Engineered t cells

Abstract

The present disclosure provides an improved composition for adoptive T cell therapy for treating, preventing, or alleviating at least one symptom of cancer, infectious disease, autoimmune disease, inflammatory disease, and immunodeficiency, or a pathological condition related thereto.

Description

Technology Field

[0001] Cross-reference regarding related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 845,311 filed on May 8, 2019, pursuant to 35 USC § 119(e), the whole of which is incorporated herein by reference.

[0003] Statement regarding the sequence list

[0004] The sequence list associated with this application is provided in text format instead of a paper copy and is incorporated into this specification by reference. The name of the text file containing the sequence list is BLBD_122_01WO_ST25. The text file, created on May 6, 2020, is 37 KB in size and is submitted electronically via EFS-Web simultaneously with the filing of this specification. Background Technology

[0005] Technology field

[0006] The present disclosure relates to an improved adoptive cell therapy. More specifically, the present disclosure relates to an improved signaling molecule, a cell, and a method of using the same.

[0007] The present disclosure relates in part to generally improved adoptive immunotherapy, and in certain embodiments relates to an immune effector cell, a composition, and a method of using the same, wherein the immune effector cell comprises: a polynucleotide encoding a human pairing-enhancing αβTCR that binds to MAGEA4, preferably to MAGEA4 (preferably MAGEA4 peptide GVYDGREHTV presented by a molecule encoding HLA-A*02:01 (SEQ ID NO. 1)); and a chimeric TGFβ receptor (CTBR).

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

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

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

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

[0012] In a specific embodiment, the immune receptor intracellular signaling domain of the first polypeptide is the IL-12Rβ2 intracellular signaling domain, and the immune receptor intracellular signaling domain of the second polypeptide is the IL-12Rβ1 intracellular signaling domain.

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

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

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

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

[0017] In a given embodiment, the transmembrane domain of the second polypeptide comprises an IL-12Rβ2 transmembrane domain.

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

[0019] In a further embodiment, the transmembrane domain of the second polypeptide comprises an IL-7Rα transmembrane domain.

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

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

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

[0023] In a specific embodiment, the transmembrane domain of the second polypeptide comprises an IL-7Rα transmembrane domain.

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

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

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

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

[0028] In a specific embodiment, the transmembrane domain of the first polypeptide comprises an IL-2Rγ transmembrane domain.

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

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

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

[0032] In a given embodiment, the transmembrane domain of the second polypeptide comprises an IL-2Rγ transmembrane domain.

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

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

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

[0036] In a further embodiment, the immune receptor intracellular signaling domain of the first polypeptide is the IL-18R1 intracellular signaling domain, and the immune receptor intracellular signaling domain of the second polypeptide is the IL-18RAP intracellular signaling domain.

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

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

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

[0040] In a specific embodiment, the transmembrane domain of the first polypeptide comprises an IL-18RAP transmembrane domain.

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

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

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

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

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

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

[0047] In a specific embodiment, the transmembrane domain of the second polypeptide comprises an IL-1R1 transmembrane domain.

[0048] In a given embodiment, the immune receptor intracellular signaling domain of the first polypeptide is the IL-1RAP intracellular signaling domain, and the immune receptor intracellular signaling domain of the second polypeptide is the IL-1RL2 intracellular signaling domain.

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

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

[0051] In a further embodiment, the immune receptor intracellular signaling domain of the first polypeptide is the IL-1RL2 intracellular signaling domain, and the immune receptor intracellular signaling domain of the second polypeptide is the IL-1RAP intracellular signaling domain.

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

[0053] In a specific embodiment, the transmembrane domain of the second polypeptide comprises an IL-1RAP transmembrane domain.

[0054] In a further embodiment, the immune receptor intracellular signaling domain of the first polypeptide is the IFNAR1 intracellular signaling domain, and the immune receptor intracellular signaling domain of the second polypeptide is the IFNAR2 intracellular signaling domain.

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

[0056] In a given embodiment, the transmembrane domain of the second polypeptide comprises an IFNAR2 transmembrane domain.

[0057] In a specific embodiment, the immune receptor intracellular signaling domain of the first polypeptide is the IFNAR2 intracellular signaling domain, and the immune receptor intracellular signaling domain of the second polypeptide is the IFNAR1 intracellular signaling domain.

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

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

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

[0061] In a given embodiment, the transmembrane domain of the first polypeptide includes a TLR1 transmembrane domain.

[0062] In a specific embodiment, the transmembrane domain of the second polypeptide includes a TLR1 transmembrane domain.

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

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

[0065] In a further embodiment, the transmembrane domain of the second polypeptide includes a TLR2 transmembrane domain.

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

[0067] In a given embodiment, the transmembrane domain of the first polypeptide includes a TLR3 transmembrane domain.

[0068] In a specific embodiment, the transmembrane domain of the second polypeptide includes a TLR3 transmembrane domain.

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

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

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

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

[0073] In a specific embodiment, the transmembrane domain of the first polypeptide includes a TLR5 transmembrane domain.

[0074] In a further embodiment, the transmembrane domain of the second polypeptide includes a TLR5 transmembrane domain.

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

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

[0077] In a given embodiment, the transmembrane domain of the second polypeptide comprises a TLR6 transmembrane domain.

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

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

[0080] In a further embodiment, the transmembrane domain of the second polypeptide includes a TLR7 transmembrane domain.

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

[0082] In a further embodiment, the transmembrane domain of the first polypeptide includes a TLR8 transmembrane domain.

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

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

[0085] In a specific embodiment, the transmembrane domain of the first polypeptide includes a TLR9 transmembrane domain.

[0086] In a given embodiment, the transmembrane domain of the second polypeptide includes a TLR9 transmembrane domain.

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

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

[0089] In a specific embodiment, the transmembrane domain of the second polypeptide includes a TLR10 transmembrane domain.

[0090] In a specific embodiment, the polypeptide cleavage signal is a viral self-cleavage polypeptide.

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

[0092] In a specific embodiment, the polypeptide cleavage signal is a viral self-cleavage 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 Tescovirus-1 (PTV-1) (P2A) peptide, Theilovirus 2A peptide, and encephalomyocarditis virus 2A peptide.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0118] In a specific embodiment, the viral self-cleavage 2A polypeptide is selected from the group consisting of: foot-and-mouth disease virus (FMDV) (F2A) peptide, equine rhinitis A virus (ERAV) (E2A) peptide, tosea atigna virus (TaV) (T2A) peptide, porcine Tescovirus-1 (PTV-1) (P2A) peptide, tailovirus 2A peptide, and encephalomyocarditis virus 2A peptide.

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

[0120] In a further embodiment, the MAGEA4 TCR binds to the peptide GVYDGREHTV presented by the HLA-A*02:01-encoded molecule.

[0121] In some embodiments, the MAGEA4 TCR comprises an alpha chain having the amino acid sequence presented in SEQ ID NO. 1 and a beta chain having the amino acid sequence presented in SEQ ID NO. 2.

[0122] In various embodiments, the MAGEA4 TCR comprises an alpha chain having the amino acid sequence presented in SEQ ID NO. 3 and a beta chain having the amino acid sequence presented in SEQ ID NO. 4.

[0123] In a specific embodiment, the cell is a hematopoietic cell.

[0124] In an additional embodiment, the cell is a T cell.

[0125] In a specific embodiment, the cell is a CD3+, CD4+, and / or CD8+ cell.

[0126] In various embodiments, the cell is an immune effector cell.

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

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

[0129] In a specific embodiment, the source of the cells is peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue derived from an infection site, ascites, pleural effusion, splenic tissue, or a tumor.

[0130] In various embodiments, the composition comprises cells expressing the MAGEA4 TCR and fusion polypeptide considered herein.

[0131] In a further embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and a cell expressing the MAGEA4 TCR and fusion polypeptide considered herein.

[0132] In a given embodiment, a method for treating a subject requiring treatment includes the step of administering an effective amount of the composition considered herein to the subject.

[0133] In various embodiments, a method for treating, preventing, or improving at least one symptom of cancer, infectious disease, autoimmune disease, inflammatory disease, and immunodeficiency, or a pathological condition related thereto, comprises the step of administering an effective amount of the composition considered herein to a subject.

[0134] In a specific embodiment, a method for treating a target with a solid tumor comprises the step of administering an effective amount of the composition considered herein to the target.

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

[0136] In a given embodiment, the solid tumor is pancreatic cancer, lung cancer, or breast cancer.

[0137] In a specific embodiment, a method for treating hematological malignancy comprises the step of administering an effective amount of the composition considered herein to a subject.

[0138] In various embodiments, the blood cancer is leukemia, lymphoma, or multiple myeloma. Brief explanation of the drawing

[0139] Fig. 1 Figure 1 illustrates the phosphorylation of STAT4 and SMAD2 / 3 in untransduced (UTD) T cells cultured for 20 minutes in the presence or absence of TGFβ1, T cells transduced with an LVV encoding MAGEA4 TCR, and T cells transduced with an LVV encoding MAGEA4 TCR and an LVV encoding IL-12-responsive chimeric TGFβ receptor (CTBR12). Fig. 2 It is cultured alone for 24 hours in the presence or absence of TGFβ1 (10 ng / ml), or A375 MAGEA4 at a 1:1 E:T ratio + It shows IFNγ secretion from UTD T cells, MAGEA4 TCR T cells, and MAGEA4 TCR / CTBR12 T cells cultured with tumor cells. Fig. 3 Figure 1 shows the volume of A375 MAGEA4+ tumor cells in an NSG tumor xenograft mouse model treated with UTD T cells, MAGEA4 TCR T cells, or MAGEA4 TCR / CTBR12 T cells. Fig. 4 This shows STAT4 and SMAD2 / 3 phosphorylation in T cells transfected with LVV encoding a MAGEA4 pairing-enhanced TCR (eTCR) and T cells transfected with LVV encoding a MAGEA4 eTCR and an IL-12-responsive chimeric TGFβ receptor (CTBR12), cultured for 20 minutes in the presence or absence of TGFβ1 for 20 minutes. Fig. 5 It is cultured alone for 24 hours in the presence or absence of TGFβ1 (10 ng / ml), or with TGFβ1 or A375 MAGEA4 at a 1:1 E:T ratio. +It shows IFNγ secretion from UTD T cells, MAGEA4 eTCR T cells, and MAGEA4 eTCR / CTBR12 T cells cultured with tumor cells. A brief explanation of sequence identifiers Sequence No. 1 ...presents the amino acid sequence of the MAGEA4 epitope. Sequence No. 2 It presents the amino acid sequence of the human MAGEA4 TCRα chain. Sequence No. 3 It presents the amino acid sequence of the human MAGEA4 TCRβ chain. Sequence No. 4 This presents the amino acid sequence of the human MAGEA4 TCR fusion polypeptide. Sequence No. 5 It presents the amino acid sequence of the human MAGEA4 eTCRα chain. Sequence number 6 It presents the amino acid sequence of the human MAGEA4 eTCRβ chain. Sequence No. 7 This presents the amino acid sequence of the human MAGEA4 eTCR fusion polypeptide. Sequence No. 8 ...presents the amino acid sequence of the IL-12-responsive chimeric TGFβ receptor (CTBR12). Sequence number 9~19 It presents the amino acid sequences of various linkers. Sequence number 20~44 It presents the amino acid sequences of the protease cleavage site and the self-cleavage polypeptide cleavage site. Sequence number 45 It presents the nucleotide sequence of the Kojak sequence. In the aforementioned sequence, if X is present, it refers to any amino acid or the absence of an amino acid. Specific details for implementing the invention

[0140] A. Overview

[0141] T cell-expressing T cell receptors (TCRs) have demonstrated limited efficacy in solid tumor indications (where present), partly due to the immunosuppressive solid tumor microenvironment (TME). The overproduction 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 invasion, suppressing immune cell function in cancer patients and resulting in a poor prognosis. TGFβ signaling via TGFβR2 in tumor-specific CTLs attenuates their function and frequency in tumors, and CD8 + Blocking TGFβ signaling on T cells with monoclonal antibodies enables more rapid tumor surveillance and leads to the presence of more CTLs at the tumor site. To date, strategies to inhibit TGFβ in a clinical setting have not resulted in significant therapeutic benefits.

[0142] The present disclosure generally relates to an immunosuppressive TGFβ signal that converts an immunosuppressive TGFβ signal into an immunostimulatory signal, to an immunostimulatory cell expressing a polypeptide, and to a cell expressing the polypeptide. Without being bound by any particular theory, the polypeptide considered herein is a chimeric TGFβ receptor (CBTR) comprising the TGFβ binding domains of TGFβR1 and TGFβR2, said receptor, when co-expressed in an immunostimulatory cell by being linked to an immunostimulatory endodomain, can convert TGFβ exposure from an immunosuppressive signal into an immunostimulatory signal that stimulates the activity and function of the immunostimulatory cell. The co-expression of the chimeric TGFβ receptor polypeptide in an immunostimulatory cell is ,For example, by restoring or increasing the secretion of pro-inflammatory cytokines, resistance to the immunosuppressive effects of TGFβ is conferred upon the cells. In a particularly preferred embodiment, the MAGEA4 TCR is a human MAGEA4 pairing-enhancing TCR (eTCR) and the chimeric TGFβ receptor is CTBR12.

[0143] In various embodiments, the present disclosure partially considers an immune effector cell expressing MAGEA4 TCR and CTBR polypeptides that converts an immunosuppressive TGFβ signal into an immunostimulatory signal through or mediated by one or more intracellular domains of one or more immune receptors.

[0144] In various embodiments, the present disclosure partially considers immune effector cells expressing MAGEA4 TCR and CTBR polypeptides that convert an immunosuppressive TGFβ signal into an immunostimulatory signal through or mediated by one or more intracellular domains of one or more cytokine receptors.

[0145] In various embodiments, the present disclosure partially considers immune effector cells expressing MAGEA4 TCR and CTBR polypeptides that convert an immunosuppressive TGFβ signal into an immunostimulatory signal through or mediated by one or more intracellular domains of one or more interleukin receptors.

[0146] In various embodiments, the present disclosure partially considers immune effector cells expressing MAGEA4 TCR and CTBR polypeptides that convert an immunosuppressive TGFβ signal into an immunostimulatory signal through or mediated by one or more intracellular domains of one or more pattern recognition receptors.

[0147] In various embodiments, the present disclosure partially considers immune effector cells expressing MAGEA4 TCR and CTBR polypeptides that convert an immunosuppressive TGFβ signal into an immunostimulatory signal through or mediated by one or more intracellular domains of one or more Toll-like receptors.

[0148] In a specific embodiment, the present disclosure partially considers an immune effector cell expressing MAGEA4 TCR, and a CTBR polypeptide comprising a TGFβR1 extracellular domain, a transmembrane domain, and one or more intracellular domains of one or more immune receptors that bind to TGFβ; and a CTBR polypeptide comprising a TGFβR2 extracellular domain, a transmembrane domain, and one or more intracellular domains of one or more immune receptors that bind to TGFβ. In one embodiment, the polypeptides are linked together by a polypeptide cleavage signal, e.g., a 2A polypeptide cleavage signal.

[0149] In certain embodiments, the present disclosure partially considers an immune effector cell expressing a MAGEA4 TCR (e.g., SEQ ID NOs 2–4), preferably a MAGEA4 pairing-enhancing TCR (eTCR; e.g., SEQ ID NOs 5–7), preferably a molecule encoding a MAGEA4 pairing-enhancing TCR that binds to a MAGEA4 peptide GVYDGREHTV presented by a molecule encoding HLA-A*02:01; a fusion polypeptide encoding a chimeric TGFβ receptor (CTBR) comprising a TGFβR1 extracellular domain, a transmembrane domain, and one or more intracellular domains of one or more immune receptors that bind to TGFβ; and a polypeptide comprising a TGFβR2 extracellular domain, a transmembrane domain, and one or more intracellular domains of one or more immune receptors that bind to TGFβ.

[0150] In a specific embodiment, the transmembrane domain and the intracellular signaling domain are isolated from the 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.

[0151] In a specific embodiment, 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.

[0152] In a preferred embodiment, the fusion polypeptide is an IL-12 reactive CTBR (CTBR12; e.g., SEQ ID NO. 8).

[0153] Techniques and related techniques and procedures for the synthesis of recombinant (i.e., engineered) DNA, peptides, and oligonucleotides, immunoassays, tissue cultures, transformations (e.g., electroporation, lipofection), enzymatic reactions, and purification may be performed as described in various general and more specific references in microbiology, molecular biology, biochemistry, molecular genetics, cell biology, virology, and immunology, as cited and discussed throughout this specification. For example, the literature of 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]; Glover의 문헌[ 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]; Anand의 문헌[ Techniques for the Analysis of Complex Genomes , (Academic Press, New York, 1992)]; Guthrie와 Fink의 문헌[ 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., NY)]; [ Gene Transfer Vectors For Mammalian Cells(JH Miller and MP Calos eds., 1987, Cold Spring Harbor Laboratory)]; Harlow and Lane's literature[ Antibodies , (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998)]; [ Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987)]; [ Handbook of Experimental Immunology , Volumes I-IV (DM Weir and CC Blackwell, eds., 1986)]; Roitt's literature[ Essential Immunology 6th Edition, (Blackwell Scientific Publications, Oxford, 1988)]; [ Current Protocols in Immunology (QE Coligan, AM Kruisbeek, DH Margulies, EM Shevach and W. Strober, eds., 1991)]; [ Annual Review of Immunology Including ] Advances in Immunology Papers within academic journals such as [the same] are also referenced.

[0154] B. Definition

[0155] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art to which this invention pertains. Any method and material similar or equivalent to that described herein may be used to carry out or test specific embodiments, but preferred embodiments of the composition, method, and material are described herein. For the purposes of this disclosure, the following terms are defined below.

[0156] Singular expressions (the articles “a,” “an,” and “the”) are used herein to refer to one or more (i.e., at least one, or more than one) of the grammatical objects expressed in the singular. For example, “an element” means one element or more than one element.

[0157] The use of alternative examples (e.g., “or”) should be understood to mean one, both, or any combination thereof.

[0158] The term “and / or” should be understood to mean either one or both of the alternative examples.

[0159] As used herein, the terms “about” or “approximately” refer to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that differs by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. As used herein, the terms “about” or “approximately” refer to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that is within the range of ± 15%, ± 10%, ± 9%, ± 8%, ± 7%, ± 6%, ± 5%, ± 4%, ± 3%, ± 2%, or ± 1% with respect to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0160] Throughout this specification, unless the context otherwise requires, the words “comprise,” “comprising,” and “comprising” are to be understood as meaning that they include the mentioned steps or elements or groups of steps or elements, but do not exclude any other steps or elements or groups of steps or elements. “Consisting of” means that it includes and is limited to the things that follow the phrase “consisting of.” Thus, the phrase “consisting of” indicates that the enumerated elements are required or essential, and that no other elements may exist. “Consisting essentially of” means that it includes any of the enumerated elements that follow the phrase, and is limited to other elements that do not interfere with or contribute to the activity or operation specified in this disclosure for the enumerated elements. Thus, the phrase “consisting essentially of” indicates that the enumerated elements are required or essential, but that no other elements that significantly affect the activity or operation of the enumerated elements exist.

