Viral vectors and use thereof in adoptive cellular therapy

Abstract

To provide gene delivery systems for safe and efficient transgene expression in adoptive cellular therapy.SOLUTION: Provided is a vector containing a first nucleotide sequence S1 encoding a protein Z1, a second nucleotide sequence S2 encoding a protein Z2, a third nucleotide sequence S3 encoding a protein Y1, and a fourth nucleotide sequence S4 encoding a protein Y2, in which Z1 and Z2 form a first dimer and Y1 and Y2 form a second dimer, in which the first dimer Z1Z2 is different from the second dimer Y1Y2.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This is an international application under the Patent Cooperation Treaty, claiming priority to U.S. Provisional Patent Application No. 62 / 853,123, filed May 27, 2019, the entire contents of which are incorporated herein by reference.

[0002] Reference to the submitted sequence listing An official copy of the Sequence Listing has been submitted electronically via EFS-Web and is being filed contemporaneously herewith as an ASCII Sequence Listing with the filename "3000011-013977_SEQLIST_ST25.txt," created on May 26, 2020, and having a size of 313,426 bytes. The Sequence Listing contained in this ASCII document is a part of the specification and is incorporated herein by reference in its entirety. [Background technology]

[0003] 1. Field The present disclosure relates to the production of T cells.In one aspect, the present disclosure relates to the production of T cells using multicistronic cassettes to express multiple proteins in a single vector.More specifically, the present disclosure relates to the production of T cells that co-express TCRαβ and CD8αβ, and their use in adoptive cell therapy.

[0004] 2.Background Genetic engineering of human lymphocytes as a potential treatment for inherited, acquired, or infectious diseases requires efficient delivery and expression of transgenes. For adoptive immunotherapy of cancer, natural and / or recombinant antitumor T cell receptors (TCRs) are used to confer antitumor reactivity to normal T cells or tumor-infiltrating lymphocytes.

[0005] (2003) discloses an anti-gp100 TCR expressed by a bicistronic RNA in which the expression of a first gene encoding the TCR β chain is controlled by a long terminal repeat (LTR) and a second gene encoding the TCR α chain is controlled by an internal ribosome entry site (IRES). CD4 T cells engineered with this anti-gp100 TCR gene were antigen-reactive.

[0006] Non-Patent Document 2 discloses a bicistronic retroviral vector for the simultaneous expression of both TCR α and TCR β chains that bind to p53 epitopes. The expression of the first gene encoding the TCR α chain is controlled by an LTR, and the second gene encoding the TCR β chain is controlled by an IRES. Lymphocytes transduced with p53 TCR could specifically recognize peptide-pulsed APCs as well as HLA-A2.1+ cells transfected with wild-type or mutant p53 proteins with high avidity.

[0007] Non-Patent Document 3 discloses various bicistronic retroviral vectors for the simultaneous expression of anti-MART-1 TCR. The expression of a first gene encoding the TCR α chain is controlled by an LTR and a second gene encoding the TCR β chain is controlled by an IRES, or vice versa. Furthermore, the expression of a first gene encoding the TCR α chain is controlled by an LTR and a second gene encoding the TCR β chain is controlled by a PGK promoter, or vice versa. T cells transduced with these vectors showed highly active T cell effector functions.

[0008] Non-Patent Document 4 discloses a bicistronic retroviral vector for simultaneous expression of NY-ESO-1 TCR. The expression of a first gene encoding a TCR α chain is controlled by an LTR and a second gene encoding a TCR β chain is controlled by an IRES, or the expression of a first gene encoding a TCR α chain is controlled by an LTR and a second gene encoding a TCR β chain is controlled by a PGK promoter. The transduced lymphocytes can efficiently recognize and kill HLA-A2 and NY-ESO-1 positive melanoma cell lines.

[0009] Non-Patent Document 5 discloses anti-gp100 TCR or anti-vector bicistronic lentiviral vectors by introducing a 2A ribosomal skip peptide after combining a furin cleavage site with an amino acid spacer (GSG or SGSG (SEQ ID NO: 8)). When the spacer sequence was augmented by the addition of a synthetic V5 peptide tag sequence, protein processing was enhanced, resulting in lentiviral vectors that could mediate high levels of TCR expression in transduced lymphocytes. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Morgan et al. J Immunol.2003 September 15;171(6):3287-32percent [Non-Patent Document 2] Cohen et al. J Immunol.2005 November 1;175(9):5799-5808 [Non-Patent Document 3] Hughes et al. Hum Gene Ther.2005 April;16(4):457-472 [Non-Patent Document 4] Zhao et al. J Immunol.2005 April 1;174(7):4415-4423 [Non-Patent Document 5] Morgan et al. Gene Therapy 2008 15,1411-1423 Summary of the Invention [Problem to be solved by the invention]

[0011] There remains a need for gene delivery systems for safe and efficient transgene expression in adoptive cell therapy. [Means for solving the problem]

[0012] In one aspect, the disclosure provides a gene delivery system comprising a vector comprising a first nucleotide sequence S1 encoding a protein Z1, a second nucleotide sequence S2 encoding a protein Z2, a third nucleotide sequence S3 encoding a protein Y1, and a fourth nucleotide sequence S4 encoding a protein Y2, wherein Z1 and Z2 form a first dimer and Y1 and Y2 form a second dimer, wherein the first dimer Z1Z2 is different from the second dimer Y1Y2, and wherein the gene delivery system is used for adaptive cell therapy.

[0013] In another aspect, S1-S2-S3-S4, S1-S2-S4-S3, S1-S3-S2-S4, S1-S3-S4-S2, S1-S4-S3-S2, S1-S4-S2-S3, S2-S1-S 3-S4, S2-S1-S4-S3, S2-S3-S1-S4, S2-S3-S4-S1, S2-S4-S3-S1, S2-S4-S1-S3, S3-S1-S2-S4, S3-S1-S4-S 2, S3-S2-S1-S4, S3-S2-S4-S1, S3-S4-S1-S2, S3-S4-S2-S1, S4-S1-S2-S3, S4-S1-S3-S2, S4-S2-S1-S3, S4-S2-S3-S1, S4-S3-S1-S2, or S4-S3-S2-S1.

[0014] In another embodiment, the vector may further comprise a fifth nucleotide sequence S5 encoding a 2A peptide and a sixth nucleotide sequence S6 encoding a linker peptide, wherein S5 and S6 are disposed between S1 and S2, S1 and S3, S1 and S4, S2 and S3, S2 and S4, and / or S3 and S4.

[0015] In another embodiment, the 2A peptide may be selected from P2A (SEQ ID NO:3), T2A (SEQ ID NO:4), E2A (SEQ ID NO:5), or F2A (SEQ ID NO:6).

[0016] In another embodiment, the linker peptide may be GSG or SGSG (SEQ ID NO:8).

[0017] In another embodiment, the vector may contain a furin peptide (SEQ ID NO:2) encoding a seventh nucleotide sequence, S7, where S7 is positioned between S1 and S2, S1 and S3, S1 and S4, S2 and S3, S2 and S4, and / or S3 and S4.

[0018] In another embodiment, the vector may further comprise a post-transcriptional regulatory element (PRE) sequence selected from a Woodchuck PRE (WPRE) or a Hepatitis B virus (HBV) PRE (HPRE).

[0019] In another embodiment, the vector may further comprise a promoter sequence controlling transcription of S1, S2, S3, S4, S5, S6 and / or S7, the promoter sequence being selected from a cytomegalovirus (CMV) promoter, a phosphoglycerate kinase (PGK) promoter, a myelin basic protein (MBP) promoter, a glial fibrillary acidic protein (GFAP) promoter, a modified MoMuLV LTR containing the myeloproliferative sarcoma virus enhancer (MNDU3), a ubiquitin C promoter, an EF-1α promoter, or a murine stem cell virus (MSCV) promoter.

[0020] In another embodiment, the first dimer Z1Z2 is selected from the group consisting of SEQ ID NOs: 13 and 14, 15 and 16, 17 and 18, 19 and 20, 21 and 22, 23 and 24, 25 and 26, 27 and 28, 29 and 30, 31 and 32, 33 and 34, 35 and 36, 37 and 38, 39 and 40, 41 and 42, 43 and 44, 45 and 46, 47 and 48, 49 and 50, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 62 and 63, 63 and 64, 64 and 65, 65 and 66, 66 and 67, 67 and 68, 68 and 69, 70 and 71, 72 and 73, 74 and 75, 76 and 77, 78 and 79, 80 and 81, 82 and 83, 84 and 85, 86 and 87, 88 and 89, 90 and 91, 92 and 93, 93 and 94, 94 and 95, 95 and 96, 96 and 97, 98 and 99, 100 and 101, 102 and 103, 103 and 104, 104 and 105, 105 and 106, 106 and 107, 108 and 109, 109 and 110, 109 and 111, 109 In one embodiment, the n-th and n-th integers may be selected from 0, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, 71 and 72, 73 and 74, 75 and 76, 77 and 78, 79 and 80, 81 and 82, 83 and 84, 85 and 86, 87 and 88, or 89 and 90.

[0021] In another embodiment, the second dimer Y1 and Y2 are set forth in SEQ ID NOs:11 and 12.

[0022] In another embodiment, the orientation is S2-S1-S4-S3.

[0023] In another embodiment, the vector has a sequence selected from PTE WPRE (SEQ ID NO:91), TPE WPRE (SEQ ID NO:92), or PTE fn WPRE (SEQ ID NO:93).

[0024] In another embodiment, the orientation is S4-S3-S2-S1.

[0025] In another embodiment, the vector has the sequence PTE CD8 TCR WPRE (SEQ ID NO:94).

[0026] In another embodiment, the viral vector is selected from an adenovirus, a poxvirus, an alphavirus, an arenavirus, a flavivirus, a rhabdovirus, a retrovirus, a lentivirus, a herpesvirus, a paramyxovirus, or a picornavirus.

[0027] In another embodiment, the vector is pseudotyped with an envelope protein of a virus selected from natural feline endogenous virus (RD114), a chimeric version of RD114 (RD114TR), gibbon ape leukemia virus (GALV), a chimeric version of GALV (GALV-TR), amphotropic murine leukemia virus (MLV4070A), baculovirus (GP64), vesicular stomatitis virus (VSV-G), fowl plague virus (FPV), Ebola virus (EboV), baboon retrovirus envelope glycoprotein (BaEV), or lymphocytic choriomeningitis virus (LCMV).

[0028] In another embodiment, the vector is pseudotyped with the envelope protein of vesicular stomatitis virus (VSV-G).

[0029] In one aspect, the disclosure relates to a method of preparing T cells for immunotherapy comprising isolating T cells from a blood sample of a human subject, activating the isolated T cells in the presence of an aminobisphosphonate, transducing the activated T cells with a vector described herein, and expanding the transduced T cells.

[0030] In another embodiment, T cells may be isolated from a leukapheresis human sample.

[0031] In another embodiment, the aminobisphosphonate may be selected from pamidronic acid, alendronic acid, zoledronic acid, risedronic acid, ibandronic acid, incadronic acid, salts thereof and / or hydrates thereof.

[0032] In another embodiment, the activating step may further be carried out in the presence of human recombinant interleukin 2 (IL-2) and human recombinant interleukin 15 (IL-15).

[0033] In another embodiment, the expanding step may be performed in the presence of IL-2 and IL-15.

[0034] In another embodiment, the T cells may be γδ T cells.

[0035] In another embodiment, the first dimer, Z1Z2, and the second dimer, Y1Y2, are co-expressed on the surface of the expanded T cells.

[0036] In another aspect, the disclosure relates to an expanded T cell population prepared by the method of the above aspect.

[0037] In one aspect, the disclosure relates to a method of treating a patient having cancer comprising administering to the patient a composition comprising a population of expanded T cells as described herein, wherein the T cells kill cancer cells that present a peptide on their surface in complex with an MHC molecule, wherein the peptide is selected from any of SEQ ID NOs: 98-255, wherein the cancer is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, melanoma, liver cancer, breast cancer, uterine cancer, Merkel cell carcinoma, pancreatic cancer, gallbladder cancer, bile duct cancer, colorectal cancer, urinary bladder cancer, kidney cancer, leukemia, ovarian cancer, esophageal cancer, brain cancer, gastric cancer, and prostate cancer.

[0038] In another embodiment, the composition further comprises an adjuvant.

[0039] In another embodiment, the adjuvant is selected from one or more of anti-CD40 antibodies, imiquimod, resiquimod, GM-CSF, cyclophosphamide, sunitinib, bevacizumab, atezolizumab, interferon-α, interferon-β, CpG oligonucleotides and derivatives, poly-(I:C) and derivatives, RNA, sildenafil, particulate formulations with poly(lactide-co-glycolide) (PLG), virosomes, interleukin (IL)-1, IL-2, IL-4, IL-7, IL-12, IL-13, IL-15, IL-21, and IL-23.

[0040] In one aspect, the disclosure relates to a method of generating an immune response in a patient having cancer comprising administering to the patient a composition comprising a population of expanded T cells as described herein, wherein the T cells kill cancer cells that present a peptide on their surface in complex with an MHC molecule, wherein the peptide is selected from any of SEQ ID NOs: 98-255, wherein the cancer is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, melanoma, liver cancer, breast cancer, uterine cancer, Merkel cell carcinoma, pancreatic cancer, gallbladder cancer, bile duct cancer, colorectal cancer, urinary bladder cancer, kidney cancer, leukemia, ovarian cancer, esophageal cancer, brain cancer, gastric cancer, and prostate cancer.

[0041] In another embodiment, the immune response comprises a cytotoxic T cell response.

[0042] In one aspect, the disclosure provides a method of preparing T cells by using a statin in the manner described herein. In another aspect, the disclosure provides a method of preparing T cells by activating the T cells in the presence of a statin.

[0043] In yet another aspect, the present disclosure relates to a method of preparing T cells for immunotherapy comprising activating T cells in the presence of a statin, transducing the activated T cells with a vector of the present disclosure, optionally pseudotyped with vesicular stomatitis virus (VSV-G) envelope protein, and expanding the transduced T cells.

[0044] In another embodiment, the T cells may include αβ T cells, γδ T cells, and / or natural killer T cells.

[0045] In another embodiment, the statins may be selected from atorvastatin, cerivastatin, dalvastatin, fluindostatin, fluvastatin, mevastatin, pravastatin, simvastatin, velostatin, and rosuvastatin.

[0046] In one aspect, the present disclosure relates to a method of preparing T cells for immunotherapy comprising activating T cells, transducing the activated T cells with a vector of the present disclosure, and expanding the transduced T cells.

[0047] In another embodiment, the activating step may further be performed in the presence of an anti-CD3 antibody and an anti-CD28 antibody.

[0048] In another embodiment, the expanding step may be carried out in the presence of IL-7 and IL-15. [Brief description of the drawings]

