Engineered gamma delta T cells and methods for making and using same - Patent Application 20070122997

Abstract

Aspects of the present disclosure relate to methods and compositions related to the preparation of immune cells, including engineered T cells that contain at least one exogenous γδ T cell receptor, such as one selected to target a particular disease or pathogen (e.g., cancer or COVID-19). T cells can be generated from human hematopoietic stem / progenitor cells and are suitable for allogeneic cell therapy because they do not induce graft-versus-host disease (GvHD) and resist host immune allorejection. Thus, such cells are suitable for off-the-shelf use in clinical treatments.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of co-pending and commonly assigned U.S. Provisional Patent Application No. 63 / 131,170, filed December 28, 2020, and entitled "ENGINEERED GAMMA DELTA(γδ)T CELLS AND METHODS OF MAKING AND USING THEREOF," which is incorporated herein by reference.

[0002] Technical Field Embodiments of the present disclosure relate to at least the fields of immunology, cell biology, molecular biology, and medicine. [Background technology]

[0003] Background of the Invention Gamma delta (γδ) T cells are a small subpopulation of T lymphocytes with the ability to bridge innate and adaptive immunity. The majority of γδ T cells in adult blood display Vγ9Vδ2 T cell receptors and respond to small, phosphorylated, non-peptide antigens, called phosphoantigens (pAgs), commonly produced by malignant cells (see, e.g., Yang et al., Immunity 50, 1043-1053.e5 (2019)). Unlike conventional αβ T cells, γδ T cells do not recognize polymorphic classical major histocompatibility complex (MHC) molecules and therefore pose no risk of graft-versus-host disease (GvHD) when adoptively transferred into an allogeneic host. Furthermore, γδ T cells possess several other unique characteristics that make them ideal cell carriers for developing off-the-shelf cell therapies for cancer. These features include: 1) γδ T cells have a role in cancer immune surveillance; 2) γδ T cells have a remarkable ability to target tumors independent of tumor antigen and major histocompatibility complex (MHC) restriction; 3) γδ T cells can use multiple mechanisms to attack tumor cells through direct killing and adjuvant effects; and 4) γδ T cells express the surface receptor FcγRIII (CD16), which participates in antibody-dependent cellular cytotoxicity (ADCC) and can potentially be combined with monoclonal antibodies for cancer therapy (see, e.g., Lepore et al., Front. Immunol. 9, 1-11 (2018); Harrer et al., Hum. Gene Ther. 29, 547-558 (2018); and Presti et al., Front. Immunol. 8, 1-11 (2017)). Unfortunately, however, the development of allogeneic, off-the-shelf γδ T cell products has been largely hindered by their availability; these cells are present in extremely low and highly variable numbers in humans (approximately 1-5% T cells in human blood), making it extremely difficult to generate therapeutic numbers of γδ T cells using allogeneic human donor blood cells (see, e.g., Silva-Santos et al., Nat. Rev. Immunol. 15, 683-691 (2015)). Traditional methods for generating γδ T cells, particularly the Vγ9Vδ2 subset, for adoptive therapy involve in vitro or in vivo expansion of peripheral blood mononuclear cells (PBMC)-derived γδ T cells using aminobisphosphonates such as zoledronic acid (ZOL). However, this methodology results in highly variable yields of γδ T cells depending on the PBMC donor. Most importantly, such γδ T cell products typically contain bystander αβ T cells, thereby posing a risk of GvHD (see, e.g., Torikai et al., Mol. Ther. 24, 1178-1186 (2016)). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Yang et al., Immunity 50, 1043-1053.e5(2019) [Non-patent document 2] Lepore et al., Front. Immunol. 9, 1-11 (2018) [Non-patent document 3] Harrer et al., Hum. Gene Ther. 29, 547-558 (2018) [Non-patent document 4] Presti et al., Front. Immunol. 8, 1-11 (2017) [Non-Patent Document 5] Silva-Santos et al., Nat.Rev.Immunol.15, 683-691 (2015) [Non-patent document 6] Torikai et al., Mol.Ther.24, 1178-1186 (2016) Summary of the Invention

[0005] Summary of the Invention Novel methods and materials that can reliably generate large quantities of homogenous monoclonal populations of γδ T cells using feeder-free differentiation systems are crucial for developing "off-the-shelf" γδ T cell therapies useful for treating a wide variety of pathological conditions. In particular, the ability to engineer cells that can be used to produce therapeutic γδ T cell populations would increase the availability and utility of new cell therapies. Embodiments of the present invention are provided to address the need for new cell therapies, more specifically, cell therapies that are not hindered by the challenges posed by personalized therapy using autologous cells.

[0006] As disclosed herein, the inventors have discovered that pluripotent cells (e.g., CD34 + We have discovered that engineered γδ T cells can be generated by γδ TCR genetic engineering of γδ T cells (stem and progenitor cells), followed by selective differentiation of the engineered cells into transgenic γδ T cells in vivo or in vitro. As discussed below, such γδ T cells can be further engineered to co-express other disease-targeting molecules (e.g., chimeric antigen receptors, "CARs") as well as immune-modulating molecules (e.g., cytokines, receptors / ligands, etc.) to modulate their capabilities. Importantly, embodiments of these in vitro-differentiated γδ T cells can be used in allogeneic "off-the-shelf" cell therapies to treat a wide range of diseases (e.g., cancer, autoimmune diseases, infectious diseases, etc.).

[0007] Embodiments of the invention include materials and methods related to the gamma and delta chain polypeptides disclosed in Table 1 below. For example, embodiments of the invention include compositions comprising gamma and / or delta chain polypeptides having the amino acid sequences set forth in Table 1 (SEQ ID NOs: 1-52). Related embodiments of the invention include compositions comprising polynucleotides encoding gamma and / or delta chain polypeptides having the amino acid sequences set forth in Table 1 (SEQ ID NOs: 1-52). In certain embodiments of the invention, these polynucleotides are placed in a vector, e.g., an expression vector designed to express these gamma and delta chain polypeptides in cells. One such embodiment of the invention is a composition comprising immune cells transduced with an expression vector comprising a polynucleotide encoding at least one T cell receptor gamma and / or delta chain polypeptide having the amino acid sequence set forth in Table 1 (SEQ ID NOs: 1-52).

[0008] Embodiments of the present invention also include methods for generating engineered functional T cells that have been modified to contain at least one exogenous nucleic acid molecule encoding, for example, a T cell receptor gamma chain polypeptide and / or a T cell receptor delta chain polypeptide (e.g., as disclosed in Table 1). Typically, these methods involve generating pluripotent cells (e.g., human CD34 + The method comprises transducing human hematopoietic stem or progenitor cells (hematopoietic stem or progenitor cells) with at least one exogenous nucleic acid molecule encoding a T cell receptor gamma chain polypeptide and / or a T cell receptor delta chain polypeptide, such that the cells transduced with the exogenous nucleic acid molecule express a T cell receptor comprising a gamma chain polypeptide and a delta chain polypeptide, and then differentiating the transduced human cells to produce engineered functional gamma delta T cells.

[0009] Methodological embodiments of the present invention may include, for example, differentiating transduced pluripotent cells in vitro. In an exemplary method, transduced CD34 +Human hematopoietic stem or progenitor cells (HSPCs) can be differentiated in vitro in the absence of feeder cells and / or cultured in medium containing cytokines, such as one or more of IL-3, IL-7, IL-6, SCF, EPO, TPO, and FLT3L, and / or in the presence of agents selected to promote nucleic acid transduction efficiency, such as retronectin. In certain embodiments, the method further comprises contacting the transduced cells with an agonist antigen or other stimulatory agent. In some embodiments of the invention, the method further comprises co-culturing the transduced cells with peripheral blood mononuclear cells, antigen-presenting cells, or artificial antigen-presenting cells. Certain embodiments of the invention further comprise expanding in vitro pluripotent cells transduced with a nucleic acid molecule encoding a T cell receptor gamma chain polypeptide or a T cell receptor delta chain polypeptide. Alternative methods of the invention may include transplanting cells transduced with nucleic acid molecules encoding a T cell receptor gamma chain polypeptide and a T cell receptor delta chain polypeptide into a subject to generate a clonal population of engineered cells in vivo.