[0161] Throughout this specification, “one embodiment, an embodiment,” “a particular embodiment, a certain embodiment,” “a related embodiment,” or “an additional embodiment or a further embodiment,” or any combination thereof, means that a specific feature, structure, or characteristic described in relation to an embodiment is included in at least one embodiment. Accordingly, not all of the foregoing phrases appearing in various places throughout this specification refer to the same embodiment. Furthermore, a specific feature, structure, or characteristic may be combined in any appropriate manner in one or more embodiments. It will also be understood that positive recitation of a feature in one embodiment serves as a basis for excluding that feature in a particular embodiment.

[0162] “Antigen (Ag)” refers to a compound, composition, or substance capable of stimulating the production of antibodies or a T cell response in an animal, including a composition injected into or absorbed by the animal (e.g., a composition containing a cancer-specific protein). Exemplary antigens include, but are not limited to, lipids, carbohydrates, polysaccharides, glycoproteins, peptides, or nucleic acids. The antigen reacts with products of specific humoral or cellular immunity, including those induced by a heterologous antigen such as the disclosed antigen.

[0163] “Target antigen” or “target of interest” is an antigen designed to bind to the binding domain considered herein. In a specific embodiment, the target antigen is selected from the group consisting of: alpha folate receptor, 5T4, αvβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, FRα, GD2, GD3, glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-A1+NY-ESO-1, HLA-A2+NY-ESO-1, HLA-A3+NY-ESO-1, IL-11Rα, IL-13Rα2, Lambda, Lewis-Y, Kappa, Mesothelin, Muc1, Muc16, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, Survivin, STn, TAG72, TEMs, VEGFR2, and WT-1. In a preferred embodiment, the target antigen is MAGEA4.

[0164] MAGE-A4 belongs to the so-called cancer / testis antigen group. Cancer / testis antigens are expressed in various malignant tumors and germ cells, but not in other adult tissues. Therefore, MAGE-A4 is an interesting immunotherapeutic target antigen. The human gene encoding MAGE-A4 is designated as MAGEA4 (ENSG00000147381).

[0165] In one embodiment, the antigen is an MHC-peptide complex, such as a class I MHC-peptide complex or a class II MHC-peptide complex.

[0167] A “linker” refers to multiple amino acid residues between various polypeptide domains added to maintain the proper spacing and shape of a molecule.

[0168] Exemplified examples of linkers suitable for use in the specific embodiments considered herein include, but are not limited to, the following amino acid sequences: GGG; DGGGS (SEQ No. 9); TGEKP (SEQ No. 10) (see, e.g., Liu et al. [PNAS 5525-5530 (1997)]); GGRR (SEQ No. 11) (see Pomerantz et al., 1995); (GGGGS) n Here, n = 1, 2, 3, 4, or 5 (Sequence No. 12) (see Kim et al. [PNAS 93, 1156-1160 (1996)]); EGKSSGSGSESKVD (Sequence No. 13) (see Chaudhary et al. [1990, Proc. Natl. Acad. Sci. USA 87:1066-1070]); KESGSVSSEQLAQFRSLD (Sequence No. 14) (see Bird et al. [1988, Science 242:423-426]), GGRRGGGS (Sequence No. 15); LRQRDGERP (Sequence No. 16); LRQKDGGGSERP (Sequence No. 17); LRQKD(GGGS)2ERP (Sequence No. 18). Alternatively, flexible linkers can use computer programs capable of modeling both the DNA binding site and the peptide itself (Desjarlalis & Berg's literature [ PNAS 90:2256-2260 (1993)], [ PNAS [Refer to 91:11099-11103 (1994)]) It can be reasonably designed by a phage display method. In one embodiment, the linker comprises the following amino acid sequence: GSTSGKPGSGEGSTKG (Sequence No. 19) (Cooper et al. [ Blood [See , 101(4): 1637-1644 (2003)]).

[0169] A “transmembrane domain” or “TM domain” is a domain that anchors a polypeptide to the cell plasma membrane. TM domains can be derived from natural, synthetic, semi-synthetic, or recombinant sources.

[0170] “Intracellular signaling domain” refers to a portion of a protein that transmits effector function signals and induces a cell to perform a specific function. Generally, the entire intracellular signaling domain can be used, but in many cases, it is not necessary to use the whole domain. Regarding the extent to which truncated portions of the intracellular signaling domain are used, these truncated portions may be used instead of the whole domain as long as they transmit effector function signals. The term “intracellular signaling domain” implies including any truncated portion of the intracellular signaling domain sufficient to transmit operator function signals.

[0171] The terms “effector function” or “effector cell function” refer to specialized functions of immune effector cells. Effector functions include, but are not limited to, activation, cytokine production, proliferation, and cytotoxic activity, and include the release of cytotoxic factors or other cellular responses induced by antigen binding to receptors expressed on immune effector cells.

[0172] “Immune disorder” refers to a disease that triggers a response from the immune system. In certain embodiments, the term “immune disorder” refers to cancer, an autoimmune disease, or immunodeficiency. In one embodiment, immune disorder includes an infectious disease.

[0173] As used herein, the term “cancer” generally refers to a class of diseases or conditions in which abnormal cells divide uncontrollably and can invade adjacent tissues.

[0174] As used herein, the term “malignant” refers to cancer that exhibits one or more of the following: uncontrolled growth of a population of tumor cells (i.e., division beyond normal limits), invasion (i.e., invasion and destruction of adjacent tissues), and metastasis (i.e., spread to other locations in the body via lymph or blood). As used herein, the term “metastasis” refers to the spread of cancer from one part of the body to another. A tumor formed by spread cells is called a “metastatic tumor” or “metastasis.” A metastatic tumor contains cells similar to those in the original (primary) tumor.

[0175] As used herein, the terms “benign” or “non-malignant” refer to tumors that may grow larger but do not spread to other parts of the body. Benign tumors are self-limiting and generally do not invade or metastasize.

[0176] “Cancer cells” refer to individual cells of cancerous growths or tissues. Cancer cells include both solid and liquid cancers. “Tumor” or “tumor cells” refer to swellings or lesions formed by the abnormal growth of cells, which can generally be benign, premature, or malignant. Most cancers form tumors, but liquid cancers, such as leukemia, do not necessarily form tumors. In the case of tumor-forming cancers, the terms cancer (cells) and tumor (cells) are used interchangeably. The amount of tumors in an individual is the “tumor burden,” which can be measured as the number, volume, or weight of the tumors.

[0177] The term “recurrence” refers to the return of cancer after a period of improvement or remission, or the diagnosis of signs and symptoms of such a return.

[0178] “Remission,” also referred to as “clinical remission,” includes both partial and complete remission. In partial remission, some, though not all, signs and symptoms of the cancer have disappeared. In complete remission, while the cancer may still be present in the body, all signs and symptoms of the cancer have vanished.

[0179] “Refractory” refers to cancer that is resistant to or does not respond to a regimen using a specific therapeutic agent. Cancer may be refractory from the start of treatment (i.e., unresponsive from the first exposure to the agent), or become refractory by developing resistance to the agent over the first treatment period or during subsequent treatment periods.

[0180] “Antigen-negative” refers to a cell that does not express an antigen or expresses a negligible amount of an antigen that is undetectable. In one embodiment, the antigen-negative cell does not bind to a receptor induced for the antigen. In one embodiment, the antigen-negative cell does not substantially bind to a receptor induced for the antigen.

[0181] “Autoimmune diseases” refer to conditions in which the body generates an immunogenic (i.e., immune system) response to certain components of its own tissues. In other words, the immune system loses the ability to recognize certain tissues or systems within the body as “self,” targeting and attacking them as if they were foreign substances. Autoimmune diseases can be classified into those where a single organ is primarily affected (e.g., hemolytic anemia and anti-immune thyroiditis) and those where the autoimmune disease process spreads through many tissues (e.g., systemic lupus erythematosus). For example, multiple sclerosis is believed to occur when T cells attack the sheath surrounding nerve fibers in the brain and spinal cord. This results in loss of coordination, weakness, and blurred vision. Autoimmune diseases are known in the art and include, for example, Hashimoto's thyroiditis, Graves' disease, lupus, multiple sclerosis, rheumatoid arthritis, hemolytic anemia, anti-immune thyroiditis, systemic lupus erythematosus, celiac disease, Crohn's disease, colitis, diabetes mellitus, scleroderma, psoriasis, etc.

[0182] “Immunodeficiency” refers to a condition in which a patient’s immune system is impaired by disease or the administration of chemicals. This pathological condition results in a deficiency of the number and types of blood cells necessary for the immune system to defend against foreign substances. 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, High-IgM Syndrome, and Diabetes Mellitus.

[0183] “Infectious disease” refers to a disease that can be transmitted from person to person or from organism to organism, and is caused by microbial or viral material (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.

[0184] As used herein, the terms “individual” and “subject” are commonly used interchangeably and refer to any animal exhibiting symptoms of cancer or other immune disorders that can be treated by the compositions and methods considered elsewhere in this invention. Suitable subjects (e.g., patients) include laboratory animals (e.g., mice, rats, rabbits, or guinea pigs), farm animals, and livestock or pets (e.g., cats or dogs). Non-human primates and preferably human patients are included. Typical subjects include human patients who have cancer or other immune disorders, have been diagnosed with them, or are at risk of developing them.

[0185] As used herein, the term “patient” refers to a subject diagnosed with cancer or other immune disorders that can be treated with compositions and methods disclosed elsewhere in this invention.

[0186] As used herein, “treatment” (or “treating”) includes any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition, and may include a very 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 proliferation. “Treatment” does not necessarily imply the complete eradication or cure of the disease or condition, or associated symptoms.

[0187] As used herein, “prevention (or similar words such as prevent, prevented, or preventing)” refers to an approach to prevent, suppress, or reduce the likelihood of onset 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 words also include reducing the intensity, effect, symptoms, and / or burden of a disease or condition before onset or recurrence.

[0188] As used herein, the phrase “alleviating at least one symptom” refers to reducing one or more symptoms of the disease or condition being treated. In certain embodiments, the disease or condition being treated is cancer, and the one or more symptoms alleviated herein include, but are not limited to, weakness, fatigue, shortness of breath, easy bruising and bleeding, frequent infections, lymph node enlargement, abdominal distension or pain (due to abdominal organ enlargement), pain of bones or joints, fractures, unintended weight loss, loss of appetite, night sweats, persistent low-grade fever, and reduced urination (due to renal dysfunction).

[0189] The terms “enhance,” “promote,” “increase,” or “expand” generally refer to the ability of the composition considered herein to produce, induce, or cause a greater physiological response (i.e., downstream effect) compared to the response caused by the vehicle or control. Measurable physiological responses, among those evident from the understanding in the art and the description herein, may include T cell proliferation, activation, persistence, cytokine secretion, and / or increased cancer cell apoptosis ability. The “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 times or more (e.g., 500 times, 1000 times) of the reaction produced by the vehicle or control composition (including all integer values ​​greater than 1 and decimal values ​​between integer values, e.g., 1.5, 1.6, 1.7, 1.8, etc.).

[0190] The term “decrease (or lessen, or reduce, or abate)” generally refers to the ability of the composition considered herein to produce, induce, or cause a smaller physiological response (i.e., downstream effect) compared to the response caused by the vehicle or control molecule / composition. The “decreased (or reduced)” amount is typically a “statistically significant” amount and may include a reduction of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 times or more (e.g., 500 times, 1000 times) of the response produced by the vehicle or control composition (reference response) or the response in a specific cell line (including all integer values ​​greater than 1 and decimal values ​​between integer values, e.g., 1.5, 1.6, 1.7, 1.8, etc.).

[0191] “Maintain or maintenance,” “preserve,” “no change,” “no substantial change,” or “no substantial decrease” refers to the ability of the composition considered herein to produce, induce, or cause a substantially similar or comparable physiological response (i.e., downstream effect) compared to the response caused by the vehicle or control molecule / composition. A comparable response is one that is not significantly different or measurably different from the reference response.

[0192] C. MAGEA4 T cell receptor

[0193] 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, namely MHC I and MHC II, that deliver peptides from different cellular compartments to the cell surface. When the TCR binds to an antigen and MHC, immune effector cells are activated through a series of biochemical events mediated by related enzymes, co-receptors, and specialized accessory molecules.

[0194] The TCR considered 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 the recombination of the variable (V) segment and the combined (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 the recombination of the variable (V) segment, the diversity (D) segment, and the combined (J) segment, and one of two constant (C) domains.

[0195] In a specific implementation, the TCR binds to MAGEA4.

[0196] In a specific embodiment, the TCR is a human TCR that binds to MAGEA4.

[0197] In a preferred embodiment, the TCR is a human pairing enhancement TCR that binds to MAGEA4.

[0198] The pairing-enhancing MAGEA4 TCR considered herein is engineered to increase TCR stability, TCR expression, specific TCR pairing, and functional binding affinity.

[0199] In certain embodiments, the constant domains of the MAGEA4 TCRα and MAGEA4 TCRβ chains are manipulated or modified to increase TCR stability, TCR expression, specific TCR pairing, and functional binding affinity.

[0200] To efficiently enhance the accurate pairing of the MAGEA4 TCR sequence and avoid pairing errors with the endogenous TCR chain, the MAGEA4 pairing-enhancing TCR considered herein includes minimally murinized TCRα and TCRβ constant domains and additionally includes hydrophobic amino acid substitutions in the TCRα transmembrane domain.

[0201] In a preferred embodiment, the MAGEA4 pairing-enhancing TCR (eTCR) comprises a MAGEA4 TCRα chain comprising an invariant domain comprising minimally saturated amino acid substitutions at positions 90, 91, 92, and 93 and hydrophobic amino acid substitutions at positions 115, 118, and 119 of the invariant region; and a MAGEA4 TCRβ chain comprising an invariant domain comprising minimally saturated amino acid substitutions at 18, 22, 133, 136, and 139.

[0202] In a preferred embodiment, MAGEA4 eTCR comprises a TCRα chain comprising a constant region comprising hydrophobic amino acid substitutions S115L, G118V, and F119L in a permembrane-penetrating domain and minimally saturated amino acid substitutions P90S, E91D, S92V, and S93P; and a TCRβ chain comprising a constant region comprising minimally saturated amino acid substitutions E18K, S22A, F133I, E / V136A, and Q139H.

[0203] In a specific preferred embodiment, the MAGEA4 TCR comprises a TCRα chain having the amino acid sequence presented at SEQ ID NO. 5 and a TCRβ chain having the amino acid sequence presented at SEQ ID NO. 6. In another specific embodiment, the MAGEA4 eTCR is expressed as a fusion polypeptide having the amino acid sequence presented at SEQ ID NO. 7.

[0204] D. Chimeric TGFβ receptor (CTBR)

[0205] In a specific embodiment, a cell comprising a human MAGEA4 TCR or a human MAGEA4 pairing-enhanced TCR (eTCR), and a polynucleotide encoding a chimeric TGFβ receptor that converts an immunostimulatory signal when exposed to TGFβ (including but not limited to TGFβ1) is considered.

[0206] 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 immune effector cell activity and function, and increasing the production and / or secretion of pro-inflammatory cytokines). In certain embodiments, the term “chimeric TGFβ receptor” is used interchangeably with the term “CTBR.”

[0207] In a specific embodiment, the CTBR polypeptide comprises the extracellular TGFβ-binding domain, transmembrane domain, and intracellular signaling domain of the immune receptor (including but not limited to cytokine receptors, interleukin receptors, pattern recognition receptors, and Toll-like receptors); polypeptide cleavage signal; and the extracellular TGFβ-binding domain, transmembrane domain, and intracellular signaling domain of the immune receptor of TGFβR1 (including but not limited to cytokine receptors, interleukin receptors, pattern recognition receptors, and Toll-like receptors).

[0208] In a specific embodiment, CTBR is a fusion polypeptide comprising: a first polypeptide comprising an extracellular TGFβ-binding domain, a transmembrane domain, and an intracellular signaling domain of an immune receptor (including but not limited to cytokine receptors, interleukin receptors, pattern recognition receptors, and Toll-like receptors); a polypeptide cleavage signal; and a second polypeptide comprising an extracellular TGFβ-binding domain, a transmembrane domain, and an intracellular signaling domain of an immune receptor (including but not limited to cytokine receptors, interleukin receptors, pattern recognition receptors, and Toll-like receptors).

[0209] In another specific embodiment, CTBR is a polypeptide complex comprising: a polypeptide comprising an extracellular TGFβ-binding domain, a transmembrane domain, and an intracellular signaling domain of an immune receptor (including, but not limited to, cytokine receptors, interleukin receptors, pattern recognition receptors, and Toll-like receptors); and a polypeptide comprising an extracellular TGFβ-binding domain, a transmembrane domain, and an intracellular signaling domain of an immune receptor (including, but not limited to, cytokine receptors, interleukin receptors, pattern recognition receptors, and Toll-like receptors) of TGFβR1.

[0210] As used herein, the term “immune receptor” refers to a receptor expressed on the surface of an immune cell that modulates an immune response when binding to a homologous ligand. Immune receptors suitable for use in certain embodiments include, but are not limited to, cytokine receptors, interleukin receptors, pattern recognition receptors, and Toll-like receptors, wherein signal transduction through immune receptors stimulates an immune response.

[0211] Examples of immune receptor transmembrane domains and intracellular signaling domains that may be used in specific embodiments considered herein include, but are not limited to, transmembrane and intracellular signaling domains isolated from the following: IL-12 receptor, IL-7 receptor, IL-15 receptor, IL-21 receptor, IL-2 receptor, IL-1 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.

[0212] Further exemplary embodiments of the immune receptor transmembrane domain and intracellular signaling domain that may be used in the specific embodiments considered herein include, but are not limited to, transmembrane and intracellular signaling domains isolated from the following: 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.

[0213] Further exemplary embodiments of the cytokine receptor transmembrane domain and intracellular signaling domain that may be used in the specific embodiments considered herein include, but are not limited to, transmembrane and intracellular signaling domains isolated from the following: IL-12Rβ2, IL-7Rα, IL-2Rγ, IL-2Rβ, IL-21R, IL-18R1, IL-18RAP, IL-1R1, IL-1RAP, IFNAR1, IFNAR2, and IL-1RL2.