[0049] [Figure 1] 1 shows a process for producing γδ T cells according to one embodiment of the present disclosure. γδ T cell production may include harvesting or obtaining leukocytes or PBMCs, e.g., leukapheresis product, depleting αβ T cells from the PBMCs or leukapheresis product, followed by activation, transduction, and expansion of γδ T cells. [Diagram 2] FIG. 1 shows a transduction strategy involving open reading frame (ORF) shuffling according to some embodiments of the present disclosure. [Diagram 3] 1 shows a lentiviral construct according to some embodiments of the present disclosure. [Figure 4] Figure 1 shows lentivirus pseudotyped with RD114TR used to transduce γδ T cells on days 3 or 6 after activation with zoledronate, IL-2, and IL-15. Transduction efficiency was assessed via flow cytometry using antibodies specific for TCR (Vβ8) and CD8 (CD8α). [Diagram 5] A shows a construct according to an embodiment of the present disclosure. B shows another construct according to an embodiment of the present disclosure. C shows another construct according to an embodiment of the present disclosure. D shows another construct according to an embodiment of the present disclosure. [Figure 6]1A shows another construct according to an embodiment of the present disclosure; FIG. 1B shows another construct according to an embodiment of the present disclosure; [Figure 7] 1 shows a schematic diagram of a construct according to some embodiments of the present disclosure. [Figure 8] A shows a construct according to an embodiment of the present disclosure. B shows another construct according to an embodiment of the present disclosure. C shows another construct according to an embodiment of the present disclosure. D shows another construct according to an embodiment of the present disclosure. [Figure 9] 1A and 1B show schematic diagrams of constructs according to some embodiments of the present disclosure. [Figure 10] Shown is the % CD8+TCR+ γδ T cells transduced with viral vectors containing PTE.CD8.TCR.WPRE, PTE.WPRE, PTE.Fn.WPRE, or TPE.WPRE. Non-transduced (NT) cells serve as a control. [Figure 11] Shown is the median fluorescence intensity (MFI) of CD8 and TCR in γδ T cells transduced with viral vectors containing PTE.CD8.TCR.WPRE, PTE.WPRE, PTE.Fn.WPRE, or TPE.WPRE. Non-transduced (NT) cells serve as a control. [Figure 12] 1 shows the tumor killing activity of γδ T cells from donor 3 transduced with viral vectors containing PTE.CD8.TCR.WPRE, PTE.WPRE, PTE.Fn.WPRE, or TPE.WPRE in high antigen expressing tumor cell lines, e.g., UACC257 (upper panel), or low antigen expressing tumor cell lines, e.g., U2OS (lower panel), as determined by Incucyte cytotoxicity assay. Targets only and non-transduced cells serve as controls. [Figure 13]AC shows the amount of interferon (IFN)-γ secretion by γδ T cells from donor 3 transduced with viral vectors containing PTE.CD8.TCR.WPRE, PTE.WPRE, PTE.Fn.WPRE, or TPE.WPRE in high antigen expressing tumor cell lines, such as, for example, UACC257 (FIG. 13A), or low antigen expressing tumor cell lines, such as, for example, U2OS (FIG. 13B), or antigen negative tumor cell lines, such as, for example, MCF-7 (FIG. 13C). Non-transduced cells serve as a control. [Figure 14] Figure 1 shows the tumor killing activity of γδ T cells from donor 4 transduced with viral vectors containing PTE.CD8.TCR.WPRE, PTE.WPRE, PTE.Fn.WPRE, or TPE.WPRE in high antigen expressing tumor cell lines, e.g., UACC257 (upper panel), or low antigen expressing tumor cell lines, e.g., U2OS (lower panel), as determined by Incucyte cytotoxicity assay. Targets only and non-transduced cells serve as controls. [Figure 15] AC shows the amount of IFN-γ secretion by γδ T cells obtained from donor 4 transduced with viral vectors containing PTE.CD8.TCR.WPRE, PTE.WPRE, PTE.Fn.WPRE, or TPE.WPRE in high antigen expressing tumor cell lines, such as, for example, UACC257 (FIG. 15A), or low antigen expressing tumor cell lines, such as, for example, U2OS (FIG. 15B), or antigen negative tumor cell lines, such as, for example, MCF-7 (FIG. 15C). Non-transduced cells serve as a control. [Figure 16] 1 shows viral vector copy numbers in γδ T cells transduced with viral vectors containing PTE.CD8.TCR.WPRE, PTE.WPRE, PTE.Fn.WPRE, or TPE.WPRE. Non-transduced cells serve as a control. [Figure 17]Shown are fold expansions of γδ T cells obtained from donor 3 (FIG. 17A) or donor 4 (FIG. 17B) transduced with viral vectors containing AB PTE.CD8.TCR.WPRE, PTE.WPRE, PTE.Fn.WPRE, or TPE.WPRE. Non-transduced (NT) cells serve as controls. [Figure 18] A shows the memory phenotype of γδ T cells as determined by flow cytometry according to some embodiments of the present disclosure. B shows the memory phenotype of γδ T cells transduced with viral vectors containing PTE.CD8.TCR.WPRE, PTE.WPRE, PTE.Fn.WPRE, or TPE.WPRE. Non-transduced (NT) cells serve as a control. [Figure 19] Comparison of transduction efficiency between γδ T cells transduced with a single lentiviral vector (LV) containing PTE.CD8.TCR.WPRE (Panel B (120 μl) and Panel C (240 μl)) or γδ T cells transduced with two separate lentiviral vectors, one containing R11KE.WPRE and the other containing CD8,WPRE (Panel D and E), using increasing amounts of viral vector, e.g., 120 μl each of R11KE.WPRE and CD8,WPRE (Panel D) and 240 μl each of R11KE.WPRE and CD8,WPRE (Panel E). Non-transduced (NT) cells serve as controls. [Figure 20] 1 shows enhanced transduction efficiency in γδ T cells transduced with increasing amounts of viral vector containing PTE.CD8.TCR.WPRE, e.g., 30 μl, 120 μl, and 240 μl. Non-transduced cells serve as a control. [Figure 21] For example, various dilutions of a lentiviral vector (LV) expressing a 4-in-1 construct of the present disclosure, such as LV-PTE.CD8.TCR.WPRE, are used to show enforced CD8 expression in CD4+ T cells obtained from donor 5 and donor 6. [Figure 22]1 shows detection of TCR expression in CD4+ T cells using various dilutions of LV expressing a 4-in-1 construct of the present disclosure, such as, for example, LV-PTE.CD8.TCR.WPRE. [Diagram 23] Shown is % target peptide / MHC complex dextramer 203 (Dex203) in CD4+ and / or CD8+ T cells from donor 5 (upper panel) and donor 6 (lower panel) transduced with a 4-in-1 construct of the present disclosure, such as, for example, LV-PTE.CD8.TCR.WPRE. [Figure 24] For example, Dex203 MFI in CD8+ and / or CD8+ T cells from donor 5 (upper panel) and donor 6 (lower panel) transduced with a 4-in-1 construct of the present disclosure, such as LV-PTE.CD8.TCR.WPRE, is shown. [Diagram 25] FIG. 1 shows the experimental design for testing the functionality of T cells transduced with a 4-in-1 construct or a TCR-only construct according to one embodiment of the present disclosure. [Figure 26] Increase in IFN-γ positive cells (upper panel) and IFN-γ MFI (lower panel) in CD4-CD8α T cells from grouped donors transduced with lentiviral vectors containing R11KE.WPRE(LV-TCR) (TCR) or PTE.CD8.TCR.WPRE(LV-CD8.TCR) (TCR+CD8) followed by co-culture with high target expressing UACC257 cells compared to co-culture with non-target expressing MCF7. Non-transduced (NT) cells serve as control (effector to target cell ratio=2:1; N=4 grouped donors). [Figure 27]Increased % granzyme B positive cells (upper panel) and increased granzyme B MFI (lower panel) in CD4-CD8α+ T cells from grouped donors transduced with LV-TCR (TCR) or LV-CD8.TCR (TCR+CD8) followed by co-culture with high target expressing UACC257 cells are shown compared to co-culture with non-target expressing MCF7. Non-transduced (NT) cells serve as control (effector to target cell ratio=2:1; N=3 donors grouped). [Figure 28] Increased % IFN-γ positive cells (upper panel) and increased IFN-γ MFI (lower panel) in CD4+CD8α+ T cells obtained from grouped donors transduced with LV-CD8.TCR (TCR+CD8) or non-transduced (NT) followed by coculture with high targeting expressing UACC257 cells compared to coculture with non-targeting expressing MCF7 (effector to target cell ratio=2:1; N=4 donors grouped). [Figure 29] Increased % Granzyme B positive cells (upper panel) and increased Granzyme B MFI (lower panel) in CD4+CD8α+ T cells obtained from grouped donors transduced with LV-CD8.TCR (TCR+CD8) or non-transduced (NT) followed by coculture with high targeting expressing UACC257 cells compared to coculture with non-targeting expressing MCF7 (effector to target cell ratio=2:1; N=4 donors grouped). [Diagram 30] Increased % IFN-γ positive cells (upper panel) and increased IFN-γ MFI (lower panel) in CD3+ T cells from grouped donors transduced with LV-TCR (TCR) or LV-CD8.TCR (TCR+CD8) followed by co-culture with high target expressing UACC257 cells are shown compared to co-culture with non-target expressing MCF7. Non-transduced (NT) cells serve as control (effector to target cell ratio=2:1; N=4 donors grouped). [Diagram 31]Increased % granzyme B positive cells (upper panel) and increased granzyme B MFI (lower panel) in CD3+ T cells from grouped donors transduced with LV-TCR (TCR) or LV-CD8.TCR (TCR+CD8) followed by co-culture with high target expressing UACC257 cells are shown compared to co-culture with non-target expressing MCF7. Non-transduced (NT) cells serve as control (effector to target cell ratio=2:1; N=3 grouped donors). [Diagram 32] Figure 1 shows increased IFN-γ secretion in CD3+ T cells from grouped donors transduced with LV-TCR (TCR) or LV-CD8.TCR (TCR+CD8) followed by coculture with high target expressing UACC257 cells compared to coculture with non-target expressing MCF7. Non-transduced (NT) cells, UACC257 cells, and MCF7 cells serve as controls (effector to target cell ratio = 2:1; grouped donors N = 4). [Diagram 33] Increased IFN-γ secretion in CD3+ T cells from individual donors 5, 6, 7, and 8 transduced with LV-TCR (TCR) or LV-CD8.TCR (TCR+CD8) followed by coculture with high target-expressing UACC257 cells compared to coculture with non-target-expressing MCF7. Non-transduced (NT) cells, UACC257 cells alone, and MCF7 cells alone serve as controls (effector to target cell ratio = 2:1). [Diagram 34] Shown are CD25+ cells (upper panel), %CD69+ cells (middle panel), and %human low-density lipoprotein receptor (hLDLR)+ cells (lower panel) in CD3+CD4+ T cells treated with atorvastatin, pravastatin, or rosuvastatin. Preactivated cells, cells activated without statins or DMSO (control), and DMSO served as controls. [Diagram 35]Shown are %CD25+ cells (upper panel), %CD69+ cells (middle panel), and %hLDLR+ cells (lower panel) in CD3+CD8+ T cells treated with atorvastatin, pravastatin, or rosuvastatin. Preactivated cells, cells activated without statins or DMSO (control), and DMSO serve as controls. [Diagram 36] 1 shows the titer of a lentiviral vector according to one embodiment of the present disclosure. [Figure 37] 1 shows a process for producing T cells according to one embodiment of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0050] As used herein, the term "self-cleaving 2A peptide" refers to a relatively short peptide (approximately 20 amino acids long, depending on the virus of origin) that acts co-translationally by preventing the formation of a normal peptide bond between glycine and the last proline, resulting in ribosomal skipping to the next codon and nascent peptide cleavage between Gly and Pro. After cleavage, the short 2A peptide remains fused to the C-terminus of the "upstream" protein, while a proline is added to the N-terminus of the "downstream" protein. The self-cleaving 2A peptide may be selected from porcine teschovirus-1 (P2A), equine rhinitis A virus (E2A), Thosea asigna virus (T2A), foot and mouth disease virus (F2A), or any combination thereof (see, e.g., Kim et al., PLOS One 6:e18556, 2011; the contents of which, including the 2A nucleic acid and amino acid sequences, are incorporated herein by reference in their entirety). By adding a linker sequence (GSG or SGSG (SEQ ID NO: 8)) before the self-cleaving 2A sequence, this may allow for efficient synthesis of biologically active proteins such as, for example, TCR.

[0051] As used herein, the term "promoter" refers to a regulatory region of DNA located generally upstream (towards the 5' region of the sense strand) of a gene that allows transcription of the gene. Promoters contain specific DNA sequences and response elements that are recognized by proteins known as transcription factors. These factors bind to the promoter sequence and recruit RNA polymerase, an enzyme that synthesizes RNA from the coding region of the gene. For example, promoter sequences used herein may be selected from the cytomegalovirus (CMV) promoter, the phosphoglycerate kinase (PGK) promoter, the myelin basic protein (MBP) promoter, the glial fibrillary acidic protein (GFAP) promoter, the modified MoMuLV LTR containing the myeloproliferative sarcoma virus enhancer (MNDU3), the ubiquitin C promoter, the EF-1α promoter, or the murine stem cell virus (MSCV) promoter.

[0052] As used herein, the term "constitutive promoter" may include a regulatory sequence that directs transcription of a gene in most cells or tissues at most times. In some non-limiting embodiments, the constitutive promoter may be selected from the group consisting of MSCV promoter, Ubiquitin C (Ubc) promoter, CMV promoter, EF-1α promoter, PGK promoter, β-actin promoter, and ROSA26 promoter.

[0053] In some embodiments, the promoter may be an inducible promoter. The activity of an inducible promoter may increase or decrease in response to a signal. For example, an inducible promoter may promote transcription in response to T cell activation or the presence of a signal, such as isopropyl β-D-1-thiogalactopyranoside (IPTG). An inducible promoter may promote transcription in response to the absence of a signal, such as phosphate. In any of these scenarios, the amount of transcription may or may not be proportional to the amount of the signal or the lack thereof. Examples of inducible promoters suitable for prokaryotic host cells may include, but are not limited to, NFAT, CD69, lac, tac, trc, trp, pho, recA, tetA, nar, phage PL, cspA, T7, and PBAD promoters (see Terpe K. 2006 Appl. Microbiol. Biotechnol. 72:211, the entire contents of which are incorporated by reference).

[0054] In some embodiments, the inducible promoter may comprise a nuclear factor of activated T cells (NFAT) / AP1 transcription response element (TRE). Upon recognition of the cognate peptide / MHC1 complex, NFAT may undergo Ca2+-dependent translocation to the nucleus, where it promotes the transcription of genes carrying the NFAT TRE. Suitable NFAT TREs are well known in the art, including the human IL2 promoter NFAT TRE (Macian et al (2001) Oncogene, 2001 Apr.30;20(19):2476-89). Zhang et al. ("Tumor-Infiltrating Lymphocytes Genetically Engineered with an Inducible Gene Encoding Interleukin-12 for the Immunotherapy of Metastatic Melanoma," Clin. Cancer Res. 21:2278-2288, 2015) describe the use of human tumor-infiltrating lymphocytes (TILs) genetically engineered to secrete single-chain IL12, whose expression is driven by an inducible NFAT promoter, in a clinical trial. The contents of these cited references are incorporated by reference in their entirety.

[0055] In some embodiments, the inducible promoter may include the CD69 promoter, for example, as disclosed in U.S. Patent No. 5,759,805, the entire contents of which are incorporated by reference. CD69 may be one of the earliest of these newly synthesized cell surface activation molecules induced in activated T cells. CD69 expression may be observed within 60 minutes of T cell stimulation, but may be absent in resting cells. CD69 expression may also be induced on thymocytes, B cells, natural killer (NK) cells, and neutrophils. Four non-coding regions, designated CNS1-4, located within 50 kb upstream of the mouse CD69 promoter may contribute to the developmental and temporal control of CD69 activation in T and B cells. The CNS2 region may function as a strong enhancer. Kulemzin et al. ("Design and analysis of stably integrated reporters for inducible transgene expression in human T cells and chimeric antigen receptor (CAR) NK cell lines," BMC Medical Genomics 2019,12(Suppl 2):44,88-95), the entire contents of which are incorporated by reference, describe that in the context of primary T cells, the activation-inducible CD69 promoter variant provides the highest fold induction. Thus, this promoter can be used to express proteins in activated but not resting human T cells or CART cells.

[0056] In some embodiments, the inducible promoter may be an IPTG-inducible promoter. An IPTG-inducible promoter may refer to any polynucleotide sequence that promotes transcription in a manner that responds to IPTG or any other lactose derivative (e.g., allolactose) that can promote transcription from the lac operon. Many examples of IPTG-inducible promoters are known in the art, including, but not limited to, tac (e.g., tacI, tacII, etc.) promoter, lac promoter, and their derivatives (e.g., lacUV5, taclac, etc.).

[0057] In one embodiment, the expression of a 4-in-1 viral vector, such as a lentiviral vector, containing sequences encoding CD8α, CD8β, TCRα, and TCRβ chains may be driven by a constitutive or inducible promoter. For example, Figure 5A shows a 4-in-1 viral vector containing PTE CD8 TCR WPRE (SEQ ID NO: 94) driven by a constitutive MSCV promoter (SEQ ID NO: 1), with codon-optimized sequences encoding CD8α (SEQ ID NO: 12) and CD8β (SEQ ID NO: 13) located upstream from sequences encoding TCRs, such as TCR R11KEα (SEQ ID NO: 13) and R11KEβ (SEQ ID NO: 14). The same coding sequences as above can also be driven by an inducible promoter, such as NFAT, CD69, or IPTG promoter.

[0058] In another embodiment, the expression of the 3-in-1 viral vector containing sequences encoding the fusion protein, TCR alpha chain, and TCR beta chain may be driven by a constitutive or inducible promoter. For example, Figure 5B shows a viral vector containing CD8aCD4Fusion.TCR WPRE (SEQ ID NO: 256) with a codon-optimized sequence encoding a fusion protein in which the CD8alpha extracellular domain is fused with the CD4 transmembrane domain and the CD4 intracellular domain, and sequences encoding the TCR R11KEalpha chain (SEQ ID NO: 13) and R11KEbeta chain (SEQ ID NO: 14) are driven by the MSCV promoter (SEQ ID NO: 1). FIG. 5C shows a viral vector containing CD8bCD4Fusion.TCR WPRE (SEQ ID NO: 257) with a codon-optimized sequence encoding a fusion protein in which the CD8β extracellular domain is fused to the CD4 transmembrane domain and the CD4 intracellular domain, and the sequences encoding the TCR R11KEα chain (SEQ ID NO: 13) and R11KEβ chain (SEQ ID NO: 14) are driven by the MSCV promoter (SEQ ID NO: 1). FIG. 5D shows a viral vector containing CD8bCD8aFusion.TCR WPRE (SEQ ID NO: 258) with a sequence encoding a fusion protein in which the CD8β extracellular domain is fused to the CD8α transmembrane domain and the CD8α intracellular domain, and the sequences encoding the TCR R11KEα chain (SEQ ID NO: 13) and R11KEβ chain (SEQ ID NO: 14) are driven by the MSCV promoter (SEQ ID NO: 1). The same coding sequences as above can also be driven by inducible promoters such as, for example, NFAT, CD69, or IPTG promoters.

[0059] In one embodiment, the expression of the 4-in-1 viral vector of the present disclosure may be driven by a bidirectional constitutive and / or inducible promoter. For example, FIG. 6A shows a 4-in-1 viral vector containing PGK.CD8.EF1a.TCR (SEQ ID NO: 259) with a codon-optimized sequence encoding CD8α and CD8β chains located upstream from a sequence encoding TCR R11KEα and R11KEβ chains, in which the sequence encoding CD8α and CD8β chains and the sequence encoding TCR R11KEα and R11KEβ chains may be separated by a bidirectional promoter, such as, for example, the PGK promoter and the EF-1α promoter. The PGK promoter may be placed at the 3' end of the codon-optimized sequence encoding CD8α and CD8β chains to drive the expression of CD8α and CD8β chains. The EF-1α promoter may be positioned 5′ to the sequence encoding the TCR R11KEα and R11KEβ chains to drive expression of the TCR11KEα and R11KEβ chains.

[0060] 6B shows another 4-in-1 viral vector containing PGK.TCR.EF1a.CD8 (SEQ ID NO: 260) with sequences encoding the TCR R11KEα and R11KEβ chains located upstream from the codon-optimized sequences encoding the CD8α and CD8β chains, in which the sequences encoding the TCR R11KEα and R11KEβ chains and the sequences encoding the CD8α and CD8β chains may be separated by a bidirectional promoter, such as, for example, the PGK promoter and the EF-1α promoter. The PGK promoter may be placed at the 3' end of the sequences encoding the TCR R11KEα and R11KEβ chains to drive expression of the TCR R11KEα and R11KEβ chains. The EF-1α promoter may be placed at the 5' end of the codon-optimized sequences encoding the CD8α and CD8β chains to drive expression of the CD8α and CD8β chains.

[0061] Some embodiments of the present disclosure may include viral vectors containing sequences encoding the TCR alpha and beta chains, as well as sequences encoding other proteins, such as cytokines (including, but not limited to, IL-1, IL-2, IL-6, IL-7, IL-10, IL-12, IL-15, IL-18, and IL-21), IL-15 / IL-15 receptor (IL-15R) fusion proteins, dominant negative TGF beta receptor (DN TGFbRII), and / or the extracellular domain of transforming growth factor beta receptor. In some embodiments, these coding sequences may be driven by a promoter or a bidirectional promoter.