[0010] In some embodiments of the present invention, the engineered T cells comprise a gene expression profile characterized by at least one of the following gene expression profiles: HLA-I negative, HLA-II negative, HLA-E positive, and / or expression of a suicide gene. Optionally, the engineered T cells further comprise an exogenous T cell receptor nucleic acid molecule encoding a T cell receptor alpha chain polypeptide or a T cell receptor beta chain polypeptide, and / or an exogenous nucleic acid molecule encoding a cytokine, and / or a suppressed endogenous TCR. In certain embodiments of the present invention, the T cell receptor gamma chain polypeptide and / or T cell receptor delta chain polypeptide expressed by these engineered cells comprise the amino acid sequences set forth in Table 1 (SEQ ID NO: 1-SEQ ID NO: 52).

[0011] Embodiments of the present invention include engineered functional gamma delta T cells produced by the methods disclosed herein. For example, embodiments of the present invention include compositions comprising engineered T cells comprising a gene expression profile characterized by HLA-I negative, HLA-II negative, HLA-E positive, expression of a suicide gene, and expression of at least one exogenous T cell receptor gamma chain polypeptide and at least one exogenous T cell receptor delta chain polypeptide. In certain embodiments, the T cell receptor gamma chain polypeptide or the T cell receptor delta chain polypeptide comprises at least one amino acid sequence set forth in SEQ ID NO: 1-52. In some embodiments of the present invention, CD34 + HSPCs can be isolated from umbilical cord blood (CB) or peripheral blood. In such embodiments of the invention, CB CD34 + HSCs can be obtained from commercial suppliers (eg, HemaCare) or established CB banks.

[0012] Because the γδ T gamma / delta cell product is an off-the-shelf product that can be used to treat patients regardless of MHC restriction, once commercially available, this cell product will have broad application in a variety of potentially life-saving therapies. In this regard, yet another embodiment of the present invention is a method of treating a subject in need of gamma delta T cells (e.g., to combat a disease such as an autoimmune disease or cancer or an infectious disease such as COVID-19), comprising administering to the subject the engineered functional T cells disclosed herein.

[0013] Other objects, features, and advantages of the present invention will become apparent to those skilled in the art from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating certain embodiments of the present invention, are given by way of illustration and not limitation. Many changes and modifications within the scope of the present invention are possible without departing from the spirit of the invention, and the invention includes all such modifications. [Brief explanation of the drawings]

[0014] [Figures 1A-1C] Figure 1A-1C. Cloning of the human γδ TCR gene. Figure 1(A) shows the experimental design for cloning the human γδ TCR. Figure 1(B) shows fluorescence-activated cell sorting (FACS) of single human γδ T cells. Figure 1(C) shows a representative DNA gel image showing human TCR γ9 and δ2 chain PCR products from five sorted single γδ T cells.

[0015] [Figure 2A-2B] Figure 2A-2B. Schematic diagram of the Lenti / G115 and Lenti / γδT vectors. The designated pMNDW lentiviral vector for HSC-based gene therapy was selected to deliver the γδ TCR gene. Figure 2(A) shows the Lenti / G115 vector encoding the G115 γδ TCR gene. Figure 2(B) shows the Lenti / γδT vector encoding the selected γδ TCR gene. The Lenti / γδT vector encoding the LYγδ1 TCR gene (see Table 1) was used in the presented study.

[0016] [Figures 3A-3E]Figures 3A-3E. Functional characterization of cloned γδ TCRs. PBMC-T cells were transduced with Lenti / γδT vectors encoding the indicated γδ TCR chains (i.e., G115, γδ1) and analyzed for their TCR expression and functionality. Figure 3(A) is a representative FACS plot showing the expression of transgenic γδ TCRs on PBMC-T cells transduced with Lenti / γδT vectors. Figure 3(B) is a FACS analysis of intracellular IFN-γ production by PBMC-T cells transduced with Lenti / γδT vectors after ZOL stimulation. Figures 3(C-E) are tumor killing studies of PBMC-T cells transduced with Lenti / γδT vectors. Figure 3(C) is the experimental design. Figure 3(D) is in vitro tumor killing of a human melanoma cell line (A375-FG) by PBMC-T cells transduced with Lenti / γδT vectors. Figure 3(E) shows in vitro tumor killing of a human multiple myeloma cell line (MM.1S-FG) by PBMC-T cells transduced with the Lenti / γδT vector. Note that the parental A375 and MM.1S human tumor cell lines were engineered to express a firefly luciferase and green fluorescent protein dual reporter (FG). Data are presented as mean ± SEM. ns is not significant; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by one-way ANOVA.

[0017] [Figure 4A-4B] Figures 4A-4B. Generation of HSC-γδ T cells in the BLT-γδT humanized mouse model. Figure 4(A) shows the experimental design for generating HSC-γδ T cells in the BLT-γδT humanized mouse model. BLT, human bone marrow-liver-thymus-transplanted NOD.Cg-PrkdcscidIl2rgtm1Wjl / SzJ (NSG) mice. BLT-γδT, human γδ TCR-engineered BLT mice. Figure 4(B) shows FACS detection of HSC-γδ T cells in various tissues of BLT-γδT mice 25 weeks after HSC transfer. BLT mice that received mock-transduced CD34+ HSCs were included as controls (denoted as BLT-Mock).

[0018] [Figure 5A-5B] Figures 5A-5B. Generation of AlloHSC-γδ T cells in ATO culture. Figure 5(A) shows the experimental design for generating AlloHSC-γδ T cells in ATO culture. Figure 5(B) shows FACS plots showing the development of AlloHSC-γδ T cells at stage 1 and the proliferation of differentiated AlloHSC-γδ T cells at stage 2 from PBSCs.

[0019] [Figures 6A-6B] Figures 6A-6D. Generation of AlloHSC-γδ T cells in feeder-free ex vivo differentiation culture. CD34+ HSCs isolated from G-CSF-mobilized peripheral blood (PBSCs) or umbilical cord blood (CB HSCs) were transduced with a Lenti / γδT vector encoding the human γδ TCR gene and then placed into feeder-free ex vivo cell culture to generate AlloHSC-γδ T cells (Figures 6A and 6B). Both PBSCs and CB HSCs could effectively differentiate into and expand as transgenic AlloHSC-γδ T cells (Figures 6C and 6D). [Figure 6C-6D] Figures 6A-6D. Generation of AlloHSC-γδ T cells in feeder-free ex vivo differentiation culture. CD34+ HSCs isolated from G-CSF-mobilized peripheral blood (PBSCs) or umbilical cord blood (CB HSCs) were transduced with a Lenti / γδT vector encoding the human γδ TCR gene and then placed into feeder-free ex vivo cell culture to generate AlloHSC-γδ T cells (Figures 6A and 6B). Both PBSCs and CB HSCs could effectively differentiate into and expand as transgenic AlloHSC-γδ T cells (Figures 6C and 6D).