[0214] Exemplary examples of interleukin receptor transmembrane and intracellular signaling domains that may be used in specific embodiments considered herein include, but are not limited to, transmembrane and intracellular signaling domains isolated from the following: IL-12Rβ2, IL-7Rα, IL-2Rγ, IL-2Rβ, IL-21R, IL-18R1, IL-18RAP, IL-1R1, IL-1RAP, and IL-1RL2.

[0215] Exemplary examples of Toll-like receptor transmembrane and intracellular signaling domains that may be used in specific embodiments considered herein include, but are not limited to, transmembrane and intracellular signaling domains isolated from the following: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, and TLR10.

[0216] 1. CTBR12 polypeptide

[0217] Interleukin-12 (IL-12) is a cytokine that promotes T cell function and activity by partially increasing IFNγ expression, increasing T cell proliferation, and enhancing IL-12 signaling. IL-12 binds to the interleukin 12 receptors beta 1 (IL-12Rβ1, also known as CD212) and beta 2 (IL-12Rβ2), which are interleukin 12 receptors.

[0218] IL-12 signaling via IL-12Rβ1 and IL-12Rβ2 phosphorylates STAT3, STAT4, and STAT5. Phosphorylated STAT3 / STAT4 translocate to the nucleus and bind to the IFNγ promoter, thereby increasing IFNγ expression. Additionally, phosphorylated STAT4 increases IFNγ expression by recruiting the Jun oncogene (c-Jun) to the IFNγ promoter and enhances IL-12 signaling by increasing the transcription of IL-12Rβ2. STAT5 phosphorylation increases T cell proliferation.

[0219] IL-12 signaling also enhances T cell proliferation by increasing the expression of the interleukin 2 receptor alpha (IL-2R) through the recruitment of STAT4 and c-Jun to the IL-2R promoter.

[0220] In various embodiments, one or more immune effector cells, including immune effector cells expressing MAGEA4 TCR or MAGEA4 eTCR, are modified by introducing one or more polynucleotides or vectors encoding one or more CTBR12 polypeptides. In various embodiments, one or more immune effector cells are modified by introducing one or more polynucleotides or vectors encoding CTBR12 and MAGEA4 TCR or MAGEA4 eTCR.

[0221] In a specific embodiment, CTBR12 converts an immunosuppressive TGFβ signal into an IL-12-mediated immunostimulatory signal. In a specific embodiment, the CTBR12 considered herein comprises: an extracellular TGFβ1-binding domain, a transmembrane domain, and an IL-12Rβ1 intracellular signaling domain of TGFβR1; a polypeptide cleavage signal; and an extracellular TGFβ1-binding domain, a transmembrane domain, and an IL-12Rβ2 intracellular signaling domain of TGFβR2. In a specific embodiment, the CTBR12 considered herein comprises: an extracellular TGFβ1-binding domain, a transmembrane domain, and an IL-12Rβ2 intracellular signaling domain of TGFβR1; a polypeptide cleavage signal; and an extracellular TGFβ1-binding domain, a transmembrane domain, and an IL-12Rβ1 intracellular signaling domain of TGFβR2.

[0222] In a specific embodiment, the CTBR12 considered herein comprises a fusion polypeptide, wherein the fusion polypeptide comprises: 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 a specific embodiment, the CTBR12 considered herein comprises a fusion polypeptide, wherein the fusion polypeptide comprises: 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.

[0223] In a specific embodiment, CTBR12 considered herein is a polypeptide complex comprising a first polypeptide comprising an extracellular TGFβ1-binding domain, a transmembrane domain, and an IL-12Rβ1 intracellular signaling domain of TGFβR1; and a second polypeptide comprising an extracellular TGFβ1-binding domain, a transmembrane domain, and an IL-12Rβ2 intracellular signaling domain of TGFβR2. In a specific embodiment, CTBR12 considered herein is a polypeptide complex comprising a first polypeptide comprising an extracellular TGFβ1-binding domain, a transmembrane domain, and an IL-12Rβ2 intracellular signaling domain of TGFβR1; and a second polypeptide comprising an extracellular TGFβ1-binding domain, a transmembrane domain, and an IL-12Rβ1 intracellular signaling domain of TGFβR2.

[0224] In a specific embodiment, the polypeptide comprises a transmembrane domain of TGFβR1 or TGFβR2. In a specific embodiment, the polypeptide comprises a transmembrane domain of IL-12Rβ1 or IL-12Rβ2. In one embodiment, the polypeptide comprises an extracellular TGFβ1 binding domain of TGFβR1, an IL-12Rβ1 transmembrane domain, and an intracellular signaling domain. In one embodiment, the polypeptide comprises an extracellular TGFβ1 binding domain of TGFβR2, an IL-12Rβ2 transmembrane domain, and an intracellular signaling domain. In one embodiment, the polypeptide comprises an extracellular TGFβ1 binding domain of TGFβR1, an IL-12Rβ2 transmembrane domain, and an intracellular signaling domain. In one embodiment, the polypeptide comprises an extracellular TGFβ1 binding domain of TGFβR2, an IL-12Rβ1 transmembrane domain, and an intracellular signaling domain.

[0225] In a specific embodiment, the polypeptide cleavage signal is a viral self-cleavage polypeptide; more preferably a viral self-cleavage 2A polypeptide; more preferably a viral self-cleavage polypeptide selected from the group consisting of: foot-and-mouth disease virus (FMDV) (F2A) peptide, equine rhinitis A virus (ERAV) (E2A) peptide, tosea acinar virus (TaV) (T2A) peptide, porcine Tescovirus-1 (PTV-1) (P2A) peptide, tailovirus 2A peptide, and encephalomyocarditis virus 2A peptide. In one embodiment, the polypeptide cleavage signal is a P2A or T2A viral self-cleavage polypeptide.

[0226] 2. CTBR7 polypeptide

[0227] Interleukin-7 (IL-7) is a cytokine that promotes T cell function and activity by partially improving the survival and proliferation of T cell precursors. IL-7 binds to the interleukin-7 receptor alpha (IL-7Rα, also known as CD127) and the common gamma chain interleukin-2 receptor (IL-2Rγ, also known as CD132 and γc). IL-7 signaling activates the JAK / STAT, PI-3K, and Src kinase pathways, leading to the transcription of anti-apoptotic genes and genes that promote the proliferation of T cell precursors.

[0228] In various embodiments, one or more immune effector cells, including immune effector cells expressing MAGEA4 TCR or MAGEA4 eTCR, are modified by introducing one or more polynucleotides or vectors encoding one or more CTBR7 polypeptides. In various embodiments, one or more immune effector cells are modified by introducing one CTBR7 and one or more polynucleotides or vectors encoding MAGEA4 TCR or MAGEA4 eTCR.

[0229] In a specific embodiment, the chimeric TGFβ receptor converts an immunosuppressive TGFβ signal into an IL-7-mediated immunostimulatory signal. In a specific embodiment, the CTBR7 considered herein comprises: the extracellular TGFβ1-binding domain, the transmembrane domain, and the IL-7Rα intracellular signaling domain of TGFβR1; polypeptide cleavage signals; and the extracellular TGFβ1-binding domain, the transmembrane domain, and the IL-2Rγ intracellular signaling domain of TGFβR2. In a specific embodiment, the CTBR7 considered herein comprises: the extracellular TGFβ1-binding domain, the transmembrane domain, and the IL-2Rγ intracellular signaling domain of TGFβR1; polypeptide cleavage signals; and the extracellular TGFβ1-binding domain, the transmembrane domain, and the IL-7Rα intracellular signaling domain of TGFβR2.

[0230] In a specific embodiment, the CTBR7 considered herein comprises a fusion polypeptide, wherein the fusion polypeptide comprises: 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 a specific embodiment, the CTBR7 considered herein comprises a fusion polypeptide, wherein the fusion polypeptide comprises: 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.

[0231] In a specific embodiment, CTBR7 considered herein is a polypeptide complex comprising a first polypeptide comprising an extracellular TGFβ1-binding domain, a transmembrane domain, and an IL-7Rα intracellular signaling domain of TGFβR1; and a second polypeptide comprising an extracellular TGFβ1-binding domain, a transmembrane domain, and an IL-2Rγ intracellular signaling domain of TGFβR2. In a specific embodiment, CTBR7 considered herein is a polypeptide complex comprising a first polypeptide comprising an extracellular TGFβ1-binding domain, a transmembrane domain, and an IL-2Rγ intracellular signaling domain of TGFβR1; and a second polypeptide comprising an extracellular TGFβ1-binding domain, a transmembrane domain, and an IL-7Rα intracellular signaling domain of TGFβR2.

[0232] In a specific embodiment, the polypeptide comprises a transmembrane domain of TGFβR1 or TGFβR2. In a specific embodiment, the polypeptide comprises a transmembrane domain of IL-7Rα or IL-2Rγ. In one embodiment, the polypeptide comprises an extracellular TGFβ1 binding domain of TGFβR1, an IL-7Rα 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-7Rα transmembrane domain, and an intracellular signaling domain.

[0233] In a specific embodiment, the polypeptide cleavage signal is a viral self-cleavage polypeptide; more preferably a viral self-cleavage 2A polypeptide; more preferably a viral self-cleavage polypeptide selected from the group consisting of: foot-and-mouth disease virus (FMDV) (F2A) peptide, equine rhinitis A virus (ERAV) (E2A) peptide, tosea acinar virus (TaV) (T2A) peptide, porcine Tescovirus-1 (PTV-1) (P2A) peptide, tailovirus 2A peptide, and encephalomyocarditis virus 2A peptide. In one embodiment, the polypeptide cleavage signal is a P2A or T2A viral self-cleavage polypeptide.

[0234] 3. CTBR15 polypeptide

[0235] Interleukin-15 (IL-15) is a cytokine that promotes T cell function and activity by partially improving T cell progenitor survival and proliferation. IL-15 binds with high affinity to IL-15Rα (also known as CD215) and then binds to a complex containing IL-2Rβ (also known as IL-15Rβ and CD122) and IL-2Rγ (also known as CD132 and γc) expressed on the same cell (cis-presentation) or on different cells (trans-presentation). IL-15 signaling activates the JAK / STAT, PI-3K, and Src kinase pathways and leads to the transcription of anti-apoptotic genes and genes that promote the proliferation of T cell progenitors.

[0236] In various embodiments, one or more immune effector cells, including immune effector cells expressing MAGEA4 TCR or MAGEA4 eTCR, are modified by introducing one or more polynucleotides or vectors encoding one or more CTBR15 polypeptides and optionally introducing a polynucleotide or vector encoding IL-15Rα. In various embodiments, one or more immune effector cells are modified by introducing one or more polynucleotides or vectors encoding one CTBR15 and MAGEA4 TCR or MAGEA4 eTCR, and optionally introducing a polynucleotide or vector encoding IL-15Rα polypeptide.

[0237] In a specific embodiment, the chimeric TGFβ receptor converts an immunosuppressive TGFβ signal into an IL-15-mediated immunostimulatory signal. In a specific embodiment, the CTBR15 considered herein comprises: the extracellular TGFβ1-binding domain, the transmembrane domain, and the IL-2Rβ intracellular signaling domain of TGFβR1; polypeptide cleavage signals; and the extracellular TGFβ1-binding domain, the transmembrane domain, and the IL-2Rγ intracellular signaling domain of TGFβR2. In a specific embodiment, the CTBR15 considered herein comprises: the extracellular TGFβ1-binding domain, the transmembrane domain, and the IL-2Rγ intracellular signaling domain of TGFβR1; polypeptide cleavage signals; and the extracellular TGFβ1-binding domain, the transmembrane domain, and the IL-2Rβ intracellular signaling domain of TGFβR2.

[0238] In a specific embodiment, the CTBR15 considered herein comprises a fusion polypeptide, wherein the fusion polypeptide comprises: a first polypeptide comprising an extracellular TGFβ1-binding domain, a transmembrane domain, and an IL-2Rβ intracellular signaling domain of TGFβR1; a polypeptide cleavage signal; and a second polypeptide comprising an extracellular TGFβ1-binding domain, a transmembrane domain, and an IL-2Rγ intracellular signaling domain of TGFβR2. In a specific embodiment, the CTBR15 considered herein comprises a fusion polypeptide, wherein the fusion polypeptide comprises: a first polypeptide comprising an extracellular TGFβ1-binding domain, a transmembrane domain, and an IL-2Rγ intracellular signaling domain of TGFβR1; a polypeptide cleavage signal; and a second polypeptide comprising an extracellular TGFβ1-binding domain, a transmembrane domain, and an IL-2Rβ intracellular signaling domain of TGFβR2.

[0239] In a specific embodiment, CTBR15 considered herein is a polypeptide complex comprising a first polypeptide comprising an extracellular TGFβ1-binding domain, a transmembrane domain, and an IL-2Rβ intracellular signaling domain of TGFβR1; and a second polypeptide comprising an extracellular TGFβ1-binding domain, a transmembrane domain, and an IL-2Rγ intracellular signaling domain of TGFβR2. In a specific embodiment, CTBR15 considered herein is a polypeptide complex comprising a first polypeptide comprising an extracellular TGFβ1-binding domain, a transmembrane domain, and an IL-2Rγ intracellular signaling domain of TGFβR1; and a second polypeptide comprising an extracellular TGFβ1-binding domain, a transmembrane domain, and an IL-2Rβ intracellular signaling domain of TGFβR2.

[0240] In a specific embodiment, the polypeptide comprises a transmembrane domain of TGFβR1 or TGFβR2. In a specific embodiment, the polypeptide comprises a transmembrane domain of IL-2Rβ or IL-2Rγ. In one embodiment, the polypeptide comprises an extracellular TGFβ1 binding domain of TGFβR1, an IL-2Rβ 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-2Rβ transmembrane domain, and an intracellular signaling domain.

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

[0242] 4. CTBR21 polypeptide

[0243] Interleukin-21 (IL-21) is a cytokine that promotes T cell function and activity by partially improving T cell progenitor survival and proliferation. IL-21 binds to the interleukin-21 receptor (IL-21R, also known as CD360) and IL-2Rγ (also known as CD132 and γc). IL-21 signaling activates the JAK / STAT, PI-3K, and Src kinase pathways, leading to the transcription of anti-apoptotic genes and genes that promote the proliferation of T cell progenitors.

[0244] In various embodiments, one or more immune effector cells, including immune effector cells expressing MAGEA4 TCR or MAGEA4 eTCR, are modified by introducing one or more polynucleotides or vectors encoding one or more CTBR21 polypeptides. In various embodiments, one or more immune effector cells are modified by introducing one or more polynucleotides or vectors encoding one CTBR21 and MAGEA4 TCR or MAGEA4 eTCR.

[0245] In a specific embodiment, the chimeric TGFβ receptor converts an immunosuppressive TGFβ signal into an IL-21-mediated immunostimulatory signal. In a specific embodiment, the CTBR21 considered herein comprises: the extracellular TGFβ1-binding domain, the transmembrane domain, and the IL-21R intracellular signaling domain of TGFβR1; polypeptide cleavage signals; and the extracellular TGFβ1-binding domain, the transmembrane domain, and the IL-2Rγ intracellular signaling domain of TGFβR2. In a specific embodiment, the CTBR21 considered herein comprises: the extracellular TGFβ1-binding domain, the transmembrane domain, and the IL-2Rγ intracellular signaling domain of TGFβR1; polypeptide cleavage signals; and the extracellular TGFβ1-binding domain, the transmembrane domain, and the IL-21R intracellular signaling domain of TGFβR2.

[0246] In a specific embodiment, CTBR21 considered herein comprises a fusion polypeptide, wherein the fusion polypeptide comprises: 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 a specific embodiment, CTBR21 considered herein comprises a fusion polypeptide, wherein the fusion polypeptide comprises: 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-21R intracellular signaling domain of TGFβR2.

[0247] In a specific embodiment, CTBR21 considered herein is a polypeptide complex comprising a first polypeptide comprising an extracellular TGFβ1-binding domain, a transmembrane domain, and an IL-21R intracellular signaling domain of TGFβR1; and a second polypeptide comprising an extracellular TGFβ1-binding domain, a transmembrane domain, and an IL-2Rγ intracellular signaling domain of TGFβR2. In a specific embodiment, CTBR21 considered herein is a polypeptide complex comprising a first polypeptide comprising an extracellular TGFβ1-binding domain, a transmembrane domain, and an IL-2Rγ intracellular signaling domain of TGFβR1; and a second polypeptide comprising an extracellular TGFβ1-binding domain, a transmembrane domain, and an IL-21R intracellular signaling domain of TGFβR2.

[0248] In a specific embodiment, the polypeptide comprises a transmembrane domain of TGFβR1 or TGFβR2. In a specific embodiment, 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.

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

[0250] 5. CTBR18 polypeptide

[0251] Interleukin-18 (IL-18) is a cytokine that promotes T cell function and activity by partially increasing IFNγ expression, increasing T cell proliferation, and protecting against activation-induced apoptosis (AICD). IL-18 binds to interleukin 18 receptor 1 (IL-18R1, also known as CD218a) and interleukin 18 receptor accessory proteins (IL-18RAP, CD218b).

[0252] IL-18 signaling via IL-18R1 and IL-18RAP leads to activation through the phosphorylation of the MyD88 adapter protein and IRAK4. Following the phosphorylation of IRAK4, the phosphorylation of IRAK1 / 2 ultimately induces the activation of NF-kappa B and AP-1 transcription factors, thereby increasing IFNγ expression and sensitivity to IL-12. The transcriptional program induced by IL-18 also increases T cell proliferation and protects against AICD.

[0253] In various embodiments, one or more immune effector cells, including immune effector cells expressing MAGEA4 TCR or MAGEA4 eTCR, are modified by introducing one or more polynucleotides or vectors encoding one or more CTBR18 polypeptides. In various embodiments, one or more immune effector cells are modified by introducing one CTBR18 and one or more polynucleotides or vectors encoding MAGEA4 TCR or MAGEA4 eTCR.

[0254] In a specific embodiment, the chimeric TGFβ receptor converts an immunosuppressive TGFβ signal into an IL-18-mediated immunostimulatory signal. In a specific embodiment, CTBR18 considered herein comprises: the extracellular TGFβ1-binding domain, the transmembrane domain, and the IL-18RAP intracellular signaling domain of TGFβR1; polypeptide cleavage signals; and the extracellular TGFβ1-binding domain, the transmembrane domain, and the IL-18R1 intracellular signaling domain of TGFβR2. In a specific embodiment, CTBR18 considered herein comprises: the extracellular TGFβ1-binding domain, the transmembrane domain, and the IL-18R1 intracellular signaling domain of TGFβR1; polypeptide cleavage signals; and the extracellular TGFβ1-binding domain, the transmembrane domain, and the IL-18RAP intracellular signaling domain of TGFβR2.