[0062] 7 shows a viral vector containing sequences encoding the TCR α and TCR β chains located upstream from a sequence encoding a cytokine, in which the sequences encoding the TCR α and TCR β chains and the sequence encoding the cytokine may be separated by a bidirectional promoter. The bidirectional promoter may be arranged in a 5' to 3' orientation, constitutive-constitutive, constitutive-inducible, inducible-constitutive, or inducible-inducible direction. For example, a constitutive promoter, such as the MSCV, PGK, or EF1 α promoter, may be placed at the 3' end of the sequences encoding the TCR α and TCR β chains to drive expression of the TCR α and TCR β chains. For example, an inducible promoter, such as the NFAT, CD69, or IPTG promoter, may be placed at the 5' end of the sequences encoding the cytokine to drive expression of the cytokine. Figure 8A shows an inducible NFAT promoter with a minimal IL-2 promoter placed at the 5' end of a sequence encoding IL-12, such as, for example, the IL-12α(p35) / IL-12β(p40) fusion protein (SEQ ID NO: 261), to drive expression of the 12α(p35) / IL-12β(p40) fusion protein in the viral vector shown in Figure 7. Figure 8B shows an inducible CD69 promoter with CNS1 and CNS2 enhancer elements placed at the 5' end of a sequence encoding IL-12, such as, for example, the IL-12α(p35) / IL-12β(p40) fusion protein (SEQ ID NO: 262), to drive expression of the 12α(p35) / IL-12β(p40) fusion protein in the viral vector shown in Figure 7. FIG. 8C shows an inducible NFAT promoter with a minimal IL-2 promoter placed at the 5' end of a sequence encoding IL-18, such as IL-18 variant 1 (sequence number 263), in the viral vector shown in FIG. 7 to drive expression of IL-18 variant 1.FIG. 8C shows an inducible CD69 promoter with CNS1 and CNS2 enhancer elements positioned at the 5' end of a sequence encoding IL-18, such as IL-18 variant 1 (SEQ ID NO: 264), in the viral vector shown in FIG. 7 to drive expression of IL-18 variant 1.

[0063] In one aspect, the disclosure provides a 4-in-1 construct having a 5' to 3' orientation of CD8β-CD8α-TCRβ-TCRα. In another aspect, the disclosure provides a 4-in-1 construct having a 5' to 3' orientation of CD8β-CD8α-TCRα-TCRβ. In another aspect, the disclosure provides a 4-in-1 construct having a 5' to 3' orientation of CD8α-CD8β-TCRβ-TCRα. In another aspect, the disclosure provides a 4-in-1 construct having a 5' to 3' orientation of CD8α-CD8β-TCRα-TCRβ.

[0064] In one aspect, the disclosure provides a 4-in-1 construct with a 5' to 3' orientation that does not include TCRβ-TCRα-CD8α-CD8β. In another aspect, the disclosure provides a 4-in-1 construct with a 5' to 3' orientation that does not include TCRβ-TCRα-CD8β-CD8α. In another aspect, the disclosure provides a 4-in-1 construct with a 5' to 3' orientation that does not include TCRα-TCRβ-CD8α-CD8β. In another aspect, the disclosure provides a 4-in-1 construct with a 5' to 3' orientation that does not include TCRα-TCRβ-CD8β-CD8α.

[0065] In one aspect, the disclosure provides a 4-in-1 construct with a 5' to 3' orientation of CD8β-CD8α-TCRβ-TCRα. In a non-limiting aspect, the disclosure provides a 4-in-1 construct with a 5' to 3' orientation that does not include TCRβ-TCRα-CD8α-CD8β.

[0066] In some embodiments, a viral vector of the present disclosure may contain sequences encoding the TCR alpha and beta chains, a TGF-beta inhibitor, such as a dominant negative TGF beta receptor (DN TGFbRII), and / or a sequence encoding the extracellular domain of a transforming growth factor-beta receptor. Figure 9A shows a viral vector containing sequences encoding the TCR alpha and beta chains located upstream from sequences encoding DN TGFbRII, in which the sequences encoding the TCR alpha and beta chains and the sequences encoding DN TGFbRII may be separated by a bidirectional promoter. For example, Figure 9A shows a constitutive promoter, such as the MSCV, Ubc, CMV, EF-1α, and PGK promoter, which may be placed at the 3' end of the sequence encoding the TCR α and TCR β chains to drive expression of the TCR α and TCR β chains; another constitutive promoter may be placed at the 5' end of the sequence encoding DN TGFbRII to drive expression of DN TGFbRII.

[0067] Alternatively, Figure 9B shows a viral vector containing a constitutive promoter, such as the MSCV, Ubc, CMV, EF-1α, and PGK promoter, located at the 5' end of the DN TGFbRII coding sequence, which is located upstream from the TCR α and β chain coding sequences and drives expression of the DN TGFbRII, TCR α, and TCR β chains. The same coding sequences as above can also be driven by an inducible promoter, such as the NFAT, CD69, or IPTG promoter.

[0068] As used herein, the term "cistron" refers to a portion of a DNA molecule that specifies the formation of one polypeptide chain, i.e., encodes one polypeptide chain. For example, a "bicistron" refers to two sections of a DNA molecule that specifies the formation of two polypeptide chains, i.e., encodes two polypeptide chains; a "tricistron" refers to three sections of a DNA molecule that specifies the formation of three polypeptide chains, i.e., encodes three polypeptide chains.

[0069] As used herein, the term "multicistronic RNA" or "multicistronic RNA" refers to an RNA that contains genetic information for translation into several proteins. In contrast, monocistronic RNA contains genetic information for translation of only a single protein. In the context of the present disclosure, the multicistronic RNA transcribed from the lentivirus in Examples 2-4 may be translated into four proteins (4-in-1): TCR α chain, TCR β chain, CD8 α chain, and CD8 β chain; or two proteins (2-in-1): TCR α chain and TCR β chain or CD8 α chain and CD8 β chain.

[0070] As used herein, the term "arranged in tandem" refers to genes being adjacent in a single file on a nucleic acid sequence, one following or following the other. The genes are ligated adjacently on the nucleic acid sequence, and the coding strands (sense strands) of each gene are ligated together on the nucleic acid sequence.

[0071] As used herein, the term "sense strand" refers to the DNA strand of a gene that is translated or translatable into a protein. When a gene is oriented in a "sense orientation" relative to a promoter of a nucleic acid sequence, the "sense strand" is located at the 5' end of the promoter, where the first codon of the nucleic acid that encodes the protein is proximal to the promoter and the last codon is distal to the promoter.

[0072] As used herein, the term "viral vector" refers to a nucleic acid vector construct that contains at least one element of viral origin, has the ability to be packaged into a viral vector particle, and encodes at least an exogenous nucleic acid. The vector and / or particle can be utilized to transfer any nucleic acid into a cell, either in vitro or in vivo. Numerous forms of viral vectors are known in the art. The term "virion" is used to refer to a single infectious viral particle. "Viral vector", "viral vector particle", and "viral particle" also refer to a complete viral particle with its DNA or RNA core and protein coat, as it exists outside a cell. For example, the viral vector may be selected from adenovirus, poxvirus, alphavirus, arenavirus, rhabdovirus, retrovirus, lentivirus, herpesvirus, paramyxovirus, or picornavirus.

[0073] The term "T cells" or "T lymphocytes" is art-recognized and is intended to include thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. Exemplary T cell populations suitable for use in certain embodiments include, but are not limited to, helper T cells (HTL; CD4+ T cells), cytotoxic T cells (CTL; ​​CD8+ T cells), CD4+CD8+ T cells, CD4-CD8- T cells, natural killer T cells, T cells expressing αβTCR (αβT cells), T cells expressing γδTCR (γδT cells), or any other T cell subset. Other exemplary T cell populations suitable for use in certain embodiments include, but are not limited to, T cells expressing one or more of the following markers: CD3, CD4, CD8, CD27, CD28, CD45RA, CD45RO, CD62L, CD127, CD197, and HLA-DR, and can be further isolated, if desired, by positive or negative selection techniques.

[0074] The terms "statin", "vastatin", or "3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor", as used interchangeably herein, refer to pharmaceutical agents that inhibit the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. This enzyme is involved in the conversion of HMG-CoA to mevalonate, which is one of the steps in cholesterol biosynthesis. Such inhibition is readily determined according to standard assays well known to those of skill in the art.

[0075] Preferred statins that may be used in accordance with the present disclosure include atorvastatin, disclosed in U.S. Pat. No. 4,681,893; atorvastatin calcium, disclosed in U.S. Pat. No. 5,273,995; cerivastatin, disclosed in U.S. Pat. No. 5,502,199; dalvastatin, disclosed in U.S. Pat. No. 5,316,765; fluindostatin, disclosed in U.S. Pat. No. 4,915,954; fluvastatin, disclosed in U.S. Pat. No. 4,739,073; No. 4,231,938; mevastatin, disclosed in U.S. Pat. No. 3,983,140; pravastatin, disclosed in U.S. Pat. No. 4,346,227; simvastatin, disclosed in U.S. Pat. No. 4,444,784; velostatin, disclosed in U.S. Pat. Nos. 4,448,784 and 4,450,171; and rosuvastatin, disclosed in U.S. Pat. Nos. 6,858,618 and 7,511,140. The contents of each of these references are incorporated herein by reference in their entirety. Representative 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors may include atorvastatin, atorvastatin calcium, also known as Liptor®, lovastatin, also known as Mevacor®, pravastatin, also known as Pravachol®, simvastatin, also known as Zocor®, and rosuvastatin.

[0076] In one aspect, the disclosure relates to activation, transduction, and / or expansion of T cells, such as, for example, tumor infiltrating lymphocytes, CD8+ T cells, CD4+ T cells, and γδ T cells, which may be used for transgene expression. In another aspect, the disclosure relates to γδ T cell activation, transduction, and expansion while depleting α- and / or β-TCR positive cells.

[0077] In one embodiment, γδ T cells may be isolated from a complex sample cultured in vitro. In another embodiment, the entire PBMC population may be activated and expanded without prior depletion of specific cell populations such as monocytes, αβ T cells, B cells, and NK cells. In another embodiment, enriched γδ T cell populations may be generated prior to their specific activation and expansion. In another embodiment, activation and expansion of γδ T cells may be performed without the presence of naive or engineered APCs. In other embodiments, isolation and expansion of γδ T cells from tumor specimens may be performed using immobilized γδ T cell mitogens, including antibodies specific for γδ TCR, and other γδ TCR activators, including lectins. In another embodiment, isolation and expansion of γδ T cells from tumor specimens may be performed in the absence of immobilized γδ T cell mitogens, including antibodies specific for γδ TCR, and other γδ TCR activators, including lectins.

[0078] In one embodiment, the γδ T cells are isolated from leukapheresis of a subject, such as a human subject. In another embodiment, the γδ T cells are not isolated from peripheral blood mononuclear cells (PBMCs).

[0079] In one embodiment, isolated γδ T cells can rapidly proliferate in response to contact with one or more antigens. Some γδ T cells, such as Vγ9Vδ2+ T cells, can rapidly proliferate ex vivo in response to contact with some antigens, such as prenyl pyrophosphate, alkylamines, and metabolites or microbial extracts, in tissue culture. Stimulated γδ T cells can exhibit multiple antigen-presenting, co-stimulatory, and adhesion molecules that can facilitate isolation of γδ T cells from a complex sample. γδ T cells in a complex sample can be stimulated ex vivo with at least one antigen for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or another suitable period of time. Stimulation of γδ T cells with appropriate antigens can expand a γδ T cell population ex vivo.

[0080] Non-limiting examples of antigens that may be used to stimulate proliferation of γδ T cells from a complex sample in vitro include prenyl pyrophosphates such as isopentenyl pyrophosphate (IPP), alkylamines, metabolites of human microbial pathogens, metabolites of commensal bacteria, methyl-3-butenyl-1-pyrophosphate (2M3B1PP), (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), ethyl pyrophosphate (EPP), farnesyl pyrophosphate (FPP), dimethylallyl phosphate (DMAP), dimethylallyl pyrophosphate (DMAPP), ethyl-adenosine triphosphate (EPPPA), geranyl pyrophosphate (GPP), geranylgeranyl pyrophosphate (GGPP), Isopentenyl-adenosine triphosphate (IPPPA), monoethyl phosphate (MEP), monoethyl pyrophosphate (MEPP), 3-formyl-1-butyl-pyrophosphate (TUBAg1), X-pyrophosphate (TUBAg2), 3-formyl-1-butyl-uridine triphosphate (TUBAg3), 3-formyl-1-butyl-deoxythymidine triphosphate (TUBAg4), monoethyl alkylamines, allyl pyrophosphate, clotoyl pyrophosphate, dimethylallyl-γ-uridine triphosphate, clotoyl-γ-uridine triphosphate, allyl-γ-uridine triphosphate, ethylamine, isobutylamine, sec-butylamine, iso-amylamine, and nitrogen-containing bisphosphonates may be mentioned.

[0081] Activation and expansion of γδ T cells can be performed using activators and costimulators described herein to trigger specific γδ T cell proliferation and persistence populations. In one embodiment, activation and expansion of γδ T cells from different cultures can achieve different clonal population subsets or mixed polyclonal population subsets. In another embodiment, different agonists can be used to identify agents that provide specific γδ activation signals. In another embodiment, agents that provide specific γδ activation signals can be different monoclonal antibodies (MAbs) against γδ TCR. In another embodiment, companion costimulators can be used that help trigger specific γδ T cell proliferation without induction of cellular energy and apoptosis. These costimulators can include ligands that bind to receptors expressed on γδ cells, such as NKG2D, CD161, CD70, JAML, DNAX accessory molecule-1 (DNAM-1), ICOS, CD27, CD137, CD30, HVEM, SLAM, CD122, DAP, and CD28. In another embodiment, the costimulatory agent can be an antibody specific for a unique epitope on the CD2 and CD3 molecules. CD2 and CD3 can have different conformations when expressed on αβ or γδ T cells. In another embodiment, specific antibodies against CD3 and CD2 can result in distinct activation of γδ T cells.

[0082] Prior to engineering γδ T cells, a population of γδ T cells may be expanded in vitro. Non-limiting examples of reagents that may be used to promote the expansion of γδ T cell populations in vitro may include anti-CD3 or anti-CD2, anti-CD27, anti-CD30, anti-CD70, anti-OX40 antibodies; IL-2, IL-15, IL-12, IL-9, IL-33, IL-18, or IL-21, CD70 (CD27 ligand), phytohemagglutinin (PHA), concavalin A (ConA), pokeweed (PWM), protein peanut agglutinin (PNA), soybean agglutinin (SBA), Lens culinaris agglutinin (LCA), Pisum sativum agglutinin (PSA), Helix pomatia agglutinin (HPA), Vicia graminea lectin (VGA), or another suitable mitogen capable of stimulating T cell proliferation.

[0083] The ability of γδ T cells to recognize a broad spectrum of antigens can be enhanced by genetic engineering of γδ T cells. In one embodiment, γδ T cells can be engineered to provide universal allogeneic therapy that recognizes selected antigens in vivo. Genetic engineering of γδ T cells may include stably integrating into the genome of isolated γδ T cells, cytokines (e.g., IL-15, IL-12, IL-2.IL-7.IL-21, IL-18, IL-19, IL-33, IL-4, IL-9, IL-23, or IL1β) expressing tumor recognition moieties such as αβ TCR, γδ TCR, chimeric antigen receptors (CARs), or lymphocyte activation domains, combining both antigen binding and T cell activation functions in a single receptor, to enhance T cell proliferation, survival, and function in vitro and in vivo. Genetic engineering of the isolated γδ T-cell may also include deleting or disrupting gene expression from one or more endogenous genes in the genome of the isolated γδ T-cell, such as, for example, the MHC locus.

[0084] Engineered γδ T cells may be produced by various methods. For example, polynucleotides encoding expression cassettes comprising tumor recognition moieties or other types of recognition moieties may be stably introduced into γδ T cells by transposon / transposase systems; or virus-based gene transfer systems such as lentivirus or retrovirus systems; or other suitable methods such as transfection, electroporation, transduction, lipofection, nanoengineered materials such as calcium phosphate (CaPO4), ormosil; viral delivery methods including adenovirus, retrovirus, lentivirus, adeno-associated virus, or other suitable methods. Several viral methods have been used for human gene therapy, such as those described in WO1993020221, the entire contents of which are incorporated herein by reference. Non-limiting examples of viral methods that may be used to engineer γδ T cells may include gamma retrovirus, adenovirus, lentivirus, herpes simplex virus, vaccinia virus, poxvirus, or adenovirus-associated virus methods.

[0085] In one aspect, the constructs and vectors described herein are used in accordance with the methodology described in U.S. Patent No. 16 / 200,308, filed November 26, 2018, the entire contents of which are incorporated by reference.

[0086] In one aspect, virus refers to naturally occurring viruses as well as artificial viruses. Viruses according to some embodiments of the present disclosure may be either enveloped or non-enveloped viruses. Parvoviruses (such as AAV) are examples of non-enveloped viruses. In preferred embodiments, the virus may be an enveloped virus. In preferred embodiments, the virus may be a retrovirus, particularly a lentivirus. Viral envelope proteins that may facilitate viral infection of eukaryotic cells may include vesicular stomatitis virus (VSV-G), modified feline endogenous retrovirus (RD114TR) (SEQ ID NO: 97), and HIV-1 derived lentiviral vectors (LV) pseudotyped with envelope glycoproteins (GP) from modified gibbon ape leukemia virus (GALVTR). These envelope proteins may efficiently facilitate the entry of other viruses, such as parvoviruses, including adeno-associated virus (AAV), thereby demonstrating their broad efficiency. For example, Moloney murine leukemia virus (MLV) 4070 env (as described in Merten et al., J. Virol. 79:834-840, 2005, the contents of which are incorporated herein by reference), RD114 env, chimeric envelope proteins RD114pro or RDpro (RD114-HIV chimeras constructed by replacing the R peptide cleavage sequence of RD114 with the HIV-1 matrix / capsid (MA / CA) cleavage sequence, as described in Bell et al. Experimental Biology and Medicine 2010;235:1269-1276, the contents of which are incorporated herein by reference), baculovirus GP64 env (as described in Wang et al. J. Virol. 81:10869-10878, 2007, the contents of which are incorporated herein by reference), or GALV env (as described in Merten et al., J. Virol. 81:10869-10878, 2007, the contents of which are incorporated herein by reference). Other viral envelope proteins may be used, including those described in (as described in et al., J. Virol. 79:834-840, 2005) or derivatives thereof.

[0087] The embodiments of the present disclosure are based on the discovery that a single lentiviral cassette can be used to generate a single lentiviral vector to express at least four separate monomeric proteins of two different dimers from a single multicistronic mRNA and co-express the dimers on the cell surface. For example, integration of a single copy of the lentiviral vector was sufficient to transform γδ T cells to co-express TCRαβ and CD8αβ.