[0020] [Figures 7A-7B]Figures 7A-7D. CMC Study - AlloCAR-γδ T Cells. Figures 7(A-B) show the feeder-free ex vivo differentiation culture method for generating monoclonal AlloCAR-γδ T cells from PBSCs (Figure 7(A)) or umbilical cord blood (CB) HSCs (Figure 7(B)). Note the large number of AlloCAR-γδ T cells and their derivatives that can be generated from PBSCs or CB HSCs of a single random healthy donor. Figures 7(C-D) show the development of AlloCAR-γδ T cells at stage 1 and the expansion of differentiated AlloCAR-γδ T cells at stage 2 from PBSCs (Figure 7(C)) or CB HSCs (Figure 7(D)). [Figure 7C-7D] Figures 7A-7D. CMC Study - AlloCAR-γδ T Cells. Figures 7(A-B) show the feeder-free ex vivo differentiation culture method for generating monoclonal AlloCAR-γδ T cells from PBSCs (Figure 7(A)) or umbilical cord blood (CB) HSCs (Figure 7(B)). Note the large number of AlloCAR-γδ T cells and their derivatives that can be generated from PBSCs or CB HSCs of a single random healthy donor. Figures 7(C-D) show the development of AlloCAR-γδ T cells at stage 1 and the expansion of differentiated AlloCAR-γδ T cells at stage 2 from PBSCs (Figure 7(C)) or CB HSCs (Figure 7(D)).

[0021] [Figure 8A] Figure 8A-8B. Pharmacological testing of AlloHSC-γδ T cells. Figure 8(A) shows representative FACS plots demonstrating the phenotypic (surface markers) and functional (intracellular production of effector molecules) analysis of AlloHSC-γδ T cells. Endogenous human γδ T (PBMC-γδ T) cells and conventional αβ T (PBMC-T) cells isolated and expanded from healthy donor peripheral blood were included as controls. Figure 8(B) shows a representative FACS analysis of surface NK receptor expression by AlloHSC-γδ T cells. Endogenous PBMC-γδ T cells, PBMC-T, and PBMC-NK cells isolated and expanded from healthy donor peripheral blood were included as controls. [Figure 8B]Figure 8A-8B. Pharmacological testing of AlloHSC-γδ T cells. Figure 8(A) shows representative FACS plots demonstrating the phenotypic (surface markers) and functional (intracellular production of effector molecules) analysis of AlloHSC-γδ T cells. Endogenous human γδ T (PBMC-γδ T) cells and conventional αβ T (PBMC-T) cells isolated and expanded from healthy donor peripheral blood were included as controls. Figure 8(B) shows a representative FACS analysis of surface NK receptor expression by AlloHSC-γδ T cells. Endogenous PBMC-γδ T cells, PBMC-T, and PBMC-NK cells isolated and expanded from healthy donor peripheral blood were included as controls.

[0022] [Figures 9A-9E] Figures 9A-9E. In vitro efficacy and MOA testing of AlloHSC-γδ T cells. Figure 9(A) shows the experimental design for the in vitro tumor cell killing assay. Figure 9(B) shows the tumor-killing effect of AlloHSC-γδ T cells against A375-FG tumor cells (n=3). Figure 9(C) shows the tumor-killing effect of AlloHSC-γδ T cells against MM.1s-FG tumor cells (n=3). Figure 9(D) shows the tumor-killing effect of AlloHSC-γδ T cells against multiple human tumor cell lines (n=3). Figure 9(E) shows the human tumor cell lines tested in this study. Data are presented as mean ± SEM. ns indicates not significant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by one-way ANOVA. E:T is the effector-to-target ratio.

[0023] [Figures 10A-10C]Figures 10A-10C. In vivo antitumor efficacy and MOA testing of AlloHSC-γδ T cells in the A375-FG human melanoma xenograft NSG mouse model. Figure 10(A) is the experimental design. BLI, bioluminescence imaging of live animals. Figure 10(B) is BLI images showing tumor burden over time in experimental mice. Figure 10(C) is quantification of B (n=4). Data are presented as mean ± SEM. ns is not significant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by one-way ANOVA.

[0024] [Figures 11A-11D] Figures 11A-11D. In vitro efficacy and MOA testing of AlloBCAR-γδ T cells. Figure 11(A) shows the experimental design for the in vitro tumor cell killing assay. Figure 11(B) shows the tumor-killing effect of AlloBCAR-γδ T cells on A375-FG melanoma cells in the absence or presence of ZOL (n=3). Figure 11(C) shows the tumor-killing effect of AlloBCAR-γδ T cells on MM.1S-FG myeloma cells in the absence or presence of ZOL. BCAR-T cells, non-CAR-engineered PBMC-T cells, and AlloHSC-γδ T cells were included as controls (n=3). Figure 11(D) shows the triple mechanism that can be deployed by AlloBCAR-γδ T cells to target tumor cells, including the CAR-mediated pathway, the γδ TCR-mediated pathway, and the NK receptor-mediated pathway. Data are presented as mean ± SEM. ns is not significant, *P<0.05, **P<0.01, ****P<0.0001 by one-way ANOVA. E:T is effector-to-target ratio.

[0025] [Figures 12A-12D]Figures 12A-12D. In vivo antitumor efficacy of AlloBCAR-γδ T cells (n=8). Figure 12(A) is the experimental design. Figure 12(B) is a representative BLI image showing tumor burden in experimental mice over time. Figure 12(C) is quantification of B. Figure 12(D) is a Kaplan-Meier survival curve of experimental mice (n=8) over a 4-month period after tumor challenge. Data are presented as mean ± SEM. ns is not significant. ****p<0.0001 by one-way ANOVA in Figure 12(C) or log-rank (Mantel-Cox) test adjusted for multiple comparisons in Figure 12(D).

[0026] [Figures 13A-13D] Figures 13A-13D. In vivo antitumor efficacy study - AlloBCAR-γδ T cells in combination with ZOL treatment. Figure 13(A) is the experimental design. Figure 13(B) is BLI images showing tumor burden over time in experimental mice. Figure 13(C) is a quantification of 13B (n=3). Figure 13(D) is a quantification of tumor burden on day 39 after tumor challenge (n=3). Data are presented as mean ± SEM. ns is not significant, *P<0.05, **P<0.01, ****P<0.0001 by one-way ANOVA. E:T is the effector-to-target ratio.

[0027] [Figure 14A]Figures 14A-14F. CMC study and in vivo persistence of Allo15CAR-γδ T cells. Figure 14(A) shows a feeder-free ex vivo differentiation culture method for generating monoclonal Allo15CAR-γδ T cells from umbilical cord blood (CB) HSCs. Note the large number of Allo15CAR-γδ T cells that can be generated from CB HSCs from a single random healthy donor. Figure 14(B) shows the development of Allo15CAR-γδ T cells at stage 1 and the expansion of differentiated Allo15CAR-γδ T cells at stage 2 from CB HSCs. Figure 14(C) shows the experimental design for studying the in vivo dynamics of Allo / 15BCAR-γδ T cells. Note that Allo / 15BCAR-γδ T cells were labeled with the FG dual reporter. Figure 14(D) shows BLI images showing the presence of FG-labeled Allo / 15BCAR-γδ T cells over time in experimental mice. Figure 14(E) is the quantification (n=1-2) of D. Data are shown as mean ± SEM. [Figures 14B-14F] Figures 14A-14F. CMC study and in vivo persistence of Allo15CAR-γδ T cells. Figure 14(A) shows a feeder-free ex vivo differentiation culture method for generating monoclonal Allo15CAR-γδ T cells from umbilical cord blood (CB) HSCs. Note the large number of Allo15CAR-γδ T cells that can be generated from CB HSCs from a single random healthy donor. Figure 14(B) shows the development of Allo15CAR-γδ T cells at stage 1 and the expansion of differentiated Allo15CAR-γδ T cells at stage 2 from CB HSCs. Figure 14(C) shows the experimental design for studying the in vivo dynamics of Allo / 15BCAR-γδ T cells. Note that Allo / 15BCAR-γδ T cells were labeled with the FG dual reporter. Figure 14(D) shows BLI images showing the presence of FG-labeled Allo / 15BCAR-γδ T cells over time in experimental mice. Figure 14(E) is the quantification (n=1-2) of D. Data are shown as mean ± SEM.