[0255] In a specific embodiment, CTBR18 considered herein comprises a fusion polypeptide, wherein the fusion polypeptide comprises: 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 a specific embodiment, CTBR18 considered herein comprises a fusion polypeptide, wherein the fusion polypeptide comprises: 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-18R1 intracellular signaling domain of TGFβR2.

[0256] In a specific embodiment, CTBR18 considered herein is a polypeptide complex comprising a first polypeptide comprising an extracellular TGFβ1-binding domain, a transmembrane domain, and an IL-18RAP intracellular signaling domain of TGFβR1; and a second polypeptide comprising an extracellular TGFβ1-binding domain, a transmembrane domain, and an IL-18R1 intracellular signaling domain of TGFβR2. In a specific embodiment, CTBR18 considered herein is a polypeptide complex comprising a first polypeptide comprising an extracellular TGFβ1-binding domain, a transmembrane domain, and an IL-18R1 intracellular signaling domain of TGFβR1; and a second polypeptide comprising an extracellular TGFβ1-binding domain, a transmembrane domain, and an IL-18RAP intracellular signaling domain of TGFβR2.

[0257] In a specific embodiment, the polypeptide comprises a transmembrane domain of TGFβR1 or TGFβR2. In a specific embodiment, 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.

[0258] In a specific embodiment, the polypeptide cleavage signal is a viral self-cleavage polypeptide; more preferably a viral self-cleavage 2A polypeptide; more preferably a viral self-cleavage polypeptide selected from the group consisting of: foot-and-mouth disease virus (FMDV) (F2A) peptide, equine rhinitis A virus (ERAV) (E2A) peptide, tosea acinar virus (TaV) (T2A) peptide, porcine Tescovirus-1 (PTV-1) (P2A) peptide, tailovirus 2A peptide, and encephalomyocarditis virus 2A peptide. In one embodiment, the polypeptide cleavage signal is a P2A or T2A viral self-cleavage polypeptide.

[0259] 6. CTBR1 polypeptide

[0260] Interleukin-1 (IL-1) is a cytokine that promotes T cell function and activity by partially increasing IFNγ expression, increasing T cell proliferation, and enhancing protection against activation-induced apoptosis (AICD). IL-1 binds to interleukin 1 receptor 1 (IL-1R1, also known as CD121a) and interleukin 1 receptor accessory protein (IL-1RAP).

[0261] IL-1 signaling via IL-1R1 and IL-1RAP leads to activation through the phosphorylation of MyD88 adapter proteins and IRAK4. Following the phosphorylation of IRAK4, the phosphorylation of IRAK1 / 2 ultimately induces the activation of NF-kappa B and AP-1 transcription factors, thereby increasing IFNγ expression and sensitivity to IL-12. The transcriptional program induced by IL-1 also increases T cell proliferation and protects against AICD.

[0262] In various embodiments, one or more immune effector cells, including immune effector cells expressing MAGEA4 TCR or MAGEA4 eTCR, are modified by introducing one or more polynucleotides or vectors encoding one or more CTBR1 polypeptides. In various embodiments, one or more immune effector cells are modified by introducing one CTBR1 and one or more polynucleotides or vectors encoding MAGEA4 TCR or MAGEA4 eTCR.

[0263] In a specific embodiment, the chimeric TGFβ receptor converts an immunosuppressive TGFβ signal into an IL-1-mediated immunostimulatory signal. In a specific embodiment, the CTBR1 considered herein comprises: the extracellular TGFβ1-binding domain, the transmembrane domain, and the IL-1RAP intracellular signaling domain of TGFβR1; polypeptide cleavage signals; and the extracellular TGFβ1-binding domain, the transmembrane domain, and the IL-1R1 intracellular signaling domain of TGFβR2. In a specific embodiment, the CTBR1 considered herein comprises: the extracellular TGFβ1-binding domain, the transmembrane domain, and the IL-1R1 intracellular signaling domain of TGFβR1; polypeptide cleavage signals; and the extracellular TGFβ1-binding domain, the transmembrane domain, and the IL-1RAP intracellular signaling domain of TGFβR2.

[0264] In a specific embodiment, CTBR1 considered herein comprises a fusion polypeptide, wherein the fusion polypeptide comprises: 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 a specific embodiment, CTBR1 considered herein comprises a fusion polypeptide, wherein the fusion polypeptide comprises: 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.

[0265] In a specific embodiment, CTBR1 considered herein is a polypeptide complex comprising a first polypeptide comprising an extracellular TGFβ1-binding domain, a transmembrane domain, and an IL-1RAP intracellular signaling domain of TGFβR1; and a second polypeptide comprising an extracellular TGFβ1-binding domain, a transmembrane domain, and an IL-1R1 intracellular signaling domain of TGFβR2. In a specific embodiment, CTBR1 considered herein is a polypeptide complex comprising a first polypeptide comprising an extracellular TGFβ1-binding domain, a transmembrane domain, and an IL-1R1 intracellular signaling domain of TGFβR1; and a second polypeptide comprising an extracellular TGFβ1-binding domain, a transmembrane domain, and an IL-1RAP intracellular signaling domain of TGFβR2.

[0266] In a specific embodiment, the polypeptide comprises a transmembrane domain of TGFβR1 or TGFβR2. In a specific embodiment, 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.

[0267] In a specific embodiment, the polypeptide cleavage signal is a viral self-cleavage polypeptide; more preferably a viral self-cleavage 2A polypeptide; more preferably a viral self-cleavage polypeptide selected from the group consisting of: foot-and-mouth disease virus (FMDV) (F2A) peptide, equine rhinitis A virus (ERAV) (E2A) peptide, tosea acinar virus (TaV) (T2A) peptide, porcine Tescovirus-1 (PTV-1) (P2A) peptide, tailovirus 2A peptide, and encephalomyocarditis virus 2A peptide. In one embodiment, the polypeptide cleavage signal is a P2A or T2A viral self-cleavage polypeptide.

[0268] 7. CTBR.TLR polypeptide

[0269] Toll-like receptors (TLR1 to TLR10) are pattern recognition receptors that detect invading pathogens and activate innate and adaptive immune responses. Activation of TLRs by various ligands leads to the induction of pro-inflammatory transcriptional programs and the expression of numerous inflammatory cytokines.

[0270] TLR signaling occurs through the homomerization of the TLR signaling domain, leading to activation via the MyD88 adapter protein and IRAK4 phosphorylation. Following the phosphorylation of IRAK4, the phosphorylation of IRAK1 / 2 ultimately induces the activation of NF-kappa B and AP-1 transcription factors, which increases the production of inflammatory cytokines and induces proliferation. TLR activation can also lead to the activation of IRF3 and IRF7 transcription factors.

[0271] In various embodiments, one or more immune effector cells, including immune effector cells expressing MAGEA4 TCR or MAGEA4 eTCR, are modified by introducing one or more polynucleotides or vectors encoding one or more CTBR.TLR polypeptides. In various embodiments, one or more immune effector cells are modified by introducing one CTBR.TLR and one or more polynucleotides or vectors encoding MAGEA4 TCR or MAGEA4 eTCR.

[0272] In a specific embodiment, the chimeric TGFβ receptor converts an immunosuppressive TGFβ signal into a TLR-mediated immunostimulatory signal. In a specific embodiment, the CTBR.TLR considered herein comprises: the extracellular TGFβ1-binding domain, the transmembrane domain, and the intracellular signaling domain of TGFβR1; the polypeptide cleavage signal; and the extracellular TGFβ1-binding domain, the transmembrane domain, and the same TLR signaling domain of TGFβR2.

[0273] In a specific embodiment, the CTBR.TLR considered herein comprises a fusion polypeptide, wherein the fusion polypeptide comprises: a first polypeptide comprising an extracellular TGFβ1-binding domain, a transmembrane domain, and an intracellular TLR signaling domain of TGFβR1; a polypeptide cleavage signal; and a second polypeptide comprising an extracellular TGFβ1-binding domain, a transmembrane domain, and the same TLR signaling domain of TGFβR2.

[0274] In a specific embodiment, the CTBR.TLR considered herein is a polypeptide complex comprising: a first polypeptide comprising an extracellular TGFβ1-binding domain, a transmembrane domain, and an intracellular signaling domain of TGFβR1; and a second polypeptide comprising an extracellular TGFβ1-binding domain, a transmembrane domain, and the same intracellular signaling domain of TGFβR2.

[0275] In a specific embodiment, the polypeptide comprises a transmembrane domain of TGFβR1 or TGFβR2. In a specific embodiment, the polypeptide comprises a transmembrane domain of a TLR. In one embodiment, the polypeptide comprises an extracellular TGFβ1 binding domain, a TLR transmembrane domain, and an intracellular signaling domain of TGFβR1. In one embodiment, the polypeptide comprises an extracellular TGFβ1 binding domain, a TLR transmembrane domain, and an intracellular signaling domain of TGFβR2.

[0276] In a specific embodiment, the polypeptide cleavage signal is a viral self-cleavage polypeptide; more preferably a viral self-cleavage 2A polypeptide; more preferably a viral self-cleavage polypeptide selected from the group consisting of: foot-and-mouth disease virus (FMDV) (F2A) peptide, equine rhinitis A virus (ERAV) (E2A) peptide, tosea acinar virus (TaV) (T2A) peptide, porcine Tescovirus-1 (PTV-1) (P2A) peptide, tailovirus 2A peptide, and encephalomyocarditis virus 2A peptide. In one embodiment, the polypeptide cleavage signal is a P2A or T2A viral self-cleavage polypeptide.

[0277] E. Polypeptide

[0278] Various polypeptides are considered herein, including but not limited to MAGEA4 TCR, MAGEA4 eTCR, CTBR, and fusion proteins comprising the aforementioned polypeptides and fragments thereof. Unless otherwise specified, “polypeptide,” “peptide,” and “protein” are used interchangeably in the ordinary sense, i.e., as sequences of amino acids. In one embodiment, “polypeptide” includes fusion polypeptides and other variants. Polypeptides may be prepared using any one of various well-known recombinant and / or synthetic techniques. Polypeptides are not limited to a specific length and, for example, may include full-length protein sequences, fragments of full-length proteins, or fusion proteins, and may include post-translational modifications of polypeptides, such as glycosylation, acetylation, phosphorylation, etc., as well as other modifications known in the art, and may include naturally occurring and non-naturally occurring modifications.

[0279] As used herein, “isolated peptide” or “isolated polypeptide,” etc., refers to peptide or polypeptide molecules isolated and / or purified in vitro from the cellular environment and from a state bound to other components of the cell, i.e., peptide or polypeptide molecules not significantly bound to biological substances.

[0280] The polypeptide includes “polypeptide variants.” Polypeptide variants may differ from the naturally occurring polypeptide in one or more substitutions, deletions, additions, and / or insertions. Such variants may occur naturally or may be produced synthetically, for example, by modifying one or more of the polypeptide sequences. For example, in certain embodiments, it may be desirable to improve the binding affinity and / or other biological properties of the polypeptide by introducing one or more substitutions, deletions, additions, and / or insertions into the polypeptide. In a specific embodiment, the polypeptide comprises a polypeptide having at least about 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 86%, 97%, 98%, or 99% amino acid identity with respect to any one of the reference sequences considered herein, wherein the variant generally retains at least one biological activity of the reference sequence.

[0281] The polypeptide variant comprises a biologically active “polypeptide fragment.” Exemplary examples of the biologically active polypeptide fragment include a DNA binding domain, a nuclease domain, etc. As used herein, the term “biologically active fragment” or “minimal biologically active fragment” refers to a polypeptide fragment having at least 100%, at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% of the naturally occurring polypeptide activity. In certain embodiments, the polypeptide fragment may comprise a chain of amino acids of at least 5 to about 1700 amino acids in length. In a specific embodiment, the fragment is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200, It is a length of 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.

[0282] In certain embodiments, the polypeptide presented herein may comprise one or more amino acids denoted by “X”. Where “X” is present in an amino acid sequence number, it refers to any one or more amino acids. In certain embodiments, a sequence number representing a fusion protein comprises a sequence of consecutive X residues representing any amino acid sequence cumulatively.

[0283] As previously stated, polypeptides can be modified in various ways, including amino acid substitution, deletion, cleavage, and insertion. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of a reference polypeptide can be produced by mutations in DNA. Methods for mutagenesis and nucleotide sequence alteration are well known in the art. For example, the literature of Kunkel [(1985, Proc. Natl. Acad. Sci. USA. 82: 488-492)], the literature of Kunkel et al. [(1987, Methods in Enzymol, 154: 367-382)], U.S. Patent No. 4,873,192, and the literature of Watson, JD et al. [ (Molecular Biology of the Refer to Gene, Fourth Edition, Benjamin / Cummings, Menlo Park, Calif., 1987)] and the references cited therein. Guidelines for appropriate amino acid substitutions that do not affect the biological activity of the protein of interest can be found in the model of Dayhoff et al. (Dayhoff et al. [(1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found. [See , Washington, DC]).

[0284] In certain embodiments, the polypeptide variant comprises one or more conservative substitutions. Since a “conservative substitution” is the substitution of an amino acid with another amino acid having similar properties, those skilled in the art of peptide chemistry expect that the secondary structure and hydropathic nature of the polypeptide will not change substantially. The modification may be a modification of the structure of the polynucleotide and polypeptide considered in certain embodiments, and a functional molecule encoding a variant or derivative polypeptide having the desired properties may still be obtained after the modification. When it is desirable to produce an equivalent or even improved variant polypeptide by changing the amino acid sequence of the polypeptide, for example, those skilled in the art may change one or more of the codons of the encoding DNA sequence, for example, according to Table 1.

[0285] amino acid codons amino acids 1-character code 3-character code codon Alanin A Ala GCA GCC GCG GCU Cysteine C Cys UGC UGU Aspartic acid D Asp GAC GAU glutamic acid E Glu GAA GAG Phenylalanine F Phe UUC UUU Glycine G Gly GGA GGC GGG GGU Histidine H His CAC CAU isoleucine I Iso AUA AUC AUU Lee Sin K Lys AAA AAG Leucine L Leu UUA UUG CUA CUC CUG CUU methionine M Met AUG Asparagine N Asn AAC AAU Proline P Pro CCA CCC CCG CCU glutamine Q Gln CAA CAG Arginine R Arg AGA AGG CGA CGC CGG CGU Serin S Ser AGC AGU UCA UCC UCG UCU Threonine T Thr ACA ACC ACG ACU Valin V Val GUA GUC GUG GUU tryptophan W Trp UGG Tyrosine Y Tyr UAC UAU

[0286] Guidelines for determining amino acid residues that can be substituted, inserted, or deleted without impairing biological activity can be verified using computer programs well known in the art, such as DNASTAR, DNA Strider, Geneious, Mac Vector, or Vector NTI software. Preferably, amino acid changes in the protein variants disclosed herein are conservative amino acid changes, that is, substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes involve substitutions of one of the amino acid groups associated with their side chains. Naturally occurring amino acids are generally divided into the following four groups: acidic (aspartate, glutamate) amino acids, basic (lysine, arginine, histidine) amino acids, nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) amino acids, and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified together as aromatic amino acids. In peptides or proteins, appropriate 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 generally recognize that a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter its biological activity (e.g., the literature of Watson et al. [ Molecular Biology of the Gene [See , 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p.224]). In one preferred embodiment in which the expression of two or more polypeptides is desired, the polynucleotide sequences encoding them may be separated by an IRES sequence as disclosed elsewhere in this invention.

[0287] The polypeptide considered in a specific embodiment includes a fusion polypeptide. In a specific embodiment, a fusion polypeptide and a polynucleotide encoding the fusion polypeptide are provided. The fusion polypeptide and the fusion protein refer to a polypeptide having at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 polypeptide segments.

[0288] In another embodiment, two or more polypeptides may be expressed as a fusion protein comprising one or more self-cleaving polypeptide sequences as disclosed elsewhere in this invention.

[0289] The fusion polypeptide may comprise: one or more polypeptide domains or segments including, but not limited to, a signal peptide, a cell-permeable 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. The fusion polypeptide is generally a C-terminal to N-terminal linkage, but may be a C-terminal to C-terminal linkage, an N-terminal to N-terminal linkage, or an N-terminal to C-terminal linkage. In certain embodiments, the polypeptides of the fusion protein may have any order. The fusion polypeptide or fusion protein may include conservedly modified variants, polymorphic variants, alleles, mutants, subsequences, and interspecies homologs, provided that the desired activity of the fusion polypeptide is preserved. The fusion polypeptide may generally be prepared by chemical synthesis methods, by chemical linkage between two moieties, or by using other standard techniques. The linked DNA sequence containing the fusion polypeptide is operably linked to a suitable transcription or translation regulatory element as disclosed elsewhere in this invention.

[0290] The fusion polypeptide may optionally include a linker that can be used to link one or more polypeptides or domains within the polypeptide. The peptide linker sequence may be used to separate any two or more polypeptide components by a distance sufficient to ensure that each polypeptide folds into appropriate secondary and tertiary structures so that the polypeptide domains can perform the desired function. Such peptide linker sequences are incorporated into the fusion polypeptide using standard techniques in the art. Suitable peptide linker sequences may be selected based on the following factors: (1) the ability to adopt a flexible extended form; (2) the ability not to adopt a secondary structure capable of interacting with functional epitopes on the first and second polypeptides; and (3) the absence of hydrophobic or charged residues capable of reacting with polypeptide functional epitopes. Preferred peptide linker sequences contain Gly, Asn, and Ser residues. Other nearly neutral amino acids, such as Thr and Ala, may also be used in the linker sequence. Amino acid sequences that can be usefully used as linkers include those described in the following literature: the literature by Maratea et al. [ Gene 40:39-46, 1985]; Murphy et al.'s literature[ Proc. Natl. Acad. Sci. USA [83:8258-8262, 1986]; U.S. Patent No. 4,935,233, and U.S. Patent No. 4,751,180. A linker sequence is not required if a specific fusion polypeptide segment contains a non-essential N-terminal amino acid region that can be used to separate functional domains and prevent steric interference. A preferred linker is generally a flexible amino acid subsequence synthesized as part of a recombinant fusion protein. The linker polypeptide may be 1 to 200 amino acids long, 1 to 100 amino acids long, or 1 to 50 amino acids long, and includes all integer values ​​in between.

[0291] Exemplary polypeptide cleavage signals include protease cleavage sites, nuclease cleavage sites (e.g., rare restriction enzyme recognition sites, self-cleavage ribozyme recognition sites), and polypeptide cleavage recognition sites such as self-cleavage viral oligopeptides (deFelipe and Ryan's literature [2004, Traffic [ , 5(8); 616-26] see reference).

