ1-binding domain of TGFβR2; (ii) the TLR5 transmembrane domain, and (iii) a TGFβR2 polypeptide comprising a TLR5 intracellular signaling domain; (b) viral self-cleaving 2A peptide; (c) a TGFβR1 polypeptide, (i) the extracellular TGFβ1-binding domain of TGFβR1; (ii) the TLR5 transmembrane domain, and (iii) A cell comprising a TGFβR1 polypeptide comprising a TLR5 intracellular signaling domain, and a second polynucleotide encoding a fusion polypeptide comprising the TGFβR1 polypeptide. (Item 105) a cell comprising a first polynucleotide encoding a MAGEA4 TCR; (a) a TGFβR2 polypeptide, (i) the extracellular TGFβ1-binding domain of TGFβR2; (ii) the TLR6 transmembrane domain, and (iii) a TGFβR2 polypeptide comprising a TLR6 intracellular signaling domain; (b) viral self-cleaving 2A peptide; (c) a TGFβR1 polypeptide, (i) the extracellular TGFβ1-binding domain of TGFβR1; (ii) the TLR6 transmembrane domain, and (iii) A cell comprising a TGFβR1 polypeptide comprising a TLR6 intracellular signaling domain, and a second polynucleotide encoding a fusion polypeptide comprising the TGFβR1 polypeptide. (Item 106) a cell comprising a first polynucleotide encoding a MAGEA4 TCR; (a) a TGFβR2 polypeptide, (i) the extracellular TGFβ1-binding domain of TGFβR2; (ii) the TLR7 transmembrane domain, and (iii) a TGFβR2 polypeptide comprising a TLR7 intracellular signaling domain; and (b) viral self-cleaving 2A peptide; (c) a TGFβR1 polypeptide, (i) the extracellular TGFβ1-binding domain of TGFβR1; (ii) the TLR7 transmembrane domain, and (iii) A cell comprising a TGFβR1 polypeptide comprising a TLR7 intracellular signaling domain, and a second polynucleotide encoding a fusion polypeptide comprising the TGFβR1 polypeptide. (Item 107) a cell comprising a first polynucleotide encoding a MAGEA4 TCR; (a) a TGFβR2 polypeptide, (i) the extracellular TGFβ1-binding domain of TGFβR2; (ii) the TLR8 transmembrane domain, and (iii) a TGFβR2 polypeptide comprising a TLR8 intracellular signaling domain; (b) viral self-cleaving 2A peptide; (c) a TGFβR1 polypeptide, (i) the extracellular TGFβ1-binding domain of TGFβR1; (ii) the TLR8 transmembrane domain, and (iii) A cell comprising a TGFβR1 polypeptide comprising a TLR8 intracellular signaling domain, and a second polynucleotide encoding a fusion polypeptide comprising the TGFβR1 polypeptide. (Item 108) a cell comprising a first polynucleotide encoding a MAGEA4 TCR; (a) a TGFβR2 polypeptide, (i) the extracellular TGFβ1-binding domain of TGFβR2; (ii) the TLR9 transmembrane domain, and (iii) a TGFβR2 polypeptide comprising a TLR9 intracellular signaling domain; and (b) viral self-cleaving 2A peptide; (c) a TGFβR1 polypeptide, (i) the extracellular TGFβ1-binding domain of TGFβR1; (ii) the TLR9 transmembrane domain, and (iii) A cell comprising a TGFβR1 polypeptide comprising a TLR9 intracellular signaling domain, and a second polynucleotide encoding a fusion polypeptide comprising the TGFβR1 polypeptide. (Item 109) a cell comprising a first polynucleotide encoding a MAGEA4 TCR; (a) a TGFβR2 polypeptide, (i) the extracellular TGFβ1-binding domain of TGFβR2; (ii) the TLR10 transmembrane domain, and (iii) a TGFβR2 polypeptide comprising a TLR10 intracellular signaling domain; (b) viral self-cleaving 2A peptide; (c) a TGFβR1 polypeptide, (i) the extracellular TGFβ1-binding domain of TGFβR1; (ii) the TLR10 transmembrane domain, and (iii) A cell comprising a TGFβR1 polypeptide comprising a TLR10 intracellular signaling domain, and a second polynucleotide encoding a fusion polypeptide comprising the TGFβR1 polypeptide. (Item 110) 112. The cell of any one of items 88 to 111, wherein the viral self-cleaving 2A polypeptide is selected from the group consisting of a foot-and-mouth disease virus (FMDV) (F2A) peptide, an equine rhinitis A virus (ERAV) (E2A) peptide, a Thosea asigna virus (TaV) (T2A) peptide, a porcine teschovirus-1 (PTV-1) (P2A) peptide, a tylovirus 2A peptide, and an encephalomyocarditis virus 2A peptide. (Item 111) 88. The cell of item 86 or item 87, wherein the fusion polypeptide comprises the amino acid sequence set forth in SEQ ID NO:5. (Item 112) 112. The cell of any one of items 1 to 41, 43 to 111, wherein the MAGEA4 TCR binds to the peptide GVYDGREHTV presented by an HLA-A*02:01 encoded molecule. (Item 113) 13. The cell of any one of items 1 to 41 and 43 to 112, wherein the MAGEA4 TCR comprises an alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 1 and a beta chain comprising the amino acid sequence set forth in SEQ ID NO: 2. (Item 114) 14. The cell of any one of items 1 to 41 and 43 to 113, wherein the MAGEA4 TCR comprises an alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 3 and a beta chain comprising the amino acid sequence set forth in SEQ ID NO: 4. (Item 115) 15. The cell according to any one of items 1 to 41 and 43 to 114, wherein the cell is a hematopoietic cell. (Item 116) The cell according to any one of items 1 to 41 and 43 to 115, wherein the cell is a T cell. (Item 117) The cells are CD3 + , CD4 + , and / or CD8 + 117. The cell according to any one of items 1 to 41 and 43 to 116, which is a cell. (Item 118) 118. The cell according to any one of items 1 to 41 and 43 to 117, wherein the cell is an immune effector cell. (Item 119) The cell of any one of items 1 to 41, 43 to 118, wherein the cell is a cytotoxic T lymphocyte (CTL), a tumor-infiltrating lymphocyte (TIL), or a helper T cell. (Item 120) 119. The cell according to any one of items 1 to 41 and 43 to 119, wherein the cell is a natural killer (NK) cell or a natural killer T (NKT) cell. (Item 121) 121. The cell according to any one of items 1 to 41 and 43 to 120, 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. (Item 122) A composition comprising the cells according to any one of items 1 to 41 and 43 to 121. (Item 123) 122. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the cells of any one of items 1 to 41, 43 to 121. (Item 124) 124. A method of treating a subject in need thereof, comprising administering to said subject an effective amount of the composition of item 123. (Item 125) 124. A method for treating, preventing, or ameliorating at least one symptom of cancer, infectious disease, autoimmune disease, inflammatory disease, and immune deficiency, or a condition associated therewith, comprising administering to the subject an effective amount of the composition of item 123. (Item 126) 124. A method for treating a solid tumor, comprising administering to the subject an effective amount of the composition of claim 123. (Item 127) Item 127. The method of item 126, wherein 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, renal cancer, or skin cancer. (Item 128) 128. The method of claim 126 or 127, wherein the solid cancer is pancreatic cancer, lung cancer, or breast cancer. (Item 129) 124. A method for treating a malignant hematological disease, comprising administering to the subject an effective amount of the composition of item 123. (Item 130) Item 130. The method of item 129, wherein the malignant blood disease is leukemia, lymphoma, or multiple myeloma.