[0028] [Figures 15A-15F]Figures 15A-15F. Immunogenicity studies. Figure 15(A) is an in vitro mixed lymphocyte culture (MLC) assay for studying GvH responses. Figure 15(B) is IFN-γ production from 15A (n=3). Donor-mismatched PBMC-T and PBMC-γδ T cells were included as controls. PBMCs from three mismatched healthy donors were used as stimulators. N, no PBMC stimulator. Figure 15(C) is an in vitro MLC assay for studying HvG responses. Figure 15(D) is IFN-γ production from C.PBMCs from three mismatched healthy donors tested as responders. Data from one representative donor are shown (n=3). Figures 15(E-F) are FACS analyses (n=3) of B2M / HLA-I and HLA-II expression on the indicated stimulator cells. Data are presented as mean ± SEM. ns is not significant, *P<0.05, **P<0.01, ****P<0.0001 by one-way ANOVA.

[0029] [Figure 16] Figure 16. Characterization of human γδ T cell products generated using various methods. Representative FACS plots showing the characteristics of human γδ T cells derived from human PBMC cultures and AlloHSC-γδ T cell cultures are shown. Tc = conventional αβ T cells.

[0030] [Figures 17A-17D] Figures 17A-17D. AlloHSC-γδ T cells directly target and kill SARS-CoV-2 infected cells. Figure 17(A) is a schematic diagram showing the engineered 293T-FG, 293T-ACE2-FG, and Calu3-FG cell lines. Figure 17(B) is FACS detection of ACE2 in 293T-FG, 293T-ACE2-FG, and Calu3-FG cells. Figures 17(C-D) are in vitro direct killing of SARS-CoV-2 infected or uninfected target cells by AlloHSC-γδ T cells (n=3). Data are presented as mean ± SSEM. ns is not significant; *P<0.05, **P<0.01, ****P<0.0001 by one-way ANOVA. DETAILED DESCRIPTION OF THE INVENTION

[0031] Detailed Description of the Invention In describing the embodiments, reference may be made to the accompanying drawings which form a part hereof, and which show, by way of illustration, specific embodiments in which the invention may be practiced. It is to be understood that other embodiments may be utilized and structural changes may be made without departing from the scope of the present invention.

[0032] Gamma delta (γδ) T cells typically account for 1–5% of peripheral blood lymphocytes in healthy individuals. Unlike classical αβ T cells, which recognize specific peptide antigens presented by major histocompatibility complex (MHC) molecules, γδ T cells can recognize general determinants expressed by cells that have become dysregulated as a result of either malignant transformation or viral infection. As a result, γδ T cells have the innate ability to recognize and kill a wide range of tumor cell types in a manner that does not require the presence of conventional tumor-specific antigens.

[0033] There is a need in the art for methods and materials that can reliably generate homogeneous monoclonal populations of various engineered human T cells, such as large quantities of engineered γδ T cells. These techniques are crucial for the development of off-the-shelf T cell therapies. Such methods and materials can provide γδ T cells that can be used in allogeneic or autologous recipient subjects for the treatment of various pathological conditions, including, for example, viral infections, fungal infections, protozoan infections, and cancer.

[0034] As discussed below, the present inventors have demonstrated that CD34 +We have discovered that engineered γδ T cells can be generated by genetically engineering γδ TCRs in pluripotent human cells, such as stem and progenitor cells (e.g., HSCs, iPSCs, ESCs), followed by selective differentiation of the engineered stem and progenitor cells into transgenic γδ T cells in vivo and / or in vitro. As is known in the art, hematopoietic stem or progenitor cells are multipotent, allowing them to self-renew and generate mature blood cells, such as erythrocytes, leukocytes, platelets, and lymphocytes. CD34 is a marker for human HSCs, and all colony-forming activity of human bone marrow (BM) cells is determined by CD34. + See, e.g., Mata et al., Transfusion. 2019 Dec;59(12):3560-3569. doi:10.1111 / trf.15597.

[0035] This finding is unexpected because the developmental pathway of gamma delta T cells is unique and distinct from that of other T cells, such as iNKT cells and αβ T cells (see, e.g., Dolens et al., EMBO Rep. 2020 May 6;21(5):e49006. doi:10.15252 / embr.201949006. Epub 2020 and Shissier et al., Mol. Immunol. 2019;105:116-130). Importantly, the in vitro differentiated γδ T cells disclosed herein can be used for allogeneic "off-the-shelf" cell therapy to treat a wide range of diseases (e.g., cancer, infection, autoimmunity, etc.). Furthermore, γδ T cells can also be engineered to co-express other disease-targeting molecules (e.g., CARs) as well as immune-modulating molecules (e.g., cytokines, receptors / ligands) to enhance their performance.

[0036] Embodiments of the present invention include methods of generating engineered functional T cells that have been modified to contain at least one exogenous nucleic acid molecule (e.g., disposed in an expression vector, such as a lentiviral vector, discussed below) encoding, for example, a T cell receptor gamma chain polypeptide and / or a T cell receptor delta chain polypeptide, e.g., a gamma chain polypeptide and / or a delta chain polypeptide having the amino acid sequence set forth in Table 1 (SEQ ID NO:1-SEQ ID NO:52). Typically, these methods involve transducing pluripotent human cells, such as hematopoietic stem / progenitor cells (i.e., pluripotent stem cells, hematopoietic stem cells, or hematopoietic progenitor cells), with at least one exogenous nucleic acid molecule encoding a T cell receptor gamma chain polypeptide and / or a T cell receptor delta chain polypeptide, such that the human cells transduced with the exogenous nucleic acid molecule express a T cell receptor comprising the gamma chain polypeptide and the delta chain polypeptide, and then differentiating the transduced human cells (e.g., hematopoietic stem / progenitor cells) to generate engineered functional gamma delta T cells. In certain methodological embodiments of the present invention, the T cell receptor gamma chain polypeptide and T cell receptor delta chain polypeptide encoded by the exogenous nucleic acid are selected as those known to form γδ T cell receptors previously observed to target cancer cells or cells infected with viruses, bacteria, fungi, or protozoa. Certain methods of the present invention include differentiating transduced human cells in in vitro culture and then further expanding these differentiated cells in in vitro culture. In some methodological embodiments of the present invention, expanding these differentiated cells in in vitro culture is performed under conditions selected to expand the differentiated population of transduced cells by at least 2-fold, 5-fold, 10-fold, or 100-fold. In some embodiments of the present invention, the engineered functional gamma delta T cells are exposed to zoledronic acid.

[0037] Methodological embodiments of the invention involve differentiating transduced pluripotent human cells (e.g., human hematopoietic stem or progenitor cells) in vitro or in vivo, and then expanding the differentiated cell population. In certain embodiments, the method further comprises contacting the transduced cells with a stimulatory agent, such as an agonist antigen. In some methodological embodiments of the invention, the population of γδ T cells is generated by a method disclosed herein that does not include a cell sorting step (e.g., FACS or magnetic bead sorting) after transducing the human cells with nucleic acids encoding gamma and delta polypeptides. In some embodiments of the invention, the method further comprises co-culturing the transduced cells with peripheral blood mononuclear cells, antigen-presenting cells, or artificial antigen-presenting cells. Typically, in these methods, transduced human cells are differentiated in vitro in the absence of feeder cells, and / or transduced hematopoietic stem or progenitor cells are cultured in medium containing cytokines such as one or more of IL-3, IL-7, IL-6, SCF, MCP-4, EPO, TPO, FLT3L, and / or agents selected to promote nucleic acid transduction efficiency, such as retronectin. Alternative methods of the invention may include transplanting cells transduced with nucleic acid molecules encoding T cell receptor gamma chain polypeptides and T cell receptor delta chain polypeptides into a subject (i.e., in vivo) to generate a clonal population of engineered cells.