[0088] In one aspect, the present disclosure relates to vectors containing multicistronic cassettes within a single vector capable of expressing more than one, more than two, more than three, more than four, more than five, or more than six genes, in which the polypeptides encoded by these genes may interact or form dimers. The dimers may be homodimers, i.e., two identical proteins that form a dimer, or heterodimers, i.e., two structurally different proteins that form a dimer.

[0089] In one embodiment, the lentiviral vector may contain a first nucleotide sequence S1 encoding a protein Z1, a second nucleotide sequence S2 encoding a protein Z2, a third nucleotide sequence S3 encoding a protein Y1, and a fourth nucleotide sequence S4 encoding a protein Y2, wherein Z1 and Z2 form a first dimer and Y1 and Y2 form a second dimer, wherein the first dimer Z1Z2 is different from the second dimer Y1Y2.

[0090] In one embodiment, the first lentiviral vector may contain a bicistronic cassette (2-in-1) encoding the dimer Z1Z2, and the second lentiviral vector may contain a bicistronic cassette (2-in-1) encoding the dimer Y1Y2. In the 2-in-1 vector, S1 and S2 may be arranged in tandem in a 5' to 3' direction of S1-S2 or S2-S1. Similarly, in the 2-in-1 vector, S3 and S4 may be arranged in tandem in a 5' to 3' direction of S3-S4 or S4-S3. Z1 and Z2 or Y1 and Y2 may be separated by one or more self-cleaving 2A peptides.

[0091] In another embodiment, a single lentiviral vector (4-in-1) may encode both separate dimers Z1Z2 and Y1Y2, in which Z1, Z2, Y1, and Y2 may be separated by one or more self-cleaving 2A peptides, e.g., S1-S2-S3-S4, S1-S2-S4-S3, S1-S3-S2-S4, S1-S3-S4-S2, S1-S4-S3-S2, S1-S4-S2-S3, S2-S1-S3-S4, S2-S1-S4-S3, S2-S3-S1-S4, S2-S3-S4-S1, S2-S4-S3-S1, S2-S4-S1-S3, S3-S1-S2-S4, S3-S1-S4-S2 , S3-S2-S1-S4, S3-S2-S4-S1, S3-S4-S1-S2, S3-S4-S2-S1, S4-S1-S2-S3, S4-S1-S3-S2, S4-S2-S1-S3, S4-S2-S3-S1, S4-S3-S1-S2, or S4-S3-S2-S1.

[0092] In one embodiment, the dimer Z1Z2 may be a TCR having a TCR α chain and a TCR β chain.

[0093] In one aspect, TCRs and antigen binding proteins that can be used with the constructs, methods, and embodiments described herein include, for example, those described in Table 2 (SEQ ID NOs: 13-90), and those described in U.S. Patent Application Publication Nos. 20170267738, 20170312350, 20180051080, 20180164315, 20180161396, 20180162922, 20180273602, 20190016801, 20190002556, No. 10,626,160, US Patent Publication No. 20190321478, US Patent Publication No. 20190256572, US Patent No. 10,550,182, US Patent No. 10,526,407, US Patent Publication No. 20190284276, US Patent Publication No. 20190016802, and US Patent No. 10,583,573, the contents of each of which publications and the sequence listings set forth therein are incorporated herein by reference in their entirety.

[0094] In another embodiment, the dimer Z1Z2 is selected from the group consisting of R11KEA (SEQ ID NOs: 13 and 14), R20P1H7 (SEQ ID NOs: 15 and 16), R7P1D5 (SEQ ID NOs: 17 and 18), R10P2G12 (SEQ ID NOs: 19 and 20), R10P1A7 (SEQ ID NOs: 21 and 22), R4P1D10 (SEQ ID NOs: 23 and 24), R4P3F9 (SEQ ID NOs: 25 and 26), R4P3H3 (SEQ ID NOs: 27 and 28), R36P3F9 (SEQ ID NOs: 29 and 30), R52P2G11 (SEQ ID NOs: 31 and 32), R52P2G12 (SEQ ID NOs: 33 and 34), R52P2G11 (SEQ ID NOs: 35 and 36), R52P2G11 (SEQ ID NOs: 37 and 38), R52P2G12 (SEQ ID NOs: 39 and 40), R52P2G11 (SEQ ID NOs: 41 and 42), R52P2G11 (SEQ ID NOs: 43 and 44), R52P2G11 (SEQ ID NOs: 45 and 46), R52P2G11 (SEQ ID NOs: 46 and 47), R52P2G11 (SEQ ID NOs: 48 and 49), R52P2G11 (SEQ ID NOs: 49 and 50), R52P2G11 (SEQ ID NOs: 51 and 52), R52P2G12 (SEQ ID NOs: 53 and 54), 1 and 32), R53P2A9 (SEQ ID NOs: 33 and 34), R26P1A9 (SEQ ID NOs: 35 and 36), R26P2A6 (SEQ ID NOs: 37 and 38), R26P3H1 (SEQ ID NOs: 39 and 40), R35P3A4 (SEQ ID NOs: 41 and 42), R37P1C9 (SEQ ID NOs: 43 and 44), R37P1H1 (SEQ ID NOs: 45 and 46), R42P3A9 (SEQ ID NOs: 47 and 48), R43P3F2 (SEQ ID NOs: 49 and 50), R43P3G5 (SEQ ID NOs: 51 and 52) , R59P2E7 (SEQ ID NOs: 53 and 54), R11P3D3 (SEQ ID NOs: 55 and 56), R16P1C10 (SEQ ID NOs: 57 and 58), R16P1E8 (SEQ ID NOs: 59 and 60), R17P1A9 (SEQ ID NOs: 61 and 62), R17P1D7 (SEQ ID NOs: 63 and 64), R17P1G3 (SEQ ID NOs: 65 and 66), R17P2B6 (SEQ ID NOs: 67 and 68), R11P3D3KE (SEQ ID NOs: 69 and 70), R39P1C12 (SEQ ID NOs: 71 and 72), R3 The TCR α and β chains may be selected from 9P1F5 (SEQ ID NOs: 73 and 74), R40P1C2 (SEQ ID NOs: 75 and 76), R41P3E6 (SEQ ID NOs: 77 and 78), R43P3G4 (SEQ ID NOs: 79 and 80), R44P3B3 (SEQ ID NOs: 81 and 82), R44P3E7 (SEQ ID NOs: 83 and 84), R49P2B7 (SEQ ID NOs: 85 and 86), R55P1G7 (SEQ ID NOs: 87 and 88), or R59P2A7 (SEQ ID NOs: 89 and 90). In one embodiment, the sequence is at least about 90%, at least about 95%, or at least about 98% identical to any of SEQ ID NOs: 13-90.

[0095] Table 1 shows examples of peptides that are bound by the TCR when the peptide is complexed with an MHC molecule. [Table 1]

[0096] In one aspect, tumor associated antigens, TAA peptides that can be used in the methods and embodiments described herein include, for example, those listed in Table 3 and those described in, for example, U.S. Patent Application Publication No. 20160187351, U.S. Patent Application Publication No. 20170165335, U.S. Patent Application Publication No. 20170035807, U.S. Patent Application Publication No. 20160280759, U.S. Patent Application Publication No. 20160287687, U.S. Patent Application Publication No. 20160346371, U.S. Patent Application Publication No. US Patent Application Publication No. 20160368965, US Patent Application Publication No. 20170022251, US Patent Application Publication No. 20170002055, US Patent Application Publication No. 20170029486, US Patent Application Publication No. 20170037089, US Patent Application Publication No. 20170136108, US Patent Application Publication No. 20170101473, US Patent Application Publication No. 20170096461, US Patent Application Publication No. 20170165337, US Patent Application Publication No. 2017 US Patent Application Publication No. 20180189505, US Patent Application Publication No. 20170173132, US Patent Application Publication No. 20170296640, US Patent Application Publication No. 20170253633, US Patent Application Publication No. 20170260249, US Patent Application Publication No. 20180051080, US Patent Application Publication No. 20180164315, US Patent Application Publication No. 20180291082, US Patent Application Publication No. 20180291083, US Patent Application Publication No. 2019025511 0, U.S. Patent No. 9,717,774, U.S. Patent No. 9,895,415, U.S. Patent Application Publication No. 20190247433, U.S. Patent Application Publication No. 20190292520, U.S. Patent Application Publication No. 20200085930, U.S. Patent No. 10,336,809, U.S. Patent No. 10,131,703, U.S. Patent No. 10,081,664, U.S. Patent No. 10,081,664, U.S. Patent No. 10,093,715, U.S. Patent No. 10,583,573, and U.S. Patent No. 20200085930, the contents, sequences, and sequence listings of each of which are incorporated herein by reference in their entirety.

[0097] In another aspect, the dimer Z1Z2 may be a T cell dimeric signaling module such as CD3δ / ε, CD3γ / ε, and CD247ζ / ζ or ζ / η; a dimer of a TCRα variable region (Vα) and a TCRβ variable region (Vβ); a dimer of an immunoglobulin heavy chain variable region (VH) and an immunoglobulin light chain variable region (VL); a dimer of Vα and VH; a dimer of Vα and VL; a dimer of Vβ and VH; or a dimer of Vβ and VL.

[0098] In another embodiment, Y1Y2 may be the CD8 α and β chains, or any other suitable dimeric membrane receptor, preferably expressed on CD8+ T cells and / or CD4+ T cells.

[0099] Furin is a ubiquitous subtilisin-like proprotein convertase whose natural sources include certain serum proteins and growth factor receptors such as the insulin-like growth factor receptor. The consensus sequence for furin cleavage is RXXR (SEQ ID NO: 7), but the actual probability of cleavage depends on the tertiary structure of the substrate and the amino acids immediately surrounding the recognition site. Addition of a furin cleavage site and a linker sequence (GSG or SGSG (SEQ ID NO: 8)) may allow for highly efficient gene expression.

[0100] In one embodiment, the nucleotide sequences of the tandemly arranged furin linker and 2A peptide may be located between Z1 and Z2, between Z1 and Y1, between Z1 and Y2, between Z2 and Y1, between Z2 and Y2, and / or between Y1 and Y2. The furin may have a consensus sequence of RXXR (SEQ ID NO:7), such as, for example, RAKR (SEQ ID NO:2). The linker sequence may be GSG or SGSG (SEQ ID NO:8). The 2A peptide may be selected from P2A (SEQ ID NO:3), T2A (SEQ ID NO:4), E2A (SEQ ID NO:5), F2A (SEQ ID NO:6), or any combination thereof.

[0101] In another embodiment, the nucleotide sequences of the linker and 2A peptide arranged in tandem may be arranged between Z1 and Z2, between Z1 and Y1, between Z1 and Y2, between Z2 and Y1, between Z2 and Y2, and / or between Y1 and Y2. The linker sequence may be GSG or SGSG (SEQ ID NO:8). The 2A peptide may be selected from P2A (SEQ ID NO:3), T2A (SEQ ID NO:4), E2A (SEQ ID NO:5), F2A (SEQ ID NO:6), or any combination thereof.

[0102] In one embodiment, engineered (or transduced) γδ T cells can be expanded in vitro without stimulation with antigen presenting cells or aminobisphosphonates. Antigen-reactive engineered T cells of the present disclosure may be expanded in vitro and in vivo. In another embodiment, active populations of engineered γδ T cells of the present disclosure may be expanded in vitro using specific antibodies, cytokines, mitogens, or fusion proteins such as IL-17Fc fusion protein, MICAFc fusion protein, and CD70Fc fusion protein, without antigenic stimulation with antigen presenting cells, antigenic peptides, non-peptide molecules, or aminobisphosphonates such as small molecule compounds. Exemplary antibodies that may be used to expand γδ T cell populations include anti-CD3, anti-CD27, anti-CD30, anti-CD70, anti-OX40, anti-NKG2D, or anti-CD2 antibodies; exemplary cytokines may include IL-2, IL-15, IL-12, IL-21, IL-18, IL-9, IL-7, and / or IL-33; exemplary mitogens include CD70, which is a ligand for human CD27, phytohemagglutinin (PHA), concavalin A (ConA), pokeweed mitogen (PWM), protein peanut agglutinin (PNA), soybean agglutinin (SBA), Lens culinaris agglutinin (LCA), Pisum sativum agglutinin (PSA), Helix pomatia agglutinin (HPA), Vicia graminea (Vicia graminea lectin (VGA) or another suitable mitogen capable of stimulating T cell proliferation may be mentioned. In another embodiment, the population of engineered γδ T cells may be expanded for less than 60 days, less than 48 days, less than 36 days, less than 24 days, less than 12 days, or less than 6 days. In another embodiment, the population of engineered γδ T cells may be expanded for about 7 days to about 49 days, about 7 days to about 42 days, about 7 days to about 35 days, about 7 days to about 28 days, about 7 days to about 21 days, or about 7 days to about 14 days.

[0103] In another aspect, the present disclosure provides a method for ex vivo expansion of an engineered γδ T cell population for adoptive transfer therapy. The engineered γδ T cells of the present disclosure may be expanded ex vivo. The engineered γδ T cells of the present disclosure may be expanded ex vivo without activation by APCs or without co-culture with APCs and aminophosphates.

[0104] treatment method Compositions containing engineered γδ T-cells as described herein may be administered for prophylactic and / or therapeutic treatment. In therapeutic applications, the pharmaceutical compositions may be administered to a subject already suffering from a disease or condition in an amount sufficient to cure or at least partially arrest the symptoms of the disease or condition. Engineered γδ T-cells may also be administered to reduce the likelihood of onset, contraction, or worsening of the condition. Effective amounts of engineered γδ T-cell populations for therapeutic use may vary based on the severity and course of the disease or condition, previous treatments, the subject's health, weight, and / or response to drugs, and / or the judgment of the treating physician.

[0105] The compositions of the present disclosure may also include one or more adjuvants. Adjuvants are substances that non-specifically promote or enhance immune responses (e.g., immune responses to antigens mediated by CD8 positive T cells and helper T (TH) cells) and are therefore considered useful in the medicaments of the present invention. Suitable adjuvants include 1018 ISS, aluminum salts, AMPLIVAX®, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, flagellin or flagellin-derived TLR5 ligands, FLT3 ligand, GM-CSF, IC30, IC31, imiquimod (ALDARA®), resiquimod, ImuFact IMP321; interleukins such as IL-2, IL-13, IL-21, interferon-α or -β, or PEGylated derivatives thereof; IS patch, ISS, ISCOMATRIX, ISCOMs, JuvImmune®, LipoVac, MALP2, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA Adjuvants include, but are not limited to, 50V, Montanide ISA-51, water-in-oil and oil-in-water emulsions, OK-432, OM-174, OM-197-MP-EC, ONTAK, OspA, PepTel® Vector System, poly(lactide-co-glycolide) [PLG]-based and dextran microparticles, talactoferrin SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, β-glucan, Pam3Cys, Aquila's QS21 stimulon derived from saponin, mycobacterial extracts and synthetic bacterial cell wall mimics; and other proprietary adjuvants such as Ribi's Detox, Quil, or Superfos. Adjuvants such as Freund's or GM-CSF are preferred. Several immunological adjuvants (e.g., MF59) specific for dendritic cells and their preparations have been previously described (Allison and Krummel, 1995). Cytokines may also be used.Several cytokines have been directly implicated in influencing the migration of dendritic cells to lymphoid tissues (e.g., TNF-), accelerating the maturation of dendritic cells into efficient antigen-presenting cells for T lymphocytes (e.g., GM-CSF, IL-1, and IL-4) (U.S. Pat. No. 5,849,589, the entire contents of which are specifically incorporated by reference herein), and acting as immune enhancers (e.g., IL-12, IL-15, IL-23, IL-7, IFN-α, IFN-β) (Gabrilovich et al., 1996).

[0106] CpG immunostimulatory oligonucleotides have also been reported to enhance adjuvant effects in a vaccine environment. Without being bound by theory, CpG oligonucleotides act by activating the innate (non-adaptive) immune system through Toll-like receptors (TLRs), primarily TLR9. CpG-induced TLR9 activation enhances antigen-specific humoral and cellular responses to a wide variety of antigens, including peptide or protein antigens, live or killed viruses, dendritic cell vaccines, autologous cell vaccines, and polysaccharide conjugates in both prophylactic and therapeutic vaccines. More importantly, it enhances dendritic cell maturation and differentiation, leading to enhanced activation of TH1 cells and potent cytotoxic T lymphocyte (CTL) generation, even in the absence of CD4 T cell help. The TH1 bias induced by TLR9 stimulation is maintained even in the presence of vaccine adjuvants such as alum or incomplete Freund's adjuvant (IFA), which normally promote a TH2 bias. CpG oligonucleotides, when formulated or co-administered with other adjuvants, or in formulations such as microparticles, nanoparticles, lipid emulsions or similar formulations, still show higher adjuvant activity, which is especially necessary to induce strong responses when the antigen is relatively weak. They also accelerate immune responses, and in some experiments allow for almost two orders of magnitude reduction in antigen dose with antibody response equivalent to the total vaccine dose without CpG (Krieg, 2006). US Patent No. 6,406,705 B1 describes the combination of CpG oligonucleotides, non-nucleic acid adjuvants, and antigens to induce antigen-specific immune responses. The CpG TLR9 antagonist is dSLIM (double stem loop immunomodulator) from Mologen (Berlin, Germany), which is a preferred component of the pharmaceutical composition of the present invention. Other TLR binding molecules, such as RNA-binding TLR7, TLR8 and / or TLR9, may also be used.

[0107] Other examples of useful adjuvants include chemically modified CpGs (e.g., CpR, Idera), dsRNA analogs such as poly(I:C) and their derivatives (e.g., AmpliGen®, Hiltonol®, poly-(ICLC), poly(IC-R), poly(I:C12U), non-CpG bacterial DNA or RNA; and immunologically active small molecules and antibodies such as cyclophosphamide, sunitinib; ipilimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, and cemiplimab, bevacizumab®, Celebrex, NCX-4016, sildenafil, tacrolimus ... Immune checkpoint inhibitors, including, but not limited to, darafil, vardenafil, sorafenib, temozolomide, temsirolimus, XL-999, CP-547632, pazopanib, VEGF trap, ZD2171, AZD2171, anti-CTLA4; other antibodies targeting key structures of the immune system (e.g., anti-CD40, anti-TGFβ, anti-TNFα receptor); and SC58175, which may act therapeutically and / or as adjuvants. The amounts and concentrations of adjuvants and additives useful in the context of the present invention can be readily determined by those skilled in the art without undue experimentation.