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 alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 2 or 5 and a beta chain comprising the amino acid sequence set forth in SEQ ID NO: 3 or 6, and the chimeric TGFβ receptor is (a) a TGFβR2 polypeptide, (i) the extracellular TGFβ1-binding domain of TGFβR2; (ii) the IL-12Rβ2 transmembrane domain, and (iii) a TGFβR2 polypeptide comprising an IL-12Rβ2 intracellular signaling domain; and (b) a polypeptide cleavage signal; and (c) a TGFβR1 polypeptide, (i) the extracellular TGFβ1-binding domain of TGFβR1; (ii) the IL-12Rβ1 transmembrane domain, and (iii) a TGFβR1 polypeptide comprising an IL-12Rβ1 intracellular signaling domain.

2. The cell of claim 1 , wherein the polypeptide cleavage signal is a viral self-cleaving polypeptide.

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

4. The cell of any one of claims 1 to 3, wherein the MAGEA4 TCR binds to the peptide GVYDGREHTV presented by an HLA-A*02:01 encoded molecule.

5. The cell of any one of claims 1 to 4, wherein 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.

6. The cell of any one of claims 1 to 4, wherein 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.

7. The cell of any one of claims 1 to 6, wherein the MAGEA4 TCR comprises the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO:

7.

8. The cell of any one of claims 1 to 7, wherein the chimeric TGFβ receptor comprises the amino acid sequence set forth in SEQ ID NO:

8.

9. The cells a) T cells, b) CD3 ＋ , CD4 ＋ , and / or CD8 ＋ cell, c) immune effector cells; d) cytotoxic T lymphocytes (CTLs), tumor-infiltrating lymphocytes (TILs), or helper T cells; or e) Natural killer (NK) cells or natural killer T (NKT) cells The cell according to any one of claims 1 to 8,

10. The cell according to any one of claims 1 to 9, wherein the source of the cell is a peripheral blood mononuclear cell, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, or a tumor.

11. A composition comprising the cells of any one of claims 1 to 10, and optionally a pharmaceutically acceptable carrier.

12. 12. The composition of claim 11 for use in the treatment of solid cancer or hematological malignancies.

13. 13. The composition of claim 12, wherein the solid cancer comprises liver cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, bladder cancer, brain cancer, sarcoma, salivary gland cancer, head and neck cancer, bone cancer, thyroid cancer, kidney cancer, esophageal cancer or skin cancer.

14. The composition of claim 12, wherein the solid cancer is esophageal cancer, ovarian cancer, salivary gland cancer, or non-small cell lung cancer.

Images