[0292] Suitable protease cleavage sites and self-cleavage peptides are known to those skilled in the art (e.g., Ryan et al.

[1997] ). J. Gener. Virol.78, 699-722]; see Scymczak et al. [(2004) Nature Biotech. 5, 589-594]). Exemplary protease cleavage sites include, but are not limited to, cleavage sites of fortivirus NIa protease (e.g., tobacco etchant virus protease), fortivirus HC protease, fortivirus P1 (P35) protease, byovirus NIa protease, byovirus RNA-2-encoded protease, aphthovirus L protease, enterovirus 2A protease, rhinovirus 2A protease, picorna 3C protease, comovirus 24K protease, nepovirus 24K protease, RTSV (rice pest spheroid virus) 3C-like protease, PYVF (parsnip yellow blot virus) 3C-like protease, heparin, thrombin, factor Xa, and enterokinase. Due to high cleavage strictness, in one embodiment, EXXYXQ(G / S) (SEVENTEEN NO. 22), such as TEV (tobacco etching virus) protease cleavage sites, e.g., ENLYFQG (SEVENTEEN NO. 20) and ENLYFQS (SEVENTEEN NO. 21), is preferred, where X represents any amino acid (cleavage by TEV occurs between Q and G or between Q and S).

[0293] 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 virus 2A peptide is an aphthovirus 2A peptide, a fortivirus 2A peptide, or a cardiovirus 2A peptide.

[0294] In one embodiment, the virus 2A peptide is 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 Tescovirus-1 (PTV-1) (P2A) peptide, Theilovirus 2A peptide, and encephalomyocarditis virus 2A peptide.

[0295] An exemplary embodiment of part 2A is provided in Table 2.

[0296] Sequence number 23 GSGATNFSLLKQAGDVEENPGP Sequence No. 24 ATNFSLLKQAGDVEENPGP Sequence number 25 LLKQAGDVEENPGP Sequence number 26 GSGEGRGSLLTCGDVEENPGP Sequence number 27 EGRGSLLTCGDVEENPGP Sequence number 28 LLTCGDVEENPGP Sequence number 29 GSGQCTNYALLKLAGDVESNPGP Sequence number 30 QCTNYALLKLAGDVESNPGP Sequence number 31 LLKLAGDVESNPGP Sequence No. 32 GSGVKQTLNFDLLKLAGDVESNPGP Sequence number 33 VKQTLNFDLLKLAGDVESNPGP Sequence No. 34 LLKLAGDVESNPGP Sequence number 35 LLNFDLLKLAGDVESNPGP Sequence number 36 TLNFDLLKLAGDVESNPGP Sequence number 37 LLKLAGDVESNPGP Sequence number 38 NFDLLKLAGDVESNPGP Sequence number 39 QLLNFDLLKLAGDVESNPGP Sequence number 40 APVKQTLNFDLLKLAGDVESNPGP Sequence number 41 VTELLYRMKRAETYCPRPLLAIHPTEARHKQKIVAPVKQT Sequence No. 42 LNFDLLKLAGDVESNPGP Sequence No. 43 LLAIHPTEARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP Sequence No. 44 EARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP

[0297] In a preferred embodiment, the polypeptide comprises MAGEA4 TCR, MAGEA4 eTCR, or one or more CTBR polypeptides. F. Polynucleotide

[0298] In certain embodiments, a fusion polypeptide comprising a MAGEA4 TCR, CTBR, engineered TCR, the aforementioned polypeptides, and fragments thereof is provided. As used herein, the terms “polynucleotide” or “nucleic acid” refer 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: precursor 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 multimeric form of nucleotides consisting 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 (including all intermediate lengths). In this context, “intermediate length” refers to any length between the cited values, e.g., 6, 7, 8, 9, etc.; 101, 102, 103, etc.; 151, 152, 153, etc.; You will easily understand that it means 201, 202, 203, etc.In a specific embodiment, the polynucleotide or variant has at least or about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the reference sequence.

[0299] In certain embodiments, polynucleotides may be codon-optimized. As used herein, the term “codon-optimized” refers to substituting codons of a polynucleotide encoding a polypeptide to increase the expression, stability, and / or activity of the polypeptide. Factors influencing codon optimization include, but are not limited to, one or more of the following: (i) variation in codon bias between two or more organisms or genes or bias tables constructed by synthesis; (ii) variation in the degree of codon bias within an organism, gene, or set of genes; (iii) systematic variation in codons including context; (iv) variation in codons according to the decoding tRNA of the codon; (v) variation in codons according to the GC percentage (%) in the entire triplet or at one of its positions; (vi) variation in the degree of similarity to a reference sequence, such as a naturally occurring sequence; (vii) variation in the codon frequency cutoff; (viii) structural characteristics 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) isolation and removal of the false translation initiation site.

[0300] As used herein, the term “nucleotide” refers to a heterocyclic nitrogenous base linked to a phosphorylated sugar and an N-glycosidically. Nucleotides are understood to include natural bases and a wide variety of modified bases recognized in the art. These bases are generally located at the 1’ position of the nucleotide sugar moiety. Nucleotides generally contain a base, a sugar, and a phosphate group. The sugar in ribonucleic acid (RNA) is ribose, and the sugar in deoxyribonucleic acid (DNA) is deoxyribose, that is, a sugar lacking the hydroxyl group present in ribose. Exemplary natural nitrogenous bases include purines, adenosine (A) and guanidine (G), and pyrimidines, cytidine (C) and thymidine (T) (or uracil (U) in the context of RNA). The C-1 atom of deoxyribose is bonded to the N-1 of pyrimidine or the N-9 of purines. Nucleotides are generally monophosphate, diphosphate, or triphosphate. Nucleotides may be unmodified or modified at the sugar, phosphate, and / or base moiety (also referred interchangeably as nucleotide analogs, nucleotide derivatives, modified nucleotides, non-natural nucleotides, and non-standard nucleotides; see, e.g., WO 92 / 07065 and WO 93 / 15187). Examples of modified nucleic acid bases are found in the literature of Limbach et al. [1994, Nucleic Acids Res It is summarized in . 22, 2183-2196.

[0301] Nucleotides may also be considered as phosphate esters of nucleosides, where esterification occurs on the hydroxyl group attached to the C-5 of the sugar. As used herein, the term “nucleoside” refers to a heterocyclic nitrogenous base linked to a sugar and an N-glycosidic linkage. Nucleosides are recognized in the art to include natural bases and well-known modified bases. These bases are generally located at the 1’ position of the nucleoside sugar moiety. Nucleosides generally include a base and a sugar. Nucleosides may be unmodified or modified at the sugar and / or base moiety (also referred to interchangeably as nucleoside analogs, nucleoside derivatives, modified nucleosides, non-natural nucleosides, and non-standard nucleosides). As previously mentioned, examples of modified nucleic acid bases are found in the literature of Limbach et al. [1994, Nucleic Acids Res It is summarized in . 22, 2183-2196.

[0302] In various exemplary embodiments, the polynucleotides considered herein include, but are not limited to, MAGEA4 TCR, MAGEA4 eTCR, one or more CTBR polypeptides, fusion polypeptides, and expression vectors, viral vectors, and delivery plasmids comprising the polynucleotides considered herein.

[0303] As used herein, the terms “polynucleotide variant” and “variant,” etc. refer to a polynucleotide that exhibits substantial sequence identity with a reference polynucleotide sequence, or a polynucleotide that hybridizes with the reference sequence under strict conditions defined below. These terms also include polynucleotides distinguished from the reference polynucleotide by the addition, deletion, substitution, or modification of at least one nucleotide. Accordingly, the terms “polynucleotide variant” and “variant” include polynucleotides in which one or more nucleotides have been added or deleted, modified, or substituted with different nucleotides. In this regard, it is well understood in the art that certain modifications, including mutations, additions, deletions, and substitutions, may be made to the reference polynucleotide, and that the polynucleotide modified thereby retains the biological function or activity of the reference polynucleotide.

[0304] In one embodiment, the polynucleotide comprises a nucleotide sequence that hybridizes to a target nucleic acid sequence under strict conditions. Hybridizing under “strict conditions” describes a hybridization protocol in which nucleotide sequences that are at least 60% identical to each other maintain a hybridized state. Generally, strict conditions are selected to be about 5°C lower than the thermal melting point (Tm) for a specific sequence at defined ionic strength and pH. Tm is the temperature at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium (under defined ionic strength, pH, and nucleic acid concentration). Since the target sequence is generally present in excess, 50% of the probes are occupied at equilibrium at Tm.

[0305] As used herein, terms including “sequence identity” or, for example, “50% identical sequence” refer to the degree of sequence identity on a nucleotide-to-nucleotide or amino acid-to-amino acid basis on a comparison window. Accordingly, the “percentage of sequence identity” can be calculated by comparing two optimally aligned sequences on 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) occurs in both sequences to obtain the number of matching positions; dividing the number of matching positions by the total number of positions within the comparison window (i.e., the window size); and multiplying the result by 100 to obtain the percentage of sequence identity. Generally, when a polypeptide variant retains at least one biological activity of a reference polypeptide, it comprises 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 with any of the reference sequences described herein.

[0306] 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.” The “reference sequence” is a length of at least 12 monomer units, including nucleotides and amino acid residues, but frequently 15 to 18, and often at least 25 monomer units. Since two polynucleotides may each contain (1) a sequence similar between the two polynucleotides (i.e., only a part of the complete polynucleotide sequence) and (2) a sequence diverging between the two polynucleotides, sequence comparison between two (or more) polynucleotides is generally performed by comparing the sequences of the two polynucleotides on a “comparison window” and identifying local regions of sequence similarity. A “comparison window” is a conceptual interval in which one sequence is compared to a reference sequence at an equal number of consecutive positions after two sequences have been optimally aligned, and refers to a conceptual interval consisting of at least 6 consecutive positions, generally about 50 to about 100 positions, and more generally about 100 to about 150 positions. A comparison window may include about 20% or less of additions or deletions (i.e., gaps) compared to a reference sequence (without additions or deletions) for optimal alignment of two sequences. Optimal sequence alignment for aligning the comparison window may be performed by computerized algorithm implementation examples (GAP, BESTFIT, FASTA, and TFASTA included in Wisconsin Genetics Software Package Release 7.0 of Genetics Computer Group at 575 Science Drive Madison, WI, USA) or by a check and best alignment (i.e., producing the highest percentage of homology on the comparison window) generated by any one of the various selected methods.For example, the literature by Altschul et al. [1997,. Nucl. Acids Res You may also refer to BLAST-family programs such as those disclosed in . 25:3389]. For a detailed discussion of sequence analysis, refer to the literature by Ausubel et al. [ Current Protocols in Molecular Biology This can be found in Unit 19.3 of [John Wiley & Sons Inc, 1994-1998, Chapter 15].

[0307] As used herein, “isolated polynucleotide” refers to a polynucleotide purified from a sequence located adjacent to it in its natural state, for example, a DNA fragment generally removed from a sequence adjacent to the fragment. “Isolated polynucleotide” also refers to complementary DNA (cDNA), recombinant DNA, or other polynucleotides that do not exist in nature and are created by human hands.

[0308] In various embodiments, the polynucleotide comprises mRNA encoding the polynucleotide considered herein. In certain embodiments, the mRNA comprises a cap, one or more nucleotides, and a poly(A) tail.

[0309] Terms describing the orientation of polynucleotides include 5' (typically the end of a polynucleotide containing a free phosphate group) and 3' (typically the end of a polynucleotide containing a free hydroxyl (OH) group). Polynucleotide sequences may be annotated as 5' to 3' orientation or 3' to 5' orientation. For DNA and mRNA, the strand in the 5' to 3' direction is designated as the “sense,” “plus,” or “coding” strand because its sequence is identical to the sequence of the messenger RNA precursor (pre-mRNA) [except that it is uracil (U) in RNA instead of thymine (T) in DNA]. For DNA and mRNA, the complementary strand in the 3' to 5' direction, which is the strand transcribed by RNA polymerase, is designated as the “template,” “antisense,” “minus,” or “non-coding” strand. As used herein, the term “reverse direction” refers to a 5’ to 3’ direction sequence written in the 3’ to 5’ direction, or a 3’ to 5’ direction sequence written in the 5’ to 3’ direction.

[0310] The terms “complementary” and “complementarity” refer to polynucleotides (i.e., sequences of nucleotides) that are related by base pairing rules. For example, the complementary strand of the DNA sequence 5’ AGTCTG 3’ is 3’ TCAGTAC 5’. The latter sequence is often written as an inverse complement to 5’ CATGACT 3’, where the 5’ end is on the left and the 3’ end is on the right. A sequence identical to an inverse complement is called a palindromic sequence. Complementaryness can be “partial,” where only some of the bases of the nucleic acid match according to base pairing rules. Alternatively, there can be “complete” or “total” complementaryness between nucleic acids.

[0311] Furthermore, those skilled in the art will understand that, as a result of genetic code degeneration, there exist many nucleotide sequences capable of encoding fragments of polypeptides or variants thereof, as described herein. Some of these polynucleotides have at least minimal homology with the nucleotide sequences of any natural gene. Nevertheless, polynucleotides that differ due to differences in codon usage, e.g., polynucleotides optimized for human and / or primate codon selection, are specifically considered in certain embodiments. In certain embodiments, polynucleotides are codon-optimized for expression and / or stability. Additionally, alleles of a gene containing the polynucleotide sequence provided herein may also be used. An allele is an endogenous gene that has been altered as a result of one or more mutations, such as deletion, addition, and / or substitution of nucleotides.

[0312] As used herein, the terms “nucleic acid cassette” or “expression cassette” refer to a gene sequence within a vector capable of expressing a polypeptide following RNA. In one embodiment, the nucleic acid cassette contains gene(s) of interest, e.g., polynucleotide(s) 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 gene(s) of interest, e.g., polynucleotide(s) of interest. The vector may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleic acid cassettes. The nucleic acid cassette is positionally and sequentially oriented within a vector so that the nucleic acid within the cassette can be transcribed into RNA, translated into a protein or polypeptide as needed, undergo appropriate post-translational modifications necessary for activity in transformed cells, and translocated to a compartment suitable for biological activity or secretion into an extracellular compartment by targeting an appropriate intracellular compartment. Preferably, the cassette has 3' and 5' ends configured to be easily inserted into a vector, and, for example, has a restriction endonuclease site at each end. In a preferred embodiment, the nucleic acid cassette contains a sequence of a therapeutic gene used to treat, prevent, or improve a genetic disorder. The cassette can be removed and inserted into a plasmid or viral vector as a single unit.

[0313] Polynucleotides include polynucleotide(s) of interest. As used herein, the term “polynucleotide of interest” refers to a polypeptide encoding a polypeptide or a fusion polypeptide, or, as considered herein, a polypeptide acting as a template for the transcription of an inhibitory polynucleotide.

[0314] The polynucleotides considered herein may be combined with other DNA sequences, such as promoters and / or enhancers, untranslated regions (UTRs), signal sequences, Kojak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosome entry sites (IRESs), recombinase recognition sites (e.g., LoxP, FRT, and Att sites), stop codons, transcription termination signals, and polynucleotides encoding self-cleaving polypeptides, epitope tags, regardless of the length of the coding sequence itself, e.g., as described elsewhere in this application or known in the art, so their total lengths may vary significantly. Accordingly, polynucleotide fragments of almost any length may be used, and the total length is preferably considered to be limited by ease of preparation and ease of use in the intended recombinant DNA protocol.

[0315] Polynucleotides can be prepared, manipulated, expressed, and / or delivered using any one of the various well-established techniques known and available in the art. To express a desired polypeptide, a nucleotide sequence encoding the polypeptide can be inserted into a suitable vector.

[0316] Exemplary examples of vectors include, but are not limited to, plasmids, autonomous replication sequences, and transposable elements, e.g., transformable elements, e.g., Sleeping Beauty, PiggyBac.

[0317] Additional exemplary embodiments of the vector include, but are not limited to, plasmids, phagesimides, cosmids, artificial chromosomes (e.g., yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or P1-derived artificial chromosome (PAC)), bacteriophages (e.g., lambda phage or M13 phage), and animal viruses.

[0318] Exemplary examples of viruses useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), varicella-zoster viruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40).

[0319] Exemplary examples of expression vectors include, but are not limited to, the pClneo vector (Promega) for expression in mammalian cells; pLenti4 / V5-DEST™, pLenti6 / V5-DEST™, and pLenti6.2 / V5-GW / lacZ (Invitrogen) for lentivirus-mediated gene delivery and expression in mammalian cells. In certain embodiments, the coding sequence of the polypeptide disclosed herein may be coupled to these expression vectors for the expression of the polypeptide in mammalian cells.

[0320] In certain embodiments, the vector is an episomal vector or a vector maintained outside the chromosome. As used herein, the term “episome” refers to a vector that can replicate without integration into the host’s chromosomal DNA and can replicate without being progressively lost from a dividing host cell, and also means that said vector replicates outside the chromosome or in an episome.

[0321] “Expression regulatory sequences,” “regulatory elements,” or “regulatory sequences” present in an expression vector are non-translating regions of the vector—origins of replication, selection cassettes, promoters, enhancers, translation initiation signals (Shine Dalgarno sequences or Kojak sequences), introns, polyadenylation sequences, and 5’ and 3’ untranslated regions—which interact with host cell proteins to perform transcription and translation. These elements may vary in strength and specificity. Depending on the vector system and host used, any number of appropriate transcription and translation elements, including ubiquitous promoters and inducible promoters, may be used.

[0322] In certain embodiments, the polynucleotide comprises a vector including, but not limited to, an expression vector and a viral vector. The vector may include one or more exogenous, endogenous, or heterogeneous regulatory sequences, such as a promoter and / or enhancer. An “endogenous regulatory sequence” is a sequence that naturally links to a given gene within the genome. An “exogenous regulatory sequence” is a sequence placed alongside a corresponding gene by genetic engineering (i.e., molecular biological techniques) so that the transcription of the gene is induced by the linked enhancer / promoter. A “heterogeneous regulatory sequence” is an exogenous sequence derived from a species different from the genetically engineered cell. A “synthetic” regulatory sequence may include elements of one or more endogenous and / or exogenous sequences, and / or a sequence determined in vitro or in a virtual environment to provide optimal promoter and / or enhancer activity for a specific therapy.

[0323] As used herein, the term “promoter” refers to a recognition site of a polynucleotide (DNA or RNA) to which RNA polymerase binds. RNA polymerase initiates and transcribes the polynucleotide operably linked to the promoter. In a specific embodiment, a promoter operating in a mammalian cell comprises an AT-rich region located approximately 25 to 30 bases upstream from the transcription initiation site and / or another sequence found in a CNCAAT region located 70 to 80 bases upstream from the transcription start region (where N may be any nucleotide).