[0108] Preferred adjuvants are anti-CD40, imiquimod, resiquimod, GM-CSF, cyclophosphamide, sunitinib, bevacizumab, atezolizumab, interferon-α, interferon-β, CpG oligonucleotides and derivatives, poly-(I:C) and derivatives, RNA, sildenafil, and particulate formulations with poly(lactide-co-glycolide) (PLG), virosomes, and / or interleukin (IL)-1, IL-2, IL-4, IL-7, IL-12, IL-13, IL-15, IL-21, and IL-23.

[0109] In a preferred embodiment of the pharmaceutical composition according to the invention, the adjuvant is selected from the group consisting of colony stimulating factors such as granulocyte-macrophage colony stimulating factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod, resiquimod, and interferon alpha.

[0110] In a preferred embodiment of the pharmaceutical composition according to the invention, the adjuvant is selected from the group consisting of colony stimulating factors such as granulocyte macrophage colony stimulating factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod, and resiquimod. In a preferred embodiment of the pharmaceutical composition according to the invention, the adjuvant is cyclophosphamide, imiquimod or resiquimod. Even more preferred adjuvants are Montanide IMS 1312, Montanide ISA 20, Montanide ISA 50V, Montanide ISA-51, poly-ICLC (Hiltonol®), and anti-CD40mAB or combinations thereof.

[0111] The engineered γδ T-cells herein can be used to treat a subject in need of treatment for a condition, such as, for example, cancer, as described herein.

[0112] A method of treating a condition (e.g., disease) in a subject with γδ T cells may include administering a therapeutically effective amount of engineered γδ T cells to the subject. The γδ T cells of the present disclosure may be administered in various regimens (e.g., timing, concentration, dosage, treatment interval, and / or formulation). The subject may also be preconditioned, e.g., with chemotherapy, radiation, or a combination of both, before receiving the engineered γδ T cells of the present disclosure. Also, a population of engineered γδ T cells may be frozen or cryopreserved before administration to the subject. A population of engineered γδ T cells may include two or more cells expressing the same tumor recognition moiety, different tumor recognition moieties, or a combination of the same tumor recognition moiety and different tumor recognition moieties. For example, a population of engineered γδ T cells may include several different engineered γδ T cells designed to recognize different antigens or different epitopes of the same antigen.

[0113] The γδ T-cells of the present disclosure may be used to treat a variety of medical conditions, hi one embodiment, the engineered γδ T-cells of the present disclosure may be used to treat cancer, including solid tumors and hematological malignancies.Non-limiting examples of cancer include acute lymphoblastic leukemia, acute myeloid leukemia, adrenal cortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal cancer, appendix cancer, astrocytoma, neuroblastoma, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain tumors such as cerebellar astrocytoma, brain astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, visual pathway and hypothalamic glioma, breast cancer, bronchial adenoma, Burkitt's lymphoma, cancer of unknown primary, central nervous system lymphoma, cerebellar astrocytoma, cervical cancer, childhood cancer, chronic lymphocytic leukemia , chronic myeloid leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous T-cell lymphoma, desmoplastic small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, germ cell tumor, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, glioma, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, pancreatic islet cell carcinoma, Kaposi's sarcoma, kidney cancer, laryngeal cancer, lip and oral cavity cancer, liposarcoma, liver cancer, lung cancer including non-small cell and small cell lung cancer, lymphoma, leukemia , macroglobulinemia, malignant fibrous histiocytoma / osteosarcoma of bone, medulloblastoma, melanoma, mesothelioma, metastatic squamous cell neck cancer of unknown primary, oral cavity cancer, multiple endocrine neoplasia syndrome, myelodysplastic syndrome, myeloid leukemia, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oral cavity cancer, oropharyngeal cancer, osteosarcoma / malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, pancreatic cancer, pancreatic islet cell cancer, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, These include pineal germinoma, pituitary adenoma, pleuropulmonary blastoma, plasma cell neoplasms, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis and ureteral transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, skin cancer Merkel cell, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, T-cell lymphoma, throat cancer, thymoma, thymic carcinoma, thyroid cancer, trophoblastic neoplasm (gestational), cancer of unknown primary, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor.

[0114] In one embodiment, the engineered γδ T-cells of the present disclosure may be used to treat an infectious disease. In another embodiment, the engineered γδ T-cells of the present disclosure may be used to treat an infectious disease, which may be caused by a virus. In yet another embodiment, the engineered γδ T-cells of the present disclosure may be used to treat an immune disease, such as an autoimmune disease.

[0115] The γδ T-cell treatment of the present disclosure may be provided to a subject before, during, and after clinical onset of a disease condition. The treatment may be provided to a subject 1 day, 1 week, 6 months, 12 months, or 2 years after clinical onset. The treatment may be provided to a subject more than 1 day, 1 week, 1 month, 6 months, 12 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or more after clinical onset of a disease. The treatment may be provided to a subject less than 1 day, 1 week, 1 month, 6 months, 12 months, or 2 years after clinical onset of a disease. The treatment may also include treating humans in clinical trials. The treatment may include administering to a subject a pharmaceutical composition comprising an engineered γδ T-cell of the present disclosure.

[0116] In another embodiment, administration of the engineered γδ T cells of the present disclosure to a subject may modulate the activity of endogenous lymphocytes in the subject's body. In another embodiment, administration of the engineered γδ T cells to a subject may provide antigen to endogenous T cells and enhance an immune response. In another embodiment, the memory T cells may be CD4+ T cells. In another embodiment, the memory T cells may be CD8+ T cells. In another embodiment, administration of the engineered γδ T cells of the present disclosure to a subject may activate the cytotoxicity of another immune cell. In another embodiment, the another immune cell may be a CD8+ T cell. In another embodiment, the another immune cell may be a natural killer T cell. In another embodiment, administration of the engineered γδ T cells of the present disclosure to a subject may suppress a regulatory T cell. In another embodiment, the regulatory T cell may be a FOX3+ Treg cell. In another embodiment, the regulatory T cell may be a FOX3- Treg cell. Non-limiting examples of cells whose activity may be modulated by the engineered γδ T cells of the present disclosure may include hematopoietic stem cells; B cells; CD4; CD8; red blood cells; white blood cells; dendritic cells, including dendritic antigen presenting cells; leukocytes; macrophages; memory B cells; memory T cells; monocytes; natural killer cells; neutrophil granulocytes; T helper cells; and T killer cells.

[0117] During most bone marrow transplants, a combination of cyclophosphamide and total body irradiation may be routinely used to prevent rejection of hematopoietic stem cells (HSCs) during transplantation by the subject's immune system. In one embodiment, ex vivo incubation of donor bone marrow with interleukin-2 (IL-2) may be performed to enhance the generation of killer lymphocytes in the donor bone marrow. Interleukin-2 (IL-2) is a cytokine that may be necessary for the growth, proliferation, and differentiation of wild-type lymphocytes. Current studies on adoptive transfer of γδ T cells to humans may require co-administration of γδ T cells with interleukin-2. However, both low and high doses of IL-2 may have highly toxic side effects. IL-2 toxicity may be manifested in multiple organs / systems, most notably the heart, lungs, kidneys, and central nervous system. In another aspect, the disclosure provides a method of administering engineered γδ T-cells to a subject without co-administration of native cytokines or modified versions thereof such as IL-2, IL-15, IL-12, IL-21, etc. In another aspect, the engineered γδ T-cells may be administered to a subject without co-administration with IL-2. In another aspect, the engineered γδ T-cells may be administered to a subject during a treatment such as a bone marrow transplant without co-administration with IL-2.

[0118] Method of administration One or more engineered γδ T-cell populations may be administered to a subject in any order or simultaneously. If simultaneously, multiple engineered γδ T-cells may be provided in a single integrated form, such as an intravenous injection, or in multiple forms, for example, as multiple intravenous infusions, subcutaneous injections, injections, or pills. The engineered γδ T-cells may be packaged together or separately in a single package or in multiple packages. One or all of the engineered γδ T-cells may be administered in multiple doses. If not simultaneously, the timing of multiple doses may vary as much as about one week, one month, two months, three months, four months, five months, six months, or about one year. In another embodiment, the engineered γδ T-cells may be expanded in vivo within the subject's body after administration to the subject. The engineered γδ T-cells may be frozen to provide cells for multiple treatments in the same cell preparation. The engineered γδ T-cells of the present disclosure, and pharmaceutical compositions comprising the cells, may be packaged as kits. The kit may comprise the engineered γδ T-cells and instructions (eg, written instructions) for use of a pharmaceutical composition comprising the cells.

[0119] In another aspect, the method of treating cancer comprises administering a therapeutically effective amount of engineered γδ T-cells to a subject, the administration treating the cancer. In another embodiment, the therapeutically effective amount of engineered γδ T-cells may be administered for at least about 10 seconds, 30 seconds, 1 minute, 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or 1 year. In another aspect, the therapeutically effective amount of engineered γδ T-cells may be administered for at least 1 week. In another aspect, the therapeutically effective amount of engineered γδ T-cells may be administered for at least 2 weeks.

[0120] The engineered γδ T cells described herein may be administered before, during, or after the onset of a disease or condition, and the timing of administration of the pharmaceutical composition containing the engineered γδ T cells may vary. For example, the engineered γδ T cells may be used prophylactically and administered continuously to a subject with a condition or tendency to a disease to reduce the likelihood of the onset of the disease or condition. The engineered γδ T cells may be administered to a subject during or as soon as possible after the onset of symptoms. Administration of the engineered γδ T cells may begin immediately after the onset of symptoms, within the first 3 hours of the onset of symptoms, within the first 6 hours of the onset of symptoms, within the first 24 hours of the onset of symptoms, within 48 hours of the onset of symptoms, or within any period of time from the onset of symptoms. The initial administration may be via any practical route, such as any route described herein, using any formulation described herein. In another aspect, administration of the engineered γδ T cells of the present disclosure may be administered intravenously. One or more doses of engineered γδ T cells may be administered as soon as practicable after onset of cancer, infectious disease, immune disease, sepsis, or in conjunction with a bone marrow transplant, for the period required to treat the immune disease, e.g., from about 24 hours to about 48 hours, from about 48 hours to about 1 week, from about 1 week to about 2 weeks, from about 2 weeks to about 1 month, from about 1 month to about 3 months, etc. For the treatment of cancer, one or more doses of engineered γδ T cells may be administered several years after onset of the cancer, before or after other treatments. In another embodiment, the engineered γδ T-cells may be administered for at least about 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, or at least 5 years. The duration of treatment may vary depending on the subject.

[0121] keep In one embodiment, γδ T cells may be formulated in freezing medium and placed in a cryogenic storage unit, such as a liquid nitrogen freezer (−196° C.) or a cryogenic freezer (−65° C., −80° C., −120° C., or −150° C.), for long-term storage for at least about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, or at least 5 years. The freezing medium may contain dimethyl sulfoxide (DMSO), and / or sodium chloride (NaCl), and / or dextrose, and / or dextran sulfate, and / or hydroxyethyl starch (HES), with physiological pH buffers to maintain a pH between about 6.0 to about 6.5, about 6.5 to about 7.0, about 7.0 to about 7.5, about 7.5 to about 8.0, or about 6.5 to about 7.5. Cryopreserved γδ T-cells can be thawed and further processed by stimulation with antibodies, proteins, peptides, and / or cytokines, as described herein. Cryopreserved γδ T-cells can be thawed and genetically modified with viral vectors (including retroviruses, adeno-associated viruses (AAAV), and lentiviral vectors) or non-viral means (including RNA, DNA, e.g., transposons, and proteins), as described herein. Modified γδ T-cells can be further cryopreserved and maintained at a concentration of at least 10 per mL of freezing medium. 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , or at least about 10 10Cell banks can be made in quantities of at least about 1, 5, 10, 100, 150, 200, 500 vials of cells. Cryopreserved cell banks may retain their functionality and can be thawed and further stimulated and expanded. In another embodiment, thawed cells can be stimulated and expanded in a suitable closed vessel, such as a cell culture bag and / or a bioreactor, to generate a quantity of cells as an allogeneic cell product. Cryopreserved γδ T-cells can retain their biological functionality under cryogenic storage conditions for at least about 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 15 months, 18 months, 20 months, 24 months, 30 months, 36 months, 40 months, 50 months, or at least about 60 months. In another embodiment, no preservatives can be used in the formulation. Cryopreserved γδ T cells can be thawed and infused into multiple patients as an allogeneic, ready-to-use cell product.

[0122] In one aspect, the engineered γδ T cells described herein are at least 1×10 3 cells / ml, at least 2 x 10 3 cells / ml, at least 3 x 10 3 cells / ml, at least 4 x 10 3 cells / ml, at least 5 x 10 3 cells / ml, at least 6 x 10 3 cells / ml, at least 7 x 10 3 cells / ml, at least 8 x 10 3 cells / ml, at least 9 x 10 3 cells / ml, at least 1 x 10 4 cells / ml, at least 2 x 10 4 cells / ml, at least 3 x 10 4 cells / ml, at least 4 x 10 4 cells / ml, at least 5 x 10 4 cells / ml, at least 6 x 10 4 cells / ml, at least 7 x 10 4 cells / ml, at least 8 x 10 4 cells / ml, at least 9 x 104 cells / ml, at least 1 x 10 5 cells / ml, at least 2 x 10 5 cells / ml, at least 3 x 10 5 cells / ml, at least 4 x 10 5 cells / ml, at least 5 x 10 5 cells / ml, at least 6 x 10 5 cells / ml, at least 7 x 10 5 cells / ml, at least 8 x 10 5 cells / ml, at least 9 x 10 5 cells / ml, at least 1 x 10 6 cells / ml, at least 2 x 10 6 cells / ml, at least 3 x 10 6 cells / ml, at least 4 x 10 6 cells / ml, at least 5 x 10 6 cells / ml, at least 6 x 10 6 cells / ml, at least 7 x 10 6 cells / ml, at least 8 x 10 6 cells / ml, at least 9 x 10 6 cells / ml, at least 1 x 10 7 cells / ml, at least 2 x 10 7 cells / ml, at least 3 x 10 7 cells / ml, at least 4 x 10 7 cells / ml, at least 5 x 10 7 cells / ml, at least 6 x 10 7 cells / ml, at least 7 x 10 7 cells / ml, at least 8 x 10 7 cells / ml, at least 9 x 10 7 cells / ml, at least 1 x 10 8 cells / ml, at least 2 x 10 8 cells / ml, at least 3 x 10 8 cells / ml, at least 4 x 10 8 cells / ml, at least 5 x 10 8 cells / ml, at least 6 x 10 8cells / ml, at least 7 x 10 8 cells / ml, at least 8 x 10 8 cells / ml, at least 9 x 10 8 cells / ml, at least 1 x 10 9 cells / ml or more, approximately 1×10 3 cells / ml ~ at least about 1 x 10 8 cells / ml, approximately 1 x 10 5 cells / ml ~ at least about 1 x 10 8 cells / ml, or approximately 1 x 10 6 cells / ml ~ at least about 1 x 10 8 The antibody may be present in the composition in an amount of cells / ml.

[0123] In one aspect, the methods described herein may be used to generate autologous or allogeneic products according to aspects of the present disclosure.

[0124] In one embodiment, the vector, construct, or sequence described herein may comprise about 80%, about 85%, about 90%, about 85%, about 96%, about 97%, about 98%, or about 99% of any of SEQ ID NOs: 1 to 7 and 265 to 266. A sequence that is "at least 85% identical to a reference sequence" is a sequence that has 85% or more, particularly 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity over its entire length to the entire length of the reference sequence.

[0125] In the context of this application, the "percentage of identity" is calculated using global pairwise alignment (i.e., two sequences are compared over their entire length). Methods for comparing the identity of two or more sequences are well known in the art. For example, the "needle" program may be used, which uses the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch, 1970 J. Mol. Biol. 48: 443-453) to find the optimal alignment (including gaps) of two sequences considering their entire length. The Needle program is available, for example, at the ebi.ac.uk World Wide Web site and is further described in the following publications (EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, P. Longden, I. and Bleasby, A. Trends in Genetics 16, (6) pp. 276-277). The percentage of identity between two polypeptides according to the invention is calculated using the EMBOSS:needle (global) program with "Gap Open" parameters equal to 10.0, "Gap Extend" parameters equal to 0.5, and the Blosum62 matrix.

[0126] A protein consisting of an amino acid sequence that is "at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical" to a reference sequence may comprise mutations, such as deletions, insertions and / or substitutions, compared to the reference sequence. In the case of substitutions, a protein consisting of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a reference sequence may correspond to a homologous sequence derived from another species than the reference sequence.

[0127] "Amino acid substitutions" may be conservative or non-conservative. Preferably, the substitutions are conservative substitutions, in which one amino acid is replaced by another amino acid having similar structural and / or chemical properties.

[0128] In one embodiment, conservative substitutions may include those described by Dayhoff in "The Atlas of Protein Sequence and Structure. Vol. 5," Natl. Biomedical Research, the entire contents of which are incorporated by reference. For example, in one embodiment, amino acids belonging to one of the following groups may be exchanged for one another, thus constituting conservative exchanges: Group 1: alanine (A), proline (P), glycine (G), asparagine (N), serine (S), threonine (T); Group 2: cysteine ​​(C), serine (S), tyrosine (Y), threonine (T); Group 3: valine (V), isoleucine (I), leucine (L), methionine (M), alanine (A), phenylalanine (F); Group 4: lysine (K), arginine (R), histidine (H); Group 5: phenylalanine (F), tyrosine (Y), tryptophan (W), histidine (H); and Group 6: aspartic acid (D), glutamic acid (E). In one aspect, conservative amino acid substitutions may be selected from T→A, G→A, A→I, T→V, A→M, T→I, A→V, T→G, and / or T→S.

[0129] In further embodiments, conservative amino acid substitutions may include the replacement of an amino acid with another amino acid of the same class, such as, for example, (1) non-polar: Ala, Val, Leu, Ile, Pro, Met, Phe, Trp; (2) uncharged polar: Gly, Ser, Thr, Cys, Tyr, Asn, Gln; (3) acidic: Asp, Glu; and (4) basic: Lys, Arg, His. Other conservative amino acid substitutions may also be made, such as: (1) aromatic: Phe, Tyr, His; (2) proton donor: Asn, Gln, Lys, Arg, His, Trp; and (3) proton acceptor: Glu, Asp, Thr, Ser, Tyr, Asn, Gln (see, for example, U.S. Patent No. 10,106,805, the entire contents of which are incorporated by reference).