[0038] In some methodological embodiments of the present invention, engineered T cells are selected to contain a specific gene expression profile characterized by, for example, at least one of the following gene expression profiles: HLA-I negative, HLA-II negative, HLA-E positive, and / or expression of a suicide gene. Typically, the engineered T cells further comprise one or more exogenous T cell receptor nucleic acid molecules encoding T cell receptor alpha chain polypeptides and T cell receptor beta chain polypeptides, and / or one or more exogenous nucleic acid molecules encoding cytokines, and / or a suppressed endogenous TCR. In some embodiments of the invention disclosed herein, the T cell receptor gamma chain polypeptide and the T cell receptor delta chain polypeptide comprise the amino acid sequences set forth in Table 1 below. In certain embodiments, the one or more additional nucleic acids encode one or more therapeutic gene products. Examples of therapeutic gene products include at least the following: 1. antigen recognition molecules, such as CARs (chimeric antigen receptors) and / or αβ TCRs (T cell receptors), γδ T receptors, etc.; 2. costimulatory molecules, such as CD28, 4-1BB, 4-1BBL, CD40, CD40L, ICOS; and / or 3. cytokines, such as IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-7, IL-9, IL-15, IL-12, IL-17, IL-21, IL-23, IFN-γ, TNF-α, TGF-β, G-CSF, GM-CSF; and 4. transcription factors, such as T-bet, GATA-3, RORγt, FOXP3, and Bcl-6. Therapeutic antibodies are included, as well as chimeric antigen receptors, single-chain antibodies, monobodies, humanized antibodies, antibodies, bispecific antibodies, single-chain FV antibodies, or combinations thereof.

[0039] Embodiments of the present invention also include materials and methods related to the gamma and delta chain polypeptides disclosed in Table 1 below. For example, embodiments of the present invention include compositions comprising gamma and / or delta chain polypeptides having the amino acid sequences shown in Table 1 (SEQ ID NOs: 1-52). Related embodiments of the present invention include compositions comprising polynucleotides encoding gamma and / or delta chain polypeptides having the amino acid sequences shown in Table 1 (SEQ ID NOs: 1-52). In certain embodiments of the present invention, these polynucleotides are placed in a vector, e.g., an expression vector designed to express these gamma and delta chain polypeptides in a cell (e.g., a mammalian cell). Compositions of the present invention may contain preservatives and / or antimicrobial agents, as well as pharmaceutically acceptable excipients required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, surfactants, and the like. For such compositions, the term "excipient" is meant to include, but is not limited to, ingredients set forth in Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, 21st ed. (2006).

[0040] Embodiments of the present invention further include engineered functional gamma delta T cells and populations of these cells produced by the methods disclosed herein. Typically, these populations consist essentially of functional gamma delta T cells (e.g., free of conventional αβ T cells). Embodiments of the present invention include compositions comprising the engineered γδ T cells or T cell populations disclosed herein, such as those comprising a gene expression profile characterized by HLA-I negative, HLA-II negative, HLA-E positive, expression of a suicide gene, and expression of exogenous T cell receptor gamma chain polypeptide and exogenous T cell receptor delta chain polypeptide. Optionally, the engineered T cells further comprise an exogenous nucleic acid molecule encoding another polypeptide, such as a T cell receptor alpha chain polypeptide and / or a T cell receptor beta chain polypeptide and / or an iNKT receptor polypeptide, and / or a cytokine, and / or comprise a suppressed endogenous TCR. Embodiments of the present invention also include compositions comprising immune cells transduced with an expression vector comprising a polynucleotide encoding at least one exogenous T cell receptor gamma chain polypeptide and / or T cell receptor delta chain polypeptide having the amino acid sequence set forth in Table 1 (SEQ ID NO:1-SEQ ID NO:52).

[0041] Methods of treating patients with the γδ T cells or cell populations disclosed herein are also provided. Embodiments of the present invention include methods of treating a subject in need of gamma delta T cells (e.g., to combat a disease such as an autoimmune disease or cancer or an infectious disease such as COVID-19), comprising administering to the subject engineered functional gamma delta T cells disclosed herein. In this manner, the engineered gamma delta T cells can be used to treat patients in need of therapeutic intervention. In some therapeutic embodiments of the present invention, the method comprises introducing one or more additional nucleic acids into gamma delta T cells, which may or may not have been previously frozen and thawed. This use provides one of the advantages of generating pre-made gamma delta T cells.

[0042] In certain therapeutic methods of the invention, the patient has been diagnosed with cancer. In some embodiments, the patient has a disease or condition involving inflammation, excluding cancer in some embodiments. In certain embodiments, the patient has an autoimmune disease or condition. In certain aspects, the cells or cell population are allogeneic with respect to the patient. In additional embodiments, the patient does not show signs of rejection or depletion of the cells or cell population. Some therapeutic methods further comprise administering to the patient a stimulatory molecule that activates γδ T cells (e.g., alone or loaded onto APCs) or a compound that elicits a suicide gene product.

[0043] Treatment of cancer patients with γδ T cells may result in the death of tumor cells in the cancer patient after administration of the cells or cell population to the patient. Treatment of inflammatory diseases or conditions may result in a reduction of inflammation. In other embodiments, patients with autoimmune diseases or conditions may experience an improvement in the symptoms of the disease or condition or may experience other therapeutic benefits from the γδ T cells. Combination treatment of γδ T cells with a standard treatment regimen or another immunotherapy regimen may be used.

[0044] As described below, the figures contained herein provide examples of some exemplary embodiments of the present invention, as well as data derived from such embodiments of the present invention.

[0045] For convenience of expression in this disclosure, we refer to the γ9δ2 TCR gene pair as γδ TCR genes. As shown in FIG. 1, each pair of γδ TCR genes comprises a gamma chain and a delta chain. In some embodiments, an engineered γδ T cell comprises a nucleic acid under the control of a heterologous promoter, meaning that the promoter is not the same genomic promoter that controls transcription of the nucleic acid. It is contemplated that an engineered γδ T cell comprises an exogenous nucleic acid comprising one or more coding sequences, some or all of which are under the control of a heterologous promoter in many embodiments described herein.

[0046] Figure 2 shows the construction of a lentiviral vector for delivering the γδ TCR gene. As shown in Figure 2, in an exemplary embodiment of the present invention, the pMNDW lentiviral vector was selected to deliver the γδ TCR gene. This vector contains the MND retroviral LTR U2 region as an internal promoter and an additional truncated woodchuck response element (WPRE) to stabilize viral mRNA, thus mediating high-level and stable expression of the transgene in human HSCs and their progeny human immune cells. The Lenti / γδT vector was constructed by inserting a synthetic bicistronic gene encoding human TCRγ9-T2A-TCRδ2 into pMNDW. Two plasmids expressing clones G115 and γδ1 from Table 1 were constructed using this strategy (Figure 2).

[0047] Figure 3 shows the functional characterization of the cloned γδ TCR. As shown in Figure 3, the gene delivery capability of the Lenti / γδ T vector (Figure 3A), as well as the functionality of its encoded γδ TCR, were studied by transducing primary human PBMC-derived conventional αβ T (denoted as PBMC-T) cells with the lentivector, followed by functional testing. Notably, this lentivector mediated efficient expression of the human γδ TCR transgene in PBMC-T cells (Figure 3B), and the resulting transgenic human γδ TCR responded to zoledronic acid (ZOL) stimulation, as evidenced by induced interferon (IFN)-γ production (Figure 3C) and enhanced tumor killing (Figure 3D-F) when the transduced PBMC-T cells were cocultured with human tumor cells.