[0324] The term “enhancer” refers to a segment of DNA containing a sequence capable of enhancing transcription, which in some cases may act independently of orientation toward other regulatory sequences. Enhancers may act cooperatively or additively with promoters and / or other enhancer elements. The term “promoter / enhancer” refers to a segment of DNA containing a sequence capable of providing both promoter and enhancer functions.

[0325] The term “operably connected” refers to a juxtaposition in which the described components are in a relationship that enables them to function in an intended manner. In one embodiment, the term refers to a functional connection between a nucleic acid expression regulatory sequence (e.g., a promoter and / or enhancer) and a second polynucleotide sequence (e.g., a polynucleotide of interest), wherein the expression regulatory sequence induces the transcription of the nucleic acid corresponding to the second sequence.

[0326] As used herein, the term “constitutive expression regulatory sequence” refers to a promoter, enhancer, or promoter / enhancer that enables the transcription of an operably linked sequence in a continuous or sequential manner. A constitutive expression regulatory sequence may be a “ubiquitous” promoter, enhancer, or promoter / enhancer that enables expression in a wide variety of cell and tissue types, or a “cell-specific,” “cell type-specific,” “cell lineage-specific,” or “tissue-specific” promoter, enhancer, or promoter / enhancer that enables expression in a limited range of cell and tissue types, respectively.

[0327] Exemplary ubiquitous expression regulatory sequences suitable for use in specific embodiments include, but are not limited to: a cytomegalovirus (CMV) outpost promoter, viral primate virus 40 (SV40) (e.g., early or late), mouloni rodent leukemia virus (MoMLV) LTR promoter, Rouss sarcoma virus (RSV) LTR, herpes simplex virus (HSV) (thymidine kinase) promoter, H5, P7.5, and P11 promoters derived from vaccinia virus, elongation factor 1-alpha (EF1a) promoter, early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70 kDa protein 5 (HSPA5), heat shock protein 90kDa beta member 1 (HSP90B1), heat shock protein 70kDa (HSP70), β-kinesin (β-KIN), human ROSA 26 locus (Irions et al. [ Nature Biotechnology [Refer to 25, 1477-1482 (2007)]), ubiquitin C promoter (UBC), phosphoglycerate kinase-1 (PGK) promoter, cytomegalovirus enhancer / chicken β-actin (CAG) promoter, β-actin promoter and myeloproliferative sarcoma virus enhancer, U3 promoter with deletion of the negative control region and substitution of the dl587rev primer binding site (MND) (Haas et al.'s literature [ Journal of Virology. [See 2003;77(17): 9439-9450]).

[0328] In one embodiment, the vector includes an MNDU3 promoter.

[0329] In one embodiment, the vector includes an EF1a promoter comprising a first intron of the human EF1a gene.

[0330] In one embodiment, the vector includes an EF1a promoter lacking the first intron of the human EF1a gene.

[0331] In a specific embodiment, it may be desirable to achieve cell type-specific, lineage-specific, or tissue-specific expression of a desired polynucleotide sequence using a cell, cell type, cell lineage, or tissue-specific expression control sequence (e.g., expressing a specific nucleic acid encoding a polypeptide only in a subgroup of cell type, cell lineage, or tissue, or during a specific stage of development).

[0332] In a specific embodiment, it may be desirable to express a polynucleotide as a T cell-specific promoter.

[0333] As used herein, “conditional expression” may refer to any type of conditional expression, including but not limited to inducible expression; repressive expression; and expression in cells or tissues having a specific physiological, biological, or disease state. This definition is not intended to exclude cell type or tissue-specific expression. Certain embodiments provide conditional expression of a polynucleotide of interest, For example, expression is regulated by performing a treatment or undergoing a condition that induces the expression of a polynucleotide or causes an increase or decrease in the expression of a polynucleotide encoded by a polynucleotide of interest, targeting cells, tissues, organisms, etc.

[0334] Exemplary examples of inducible promoters / systems include steroid-inducible promoters, such as promoters for genes encoding glucocorticoid or estrogen receptors (inducible by treatment with the corresponding hormone), metallothione promoters (inducible by treatment with various heavy metals), MX-1 promoters (inducible by interferon), and “GeneSwitch” mifepristone-modulated systems (Sirin et al. [2003, Gene[See , 323: 67]), cuminate-inducible gene switches (WO 2002 / 088346), tetracycline-dependent regulatory systems, etc., are included but not limited to these. Inducers include but are not limited to glucocorticoids, estrogens, mifepristone (RU486), metals, interferons, small molecules, cuminates, tetracyclines, doxycyclines, and variants thereof.

[0335] As used herein, “internal ribosomal entry site” or “IRES” refers to a component that induces cap-independent translation of a gene by facilitating direct internal ribosomal entry to a start codon, such as the ATG of a cistron (protein-coding region). For example, Jackson et al. [1990. Trends in Biochem Science 15(12):477-83)] and the literature of Jackson and Kaminski [1995. RNA Refer to [1(10):985-1000]. Examples of IRES commonly used by those skilled in the art include those described in U.S. Patent No. 6,692,736. Additional examples of “IRES” known in the art include IRES obtainable from picornavirus (Jackson et al., 1990), and, for example, immunoglobulin heavy chain binding protein (BiP), vascular endothelial growth factor (VEGF) (Huez et al., 1998. Mol. Cell. Biol.[18(11):6178-6190] (see reference), fibroblast growth factor 2 (FGF-2), and insulin-like growth factor (IGFII), translation initiation factor eIF4G, and yeast transcription factors TFIID and HAP4, IRES obtained from viral or cellular mRNA sources such as commercially available encephalomyelitis virus (EMCV) from Novagen (see reference by Duke et al. [1992. J. Virol 66(3):1602-9]), and VEGF IRES (see reference by Huez et al. [1998. Mol Cell Biol 18(11):6178-90]), but not limited to these. IRES have also been reported in the viral genomes of picornavirus, dicystovirus, and flavivirus species, and in HCV, Fresnez's mouse leukemia virus (FrMLV), and Moloni's mouse leukemia virus (MoMLV).

[0336] In one embodiment, the IRES used in the polynucleotide considered herein is an EMCV IRES.

[0337] In certain embodiments, the polynucleotide comprises a polynucleotide having a common Kozak sequence and encoding the desired polypeptide. As used herein, the term “Kozak sequence” refers to a short nucleotide sequence that significantly facilitates the initial binding of mRNA to a small subunit of a ribosome and increases translation. The common Kozak sequence is (GCC)RCCATGG (SEQ No. 45), where R is a purine (A or G) (Kozak's literature [1986. Cell . 44(2):283-92] and Kozak's literature [1987. Nucleic Acids Res [See . 15(20):8125-48]).

[0338] Factors that induce efficient termination and polyadenylation of heterogeneous nucleic acid transcripts increase heterogeneous gene expression. Transcription termination signals are typically identified downstream of polyadenylation signals. In certain embodiments, the vector comprises a polyadenylation sequence at the 3' end of a polynucleotide encoding the polypeptide to be expressed. As used herein, the terms “poly-A site” or “poly-A sequence” refer to a DNA sequence that induces both termination and polyadenylation of early RNA transcription by RNA polymerase II. Since polyadenylation sequences can promote mRNA stability by adding a poly-A tail to the 3' end of the coding sequence, they contribute to an increase in translation efficiency. Cleavage and polyadenylation are induced by the poly(A) sequence within the RNA. The core poly(A) sequence for the mammalian mRNA precursor has two recognition elements located on the flanks of the cleavage-polyadenylation site. Generally, a nearly invariant AAUAAA hexamer is positioned 20 to 50 nucleotides upstream from a more variable element rich in U or GU residues. Cleaving of the initial transcript occurs between these two elements and is coupled to up to 250 adenosines added to the 5' cleavage product. In certain embodiments, the core poly(A) sequence is an ideal polyA sequence (e.g., AATAAA, ATTAAA, AGTAAA). In certain embodiments, the poly(A) sequence is an SV40 polyA sequence, a bovine growth hormone polyA sequence (BGHpA), a rabbit β-globin polyA sequence (rβgpA), or any other suitable heterogeneous or endogenous polyA sequence known in the art.

[0339] In some embodiments, the polynucleotide or the cell containing the polynucleotide uses a suicide gene (including an inducible suicide gene) to directly reduce the risk of toxicity and / or uncontrolled proliferation. In certain embodiments, the suicide gene is not immunogenic to the host containing the polynucleotide or the cell. Specific examples of suicide genes available for use are caspase-9, caspase-8, or cytosine deaminase. Caspase-9 can be activated using a specific chemoinducer (CID) for dimerization.

[0340] In certain embodiments, one or more polynucleotides encoding MAGEA4 TCRα and TCRβ chains containing an eTCR chain, and / or one or more CTBR polypeptides are introduced into a cell (e.g., an immune effector cell) by a nonviral or viral vector. The term “vector” is used herein to refer to a nucleic acid molecule capable of delivering or transporting another nucleic acid molecule. The delivered nucleic acid is generally linked to the vector nucleic acid molecule, for example, inserted into the vector nucleic acid molecule. The vector may contain a sequence that induces autonomous replication in the cell, or may contain a sequence sufficient to allow integration into host cell DNA. In certain embodiments, a nonviral vector is used to deliver one or more polynucleotides considered herein to a T cell.

[0341] Exemplary examples of nonviral vectors include, but are not limited to, mRNA, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, and bacterial artificial chromosomes.

[0342] Exemplary methods of nonviral delivery of polynucleotides considered in specific embodiments include, but are not limited to: electroporation, sonication, lipofection, microinjection, bioistics, virosome, liposome, immunoliposome, nanoparticle, polycation or lipid:nucleic acid conjugate, naked DNA, artificial virion, DEAE-dextran-mediated delivery, gene gun, and heat shock.

[0343] Exemplary examples of polynucleotide delivery systems suitable for use in the specific embodiments considered in the specific embodiments include, but are not limited to, those 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 the efficient receptor-recognized lipofection of polynucleotides are described in the literature. For example, the literature by Liu et al. [(2003) Gene Therapy. 10:180-187]; and the literature of Balazs et al.[(2011) Journal of Drug Delivery. [See 2011:1-12] Antibody-targeting, bacterial-derived, non-biological nanocell-based delivery is also considered in certain embodiments.

[0344] In various embodiments, the polynucleotide is mRNA introduced into a cell to transiently express a desired polypeptide. As used herein, “transient” refers to the expression of a non-integrated transplanted gene for a period of hours, days, or weeks, wherein the expression period is shorter than the expression period when the polynucleotide is integrated into the genome or contained within a stable plasmid replicon in the cell.

[0345] In a specific embodiment, a viral vector is used to deliver one or more polynucleotides considered herein to a T cell.

[0346] Exemplary embodiments of viral vector systems suitable for use in the specific embodiments considered herein include, but are not limited to, adeno-associated viruses (AAVs), retroviruses (including lentiviruses), herpes simplex viruses, adenoviruses, and vaccinia virus vectors.

[0347] In a specific embodiment, a polycistron polynucleotide encoding a MAGEA4 TCR (SEQN 4) comprising a TCRα chain (SEQN 2) and a TCRβ chain (SEQN 3) and a polycistron polynucleotide encoding a CTBR (SEQN 8) are introduced into a cell using a nonviral or viral vector. In a specific embodiment, a polycistron polynucleotide encoding a fusion protein encoding a MAGEA4 TCR (SEQN 7) comprising a TCRα chain (SEQN 5) and a TCRβ chain (SEQN 6), and a polycistron polynucleotide encoding a CTBR (SEQN 8) are introduced into a cell using a nonviral or viral vector.

[0348] In a specific embodiment, a polycistron polynucleotide encoding MAGEA4 TCR (SEQN 4) and CTBR (SEQN 8), comprising TCRα chain (SEQN 2) and TCRβ chain (SEQN 3), is introduced into a cell using a nonviral or viral vector. In a specific embodiment, a fusion protein encoding MAGEA4 TCR (SEQN 7), comprising TCRα chain (SEQN 5) and TCRβ chain (SEQN 6), and a polycistron polynucleotide encoding CTBR (SEQN 8) are introduced into a cell using a nonviral or viral vector.

[0349] G. Genetically modified cells

[0350] In various embodiments, cells are modified to express MAGEA4 TCR or MAGEA4 eTCR and CTBR for use in the treatment of cancer. Cells may be non-genetically modified to express the polypeptides considered herein, or, in particularly preferred embodiments, cells may be genetically modified to express the polypeptides considered 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 within a cell. The terms “genetically modified cell,” “modified cell,” and “re-induced cell” are used interchangeably in specific embodiments.

[0351] In a specific embodiment, the MAGEA4 TCR and one or more CTBR polypeptides considered herein are introduced into immune effector cells and expressed in the immune effector cells to enhance the cell's resistance to immunosuppressive signals in the TME mediated by TGFβ. In a specific embodiment, the MAGEA4 eTCR and one or more CTBR polypeptides are introduced into immune effector cells and expressed in the immune effector cells.

[0352] “Immune effector cells” are any cell of the immune system that possesses one or more effector functions (e.g., cytotoxic cell death activity, secretion of cytokines, induction of ADCC and / or CDC). Exemplary immune effector cells considered herein are T lymphocytes, in particular cytotoxic T cells (CTL; ​​CD8+ T cells), TILs, and helper T cells (HTL; CD4+ T cells). In one embodiment, immune effector cells include natural killer (NK) cells. In one embodiment, immune effector cells include natural killer T (NKT) cells. Immune effector cells may be self / spontaneous (“self”) or non-self (“non-self”), e.g., allogeneic, syngeneic, or xenogeneic.

[0353] As used herein, “autologous” refers to cells of the same subject. As used herein, “allogenic” refers to cells of the same species that are genetically different from the comparative cell. As used herein, “syngeneic” refers to cells of a different subject that are genetically identical to the comparative cell. As used herein, “xenogeneic” refers to cells of a different species from the comparative cell. In a preferred embodiment, the cell is an autologous cell.

[0354] Exemplary immune effector cells suitable for introducing the CTBR polypeptide considered herein include T lymphocytes. The terms “T cell” or “T lymphocyte” are intended to include thymus cells, immature T lymphocytes, mature T lymphocytes, quiescent T lymphocytes, or activated T lymphocytes. T cells may be T helper (Th) cells, for example, T helper 1 (Th1) or T helper 2 (Th2) cells. T cells are helper T cells (HTL; CD4 + T cells) CD4 + T cells, cytotoxic T cells (CTL; ​​CD8 + T cells), CD4 + CD8+ T cells, CD4 - CD8 - It may be T cells, or any other subset of T cells. Other exemplary T cell populations suitable for use in a particular embodiment include untreated T cells and memory T cells.

[0355] As understood by those skilled in the art, other cells may be used as immune effector cells in combination with MAGEA4 TCR or MAGEA4 eTCR and one or more CTBR polypeptides considered herein. In particular, immune effector cells also include NK cells, NKT cells, neutrophils, and macrophages. Immune effector cells also include progenitor cells of effector cells, and these progenitor cells are in vivo or They can be induced to differentiate into immune effector cells in vitro. Thus, in a specific embodiment, the immune effector cells are CD34 cells derived from umbilical cord blood, bone marrow, or mobilized peripheral blood that differentiate into mature immune effector cells upon administration to a subject, or can be induced in vitro to differentiate into mature immune effector cells. + It includes precursor cells of immune effector cells, such as hematopoietic stem cells (HSCs), contained within the population.

[0356] As used herein, immune effector cells genetically engineered to contain specific chimeric receptors may be referred to as “re-induced antigen-specific immune effector cells.”

[0357] Terms used at this institution “CD34 + “Cell” refers to a cell expressing the CD34 protein on its cell surface. As used herein, “CD34” often refers to a cell surface glycoprotein (e.g., sialomusin protein) that acts as a cell-cell adhesion factor and is involved in the entry of T cells into lymph nodes. CD34 + The cell population contains hematopoietic stem cells (HSCs), which differentiate when administered to a patient and contribute to all hematopoietic lines, including T cells, NK cells, NKT cells, neutrophils, and monocyte / macrophage lineages.

[0358] A method for producing immune effector cells expressing the MAGEA4 TCR or MAGEA4 eTCR and chimeric TGFβ receptor polypeptide considered herein is provided in a specific embodiment. In one embodiment, the method comprises the step of transfecting or transducing immune effector cells isolated from an individual so that the immune effector cells express the MAGEA4 TCR or MAGEA4 eTCR and one or more chimeric TGFβ receptor polypeptides considered herein. In one embodiment, the method comprises the step of transfecting or transducing immune effector cells isolated from an individual so that the immune effector cells express the MAGEA4 TCR or MAGEA4 eTCR and one or more chimeric TGFβ receptor polypeptides considered herein, as well as the MAGEA4 TCR and MAGEA4 eTCR. In a specific embodiment, the immune effector cells are isolated from an individual and genetically modified in vitro without further manipulation. Then, these cells may be directly re-administered into the individual. In a further embodiment, immune effector cells are first activated, stimulated, and proliferated in vitro before being genetically modified. In this regard, immune effector cells may be cultured before and / or after genetic modification.

[0359] In a specific embodiment, the source of cells is obtained from a subject prior to the in vitro manipulation or genetic modification of the immune effector cells described herein. In a specific embodiment, the modified immune effector cells include T cells.

[0361] *T cells may be obtained from a number of sources including, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue of the infection site, ascites, pleural effusion, splenic tissue, and tumors. In certain embodiments, T cells are obtained from any number of technologies known to those skilled in the art, e.g., It can be obtained from blood units collected from a subject using sedimentation such as FICOLL™ separation.

[0362] In other embodiments, isolated or purified T cell populations are used. In some embodiments, after isolation of PBMCs, both cytotoxic T lymphocytes and helper T lymphocytes may be classified into untreated, memory, and effector T cell subpopulations before or after activation, proliferation, and / or genetic modification.

[0363] In one embodiment, the isolated or purified T cell population is CD3 + , CD4 + , CD8 + , or expresses one or more markers including but not limited to combinations thereof.

[0364] In a specific embodiment, T cells are isolated from an individual and then first activated and stimulated to proliferate in vitro before being modified to express a chimeric TGFβ receptor polypeptide.