[0130] In another embodiment, conservative substitutions may be made according to Table 1. Methods for predicting tolerance to protein modifications can be found, for example, in Guo et al., Proc. Natl. Acad. Sci., USA, 101(25):9205-9210 (2004), the entire contents of which are incorporated by reference.

[0131] Table A: Conservative Amino Acid Substitutions [Table 2]

[0132] In one aspect, the sequences described herein may contain 1, 2, 3, 4, 5, 10, 15, 20, 25, or 30 amino acid or nucleotide mutations, substitutions, deletions. In one aspect, any one of SEQ ID NOs: 1-97 and 265-266 may contain 1, 2, 3, 4, 5, 10, 15, 20, 25, or 30 mutations, substitutions, or deletions. In yet another aspect, the mutations or substitutions are conservative amino acid substitutions.

[0133] In another embodiment, conservative substitutions may be those shown in Table B under the heading of "conservative substitutions." If such substitutions result in altered biological activity, more substantial changes, designated "exemplary substitutions" in Table B, may be introduced and the products screened, if necessary.

[0134] Table B: Amino acid substitutions [Table 3] EXAMPLES

[0135] Example 1 DNA and protein sequences [Table 4-1] [Table 4-2]

Table 4-3

Table 4-4

Table 4-5

Table 4-6

Table 4-7

Table 4-8

Table 4-9

Table 4-10

Table 4-11

Table 4-12

Table 4-13

Table 4-14

Table 4-15

Table 4-16

Table 4-17

Table 4-18

Table 4-19

[0136] TAA peptide sequence [Table 5]

[0137] Example 2 γδT cell production To isolate γδ T cells, in one embodiment, γδ T cells may be isolated from a subject or from a composite sample of a subject. In one embodiment, the composite sample may be a peripheral blood sample, a cord blood sample, a tumor, a stem cell precursor, a tumor biopsy, a tissue, lymph, or from an epithelial site of a subject in direct contact with the external environment, or derived from stem precursor cells. γδ T cells may be directly isolated from a composite sample of a subject, for example, by sorting γδ T cells expressing one or more cell surface markers using flow cytometry techniques. Wild-type γδ T cells may exhibit a number of antigen recognition, antigen presentation, co-stimulation, and adhesion molecules that may be associated with γδ T cells. Wild-type γδ T cells may be isolated from a composite sample using one or more cell surface markers, such as a specific γδ TCR, antigen recognition, antigen presentation, ligand, adhesion molecule, or co-stimulation molecule. Various molecules associated with or expressed by γδ T cells may be used to isolate γδ T cells from a complex sample, such as, for example, isolating a mixed population of Vδ1, Vδ2, Vδ3 cells or any combination thereof.

[0138] For example, peripheral blood mononuclear cells can be collected from a subject using an apheresis device, including, for example, the Ficol-Paque™ PLUS (GE Healthcare) system, or another suitable device / system. γδ T cells or a desired subpopulation of γδ T cells can be purified from the collected sample, for example, using flow cytometry techniques. Umbilical cord blood cells can also be obtained from umbilical cord blood at the time of the subject's birth.

[0139] Using positive and / or negative selection of cell surface markers expressed on harvested γδ T cells, γδ T cells or γδ T cell populations expressing similar cell surface markers can be isolated directly from peripheral blood samples, umbilical cord blood samples, tumors, tumor biopsies, tissues, lymph, or from epithelial samples of a subject. For example, γδ T cells can be isolated from composite samples based on positive or negative expression of CD2, CD3, CD4, CD8, CD24, CD25, CD44, Kit, TCRα, TCRβ, TCRα, TCRδ, NKG2D, CD70, CD27, CD30, CD16, CD337 (NKp30), CD336 (NKp46), OX40, CD46, CCR7, and other suitable cell surface markers.

[0140] FIG. 1 illustrates γδ T-cell production according to one embodiment of the present disclosure. The process may include harvesting or obtaining leukocytes or PBMCs from a leukapheresis product. Leukapheresis may include harvesting whole blood from a donor and separating the components using an apheresis device. The pheresis device separates the desired blood components and the remainder is returned to the donor's circulation. For example, white blood cells, plasma, and platelets may be harvested using an apheresis device, and red blood cells and neutrophils are returned to the donor's circulation. A commercially available leukapheresis product may be used in this process. Another way to obtain leukocytes is to obtain them from the buffy coat. To isolate the buffy coat, anticoagulated whole blood is obtained from a donor and centrifuged. After centrifugation, the blood is separated into plasma, red blood cells, and buffy coat. The buffy coat is a layer located between the plasma layer and the red blood cell layer. Leukapheresis harvesting may result in greater purity and a greater increase in mononuclear cell content than can be achieved by buffy coat harvesting. The mononuclear cell content possible by leukapheresis may typically be 20-fold that obtained from buffy coat. The use of a Ficoll gradient for further separation may be necessary to enrich for mononuclear cells.

[0141] To deplete αβT cells from PBMCs, αβTCR expressing cells may be separated from PBMCs, for example by magnetic separation using CliniMACS® magnetic beads coated with anti-αβTCR antibodies, followed by cryopreservation of αβTCR-T cell depleted PBMCs. To produce an "off-the-shelf" T cell product, cryopreserved αβTCR-T cell depleted PBMCs may be thawed and activated in the presence of an aminobisphosphonate, e.g., zoledronate; and / or isopentenyl pyrophosphate (IPP); and / or cytokines, e.g., interleukin 2 (IL-2), interleukin 15 (IL-15), and / or interleukin 18 (IL-18); and / or other activating agents, e.g., Toll-like receptor 2 (TLR2) ligands, for 1-10 days, e.g., 2-7 days, on a small / medium scale, e.g., in 24 to 4-6 well plates or T75 / T175 flasks, or on a larger scale, e.g., in 50 ml to 100 liter bags.

[0142] Figure 1 shows that activated T cells may be engineered by transducing isolated γδ T cells with a viral vector, such as a lentiviral vector, expressing an exogenous gene of interest, such as an αβ TCR for a specific cancer antigen and CD8. Transduction may be performed once or multiple times, on a small scale, e.g., in 24- to 4- to 6-well plates, or on a medium / large scale, e.g., over ½- to 5-day periods, e.g., 1 day, to achieve stable transgene expression.

[0143] Figure 1 further illustrates that expansion of transduced or engineered γδ T cells may be carried out in small / medium scale, e.g., flasks / G-Rex, or in large scale, e.g., 50ml-100 liter bags, in the presence of cytokines, e.g., IL-2, IL-15, IL-18, for a period of, e.g., 7-35 days, e.g., 7-28 days. The expanded transduced T cell product may then be cryopreserved as an "off the shelf" T cell product for infusion into patients.

[0144] Example 3 Lentivirus vectors The lentiviral vectors used herein contain several elements previously shown to enhance vector function, including a central polypurine tract (cPPT) to improve self-replication and nuclear import; a promoter from murine stem cell virus (MSCV) (SEQ ID NO: 1), which has been shown to reduce vector silencing in some cell types; and a woodchuck hepatitis virus post-transcriptional response element (WPRE) (SEQ ID NO: 9) to improve transcription termination, and the backbone was a deleted 3'-LTR self-inactivating (SIN) vector design that may have improved safety, sustained gene expression, and anti-silencing properties (Yang et al. Gene Therapy (2008) 15, 1411-1423, the entire contents of which are incorporated by reference).

[0145] In one aspect, the vectors, constructs, or sequences described herein comprise a mutated form of the WPRE. In another aspect, the sequences or vectors described herein comprise a mutation in WPRE version 1, e.g., WPREmut1 (SEQ ID NO: 265), or WPRE version 2, e.g., WPREmut2 (SEQ ID NO: 266). In one aspect, the WPRE mutant comprises at most one mutation, at most two mutations, at most three mutations, at least four mutations, or at most five mutations. In one aspect, the vectors, constructs, or sequences described herein do not comprise a WPRE. In another aspect, the disclosure provides for 1, 2, 3, 4, 5, 10, or 20 substitutions in one of SEQ ID NOs: 91-96.

[0146] In another embodiment, the vectors, constructs, or sequences described herein do not comprise an X protein promoter.

[0147] To obtain optimal co-expression levels of TCRαβ and CD8αβ in transduced γδ T cells, lentiviral vectors of various designs were generated. Figure 2 shows that T cells may be transduced with two separate lentiviral vectors expressing TCRαβ or CD8αβ (2-in-1), and with a single lentiviral vector co-expressing TCRαβ and CD8αβ (4-in-1). In the 4-in-1 vector, the nucleotides encoding the TCRα chain, TCRβ chain, CD8α chain, and CD8β chain may be shuffled in various orders. The various 4-in-1 vectors thus generated may be used to transduce γδ T cells, followed by measuring the TCR / CD8 co-expression levels of the transduced cells using techniques known in the art, such as, for example, flow cytometry.

[0148] To generate lentiviral vectors that co-express TCRαβ and CD8αβ, nucleotides encoding the furin linker (GSG or SGSG (SEQ ID NO: 8))-2A peptide may be placed between the TCRα and TCRβ chains, between the CD8α and CD8β chains, or between the TCR and CD8 chains to allow for highly efficient gene expression. The 2A peptide may be selected from P2A (SEQ ID NO: 3), T2A (SEQ ID NO: 4), E2A (SEQ ID NO: 5), or F2A (SEQ ID NO: 6).

[0149] Lentiviral vectors may also contain a post-transcriptional regulatory element (PRE), such as the woodchuck PRE (WPRE) (SEQ ID NO: 9), to enhance transgene expression by increasing both nuclear and cytoplasmic mRNA levels. One or more regulatory elements, including the mouse RNA transport element (RTE), the constitutive transport element (CTE) of simian retrovirus type 1 (SRV-1), and the 5' untranslated region of human heat shock protein 70 (Hsp70 5'UTR), may also be used and / or combined with the WPRE to increase transgene expression.

[0150] Lentiviral vectors may be pseudotyped with RD114TR (SEQ ID NO: 97), a chimeric glycoprotein that contains the extracellular and transmembrane domains of feline endogenous virus (RD114) fused to the cytoplasmic tail (TR) of murine leukemia virus. Other viral envelope proteins, such as VSV-G env, MLV4070A env, RD114 env, chimeric envelope protein RD114pro, baculovirus GP64 env, or GALV env, or derivatives thereof, may also be used.

[0151] 3 shows four different 4-in-1 vectors co-expressing TCRαβ(R11KEA) and CD8αβ, namely PTE WPRE (SEQ ID NO: 91), TPE WPRE (SEQ ID NO: 92), PTE fn WPRE (SEQ ID NO: 93), and PTE CD8 TCR WPRE (SEQ ID NO: 94), and two 2-in-1 vectors, namely R11KE WPRE (SEQ ID NO: 95) expressing TCRαβ(R11KEA) and CD8 WPRE (SEQ ID NO: 96) expressing CD8αβ. TCRαβ(R11KEA) binds to PRAME-004(SLLQHLIGL)(SEQ ID NO: 147) in a complex with an MHC molecule.

[0152] Example 4 Co-expression of TCR and CD8 γδ T cells obtained from donor 1 and donor 2 were produced as shown in Figure 1. On day 3 or 6 after activation with zoledronate, IL2, and IL15, γδ T cells were transduced with lentivirus pseudotyped with RD114TR, e.g., PTE WPRE (SEQ ID NO: 91), TPE WPRE (SEQ ID NO: 92), PTE fn WPRE (SEQ ID NO: 93), and PTE CD8 TCR WPRE (SEQ ID NO: 94), followed by measuring the co-expression levels of R11KEA and CD8 using flow cytometry. Antibodies (CD8) specific for TCR (Vβ8) and CD8 (CD8α) were used to assess transduction efficiency via flow cytometry.

[0153] Figure 4 shows that in γδ T cells from donor 1, the co-expression levels of R11KEA and CD8 obtained by transduction with PTE CD8 TCR WPRE, i.e., 40.5% (day 3) and 18.5% (day 6), are higher than those obtained by transduction with PTE WPRE (29.6% (day 3), 16.2% (day 6)), TPE WPRE (30.8% (day 3), 11.0% (day 6)), and PTE fn WPRE (33.0% (day 3), 15.0% (day 6)). In γδ T cells from donor 2, the co-expression level of R11KEA and CD8 at day 6 post activation obtained by transduction with PTE WPRE, i.e. 18.8%, is higher than that obtained by transduction with TPE WPRE (14.2%), PTE fn WPRE (14.7%) and PTE CD8 TCR WPRE (17.2%). As a control, background levels of R11KEA and CD8 were detected in γδ T cells transduced separately with the 2-in-1 vectors, i.e. TCRαβ(R11KEA) or CD8 WPRE.

[0154] Example 5 The effect on transgene expression and functionality of 4-in-1 viral vectors, e.g., lentiviral vectors, in which the sequences encoding the CD8αβ chain and the TCRαβ chain are placed in different locations within the vector.

[0155] WO 2019 / 204662 describes CD4+ cells expressing an exogenous CD8αβ co-receptor and one or more exogenously engineered antigen receptors, such as, for example, TCRs. Table 4 shows a comparison of the 4-in-1 constructs described in WO 2019 / 204662 with those according to embodiments of the present disclosure.

[0156] [Table 6]

[0157] The open reading frame (ORF) of the nucleic acid molecule of the present disclosure may be at least partially codon-optimized. Codon optimization is based on the discovery that translation efficiency may be determined by the different frequencies of occurrence of transfer RNAs (tRNAs) in a cell. Thus, the open reading frame of the nucleic acid molecule of the present disclosure may be modified compared to the corresponding wild-type coding region such that at least one codon of the wild-type sequence encoding a tRNA that is relatively rare in a cell may be replaced with a codon that encodes a tRNA that is relatively frequent in a cell and may carry the same amino acid as the relatively rare tRNA. By this modification, the open reading frame of the nucleic acid molecule of the present disclosure may be modified such that a codon available for a frequent tRNA may replace a codon that corresponds to the rare tRNA. In other words, according to the present disclosure, by such modification, all codons of the wild-type open reading frame encoding a rare tRNA may be replaced with a codon that encodes a tRNA that is relatively frequent in a cell and may carry the same amino acid as the relatively rare tRNA. Those skilled in the art know which tRNAs are relatively frequent in cells and, in contrast, which tRNAs are relatively rare; see, for example, Akashi, Curr. Opin. Genet. Dev. 2001, 11(6):660-666, the contents of which are incorporated by reference. In some embodiments, the open reading frame of the nucleic acid molecule of the present disclosure may be codon-optimized, preferably for the system in which the nucleic acid molecule of the present disclosure is expressed, preferably for the system in which the nucleic acid molecule of the present disclosure is translated. Preferably, the open reading frame of the nucleic acid molecule of the present disclosure may be codon-optimized according to mammalian codon usage, more preferably according to human codon usage. Preferably, the open reading frame may be codon-optimized and G / C content may be corrected.

[0158] To determine which transgene orientation provides better transgene expression and function, γδ T cells were transduced with three 4-in-1 viral vectors each containing a sequence encoding the TCR αβ chain located upstream of a sequence encoding the CD8 αβ chain, e.g., PTE.WPRE (sequence number 91), TPE.WPRE (sequence number 92), and PTE.fn.WPRE (sequence number 93), and a 4-in-1 viral vector containing a sequence encoding the CD8 αβ chain located upstream of a sequence encoding the TCR αβ chain, e.g., PTE.CD8.TCR.WPRE (sequence number 94), followed by fluorescence-activated cell sorting (FACS) analysis using fluorescently tagged anti-CD8 and fluorescently tagged anti-TCRVβ8 (Vb8) antibodies to detect CD8 and TCR expression on the cell surface, respectively.

[0159] Figures 10 and 11 show that γδ T cells obtained from donor 3 and donor 4 transduced with a 4-in-1 viral vector containing PTE.CD8.TCR.WPRE provide the highest expression of both CD8 and TCR on the cell surface on day 14 of manufacture compared to those transduced with a 4-in-1 viral vector containing PTE.WPRE, TPE.WPRE, or PTE.fn.WPRE based on % of CD8+Vb8+ double-positive cells (Figure 10) and MFI of CD8 or MFI of Vb8 (Figure 11).

[0160] High expression of both CD8 and TCR on the cell surface of γδ T cells transduced with a 4-in-1 viral vector containing PTE.CD8.TCR.WPRE correlates well with their in vitro killing activity. For example, FIG. 12 shows that γδ T cells obtained from donor 3 transduced with 4-in-1 viral vectors containing PTE.CD8.TCR.WPRE exhibited the best killing activity against both high target peptide presenting cell line UACC257 (upper panel) and low target peptide presenting cell line U2OS (lower panel) compared to those transduced with 4-in-1 viral vectors containing PTE.WPRE, TPE.WPRE, or PTE.fn.WPRE, and FIGS. 13A-13C show the amount of IFN-γ secretion by corresponding γδ T cells transduced with 4-in-1 viral vectors containing PTE.CD8.TCR.WPRE, PTE.WPRE, TPE.WPRE, or PTE.fn.WPRE in the presence of target cells such as UACC257 (FIG. 13A), U2OS (FIG. 13B), and the target negative cell line MCF-7 (FIG. 13C).

[0161] FIG. 14 shows that γδ T cells obtained from donor 4 transduced with the 4-in-1 containing viral vector PTE.CD8.TCR.WPRE also exhibited the best killing activity against both UACC257 (upper panel) and U2OS (lower panel) compared to those transduced with 4-in-1 viral vectors containing PTE.WPRE, TPE.WPRE, or PTE.fn.WPRE, and FIGS. 15A-15C show the amount of IFN-γ secretion by corresponding γδ T cells transduced with the 4-in-1 viral vectors containing PTE.CD8.TCR.WPRE, PTE.WPRE, TPE.WPRE, or PTE.fn.WPRE in the presence of target cells such as UACC257 (FIG. 15A), U2OS (FIG. 15B), and MCF-7 (FIG. 15C).

[0162] Figure 16 shows that γδ T cells from donor 3 and donor 4 transduced with a 4-in-1 viral vector containing PTE.CD8.TCR.WPRE yield less than 0.6 copies of integrated vector per cell, similar to those transduced with PTE.WPRE, TPE.WPRE, and PTE.fn.WPRE. This low copy number of integrated vector per cell is within the safety requirement, i.e., less than 5 copies of integrated vector per cell.