[0048] Figure 4 shows the long-term in vivo provision of transgenic γδ T cells by adoptive transfer of γδ TCR-engineered HSCs. Increasing the number of functional γδ T cells in cancer patients can enhance antitumor immunity, which can potentially be achieved by adoptively transferring γδ TCR-engineered autologous HSCs into cancer patients. As shown in Figure 4, to demonstrate the feasibility of generating HSC-engineered γδ T cells in vivo, we isolated human CD34 T cells from G-CSF-mobilized healthy donor PBMCs (denoted as PBSCs). + After isolating HSCs and transducing them with the Lenti / γδT vector, the engineered HSCs were adoptively transferred into a BLT (bone marrow-liver-thymus) humanized mouse model. Large numbers of human HSC-γδT cells (e.g., >15% of total blood cells) were generated in the mice and detected in multiple tissues and organs over an 8-week period. High levels of transgenic HSC-γδT cells were maintained long-term for up to 6 months, as long as the experiment was performed.

[0049] Figure 5 shows the expression of allogeneic hematopoietic stem cell-engineered human γδ T (in artificial thymic organoid (ATO) cultures) for off-the-shelf cell therapy applications. Allo This figure shows the in vitro generation of HSC-γδ T cells. Autologous cell therapy has shown great promise in the treatment of both hematological cancers and solid tumors, but it has several limitations. Autologous cell production, particularly T cells collected from patients, is time-consuming, logistically challenging, and expensive. Furthermore, patients who undergo severe lymphopenic conditioning may not always be able to generate sufficient autologous cell products. There is a great need for allogeneic cell products that can be produced on a large scale and easily distributed to treat a wide range of cancer patients. As shown in Figure 5, embodiments of the present invention build on the HSC manipulation approach and develop two in vitro culture methods (feeder-dependent and feeder-independent cultures) to generate large numbers of pre-made human γδ T cells for allogeneic cell therapy applications.

[0050] Figure 6. Feeder-free ex vivo differentiation cultureAllo Generation of HSC-γδ T cells. As shown in Figure 6, CD34 T cells isolated from G-CSF-mobilized peripheral blood (denoted as PBSCs) or umbilical cord blood (denoted as CB HSCs) were used. + HSCs were transduced with a Lenti / γδT vector encoding the human γδ TCR gene and then placed into feeder-free ex vivo cell culture. Allo HSC-γδ T cells were generated (Figures 6A and 6B). Both PBSCs and CB HSCs were transgenic. Allo Efficient differentiation into HSC-γδT cells and transgenic Allo These cells can be expanded as HSC-γδ T cells (Figures 6C and 6D). Similarly, transduction of HSCs with a lentiviral vector encoding the human γδ TCR gene together with a CAR gene results in the development of HSC-γδ T cells. Allo CAR-γδ T cells can be generated (Figure 7). Approximately 10 13 Scale Allo It is estimated that HSC-γδ T cells can be generated from either PBSCs from healthy donors or HSCs from CB samples, which requires 10,000–100,000 doses (approximately 10 per dose). 8 ~10 9 Despite differences in proliferation folds, the cells generated from PBSCs and CB HSCs were Allo HSC-γδ T cells and their derivatives showed similar phenotypes and functionality. Unless otherwise indicated, CB HSC-derived Allo HSC-γδ T cells and their derivatives were utilized for the proof-of-principle studies described below. Figure 7 then shows that HSC-γδ T cells differentiate in feeder-free ex vivo cultures. Allo 1 shows the generation of CAR-γδT cells. Figure 8 shows Allo 1 shows data from a pharmacological study of HSC-γδ T cells. Allo The phenotype and functionality of HSC-γδ T cells were studied using flow cytometry (Figure 8). Three controls were identified: 1) HSC-γδ T cells isolated from peripheral blood of healthy donors (denoted as PBMC-γδ T cells), expanded in vitro by ZOL stimulation, and expressed CD3+ TCRVδ2 + 2) endogenous human γδ T cells identified as PBMC-T cells isolated from peripheral blood of healthy donors (denoted as PBMC-T cells) and expanded in vitro by anti-CD3 / CD28 stimulation to express CD3 + TCRαβ + 3) endogenous human conventional αβ T cells identified as CD3 T cells, isolated from peripheral blood of healthy donors (denoted as PBMC-NK cells), expanded in vitro with K562-based artificial antigen-presenting cell (aAPC) stimulation, and - CD56 + Endogenous human NK cells identified as Allo HSC-γδ T cells produced very high levels of multiple cytotoxic molecules (e.g., perforin and granzyme B) and expressed memory T cell markers CD27 and CD45RO, similar to those of endogenous γδ T cells (Figure 8A). Allo HSC-γδ T cells expressed high levels of NK activating receptors (e.g., NKG2D) and (e.g., DNAM-1) at levels similar to those of endogenous γδ T cells (Figure 8B). Allo HSC-γδ T cells expressed higher levels of NKp30 and NKp44 than endogenous γδ T cells (Fig. 8B ). Allo These findings suggest that HSC-γδ T cells may have a more potent NK pathway tumor-killing ability than endogenous γδ T cells and even endogenous NK cells.

[0051] Figure 9 shows data from in vitro efficacy and MOA testing of AlloHSC-γδ T cells. One of the most attractive features of γδ T cells is their ability to attack tumors via multiple mechanisms, including γδ TCR-mediated pathways and NK receptor-mediated pathways. Therefore, we established an in vitro tumor cell killing assay to test their tumor-killing ability (Figure 9A). We engineered a human tumor cell line to overexpress a firefly luciferase (Fluc) and enhanced green fluorescent protein (EGFP) dual reporter, allowing for sensitive measurement of tumor cell killing using luminescence readout or flow cytometry assays. In this study, multiple engineered human tumor cell lines were used as target cells, including a melanoma cell line (A375), a multiple myeloma cell line (MM.1S), a lung cancer cell line (H292-FG), a breast cancer cell line (MDA-MB-231), a prostate cancer cell line (PC3-FG), an ovarian cancer cell line (OVCAR3 and OVCAR8), and a leukemia cell line (K562) (Figure 9E). As expected, Allo HSC-γδ T cells by themselves effectively killed tumor cells via the NK pathway, and the tumor-killing effect could be further enhanced by the addition of ZOL, indicating the existence of a γδ TCR-mediated killing mechanism (Figures 9B, 9C, and 9D).

[0052] Figure 10 shows Allo Figure 10 shows data from an in vivo antitumor efficacy and MOA study of HSC-γδ T cells. As shown in Figure 10, we used a human ovarian cancer xenograft NSG mouse model to demonstrate Allo The in vivo antitumor efficacy of HSC-γδ T cells was evaluated. OVCAR3-FG tumor cells were inoculated intraperitoneally (ip) into NSG mice to allow tumor formation, followed by PBMC-NK or Allo HSC-γδ T cells were injected ip (Fig. 10A). Allo HSC-γδ T cells effectively suppressed tumor growth with efficacy similar to or even greater than that of PBMC-NK cells, as evidenced by time-lapse bioluminescence imaging (BLI) monitoring in living animals (Figures 10B and 10C).