[0365] To obtain a sufficient therapeutic dose of a T cell composition, T cells are often subjected to one or more stimulations, activations, and / or proliferations. T cells are generally, for example, U.S. Patents No. 6,352,694; No. 6,534,055; No. 6,905,680; No. 6,692,964; No. 5,858,358; No. 6,887,466; No. 6,905,681; No. 7,144,575; No. 7,067,318; No. 7,172,869; No. 7,232,566; No. 7,175,843; No. 5,883,223; No. 6,905,874; No. 6,797,514; and may be activated and proliferated using methods such as those described in No. 6,867,041, each of which is incorporated herein by reference in its entirety. In certain embodiments, T cells are activated and proliferated for about 6 hours, about 12 hours, about 18 hours, or about 24 hours before being introduced into a vector or polynucleotide encoding a MAGEA4 TCR or MAGEA4 eTCR and a chimeric TGFβ receptor polypeptide.

[0366] In one embodiment, T cells are simultaneously activated when they are modified.

[0367] In various embodiments, a method for generating immune effector cells comprises the steps of activating a cell population containing T cells and proliferating the T cell population. T cell activation can be achieved by providing a primary stimulating signal through the T cell TCR / CD3 complex and providing a secondary co-stimulating signal through an accessory molecule (e.g., CD28).

[0368] The TCR / CD3 complex binds T cells to an appropriate CD3 binder, for example, It can be stimulated by contact with a CD3 ligand or an anti-CD3 monoclonal antibody. Exemplary examples of CD3 antibodies include, but are not limited to, OKT3, G19-4, BC3, and 64.1.

[0369] In addition to the primary stimulating signal provided via the TCR / CD3 complex, a secondary co-stimulating signal is required for the induction of a T cell response. In certain embodiments, a CD28 binder may be used to provide the co-stimulating signal. Exemplary examples of CD28 binders include, but are not limited to: natural CD28 ligands, e.g., 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 CD28 molecules, e.g., monoclonal antibodies 9.3, B-T3, XR-CD28, KOLT-2, 15E8, 248.23.2, and EX5.3D10.

[0370] In one embodiment, a molecule providing a primary stimulus signal, e.g., a molecule providing stimulation via a TCR / CD3 complex, and a co-stimulator are bound to the same surface.

[0371] In a specific embodiment, a binder providing a stimulating signal and a co-stimulating signal is localized to the surface of a cell. This can be achieved by transfecting or transducing the cell with a nucleic acid encoding the binder in a form suitable for expression on the cell surface, or alternatively by binding the binder to the cell surface.

[0372] In another embodiment, a molecule providing a primary stimulus signal, for example, a molecule providing stimulation through a TCR / CD3 complex and a co-stimulatory molecule are displayed on an antigen-presenting cell.

[0373] In one embodiment, a molecule providing a primary stimulation signal, e.g., a molecule providing stimulation via a TCR / CD3 complex, and a co-stimulator are provided on separate surfaces.

[0374] In a specific embodiment, one of the binders providing the stimulating signal and the co-stimulating signal is soluble (provided as a solution), and the other agent(s) are provided on one or more surfaces.

[0375] In a specific embodiment, the binder providing the stimulating signal and the co-stimulating signal is provided in a soluble form (provided as a solution).

[0376] In various embodiments, the method for producing T cells considered herein includes the step of activating T cells with anti-CD3 and anti-CD28 antibodies.

[0377] In one embodiment, the step of proliferating T cells activated by the method considered herein further comprises the step of culturing a cell population containing T cells for several hours (about 3 hours) to about 7 days to about 28 days, or any integer value in between. 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 to 3 days. Multiple cycles of stimulation / activation / proliferation may be preferred so that the culture time of the T cells can be 60 days or more.

[0378] In certain embodiments, conditions suitable for T cell culture comprise a suitable medium (e.g., Minimal Essential Media or RPMI Media 1640 or X-vivo 15 (Lonza)) and one or more factors necessary for proliferation and survival, including but not limited to: serum (e.g., bovine fetal 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 additive suitable for cell growth known to those skilled in the art.

[0379] Additional exemplary examples of cell culture media include, but are not limited to, RPMI 1640, Clicks, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer supplemented with amino acids, sodium pyruvate, and vitamins, and these media may be supplemented with no serum or an appropriate amount of serum (or plasma), and may be supplemented with a defined set of hormones and / or a sufficient amount of cytokine(s) for the growth and proliferation of T cells.

[0380] Antibiotics such as penicillin and streptomycin are included only in the experimental culture and not in the cell culture to be injected into the subject. Target cells are maintained under conditions necessary to support growth, e.g., an appropriate temperature (e.g., 37°C) and atmosphere (e.g., air + 5% CO2).

[0381] In a specific embodiment, PBMCs or isolated T cells are contacted with stimulants and co-stimulants, such as anti-CD3 and anti-CD28 antibodies attached to beads or other surfaces, in a culture medium containing appropriate cytokines such as IL-2, IL-7, and / or IL-15.

[0382] In other embodiments, artificial APCs (aAPCs) are created by manipulating K562, U937, 721.221, T2, and C1R cells to induce stable expression and secretion of various co-stimulatory molecules and cytokines. In specific embodiments, K32 or U32 aAPCs are used to induce the display of one or more antibody-based stimulatory molecules on the surface of AAPC cells. T cell populations may be proliferated by aAPCs expressing various co-stimulatory molecules, including but not limited to CD137L (4-1BBL), CD134L (OX40L), and / or CD80 or CD86. Finally, aAPCs provide an efficient platform for proliferating genetically modified T cells and maintaining CD28 expression in CD8 T cells. The aAPCs provided in WO 03 / 057171 and US2003 / 0147869 are incorporated herein by reference in their entirety.

[0383] In a specific embodiment, one or more polynucleotides encoding MAGEA4 TCR or MAGEA4 eTCR and a chimeric TGFβ receptor are introduced into a population of T cells. In a specific embodiment, a polynucleotide encoding a chimeric TGFβ receptor is introduced into a population of T cells expressing MAGEA4 TCR or MAGEA4 eTCR. In a specific embodiment, a polynucleotide encoding a chimeric MAGEA4 TCR or MAGEA4 eTCR is introduced into a population of T cells expressing a chimeric TGFβ receptor. In a specific embodiment, a polynucleotide encoding a chimeric MAGEA4 TCR or MAGEA4 eTCR and a chimeric TGFβ receptor is introduced into a population of T cells. In a specific embodiment, a polynucleotide encoding a chimeric MAGEA4 TCR or MAGEA4 eTCR and a polynucleotide encoding a chimeric TGFβ receptor are simultaneously introduced into a population of T cells. Polynucleotides can be introduced into T cells by microinjection, transfection, lipofection, heat shock, electroporation, transduction, gene gun, microinjection, DEAE-dextran-mediated delivery, etc.

[0384] In a preferred embodiment, the polynucleotide is introduced into the T cell by viral transduction.

[0385] Polynucleotides on immune effector cells or CD34 + Exemplary examples of viral vector systems suitable for introduction into cells include, but are not limited to, adeno-associated viruses (AAVs), retroviruses, herpes simplex viruses, adenoviruses, and vaccinia virus vectors for gene transfer.

[0386] In one embodiment, the polynucleotide is introduced into the T cell by AAV transduction.

[0387] In one embodiment, the polynucleotide is introduced into the T cell by retroviral transduction.

[0388] In one embodiment, the polynucleotide is introduced into the T cell by lentivirus transduction.

[0389] In one embodiment, the polynucleotide is introduced into the T cell by an adenovirus.

[0390] In one embodiment, the polynucleotide is introduced into the T cell by herpes simplex virus transduction.

[0391] In one embodiment, the polynucleotide is introduced into the T cell by vaccinia virus transduction.

[0392] H. Composition and Formulation

[0393] The compositions considered herein may include one or more MAGEA4 TCR polypeptides, MAGEA4 eTCR polypeptides, CTBR polypeptides, polynucleotides, vectors containing the same, genetically modified immune effector cells, etc., such as those considered herein. The 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 TCR and CTBR12.

[0394] “Pharmaceutical composition” refers to a composition formulated into a pharmaceutically acceptable or physiologically acceptable solution to be administered to cells or animals alone or in combination with one or more other therapies. Additionally, it will be understood that, if desired, the composition may be administered in combination with other agents, such as, for example, cytokines, growth factors, hormones, small molecules, chemotherapeutic agents, prodrugs, drugs, antibodies, or various other pharmaceutical active agents. There are virtually no limitations on other components that may be included in the composition, provided that the additional agent does not adversely affect the composition’s ability to deliver the intended therapy. In a preferred embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, a diluent, or an excipient, and one or more cells modified to express MAGEA4 TCR and CTBR, preferably MAGEA4 eTCR and CTBR12 polypeptides.

[0395] The phrase “pharmacologically acceptable” is used herein to refer to compounds, substances, compositions, and / or dosage forms that, within the scope of sound medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, and correspond to a reasonable benefit / risk ratio.

[0396] As used herein, “pharmaceutically acceptable carriers, diluents, or excipients” include, but are not limited to, isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer solution; and any other compatible material used in pharmaceutical formulations.

[0397] In a specific embodiment, the composition comprises an amount 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 amount” of genetically modified therapeutic cells, e.g., T cells, to achieve beneficial or desired preventive or therapeutic outcomes, including clinical outcomes.

[0398] The “prophylactic effective dose” refers to the amount of genetically modified therapeutic cells effective in achieving the desired prophylactic outcome. Although not always the case, generally, the prophylactic effective dose is lower than the therapeutic effective dose because it is administered to the subject prior to disease or during the early stages of the disease.

[0399] The “therapeutic effective dose” of genetically modified therapeutic cells may vary depending on factors such as the individual’s disease state, age, sex, and weight, and the ability of stem and progenitor cells to induce a desired response in the individual. The therapeutic effective dose is also an amount in which the therapeutically beneficial effect outweighs any toxic or adverse effects of the virus or transduced therapeutic cells. The term “therapeutic effective dose” includes an amount effective in “treating” a subject (e.g., a patient). Where a therapeutic dose is indicated, the exact amount of the composition to be administered may be determined by a physician taking into account the individual differences in the subject’s age, weight, tumor size, degree of infection or metastasis, and condition.

[0400] Generally, pharmaceutical compositions containing T cells described herein contain 10 per 1 kg of body weight. 6 to 10 13 Dog cells, preferably 10 8 to 10 13It may be administered in doses of cells (including all integer values ​​within these ranges). The number of cells will depend on the composition as well as the intended end use of the cell types contained therein. For the uses provided herein, the cell volume is generally 1 liter or less, and may be 500 ml or less, even 250 ml or 100 ml or less. Accordingly, the desired cell density is generally 10 6 Higher than individual cells / ml, generally 10 7 Higher than individual cells / ml, generally 10 8 It is greater than 1 cell / ml. The clinically flexible number of immune cells can be divided for multiple infusions, and the cumulative number is 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , or 10 13 It consists of one or more cells. The composition may be administered multiple times in doses within this range. The cells may be allogeneic, homologous, xenogeneic, or autologous for the patient receiving treatment.

[0401] In a specific embodiment, the composition is preferably formulated for parenteral administration, e.g., intravascular (intravenous or intra-arterial) administration.

[0402] The liquid pharmaceutical composition, whether in the form of a solution, suspension, or other similar form, may comprise one or more of the following: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, or isotonic sodium chloride. The parenteral formulation may be enclosed in ampoules, disposable syringes, or multi-dose vials made of glass or plastic. The injectable pharmaceutical composition is preferably sterile.

[0403] In one embodiment, the T cell composition considered herein is formulated in a pharmaceutically acceptable cell culture medium. Such composition is suitable for administration to human subjects. In a specific embodiment, the pharmaceutically acceptable cell culture medium is a serum-free medium.

[0404] Serum-free media have several advantages over serum-containing media, including a simplified and better-defined composition, reduced contamination, elimination of potential sources of infectious material, and lower cost. In various embodiments, serum-free media are animal-free and may optionally be protein-free. Optionally, the media may contain biopharmaceutical acceptable recombinant proteins. “Animal-free” media refer to media whose components are derived from non-animal sources. Recombinant proteins replace natural animal proteins in animal-free media, and nutrients are obtained from synthetic, plant, or microbial sources. In contrast, “protein-free” media are defined as being substantially protein-free.

[0405] Exemplary examples of serum-free media used in specific compositions include, but are not limited to, QBSF-60 (Quality Biological, Inc.), StemPro-34 (Life Technologies), and X-VIVO 10.

[0406] In a preferred embodiment, the composition comprising the immune effector cells considered herein is formulated as a solution comprising PlasmaLyte A.

[0407] In another preferred embodiment, the composition comprising the immune effector cells considered herein is formulated as a solution containing a cryopreservation agent. For example, high cell viability results after thawing can be maintained by using a cryopreservation medium containing a cryopreservation agent. Exemplary examples of cryopreservation media used in a particular composition include, but are not limited to, CryoStor CS10, CryoStor CS5, and CryoStor CS2.

[0408] In a more preferred embodiment, a composition comprising the immune effector cells considered herein is formulated in a solution comprising PlasmaLyte A and CryoStor CS10 in a 50:50 ratio.

[0409] In certain embodiments, the composition comprises, alone or in combination with one or more therapeutic agents, an effective amount of genome-edited immune effector cells modified to express MAGEA4 TCR and CTBR, preferably MAGEA4 eTCR and CTBR12 polypeptides. Accordingly, the immune effector cell composition may be administered alone or in combination with other known cancer therapeutic agents, such as radiotherapy, chemotherapy, transplantation, immunotherapy, hormone therapy, photodynamic therapy, etc. The composition may also be administered in combination with antibiotics. Such therapeutic agents may be accepted in the art as standard therapeutic agents for specific pathological conditions, e.g., specific cancers, as described herein. Exemplary therapeutic agents considered in certain embodiments include cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory agents, chemotherapy agents, radiotherapy agents, therapeutic antibodies, or other activators and adjuvants.

[0410] In a given embodiment, a composition comprising genome-edited immune effector cells modified to express MAGEA4 TCR and CTBR, preferably MAGEA4 eTCR and CTBR12 polypeptides, may be administered with any number of chemotherapeutic agents.

[0411] In a specific embodiment, a composition comprising immune effector cells modified to express MAGEA4 TCR and CTBR, preferably MAGEA4 eTCR and CTBR12 polypeptides, is administered together with a therapeutic antibody. Exemplary examples of therapeutic antibodies suitable for use in combination with modified T cells considered in specific embodiments include, but are not limited to: atezolizumab, avelumab, babituximab, bevacizumab (Avastin), vivatuzumab, blinatumomab, conatumomab, crizotinib, daratumumab, duligotumab, dasetuzumab, dalotuzumab, durvalumab, elotuzumab (HuLuc63), gemtuzumab, ibritumomab, indatuximab, innotuzumab, ipilimumab, lorbotuzumab, lucatuzumab, milatuzumab, moxetumomab, nivolumab, ocaratuzumab, ofatumomab, pembrolizumab, rituximab, siltuximab, teprotumumab, and ublituximab.

[0412] In certain embodiments, formulations of pharmaceutically acceptable carrier solutions are well known to those skilled in the art, as are appropriate administration and therapeutic regimens for the use of the specific compositions described herein in various therapeutic regimens, and appropriate administration and therapeutic regimens include, for example, enteral and parenteral administration and formulations, e.g., intravascular, intravenous, intra-arterial, intraosseous, intraventricular, intracerebral, intracranial, intraspinal, intradural, and intramedullary administration and formulations. Those skilled in the art will understand that the specific embodiments considered herein may include other formulations well known in the pharmaceutical field and, for example, those described in the following literature: Remington: The Science and Practice of Pharmacy, volume I and volume II. 22nd Edition. Edited by Loyd V. Allen Jr. Philadelphia, PA: Pharmaceutical Press; 2012 (the entirety of which is incorporated herein by reference).

[0413] I. Treatment Methods

[0414] Immuno-effector cells including CTBRs considered herein, including MAGEA4 TCR T cells or MAGEA4 eTCR T cells, provide an improved adoptive immunotherapy method for use to prevent, treat, and alleviate cancer, or to prevent, treat, and alleviate at least one symptom associated with cancer.

[0415] Immuno-effector cells comprising MAGEA4 TCR or MAGEA4 eTCR and CTBR considered herein provide an improved drug product for use in the prevention, treatment, or alleviation of at least one symptom of cancer, GVHD, infectious disease, autoimmune disease, inflammatory disease, or immunodeficiency. As used herein, the term “drug product” refers to modified cells produced using the composition and method considered herein. In certain embodiments, the drug product comprises genetically modified immune-effector cells, T cells modified to express MAGEA4 TCR or MAGEA4 eTCR and further modified to express the CTBR polypeptide. Additionally, the modified T cells considered in certain embodiments provide safer and more effective adoptive cell therapy because they resist modified T cell depletion and exhibit increased durability and persistence that can lead to sustained treatment in the tumor microenvironment.

[0416] In a specific embodiment, an effective amount of modified immune effector cells or T cells comprising or expressing MAGEA4 TCR or MAGEA4 eTCR and CTBR is administered to a subject to prevent, treat, or alleviate at least one symptom of cancer, GVHD, infectious disease, autoimmune disease, inflammatory disease, or immunodeficiency.

[0417] In a specific embodiment, a method for preventing, treating, or alleviating at least one symptom of cancer comprises the step of administering to a subject an effective amount of modified immune effector cells or T cells comprising or expressing CTBR and MAGEA4 TCR or MAGEA4 eTCR. Genetically modified cells are a more durable and sustained finished product because said cells are more resistant to immunosuppressive signals from the tumor microenvironment by converting immunosuppressive TGFβ signals into immunostimulatory signals.

[0418] In a specific embodiment, the modified immune effector cells considered herein are used in the treatment of solid tumors or cancer.

[0419] In certain embodiments, the modified immune effector cells considered herein are used to treat solid tumors or cancers, including but not limited to: adrenal cancer, adrenocortical carcinoma, anal cancer, appendiceal cancer, astrocytoma, atypical anomaly / rhabdomyotumor, basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone cancer, brain / CNS cancer, breast cancer, bronchial tumor, heart tumor, cervical cancer, cholangiocarcinoma, chondrosarcoma, chordoma, colon cancer, colorectal cancer, craniopharyngioma, carcinoma in situ (DCIS), endometrial cancer, ependymoma, esophageal cancer, sciatic neuroblastoma, Ewing sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, ocular cancer, fallopian tube cancer, fibrous histosarcoma, fibrosarcoma, gallbladder cancer, gastric cancer, gastric carcinoid tumor, gastric stromal tumor (GIST), germ cell tumor, glioma, glioblastoma, head and neck cancer, hemangioma, hepatocellular carcinoma, Hypopharyngeal cancer, intraocular melanoma, Kaposi's sarcoma, kidney cancer, laryngeal cancer, leiomyosarcoma, lip cancer, liposarcoma, liver cancer, lung cancer, non-small cell lung cancer, pulmonary carcinoid tumor, malignant mesothelioma, medullary carcinoma, medulloblastoma, meningioma, melanoma, Merkel cell carcinoma, midline duct carcinoma, oral cancer, myxosarcoma, myelodysplastic syndrome, myeloproliferative neoplasm, nasal and sinus cancer, nasopharyngeal cancer, neuroblastoma, oligodendroma, oral cancer, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic islet cell tumor, papillary carcinoma, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal tumor, pituitary tumor, pleuropulmonary blastoma, primary peritoneal cancer, prostate cancer, rectal cancer, retinoblastoma, renal cell carcinoma, renal pelvis and ureteral cancer, Rhabdomyosarcoma, salivary gland cancer, sebaceous gland carcinoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, small cell lung cancer, small intestine cancer, stomach cancer, sweat gland carcinoma, synovial tumor, testicular cancer, throat cancer, thymic cancer, thyroid cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vascular cancer, vulvar cancer, and Wilms' tumor.