[0163] Figures 17A and 17B show that γδ T cells obtained from donor 3 and donor 4, respectively, transduced with a 4-in-1 viral vector containing PTE.CD8.TCR.WPRE achieve comparable levels of cell proliferation at day 14 of manufacture as those transduced with 4-in-1 viral vectors containing PTE.WPRE, TPE.WPRE, or PTE.fn.WPRE.

[0164] To determine the memory cell phenotype of transduced γδ T cells, cells were stained with allophycocyanin (APC)-Cy7-tagged anti-CD45RA and BV421-tagged anti-CCR7 antibodies, followed by FACS analysis to determine the percentage of Tcm, naive T cells, TemRA, and Teff present in the transduced γδ T cells. Figure 18A shows an example of such an analysis.

[0165] FIG. 18B shows that γδ T cells from donor 3 and donor 1 transduced with 4-in-1 viral vectors containing PTE.CD8.TCR.WPRE achieve comparable levels of memory T cell phenotype to those transduced with 4-in-1 viral vectors containing PTE.WPRE, TPE.WPRE, or PTE.fn.WPRE on day 14 of manufacture.

[0166] Example 6 Effect on transgene expression in cells transduced with one 4-in-1 viral vector versus two 2-in-1 viral vectors

[0167] Figure 19 shows that more CD8+TCR+ γδ T cells were obtained from transduction with a 4-in-1 lentiviral vector containing PTE.CD8.TCR.WPRE (120 μl) than from transduction with a mixture of 2-in-1 lentiviral vectors containing CD8.WPRE (120 μl) and R11KE.WPRE (120 μl) (panel B, 20.9%). On the other hand, more CD8+TCR- γδ T cells were obtained from transduction with a mixture of 2-in-1 lentiviral vectors containing CD8.WPRE (120 μl) and R11KE.WPRE (120 μl) than from transduction with a 4-in-1 lentiviral vector containing PTE.CD8.TCR.WPRE (panel B, 21.2%). Similarly, more CD8+TCR+ γδ T cells were obtained from transduction with a 4-in-1 lentiviral vector containing PTE.CD8.TCR.WPRE (240 μl) than from transduction with a mixture of 2-in-1 lentiviral vectors containing CD8.WPRE (240 μl) and R11KE.WPRE (240 μl) (panel C, 27.7%). On the other hand, more CD8+TCR- γδ T cells were obtained from transduction with a mixture of 2-in-1 lentiviral vectors containing CD8.WPRE (240 μl) and R11KE.WPRE (240 μl) than from transduction with a 4-in-1 lentiviral vector containing PTE.CD8.TCR.WPRE (panel C, 24.4%). Non-transduced (NT) γδ T cells served as a control (Panel A). Two-color staining was performed using APC-tagged anti-CD8β antibody and phycoerythrin (PE)-tagged target peptide / MHC complex tetramer.These results suggest that transduction with a 4-in-1 lentiviral vector containing sequences encoding CD8αβ and TCRαβ, such as PTE.CD8.TCR.WPRE, may result in a greater number of CD8+TCR+ T cells than transduced with a mixture of a 2-in-1 lentiviral vector containing sequences encoding CD8αβ, such as CD8.WPRE, and a 2-in-1 lentiviral vector containing sequences encoding TCRαβ, such as R11KE.WPRE. On the other hand, transduction with a mixture of a 2-in-1 lentiviral vector containing sequences encoding CD8αβ, such as CD8.WPRE, and a 2-in-1 lentiviral vector containing sequences encoding TCRαβ, such as R11KE.WPRE, may result in a greater number of CD8+TCR- T cells than transduced with a 4-in-1 lentiviral vector containing sequences encoding CD8αβ and TCRαβ, such as PTE.CD8.TCR.WPRE.

[0168] Figure 20 shows that increasing the amount of 4-in-1 viral vector containing PTE.CD8.TCR.WPRE for transduction, e.g., 30μl, 120μl, 240μl, improves transduction efficiency, e.g., % of CD8+TCR+γδ T cells increases, e.g., 9.6% at 30μl, 20.9% at 120μl, 27.7% at 240μl. Non-transduced γδ T cells serve as control. Two-color staining was performed using APC-tagged anti-CD8β antibody and PE-tagged target peptide / MHC complex tetramer.

[0169] Example 7 Expression of 4-in-1 constructs in αβ T cells For example, engineered lymphocytes, including engineered αβ T cells expressing recombinant proteins such as CD8αβ and / or TCRαβ, may be produced according to the methods disclosed in US 2019 / 0247433, the entire contents of which are incorporated by reference. For example, FIG. 37 shows a T cell production process 370, which may include: isolation of PBMCs (371), in which the PBMCs may be used fresh or may be stored frozen until ready for use, or may be a leukopak, e.g., leukophaeresis product, or may be used as the starting material for T cell production and selection of lymphocyte populations (e.g., αβTCR+ T cells, CD8+, CD4+, or both); thawing and resting of lymphocytes (372), e.g., overnight, e.g., for about 16 hours or about 4 to 6 hours, which may kill apoptotic cells and restore T cell function (this step may not be necessary if fresh material is used); activation of lymphocytes (373), which may include thawing and resting (374), in which the PBMCs are frozen until ready for use, or may be a leukopak, e.g., leukophaeresis product, or may be used as the starting material for T cell production and selection of lymphocyte populations (e.g., αβTCR+ T cells, CD8+, CD4+, or both); 373) (which may use anti-CD3 and anti-CD28 antibodies (soluble or surface-bound, e.g., magnetic or biodegradable beads, antibodies immobilized to the culture vessel); transduction with viral vectors containing sequences encoding polypeptide recombinant proteins, e.g., CD8αβ and / or TCRαβ (374) (wherein the viral vectors may be lentiviral or retroviral vectors, or transfection may be performed by non-viral methods); expansion, harvesting, and cryopreservation of lymphocytes (375) (which may be performed in the presence of, e.g., cytokines, e.g., IL-7 and IL-15, serum (ABS or FBS), and / or cryopreservation medium).

[0170] Exogenous CD8 expression To determine exogenous CD8 expression in αβ T cells transduced with a viral vector containing a 4-in-1 construct encoding CD8 and TCR, T cells obtained from donor 5 and donor 6 were transduced while increasing the amount of LV-PTE.CD8.TCR.WPRE, and following the lymphocyte < singlet < live cell < CD3+ population, % CD8α+ cells in the CD4+ cells were detected. Figure 21 shows that % CD8α+ CD4+ cells from donor 5 increased from 2.87% (non-transduced) to 14.7% (2.5 μl / 1×10 6 cells), 19.5% (5 μl / 1×10 6 cells), 21.7% (7.5 μl / 1×10 6 cells), and 24.1% (10 μl / 1×10 6 cells); and % CD8α+ CD4+ cells from donor 6 increased from 1.93% (non-transduced) to 12.5% (2.5 μl / 1×10 6 cells), 17.2% (5 μl / 1×10 6 cells), 19.6% (7.5 μl / 1×10 6 cells), and 20.8% (10 μl / 1×10 6 cells).

[0171] Exogenous TCR expression To determine exogenous TCR expression in αβ T cells transduced with a viral vector containing a 4-in-1 construct encoding CD8 and TCR, T cells obtained from donor 5 and donor 6 were transduced while increasing the amount of LV-PTE.CD8.TCR.WPRE, and following the lymphocyte < singlet < live cell < CD3+ < CD4+CD8+ population, % target peptide / MHC complex dextramer 203+ (i.e., TCR+) cells in the CD4+CD8+ cell population were detected. Figure 22 shows that % dextramer 203+ cells from donor 5 increased from 0.32% (non-transduced) to 41.9% (2.5 μl / 1×10 6 cells), 48.3% (5 μl / 1×10 6 cells), 54.5% (7.5 μl / 1×106 cells), and 49.5% (10 μl / 1×10 6 cells) in donor 6; the % dextramer 203+ cells from donor 6 increased from 0.19% (untransduced) to 35.5% (2.5 μl / 1×10 6 cells), 41.2% (5 μl / 1×10 6 cells), 44.6% (7.5 μl / 1×10 6 cells), and 44.0% (10 μl / 1×10 6 cells), indicating an increase.

[0172] To detect TCR expression in various αβ T cell populations, αβ T cells transduced with LV-PTE.CD8.TCR.WPRE were analyzed by FACS gating on lymphocytes < singlet < live cells < CD3+ < CD4+ / -CD8+ / -). Figure 23 shows that the % dextramer 203 (Dex203)+ (i.e., TCR+) cells obtained from donor 5 (upper panel) and donor 6 (lower panel) are generally higher in the CD4+CD8α+ cell population than in the CD4-CD8α+ cell population. In contrast, the % Dex203 (i.e., TCR) cells are minimal in the CD4CD8α- cell population. Similarly, Figure 24 shows that the % dextramer 203 (Dex203) MFI cells obtained from donor 5 (upper panel) and donor 6 (lower panel) are generally higher in the CD4+CD8α+ cell population than in the CD4-CD8α+ cell population. In contrast, the % Dex203 MFI is minimal in the CD4CD8α- cell population. These results suggest that the exogenous TCR and CD8 encoded by LV-PTE.CD8.TCR.WPRE can be co-expressed simultaneously in both CD4 T cells and CD4- T cells.

[0173] Example 8 Functional analysis of αβ T cells expressing a 4-in-1 construct or a TCR-only construct FIG. 25 shows the experimental design for testing the functionality of αβ T cells transduced with lentiviral vectors (LV), e.g., containing a 4-in-1 construct (LV-CD8.TCR) such as PTE.CD8.TCR.WPRE, or a TCR-only construct (LV-TCR) such as R11KE.WPRE. Briefly, on day -1, target cells such as high antigen expressing UACC257+RFP cell line (positive control) and antigen-negative MCF7+GFP cell line (negative control) were seeded in 96-well plates. LV-CD8.TCR (5 μl / 1×10 6 cells) or LV-TCR (2.5 μl / 1 × 10 6 Donor cell products, e.g., PBMCs (obtained from donors 5, 6, 7, 8) transduced with 100,000 cells each, were thawed and rested overnight in 24-well G-Rex® gas-permeable rapid growth devices. On day 0, donor cell products (effector cells) were co-cultured with target cells, e.g., UACC257+RFP and MCF7+GFP, at an effector to target cell (E / T) ratio of 2:1, e.g., 200,000 effector cells:100,000 target cells. After 5 hours of incubation at 37°C, a protein transport inhibitor, e.g., GolgiStop™ (BD Biosciences), was added to each well at 0.5 μl / well, followed by 4 hours of incubation at 37°C. The cells were then centrifuged and the supernatant collected for ELISA to detect IFN-γ expression, and the cells were collected for staining using an intracellular cytokine staining (ICS) panel, e.g., CD3, CD4, CD8, IFN-γ, Granzyme B, and Live / Dead.

[0174] CD4-CD8+ T cell population FIG. 26 shows that co-culture of CD4-CD8α+ T cells from grouped donors transduced with LV-TCR (TCR) or LV-CD8.TCR (TCR+CD8) with high target expressing UACC257 cells resulted in higher %IFN-γ positive cells (upper panel) and higher IFN-γ MFI (lower panel) than non-transduced (NT). In contrast, when CD4-CD8α+ T cells transduced with LV-TCR (TCR) or LV-CD8.TCR (TCR+CD8) were co-cultured with antigen-negative MCF7, no significant difference was observed in %IFN-γ positive cells and IFN-γ MFI between transduced and non-transduced cells. FACS was gated on CD4-CD8α+IFN-γ+ T cells. Non-transduced (NT) cells served as a control. (Effector to target cell ratio = 2:1; N donors per group = 4). These results suggest that CD4-CD8α+ T cells transduced with LV-TCR or LV-CD8.TCR are functionally activated, e.g., express IFN-γ, upon contact with high antigen-expressing target cells, e.g., UACC257 cells, whereas the transduced cells have little effect on antigen-negative cells, e.g., MCF7 cells.

[0175] FIG. 27 shows that co-culture of CD4-CD8α+ T cells from grouped donors transduced with LV-TCR (TCR) or LV-CD8.TCR (TCR+CD8) with high target expressing UACC257 cells resulted in higher % granzyme B positive cells (upper panel) and higher granzyme B MFI (lower panel) than non-transduced (NT). In contrast, when CD4-CD8α+ T cells transduced with LV-TCR (TCR) or LV-CD8.TCR (TCR+CD8) were co-cultured with antigen-negative MCF7, no significant difference was observed in % granzyme B positive cells and granzyme B MFI between transduced and non-transduced cells. FACS was gated on CD4-CD8α+ granzyme B+ T cells. Non-transduced (NT) cells served as a control. (Effector to target cell ratio = 2:1; N donors per group = 3). These results suggest that CD4-CD8α+ T cells transduced with LV-TCR or LV-CD8.TCR are functionally activated, e.g., express granzyme B, upon contact with high antigen-expressing target cells, e.g., UACC257 cells, and that the transduced cells may have little effect on antigen-negative cells, e.g., MCF7 cells.

[0176] CD4-CD8+ T cell population FIG. 28 shows that co-culture of CD4+CD8α+ T cells from grouped donors transduced with LV-CD8.TCR (TCR+CD8) with high target expressing UACC257 cells resulted in higher %IFN-γ positive cells (upper panel) and higher IFN-γ MFI (lower panel) than non-transduced (NT). In contrast, when CD4-CD8α+ T cells transduced with thLV-CD8.TCR (TCR+CD8) were co-cultured with antigen-negative MCF7, no significant difference was observed in %IFN-γ positive cells and IFN-γ MFI between transduced and non-transduced cells. FACS gated on CD4+CD8α-IFN-γ+ for NT cells and CD4+CD8α+IFN-γ+ for LV-CD8.TCR-transduced cells. Non-transduced (NT) cells served as control. (Effector to target cell ratio = 2:1; N donors per group = 4). These results suggest that CD4+CD8α+Tc cells transduced with LV-CD8.TCR are functionally activated, e.g., express IFN-γ, upon contact with high antigen-expressing target cells, e.g., UACC257 cells, and that the transduced cells may have little effect on antigen-negative cells, e.g., MCF7 cells.

[0177] FIG. 29 shows that co-culture of CD4+CD8α+ T cells from grouped donors transduced with LV-CD8.TCR (TCR+CD8) with high target expressing UACC257 cells resulted in higher % granzyme B positive cells (upper panel) and higher granzyme B MFI (lower panel) than non-transduced (NT). In contrast, when LV-CD8.TCR (TCR+CD8) transduced CD4+CD8α+ T cells were co-cultured with antigen-negative MCF7, no significant difference was observed in % granzyme B positive cells and granzyme B MFI between transduced and non-transduced cells. FACS gated on CD4+CD8α- granzyme B+ for NT cells and CD4+CD8α+ granzyme B+ for LV-CD8.TCR transduced T cells. Non-transduced (NT) cells served as control. (Effector to target cell ratio = 2:1; N donors per group = 3). These results suggest that CD4+CD8α+ T cells transduced with LV-CD8.TCR are functionally activated, e.g., express granzyme B, upon contact with high antigen-expressing target cells, e.g., UACC257 cells, and that the transduced cells may have little effect on antigen-negative cells, e.g., MCF7 cells.

[0178] CD3+T cells FIG. 30 shows that co-culture of CD3+ T cells from grouped donors transduced with LV-TCR (TCR) or LV-CD8.TCR (TCR+CD8) with high target expressing UACC257 cells resulted in higher %IFN-γ positive cells (upper panel) and higher IFN-γ MFI (lower panel) than non-transduced (NT). In contrast, when CD4-CD8α+ T cells transduced with LV-TCR (TCR) or LV-CD8.TCR (TCR+CD8) were co-cultured with antigen-negative MCF7, no significant difference was observed in %IFN-γ positive cells and IFN-γ MFI between transduced and non-transduced cells. FACS was gated on CD3+ T cells. Non-transduced (NT) cells serve as a control. (Effector to target cell ratio=2:1; grouped donors N=4). These results suggest that CD3+ T cells transduced with LV-TCR or LV-CD8.TCR are functionally activated, e.g., by expressing IFN-γ, upon contact with highly antigen-expressing target cells, e.g., UACC257 cells, and that the transduced cells have little effect on antigen-negative cells, e.g., MCF7 cells.

[0179] FIG. 31 shows that co-culture of CD3+ T cells from grouped donors transduced with TCR (TCR) or LV-CD8.TCR (TCR+CD8) with high target expressing UACC257 cells resulted in higher % granzyme B positive cells (upper panel) and higher granzyme B MFI (lower panel) than non-transduced (NT). In contrast, when CD3+ T cells transduced with LV-TCR (TCR) or LV-CD8.TCR (TCR+CD8) were co-cultured with antigen-negative MCF7, no significant difference was observed in % granzyme B positive cells and granzyme B MFI between transduced and non-transduced cells. FACS was gated on CD3+ T cells. Non-transduced (NT) cells served as a control. (Effector to target cell ratio=2:1; grouped donors N=3). These results suggest that CD3+ T cells transduced with LV-TCR or LV-CD8.TCR are functionally activated, e.g., by expressing granzyme B, upon contact with highly antigen-expressing target cells, e.g., UACC257 cells, and that the transduced cells may have little effect on antigen-negative cells, e.g., MCF7 cells.

[0180] Figure 32 shows that co-culture of CD3+ T cells from group donors transduced with LV-TCR (TCR) or LV-CD8.TCR (TCR+CD8) with high target expressing UACC257 cells results in higher secretion of IFN-γ than non-co-transduced (NT), MCF7 cells alone, or UACC257 cells alone. In contrast, when CD4-CD8α+ T cells transduced with LV-TCR (TCR) or LV-CD8.TCR (TCR+CD8) were co-cultured with antigen negative MCF7, no significant difference was observed in the level of IFN-γ secretion between transduced and non-transduced cells. (Effector to target cell ratio = 2:1; group donors N = 4). These results suggest that CD3+ T cells transduced with LV-TCR or LV-CD8.TCR are functionally activated, e.g., by secreting IFN-γ, upon contact with highly antigen-expressing target cells, e.g., UACC257 cells, and that the transduced cells have little effect on antigen-negative cells, e.g., MCF7 cells.