[0053] Figure 11 shows Allo Data from in vitro efficacy and MOA testing of BCAR-γδ T cells are shown. As shown in Figure 11, allogeneic HSC-engineered B-cell maturation antigen (BCMA)-targeted CAR-modified γδ T cells ( Allo The tumor-aggressive potential of BCAR-γδT) cells was investigated using an established in vitro tumor-killing assay as previously described (Figure 11A). Two human tumor cell lines were investigated: 1) BCMA; + 1) a human MM cell line, MM.1S, which acts as a target for CAR-mediated killing; and 2) BCMA. - The human melanoma cell line, A375, which acts as a negative control target for CAR-mediated killing, was included in this study. Both human tumor cell lines were engineered to overexpress a firefly luciferase (Fluc) and enhanced green fluorescent protein (EGFP) dual reporter, and the resulting MM.1S-FG and A375-FG cell lines were then utilized in the study. Allo Similar to HSC-γδT cells, Allo BCAR-γδ T cells are able to target BCMA, likely via a CAR-independent NK killing pathway. - ZOL killed A375-FG cells with consistent efficacy, and the tumor-killing effect was further enhanced in the presence of ZOL, likely due to the addition of the gdTCR killing pathway (Figure 11B). More importantly, BCMA + When tested using the tumor line MM, Allo BCAR-γδ T cells effectively killed tumor cells with efficacy superior to that of HSC-γδ T cells and comparable to that of conventional BCAR-T cells (Figure 11C). Allo We provide evidence that BCAR-γδ T cells can target human tumor cells using three mechanisms: 1) the CAR-dependent pathway, 2) the γδ TCR-dependent pathway, and 3) the NK pathway (Figure 11D). Allo This unique triple targeting ability of BCAR-γδ T cells is attractive because it could potentially circumvent antigen escape, a phenomenon reported in autologous CAR-T treatment clinical trials in which tumor cells downregulated the expression of CAR-targeted antigens to escape attack by CAR-T cells.

[0054] Figure 12 shows Allo Data from an in vivo anti-tumor efficacy study of BCAR-γδ T cells are shown in Figure 12. Using an established MM.1S-FG xenograft NSG mouse model, Allo The in vivo antitumor efficacy of BCAR-γδ T cells was tested, and conventional BCAR-T cells were included as a control. Under low tumor burden conditions (Figure 12A), Allo BCAR-γδ T cells eliminated MM tumor cells as effectively as BCAR-T cells (Figures 12B and 12D). However, experimental mice treated with BCAR-T cells eventually died of graft-versus-host disease (GvHD) despite being tumor-free. Allo Experimental mice treated with BCAR-γδ T cells were tumor-free, did not develop GvHD, and survived for a long period of time (FIG. 12C).

[0055] Figure 13 shows the effect of ZOL treatment in combination with Allo 13 shows data from an in vivo antitumor efficacy study of BCAR-γδ T cells. As shown in FIG. 13, in combination with ZOL treatment Allo The in vivo antitumor efficacy of BCAR-γδ T cells was also tested using an established MM.1S-FG xenograft NSG mouse model under high tumor burden conditions. Allo To test the potential enhancement of the antitumor effect of BCAR-γδ T cells by γδ TCR stimulation, ZOL treatment was performed. Allo BCAR-γδ T cells significantly suppressed tumor growth (Figure 13A), and ZOL treatment further enhanced the efficacy (Figures 13B-13D). Allo These results suggest that the antitumor effects of BCAR-γδ T cells may be further enhanced. ZOL is a clinically available small molecule drug. Allo The possibility of a combined therapy of BCAR-γδ T cells and ZOL is feasible and attractive.

[0056] Figure 14 shows that IL-15 enhanced Allo BCAR-γδT cells ( Allo15IL-15 is a key cytokine that supports the in vivo persistence and functionality of many immune cells, including many subtypes of T cells and NK cells. Therefore, we have demonstrated that IL-15 is a key cytokine that supports the in vivo persistence and functionality of many immune cells, including many subtypes of T cells and NK cells. Allo We investigated the potential benefits of including CB-derived CD34 in BCAR T cell products. A Lenti / BCAR-IL15-γδT lentivector was constructed to co-deliver BCAR, IL-15, and γδ TCR genes (Figure 14A). + HSCs were transduced with the Lenti / BCAR-IL15-γδT vector and then placed into established feeder-free ex vivo HSC-γδT differentiation cultures (FIG. 14A). Allo yields similar to BCAR-γδ T cells Allo15 BCAR-γδ T cells were successfully generated (Figures 14A and 14B). Allo Enhanced by IL-15 compared with BCAR-γδ T cells Allo15 BCAR-γδ T cells demonstrated significantly improved in vivo persistence and, when encountering pre-established MM tumors, demonstrated significantly improved anti-tumor responses (e.g., in vivo clonal expansion, Figures 14C-14E).

[0057] Figure 15 shows Allo HSC-γδT and Allo Data from immunogenicity studies of BCAR-γδ T cells are shown. As shown in Figure 15, there are two immunogenicity concerns for allogeneic cell therapy: a) graft-versus-host (GvH) responses and b) host-versus-graft (HvG) responses. GvHD is a major safety concern. However, γδ T cells do not respond to mismatched HLA molecules and protein autoantigens, so they are not expected to induce GvHD. This concept is evidenced by the lack of GvHD in human clinical experience with allogeneic HSC transfer and autologous γδ T cell transfer, and is supported by our in vitro mixed lymphocyte culture (MLC) assay (Figure 15A). PBMC-γδ T cells also AlloNote that HSC-γδ T cells also failed to respond to allogeneic PBMCs, in stark contrast to conventional PBMC-T cells (Figure 15B). On the other hand, HvG risk is primarily an efficacy concern, mediated through host immune cell elimination of allogeneic therapeutic cells primarily by conventional CD8 and CD4 αβ T cells that recognize mismatched HLA-I and HLA-II molecules. Indeed, in an in vitro mixed lymphocyte culture (MLC) assay (Figure 15C), both conventional PBMC-T cells and PBMC-γδ T cells significantly induced responses from PBMC-T cells from multiple mismatched donors (Figure 15D). Interestingly, Allo HSC-γδ T cells may exhibit reduced immunogenicity and express lower levels of HLA-I / II (Figures 15E and 15F). Allo HSC-γδ T cells are strongly supported as ideal candidates for off-the-shelf cell therapy that does not develop GvHD and is HvG-resistant.

[0058] Figure 16 shows Allo We provide data from a comparative study of the unique properties of HSC-γδ T cell products. Existing methods for generating human γδ T cell products primarily respond to the expansion of γδ T cells from human PBMCs. This culture method starts and ends with a mixed cell population containing human γδ T cells as well as other cells, particularly heterogeneous conventional αβ T (Tc) cells, which can cause GvHD when transferred into an allogeneic recipient ( FIG. 16 ). As a result, this method requires a purification step to generate a “ready-made” γδ T cell product to avoid GvHD. Herein, Allo HSC-γδ T cell cultures are unique in two aspects: 1) they do not support the generation of randomly rearranged αβ TCR recombinants, but produce randomly rearranged endogenous αβ TCRs, thus eliminating the risk of GvHD; and 2) they are transgenic. Allo Supports synchronized differentiation of HSC-γδ T cells, thereby eliminating the presence of undifferentiated progenitor cells and other lineages of immune cells. Allo The HSC-γδ T cell product is pure, homogeneous, and free of GvHD risk; therefore, no cell purification / sorting steps are required with this methodology.

[0059] The inventors have demonstrated that SARS-CoV-2 Allo To investigate the therapeutic potential of HSC-γδ T cells, we established an in vitro COVID-19 infection model (Figures 17A-D). SARS-CoV-2 primarily enters host human cells by binding to cell surface ACE2 (angiotensin-converting enzyme 2) using the viral spike (S) protein. Therefore, we used two ACE2-positive human cells as target cells: the 293T human epithelial cell line engineered to overexpress ACE2, and the Calu-3 human lung epithelial cell line, which naturally expresses ACE2 (Figures 17A and 17B). These cell lines were further engineered to overexpress a firefly luciferase and enhanced green fluorescent protein (FG) dual reporter, enabling sensitive measurement of cell viability using luminescence readout (Figure 17A). Allo HSC-γδ T cells effectively killed both 293T-ACE2-FG and Calu-3-FG target cells upon SARS-CoV-2 infection, whereas no target cell killing was observed without viral infection (Figure S17D). Notably, SARS-CoV-2 infection alone did not affect the viability of ACE2-positive target cells (Figure S17D).