[0420] In certain embodiments, the modified immune effector cells considered 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.

[0421] In certain embodiments, the modified immune effector cells considered herein are used in the treatment of various cancers, including but not limited to pancreatic cancer, bladder cancer, and lung cancer.

[0422] In a specific embodiment, the modified immune effector cells considered herein are used in the treatment of liquid cancer or blood cancer.

[0423] In certain embodiments, the modified immune effector cells considered herein are used in the treatment of B-cell malignancies, including but not limited to leukemia, lymphoma, and multiple myeloma.

[0424] In certain embodiments, the modified immune effector cells considered herein are used to treat liquid cancers, including but not limited to: leukemias, lymphomas, and multiple myelomas: acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, erythroleukemia, blastocyst leukemia (HCL), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML) and polycythemia vera, Hodgkin lymphoma, nodular lymphocyte-dominant Hodgkin lymphoma, Burkitt 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, subacute multiple myeloma, plasma cell leukemia, nonsecretory myeloma, IgD myeloma, osteosclerotic myeloma, osteoisoplastic plasmacytoma, and extramedullary plasmacytoma.

[0425] Cells preferred for use in the method considered herein include autologous / autogenic (“self”) cells, preferably hematopoietic cells, more preferably T cells, and more preferably immune effector cells.

[0426] In a specific embodiment, a method is provided comprising the step of administering a therapeutically effective amount of the modified immune effector cells considered herein or a composition containing such cells to a patient in need thereof, either alone or in combination with one or more therapeutic agents. In a specific embodiment, the cells are used to treat a patient at risk of developing cancer, GVHD, an infectious disease, an autoimmune disease, an inflammatory disease, or immunodeficiency. Accordingly, a specific embodiment comprises the treatment, prevention, or alleviation of at least one symptom of cancer, an infectious disease, an autoimmune disease, an inflammatory disease, or immunodeficiency, said treatment comprising the step of administering a therapeutically effective amount of the genome-edited cells considered herein to a subject in need thereof.

[0427] In one embodiment, a method for treating a subject requiring treatment for cancer, GVHD, infectious disease, autoimmune disease, inflammatory disease, or immunodeficiency comprises the step of administering an effective amount, e.g., a therapeutically effective amount, of a composition comprising modified immune effector cells considered herein. Although appropriate dosage and administration schedules may be determined by clinical trials, the dosage and frequency of administration are determined by factors such as the patient's condition and the type and severity of the patient's disease.

[0428] In one exemplary embodiment, the effective amount of modified immune effector cells provided to the subject is at least 2 x 10 6 Cells / kg (body weight), at least 3 x 10 6 Cells / kg, at least 4 x 10 6 Cells / kg, at least 5 x 10 6 Cells / kg, at least 6 x 106 Cells / kg, at least 7 x 10 6 Cells / kg, at least 8 x 10 6 Cells / kg, at least 9 x 10 6 Cells / kg, or at least 10 x 10⁻⁶ 6 Cells / kg or more, including all intervened cell doses.

[0429] In another exemplary embodiment, the effective amount of modified immune effector cells provided to the subject is approximately 2 x 10 6 Cells / kg (body weight), approx. 3 x 10 6 Cells / kg, approx. 4 x 10 6 Cells / kg, approx. 5 x 10 6 Cells / kg, approx. 6 x 10 6 Cells / kg, approx. 7 x 10 6 Cells / kg, approx. 8 x 10 6 Cells / kg, approx. 9 x 10 6 Cells / kg, or about 10 x 10⁶ 6 Cells / kg or more, including all intervened cell doses.

[0430] In another exemplary embodiment, the effective amount of modified immune effector cells provided to the subject is approximately 2 x 10 6 Cells / kg (body weight) to about 10 x 10⁶ 6 Cells / kg, approx. 3 x 10 6 cells / kg to about 10 x 10⁶ 6 Cells / kg, approx. 4 x 10 6 cells / kg to about 10 x 10⁶ 6 Cells / kg, approx. 5 x 10 6 cells / kg to about 10 x 10⁶ 6 Cells / kg, 2 x 10⁻⁶ 6 cells / kg to about 6 x 10⁶ 6 Cells / kg, 2 x 10⁻⁶ 6 cells / kg to about 7 x 10⁶ 6 Cells / kg, 2 x 10⁻⁶ 6cells / kg to about 8 x 10⁶ 6 Cells / kg, 3 x 10⁻⁶ 6 cells / kg to about 6 x 10⁶ 6 Cells / kg, 3 x 10⁻⁶ 6 cells / kg to about 7 x 10⁶ 6 Cells / kg, 3 x 10⁻⁶ 6 cells / kg to about 8 x 10⁶ 6 Cells / kg, 4 x 10 6 cells / kg to about 6 x 10⁶ 6 Cells / kg, 4 x 10 6 cells / kg to about 7 x 10⁶ 6 Cells / kg, 4 x 10 6 cells / kg to about 8 x 10⁶ 6 Cells / kg, 5 x 10 6 cells / kg to about 6 x 10⁶ 6 Cells / kg, 5 x 10 6 cells / kg to about 7 x 10⁶ 6 Cells / kg, 5 x 10 6 cells / kg to about 8 x 10⁶ 6 cells / kg, or 6 x 10⁶ 6 cells / kg to about 8 x 10⁶ 6 It is cells / kg and includes all cell doses included.

[0431] Those skilled in the art will recognize that multiple administrations of the composition considered in a particular embodiment may be required to affect the desired therapy. For example, the composition may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more 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 longer.

[0432] In a specific embodiment, it may be desirable to administer activated T cells to a subject, then collect blood again (or perform apheresis), activate T cells from it, and re-inject these activated and proliferated T cells and expanded T cells into the patient. This process may be performed several times every few weeks. In a specific embodiment, T cells may be activated from 10 cc to 400 cc of collected blood. In a specific embodiment, T cells are activated from 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 of collected blood. Without being bound by theory, using such multiple blood draws / multiple re-infusion protocols can serve to select specific T cell populations.

[0433] In one embodiment, a method for treating a subject diagnosed with cancer comprises the steps of: extracting immune effector cells from the subject; introducing one or more vectors encoding MAGEA4 TCR or MAGEA4 eTCR and a chimeric TGFβ receptor into the immune effector cells to produce a population of modified immune effector cells; and administering the population of modified immune effector cells to the same subject. In a preferred embodiment, the immune effector cells comprise T cells.

[0434] A method for administering a cell composition considered in a specific embodiment comprises any method effective for reintroducing immune effector cells modified in vitro or for reintroducing modified progenitor cells of immune effector cells that differentiate into mature immune effector cells when introduced into a subject. One method comprises the steps of modifying peripheral blood T cells in vitro by introducing one or more vectors encoding MAGEA4 TCR or MAGEA4 eTCR and a chimeric TGFβ receptor, and reinjecting the transduced cells into a subject.

[0435] All publications, patent applications, and issued patents cited in this specification are incorporated herein by reference as each individual publication, patent application, or issued patent is specifically and individually included by reference.

[0436] Although the foregoing embodiments have been described in some detail as examples and embodiments for the purpose of clarifying understanding, it will be readily apparent to those skilled in the art, in light of the teachings taken herein, that certain changes and variations may be made without departing from the spirit or scope of the appended claims. The following embodiments are provided merely as examples and are not intended to be limiting. Those skilled in the art will readily recognize various insignificant parameters that may be changed or modified to obtain essentially similar results.

[0437] Examples

[0438] Example 1

[0439] MAGEA4 TCR T cells expressing TGFβ signal transducers convert IL-12 signals in response to MAGEA4 and TGFβ1 and secrete increased IFNγ.

[0440] Peripheral blood mononuclear cells (PBMCs) from healthy donors were activated with soluble anti-CD3 (50 ng / ml) and anti-CD28 (50 ng / ml) and transduced with a lentiviral vector (LVV) expressing (i) MAGEA4 TCR (e.g., SEQ NO. 4) or (ii) MAGEA4 TCR and IL-12-responsive chimeric TGFβ signal transducer (CTBR12) encoded in a separate vector (e.g., SEQ NO. 4 and SEQ NO. 8) (MOI=20). After culturing in a medium containing IL2 for 10 days, the cell products were collected and cryopreserved for in vitro analysis.

[0441] CTBR12 signaling

[0442] IL-12 signaling involves the activation of STAT4 through receptor dimerization and phosphorylation. STAT4 phosphorylation in response to TGFβ was evaluated. Additionally, Smad2 / 3 phosphorylation was assessed to determine whether CTBR12 blocks intrinsic TGFβ signaling. MAGEA4 TCR T cells and MAGEA4 TCR / CTBR12 T cells were incubated overnight in serum-free medium and then exposed to TGFβ1 (10 ng / ml) for 20 minutes. Cells were fixed, permeated, and stained with anti-phospho-Smad2 / 3 (pS465 / 467) and phospho-STAT4 (pY693). CTBR12 blocked Smad2 / 3 phosphorylation and activated STAT4 in T cells expressing MAGEA4 TCR (Fig. 1, far right panel). These data indicate that when CTBR12 is co-expressed with MAGEA4 TCR, it can block intrinsic TGFβ signaling and convert IL-12 signaling.

[0443] MAGEA4 TCR signal transduction

[0444] Functional TCRs secrete IFNγ in response to antigens, and secretion can be enhanced by IL-12 signaling. In the presence or absence of TGFβ1 (10 ng / ml), untransduced (UTD) T cells, MAGEA4 TCR T cells, and MAGEA4 TCR / CTBR12 T cells were transfused in a 1:1 E:T ratio to A375 MAGEA4 + Tumor cells were cultured for 24 hours. After 24 hours, the amount of IFNγ secreted into the medium was determined. MAGEA4 TCR / CTBR12 T cells produced significantly higher 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.

[0445] Example 2

[0446] CTBR12 expression enhances MAGEA4 TCR T cell efficacy in vivo.

[0447] We evaluated whether CTBR12 expression enhances MAGEA4 TCR T cell efficacy in vivo using a xenograft NOD.Cg-Prkdcscid IL2rgtm1Wjl / SzJ (NSG) mouse model. A375 MAGEA4 in NSG mice + Tumor cells were subcutaneously transplanted. Tumor volume was measured twice weekly using a caliper and calculated using the formula: Tumor Volume = Length x Width x Height x 0.52. The tumor was 50 mm 3 When the average volume was reached, the mouse 0.625x10 6 Dog UTD T cells, 0.625x10 6 Canine GVY tetramer-positive MAGEA4 TCR T cells, or 0.625x10 6Canine GVY tetramer-positive MAGEA4 TCR / CTBR12 T cells were intravenously injected. MAGEA4 TCR / CTBR12 T cells regulate 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.

[0448] Example 3

[0449] Enhanced MAGEA4 TCR T cells expressing TGFβ signaling transducers convert IL-12 signals in response to MAGEA4 and TGFβ1 and secrete increased IFNγ.

[0450] Peripheral blood mononuclear cells (PBMCs) from healthy donors were activated with soluble anti-CD3 (50 ng / ml) and anti-CD28 (50 ng / ml) and transduced with (i) a MAGEA4 TCR pairing-enhancing TCR (eTCR) (e.g., SEQ ID NO. 7) or (ii) a lentiviral vector (LVV) expressing a MAGEA4 eTCR and CTBR12 receptor encoded in the same vector (e.g., SEQ ID NO. 7 and SEQ ID NO. 8) (MOI=20). After incubation in a medium containing IL2 for 10 days, cell products were collected and cryopreserved for in vitro analysis.

[0451] CTBR12 signaling

[0452] IL-12 signaling involves the activation of STAT4 through receptor dimerization and phosphorylation. STAT4 phosphorylation in response to TGFβ was evaluated. Additionally, Smad2 / 3 phosphorylation was assessed to determine whether CTBR12 blocks intrinsic TGFβ signaling. MAGEA4 eTCR T cells and MAGEA4 eTCR / CTBR12 T cells were incubated overnight in serum-free medium and then exposed to TGFβ1 (10 ng / ml) for 20 minutes. Cells were fixed, permeated, and stained with anti-phospho-Smad2 / 3 (pS465 / 467) and phospho-STAT4 (pY693). CTBR12 blocked Smad2 / 3 phosphorylation and activated STAT4 in T cells expressing MAGEA4 eTCR (Fig. 4, far right panel). These data indicate that CTBR12 can block intrinsic TGFβ signaling and convert IL-12 signaling when co-expressed with MAGEA4 eTCR.

[0453] MAGEA4 TCR signal transduction

[0454] Functional TCRs secrete IFNγ in response to antigens, and secretion can be enhanced by IL-12 signaling. In the presence or absence of TGFβ1 (10 ng / ml), untransduced (UTD) T cells, MAGEA4 eTCR T cells, and MAGEA4 eTCR / CTBR12 T cells were transfused in a 1:1 E:T ratio to A375 MAGEA4 + Tumor cells were cultured together for 24 hours. After 24 hours, the amount of IFNγ secreted into the medium was determined. TGFβ1 treatment inhibited 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 against TGFβ immunosuppression and promotes enhanced effector function in vitro.

[0455] In general, in the following claims, the terms used should not be interpreted as limiting the claims to the specific embodiments disclosed in this specification and claims, but should be interpreted as including all possible embodiments together with the full scope of equivalents to which such claims are granted. Accordingly, the claims are not limited by this disclosure.

Claims

Claim 1 One or more polynucleotides encoding a MAGEA4 TCR and a chimeric TGFβ receptor, wherein the MAGEA4 TCR comprises an alpha chain comprising the amino acid sequence presented in SEQ ID NO. 2 or SEQ ID NO. 5 and a beta chain comprising the amino acid sequence presented in SEQ ID NO. 3 or SEQ ID NO. 6, and the chimeric TGFβ receptor comprises: (a) a TGFβR2 polypeptide comprising the following: (i) Extracellular TGFβ1-binding domain of TGFβR2; (ii) IL-12Rβ2 penetrating domain; and (iii) an IL-12Rβ2 intracellular signaling domain; and (b) a TGFβR1 polypeptide comprising the following: (i) Extracellular TGFβ1-binding domain of TGFβR1; (ii) IL-12Rβ1 penetrating domain; and (iii) IL-12Rβ1 intracellular signaling domain. Claim 2 In claim 1, the chimeric TGFβ receptor comprises one or more polynucleotides further comprising a polypeptide cleavage signal between the TGFβR2 polypeptide and the TGFβR1 polypeptide. Claim 3 In paragraph 2, the polypeptide cleavage signal is one or more polynucleotides that are viral self-cleavage polypeptides. Claim 4 In paragraph 3, one or more polynucleotides in which the viral self-cleaving polypeptide is selected from the group consisting of foot-and-mouth disease virus (FDV) (F2A) peptide, equine rhinitis A virus (ERAV) (E2A) peptide, Thosea asigna virus (TaV) (T2A) peptide, porcine teschovirus-1 (PTV-1) (P2A) peptide, Theilovirus 2A peptide, and encephalomyocarditis virus 2A peptide. Claim 5 One or more polynucleotides, wherein in any one of claims 1 to 4, MAGEA4 TCR binds to the peptide GVYDGREHTV presented by a molecule encoding HLA-A*02:

01. Claim 6 One or more polynucleotides according to any one of claims 1 to 4, wherein the MAGEA4 TCR comprises an alpha chain having the amino acid sequence presented in SEQ ID NO. 2 and a beta chain having the amino acid sequence presented in SEQ ID NO.

3. Claim 7 One or more polynucleotides according to any one of claims 1 to 4, wherein the MAGEA4 TCR comprises an alpha chain having the amino acid sequence presented in SEQ ID NO. 5 and a beta chain having the amino acid sequence presented in SEQ ID NO.

6. Claim 8 One or more polynucleotides according to any one of claims 1 to 4, wherein the MAGEA4 TCR comprises the amino acid sequence presented in SEQ ID NO. 4 or SEQ ID NO.

7. Claim 9 In any one of claims 1 to 4, one or more polynucleotides wherein the chimeric TGFβ receptor comprises the amino acid sequence presented in SEQ ID NO.

8. Claim 10 One or more polynucleotides encoding a MAGEA4 TCR and a chimeric TGFβ receptor, wherein (a) the MAGEA4 TCR comprises an alpha chain comprising the amino acid sequence presented in SEQ ID NO. 2 and a beta chain comprising the amino acid sequence presented in SEQ ID NO. 3; and (b) the chimeric TGFβ receptor comprises the amino acid sequence presented in SEQ ID NO.

8. Claim 11 One or more polynucleotides encoding a MAGEA4 TCR and a chimeric TGFβ receptor, wherein (a) the MAGEA4 TCR comprises an alpha chain comprising the amino acid sequence presented in SEQ ID NO. 5 and a beta chain comprising the amino acid sequence presented in SEQ ID NO. 6; and (b) the chimeric TGFβ receptor comprises the amino acid sequence presented in SEQ ID NO.

8. Claim 12 One or more polynucleotides encoding a MAGEA4 TCR and a chimeric TGFβ receptor, wherein (a) the MAGEA4 TCR comprises the amino acid sequence presented in SEQ ID NO. 4 or SEQ ID NO. 7; and (b) the chimeric TGFβ receptor comprises the amino acid sequence presented in SEQ ID NO.

8. Claim 13 A vector comprising one or more polynucleotides of any one of claims 1 to 4. Claim 14 In paragraph 13, a vector that is a lentivirus vector. Claim 15 A vector comprising one or more polynucleotides of claim 5. Claim 16 In paragraph 15, a vector that is a lentivirus vector. Claim 17 A lentivirus vector comprising one or more polynucleotides of claim 10. Claim 18 A lentivirus vector comprising one or more polynucleotides of claim 11. Claim 19 A lentivirus vector comprising one or more polynucleotides of claim 12.

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