[0181] Figure 33 shows that co-culture of CD3+ T cells from individual donors 5, 6, 7, and 8 transduced with LV-TCR (TCR) or LV-CD8.TCR (TCR+CD8) with high target expressing UACC257 cells secreted more IFN-γ than non-co-transduced (NT), MCF7 cells alone, or UACC257 cells alone. In contrast, when CD4-CD8α+ T cells transduced with LV-TCR (TCR) or LV-CD8.TCR (TCR+CD8) were co-cultured with antigen-negative MCF7, no significant difference in the level of IFN-γ secretion was observed between transduced and non-transduced cells (effector to target cell ratio = 2:1). These results suggest that CD3+ T cells from individual donors transduced with LV-TCR or LV-CD8.TCR are functionally activated, e.g., by secreting IFN-γ, upon contact with highly antigen-expressing target cells, e.g., UACC257 cells, whereas the transduced cells have little effect on antigen-negative cells, e.g., MCF7 cells.

[0182] Example 9 Effect of statins on the expression of T cell activation markers To determine the effect of statins on the expression of T cell activation markers, T cells were treated with statins, e.g., atorvastatin, pravastatin, rosuvastatin, etc., followed by FACS analysis to measure the expression of T cell activation markers, e.g., CD25, CD69, and hLDLR.

[0183] CD4+ T cell population FIG. 34 shows CD25+ cells (upper panel), %CD69+ cells (middle panel), and %hLDLR+ cells (lower panel) in CD3+CD4+ T cells treated with atorvastatin, pravastatin, or rosuvastatin. Preactivated cells, cells activated without statins or DMSO (control), and DMSO serve as controls. These results show that statins such as atorvastatin, for example, may increase %CD4+hLDLR+ cells, while atorvastatin, pravastatin, and rosuvastatin have little effect on %CD4+CD25+ cells and %CD4+CD69+ cells. FACS gated on lymphocytes>singlets>live / dead>CD3+>CD4+.

[0184] CD8+ T cell population FIG. 35 shows %CD25+ cells (upper panel), %CD69+ cells (middle panel), and %hLDLR+ cells (lower panel) in CD3+CD8+ T cells treated with atorvastatin, pravastatin, or rosuvastatin. Preactivated cells, cells activated without statins or DMSO (control), and DMSO serve as controls. These results show that statins such as atorvastatin, for example, may increase %CD8+hLDLR+ cells, while atorvastatin, pravastatin, and rosuvastatin have little effect on %CD8+CD25+ cells and %CD8+CD69+ cells. FACS gated on lymphocytes>singlets>live / dead>CD3+>CD8+.

[0185] Example 10 Effect of WPRE on lentiviral titers To determine the effect of WPRE on lentiviral titer, lentiviral vectors (LV) containing wild type (wt) WPRE (SEQ ID NO: 9) (LV-A), no WPRE (LV-B), WPREmut1 (SEQ ID NO: 265) (LV-C), or WPREmut2 (SEQ ID NO: 266) (LV-D) were generated. HEK293T cells were transfected with LV-A, LV-B, LV-C, or LV-D, followed by titer determination using methods known in the art. Figure 36 shows that the titers of these lentiviral vectors are in the order LV-C>LV-D≧LV-A>LV-B. These results suggest that WPREmut1 and WPREmut2 may be useful for improving lentiviral vector production.

[0186] Advantages of the present disclosure may include the generation of viral vectors that co-express multiple transgenes, e.g., four polypeptides, in a single vector, and the generation of γδ T cells that co-express TCRαβ and CD8αβ as a safe, target-specific, "off-the-shelf" T cell product for adoptive cell therapy.

[0187] All references mentioned herein are incorporated by reference as if each reference was specifically and individually indicated to be incorporated by reference. The citation of any reference is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such reference by virtue of prior invention.

Claims

1. 1. A vector comprising a nucleotide sequence S1 encoding a CD8 α polypeptide, a nucleotide sequence S2 encoding a CD8 β polypeptide, a nucleotide sequence S3 encoding a T cell receptor (TCR) α polypeptide, and a nucleotide sequence S4 encoding a TCR β polypeptide, wherein said nucleotide sequences are arranged in tandem in a 5' to 3' direction selected from S1-S2-S3-S4, S1-S2-S4-S3, S2-S1-S3-S4, S2-S1-S4-S3, S3-S4-S1-S2, S3-S4-S2-S1, S4-S3-S1-S2, and S4-S3-S2-S1.

2. 2. The vector of claim 1, wherein the nucleotide sequences are arranged in tandem in a 5' to 3' direction selected from S1-S2-S3-S4, S1-S2-S4-S3, S2-S1-S3-S4, and S2-S1-S4-S3.

3. 3. The vector of claim 2, wherein the nucleotide sequences are arranged in tandem in the 5' to 3' direction of S2-S1-S4-S3.

4. The vector according to any one of claims 1 to 3, wherein the CD8αβ polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:11 and at least 90% identical to SEQ ID NO:

12.

5. The TCRαβ polypeptide is selected from the group consisting of SEQ ID NOs: 13 and 14, 15 and 16, 17 and 18, 19 and 20, 21 and 22, 23 and 24, 25 and 26, 27 and 28, 29 and 30, 31 and 32, 33 and 34, 35 and 36, 37 and 38, 39 and 40, 41 and 42, 43 and 44, 45 and 46, 47 and 48, 49 and 50, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, 71 and 72, 73 and 74, 75 and 76, 77 and 78, 79 and 80, 81 and 82, 82 and 83, 84 and 85, 86 and 87, 88 and 89, 90 and 91, 92 and 93, 94 and 95, 96 and 97, 98 and 99, 99 and 100, 101 and 102, 103 and 104, 105 and 106, 106 and 107, 108 and 109, 109 and 101, 109 and 102, 109 and 103, 110 and 114, 111 and 115, 112 and 116, 113 and 117, 114 and 118, 5. The vector of claim 1, comprising an amino acid sequence that is at least 90% identical to an amino acid sequence selected from: 9 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, 71 and 72, 73 and 74, 75 and 76, 77 and 78, 79 and 80, 81 and 82, 83 and 84, 85 and 86, 87 and 88, or 89 and 90.

6. The vector of any one of claims 1 to 5, wherein the TCRαβ polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:13 and at least 90% identical to SEQ ID NO:

14.

7. 7. The vector of claim 1, further comprising a nucleotide sequence S5 encoding a 2A peptide and a nucleotide sequence S6 encoding a linker peptide, wherein S5 and S6 are positioned between S1 and S2, S1 and S3, S1 and S4, S2 and S3, S2 and S4, and / or S3 and S4.

8. 8. The vector of claim 7, wherein the 2A peptide is selected from P2A (SEQ ID NO: 3), T2A (SEQ ID NO: 4), E2A (SEQ ID NO: 5), or F2A (SEQ ID NO: 6).

9. 9. The vector of claim 7 or 8, wherein the linker peptide is GSG or SGSG (SEQ ID NO: 8).

10. 10. The vector of any one of claims 1 to 9, further comprising a nucleotide sequence S7 encoding a furin peptide (SEQ ID NO:2), wherein S7 is positioned between S1 and S2, S1 and S3, S1 and S4, S2 and S3, S2 and S4, and / or S3 and S4.

11. 1. A vector comprising a first nucleotide sequence S1 encoding a protein Z1, a second nucleotide sequence S2 encoding a protein Z2, a third nucleotide sequence S3 encoding a protein Y1, and a fourth nucleotide sequence S4 encoding a protein Y2, wherein Z1 and Z2 form a first dimer and Y1 and Y2 form a second dimer, said first dimer Z1Z2 being structurally distinct from said second dimer Y1Y2, and wherein said vector is a gene delivery system used for adaptive cell therapy.

12. Said S1, S2, S3, and S4 are S1-S2-S3-S4, S1-S2-S4-S3, S1-S3-S2-S4, S1-S3-S4-S2, S1-S4-S3-S2, S1-S4-S2- S3, S2-S1-S3-S4, S2-S1-S4-S3, S2-S3-S1-S4, S2-S3-S4-S1, S2-S4-S3-S1, S2-S4-S1-S3, S3-S1-S2-S4, S 12. The vector of claim 11, wherein the 5' to 3' orientation of the ribozymes is selected from 3-S1-S4-S2, S3-S2-S1-S4, S3-S2-S4-S1, S3-S4-S1-S2, S3-S4-S2-S1, S4-S1-S2-S3, S4-S1-S3-S2, S4-S2-S1-S3, S4-S2-S3-S1, S4-S3-S1-S2, or S4-S3-S2-S1.

13. 13. The vector of claim 11 or 12, further comprising a fifth nucleotide sequence S5 encoding a 2A peptide and a sixth nucleotide sequence S6 encoding a linker peptide, wherein S5 and S6 are positioned between S1 and S2, S1 and S3, S1 and S4, S2 and S3, S2 and S4, and / or S3 and S4.

14. 14. The vector of claim 13, wherein the 2A peptide is selected from P2A (SEQ ID NO: 3), T2A (SEQ ID NO: 4), E2A (SEQ ID NO: 5), or F2A (SEQ ID NO: 6).

15. 15. The vector of claim 13 or 14, wherein the linker peptide is GSG or SGSG (SEQ ID NO: 8).

16. 16. The vector of any one of claims 11 to 15, further comprising a seventh nucleotide sequence S7 encoding a furin peptide (SEQ ID NO:2), wherein S7 is positioned between S1 and S2, S1 and S3, S1 and S4, S2 and S3, S2 and S4, and / or S3 and S4.

17. 17. The vector of any one of claims 11 to 16, further comprising a post-transcriptional regulatory element (PRE) sequence selected from a woodchuck PRE (WPRE) or a Hepatitis B virus (HBV) PRE (HPRE).

18. 18. The vector according to claim 11, further comprising a promoter sequence controlling transcription of S2 to S7, wherein the promoter sequence is selected from a cytomegalovirus (CMV) promoter, a phosphoglycerate kinase (PGK) promoter, a myelin basic protein (MBP) promoter, a glial fibrillary acidic protein (GFAP) promoter, a modified MoMuLV LTR containing myeloproliferative sarcoma virus enhancer (MNDU3), a ubiquitin C promoter, an EF-1α promoter, or a murine stem cell virus (MSCV) promoter.

19. The first dimer Z1Z2 is selected from the group consisting of SEQ ID NOs: 13 and 14, 15 and 16, 17 and 18, 19 and 20, 21 and 22, 23 and 24, 25 and 26, 27 and 28, 29 and 30, 31 and 32, 33 and 34, 35 and 36, 37 and 38, 39 and 40, 41 and 42, 43 and 44, 45 and 46, 47 and 48, 49 and 50, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 62 and 63, 63 and 64, 64 and 65, 65 and 66, 66 and 67, 67 and 68, 68 and 69, 70 and 71, 72 and 73, 74 and 75, 76 and 77, 78 and 79, 80 and 81, 82 and 82, 83 and 84, 85 and 86, 87 and 89, 88 and 90, 89 and 91, 92 and 93, 94 and 95, 95 and 96, 96 and 97, 97 and 98, 98 and 99, 100 and 101, 102 and 103, 103 and 104, 104 and 105, 105 and 106, 106 and 107, 108 and 109, 109 and 108, 109 and 109, 19. The vector of any one of claims 11 to 18, selected from: 4, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, 71 and 72, 73 and 74, 75 and 76, 77 and 78, 79 and 80, 81 and 82, 83 and 84, 85 and 86, 87 and 88, or 89 and 90.

20. The vector according to any one of claims 11 to 19, wherein the second dimers Y1 and Y2 are SEQ ID NOs: 11 and 12.

21. The vector according to any one of claims 11 to 20, wherein the orientation is S2-S1-S4-S3.

22. 22. The vector of claim 21, comprising a sequence selected from PTE WPRE (SEQ ID NO:91), TPE WPRE (SEQ ID NO:92), or PTE fn WPRE (SEQ ID NO:93).

23. The vector according to any one of claims 11 to 20, wherein the orientation is S4-S3-S2-S1.

24. 24. The vector of claim 23, comprising the sequence PTE CD8 TCR WPRE (SEQ ID NO: 94).

25. 25. The vector of any one of claims 11 to 24, which is a viral vector selected from an adenovirus, a poxvirus, an alphavirus, an arenavirus, a flavivirus, a rhabdovirus, a retrovirus, a lentivirus, a herpesvirus, a paramyxovirus, or a picornavirus.

26. 26. The vector of claim 25, wherein the vector is pseudotyped with an envelope protein of a virus selected from the natural feline endogenous virus (RD114), a chimeric version of RD114 (RD114TR), gibbon ape leukemia virus (GALV), a chimeric version of GALV (GALV-TR), amphotropic murine leukemia virus (MLV4070A), baculovirus (GP64), vesicular stomatitis virus (VSV-G), fowl plague virus (FPV), Ebola virus (EboV), or baboon retrovirus envelope glycoprotein (BaEV), lymphocytic choriomeningitis virus (LCMV).

27. Isolating T cells from a blood sample of a human subject; activating the isolated T cells in the presence of an aminobisphosphonate; transducing the activated T cells with a vector according to any one of claims 11 to 26; Expanding the transduced T cells.

2. A method for preparing T cells for immunotherapy comprising:

28. 28. The method of claim 27, wherein the T cells are isolated from a leukapheresis human sample.

29. 29. The method of claim 27 or 28, wherein the aminobisphosphonate is selected from pamidronic acid, alendronic acid, zoledronic acid, risedronic acid, ibandronic acid, incadronic acid, salts of any of the foregoing and / or hydrates thereof.

30. The method according to any one of claims 27 to 29, wherein the activating step is further carried out in the presence of human recombinant interleukin 2 (IL-2) and human recombinant interleukin 15 (IL-15).

31. The method of any one of claims 27 to 30, wherein the expanding step is carried out in the presence of IL-2 and IL-15.

32. The method of any one of claims 27 to 31, wherein the T cells are γδ T cells.

33. The method of any one of claims 27 to 32, wherein the first dimer Z1Z2 and the second dimer Y1Y2 are co-expressed on the surface of the expanded T cells.

34. 33. An expanded population of T cells prepared by the method of any one of claims 27 to 32.

35. 35. A method of treating a patient with cancer comprising administering to the patient a composition comprising the expanded T cell population of claim 34, the T cells kill cancer cells that present the peptide on their surface in complex with an MHC molecule; the peptide is selected from SEQ ID NOs: 98-255; The method, wherein the cancer is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, melanoma, liver cancer, breast cancer, uterine cancer, Merkel cell carcinoma, pancreatic cancer, gallbladder cancer, bile duct cancer, colorectal cancer, urinary bladder cancer, kidney cancer, leukemia, ovarian cancer, esophageal cancer, brain cancer, gastric cancer, and prostate cancer.

36. 36. The method of claim 35, wherein the composition further comprises an adjuvant.

37. 37. The method of claim 36, wherein the adjuvant is selected from anti-CD40 antibodies, imiquimod, resiquimod, GM-CSF, cyclophosphamide, sunitinib, bevacizumab, atezolizumab, interferon-α, interferon-β, CpG oligonucleotides and derivatives, poly-(I:C) and derivatives, RNA, sildenafil, particulate formulations with poly(lactide-co-glycolide) (PLG), virosomes, interleukin (IL)-1, IL-2, IL-4, IL-7, IL-12, IL-13, IL-15, IL-21, and IL-23.

38. 35. A method of generating an immune response in a patient with cancer comprising administering to the patient a composition comprising the expanded T cell population of claim 34, the T cells kill cancer cells that present the peptide on their surface in complex with an MHC molecule; the peptide is selected from SEQ ID NOs: 98-255; The method, wherein the cancer is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, melanoma, liver cancer, breast cancer, uterine cancer, Merkel cell carcinoma, pancreatic cancer, gallbladder cancer, bile duct cancer, colorectal cancer, urinary bladder cancer, kidney cancer, leukemia, ovarian cancer, esophageal cancer, brain cancer, gastric cancer, and prostate cancer.

39. 39. The method of claim 38, wherein the composition further comprises an adjuvant.

40. 40. The method of claim 39, wherein the adjuvant is selected from anti-CD40 antibodies, imiquimod, resiquimod, GM-CSF, cyclophosphamide, sunitinib, bevacizumab, atezolizumab, interferon-α, interferon-β, CpG oligonucleotides and derivatives, poly-(I:C) and derivatives, RNA, sildenafil, particulate formulations with poly(lactide-co-glycolide) (PLG), virosomes, interleukin (IL)-1, IL-2, IL-4, IL-7, IL-12, IL-13, IL-15, IL-21, and IL-23.

41. The method of any one of claims 38 to 40, wherein the immune response comprises a cytotoxic T cell response.

42. activating the T cells in the presence of a statin; Transducing the activated T cells with a vector according to any one of claims 11 to 26, which is pseudotyped with the envelope protein of VSV-G; Expanding the transduced T cells.

2. A method for preparing T cells for immunotherapy comprising:

43. 43. The method of claim 42, wherein the T cells comprise αβ T cells, γδ T cells, and / or natural killer T cells.

44. 44. The method of claim 42 or 43, wherein the statin is selected from atorvastatin, cerivastatin, dalvastatin, fluindostatin, fluvastatin, mevastatin, pravastatin, simvastatin, velostatin, and rosuvastatin.

45. activating the T cells; transducing the activated T cells with a vector according to any one of claims 11 to 26; Expanding the transduced T cells.

2. A method for preparing T cells for immunotherapy comprising:

46. 46. ​​The method of claim 45, wherein the T cells comprise αβ T cells, γδ T cells, and / or natural killer T cells.

47. 47. The method of claim 45 or 46, wherein the T cells comprise αβ T cells.

48. 48. The method of claim 47, wherein the activating step is carried out in the presence of an anti-CD3 antibody and an anti-CD28 antibody.

49. 49. The method of claim 47 or 48, wherein the expanding step is carried out in the presence of IL-7 and IL-15.

50. 50. An expanded population of T cells prepared by the method of any one of claims 42 to 49.

51. 51. A method of treating a patient with cancer comprising administering to the patient a composition comprising the expanded T cell population of claim 50, the T cells kill cancer cells that present the peptide on their surface in complex with an MHC molecule; the peptide is selected from SEQ ID NOs: 98-255; The method, wherein the cancer is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, melanoma, liver cancer, breast cancer, uterine cancer, Merkel cell carcinoma, pancreatic cancer, gallbladder cancer, bile duct cancer, colorectal cancer, urinary bladder cancer, kidney cancer, leukemia, ovarian cancer, esophageal cancer, brain cancer, gastric cancer, and prostate cancer.

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