[0060] It is particularly noted that any embodiment discussed in the context of a particular cell or cell population embodiment can be used with respect to any other cell or cell population embodiment. Furthermore, any embodiment used in the context of a particular method can be implemented in the context of any other method described herein. Furthermore, aspects of different methods described herein can be combined to achieve other methods, as well as to create or describe uses for any cell or cell population. It is specifically contemplated that aspects of one or more embodiments can be combined with aspects of one or more other embodiments described herein. Furthermore, any method described herein can be rephrased as describing one or more uses of the cells or cell populations described herein. For example, the use of engineered γδ T cells or γδ T cell populations can be described from any method described herein.

[0061] In certain embodiments, engineered γδ T cells are present that express at least one γδ T cell receptor (γδ TCR) and an exogenous suicide gene product, wherein the at least one γδ TCR is expressed from an endogenous γδ TCR gene under the transcriptional control of an exogenous nucleic acid and / or a recombinantly modified promoter region. Methods in the art for using suicide genes may be used, such as those described in U.S. Patent No. 8,628,767, U.S. Patent Application Publication No. 20140369979, U.S. Patent Application Publication No. 20140242033, and U.S. Patent Application Publication No. 20040014191, all of which are incorporated by reference in their entireties. In further embodiments, the TK gene is a viral TK gene, i.e., a TK gene derived from a virus. In certain embodiments, the TK gene is a herpes simplex virus TK gene. In some embodiments, the suicide gene product is activated by a substrate. Thymidine kinase is a suicide gene product activated by ganciclovir, penciclovir, or derivatives thereof. In certain embodiments, the substrate that activates the suicide gene product is labeled for detection. In some cases, the substrate may be labeled for imaging. In some embodiments, the suicide gene product may be encoded by the same or different nucleic acid molecules encoding one or both of TCR-gamma and TCR-delta. In certain embodiments, the suicide gene is sr39TK or inducible caspase 9. In alternative embodiments, the cells do not express an exogenous suicide gene.

[0062] In additional embodiments, the engineered γδ T cells lack surface expression of at least one HLA-I or HLA-II molecule or have reduced surface expression of at least one HLA-I or HLA-II molecule. In some embodiments, the lack of surface expression of HLA-I and / or HLA-II molecules is achieved by disrupting genes encoding individual HLA-I / II molecules, or by disrupting the gene encoding B2M (beta 2 microglobulin), a common component of all HLA-I complex molecules, or by disrupting the gene encoding CIITA (class II major histocompatibility complex transactivator), a key transcription factor that controls the expression of all HLA-II genes. In certain embodiments, the cells lack surface expression of one or more HLA-I and / or HLA-II molecules or have a reduced level of expression of such molecules by (at least) 50, 60, 70, 80, 90, 100% (or any derivable range therein). In some embodiments, HLA-I or HLA-II is not expressed in γδ T cells because the cells have been engineered by gene editing. In some embodiments, the gene editing involved is CRISPR-Cas9. Instead of Cas9, CasX or CasY may be involved. Zinc finger nucleases (ZFNs) and TALENs are other gene editing techniques similar to Cpf1, all of which may be used. In other embodiments, the γδ T cells contain one or more different siRNA or miRNA molecules targeted to reduce the expression of HLA-I / II molecules, B2M, and / or CIITA.

[0063] In some embodiments, the γδ T cells of the invention comprise a recombinant vector or nucleic acid sequences from a recombinant vector introduced into the cell. In certain embodiments, the recombinant vector is or was a viral vector. In further embodiments, the viral vector is or was a lentivirus, retrovirus, adeno-associated virus (AAV), herpesvirus, or adenovirus. It is understood that the nucleic acid of certain viral vectors is integrated into the host genome sequence.

[0064] In some embodiments, the γδ T cells of the invention are placed in selected media conditions during proliferation and differentiation (e.g., not in media containing animal serum). In further embodiments, the γδ T cells are frozen or have been frozen. In some embodiments, the γδ T cells are pre-frozen and the pre-frozen cells are stable at room temperature for at least 1 hour. In some embodiments, the γδ T cells are pre-frozen and the pre-frozen cells are stable at room temperature for at least 1, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, 24, 30, or 48 hours (or any derivable range therein). In certain embodiments, the γδ T cells or population of γδ T cells in solution comprise dextrose, one or more electrolytes, albumin, dextran, and / or DMSO. In further embodiments, the cells are present in a solution that is sterile, non-pyrogenic, and isotonic.

[0065] In embodiments comprising a plurality of cells, the γδ T cell population comprises about 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15or more (or any range derivable therein), may contain at least these numbers of cells, or may contain up to these numbers of cells. In some cases, the cell population is at least about 10 6 ~10 12 engineered γδ T cells. In some embodiments, it is contemplated that populations of cells having these numbers are generated from a single batch of cells and are not the result of pooling batches of cells generated separately.

[0066] In certain embodiments, there is a T cell population comprising clonal γδ T cells comprising a γδ T cell receptor and one or more exogenous nucleic acids encoding a thymidine kinase suicide gene product, wherein the clonal γδ T cells have been engineered to not express functional beta 2 microglobulin (B2M) and / or class II, major histocompatibility complex or transactivator (CIITA), and the cell population comprises at least about 10 total cells. 6 ~10 12 At least about 10 2 ~10 6 In certain cases, the cells are frozen in solution.

[0067] Some embodiments relate to methods of preparing γδ T cells or populations of cells, particularly populations in which some or all of the cells are clonal, hi certain embodiments, the cell population comprises at least or up to 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% (or any range derivable therein) of the cells that are clonal, i.e., the percentage of cells that are derived from the same ancestral cell as another cell in the population. In other embodiments, the cell population comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 7, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 (or any range derivable therein) different parent cells, including a cell population comprised of cells that originate from at least this number of different parent cells or that originate from up to this number of different parent cells.

[0068] Methods are provided for preparing, generating, manufacturing and using engineered γδ T cells and γδ T cell populations, which methods, in embodiments, include the steps of obtaining pluripotent cells, obtaining hematopoietic progenitor cells, obtaining progenitor cells capable of becoming one or more hematopoietic cells, obtaining progenitor cells capable of becoming γδ T cells, selecting cells from a population of mixed cells using one or more cell surface markers, selecting cells from the population of cells using CD34 + a step of selecting cells, and + Isolating cells, CD34 + and CD34 -separating the cells from each other, selecting the cells based on a cell surface marker other than or in addition to CD34, introducing into the cells one or more nucleic acids encoding a γδ T cell receptor (TCR), infecting the cells with a viral vector encoding a γδ T cell receptor (TCR), transfecting the cells with one or more nucleic acids encoding a γδ T cell receptor (TCR), transfecting the cells with an expression construct encoding a γδ T cell receptor (TCR), Integrating an exogenous nucleic acid encoding a T cell receptor (TCR) into the genome of a cell, introducing one or more nucleic acids encoding a suicide gene product into a cell, infecting a cell with a viral vector encoding a suicide gene product, transfecting a cell with one or more nucleic acids encoding a suicide gene product, transfecting a cell with an expression construct encoding a suicide gene product, integrating an exogenous nucleic acid encoding a suicide gene product into the genome of a cell, introducing one or more nucleic acids encoding one or more polypeptides and / or nucleic acid molecules for gene editing into a cell, infecting a cell with a viral vector encoding one or more polypeptides and / or nucleic acid molecules for gene editing, transfecting a cell with one or more nucleic acids encoding one or more polypeptides and / or nucleic acid molecules for gene editing, transfecting a cell with an expression construct encoding one or more polypeptides and / or nucleic acid molecules for gene editing, integrating an exogenous nucleic acid encoding one or more polypeptides and / or nucleic acid molecules for gene editing, editing the genome of a cell, editing a promoter region of a cell, Editing the promoter and / or enhancer regions for TCR genes, eliminating expression of one or more genes, isolated human CD34 +Eliminating expression of one or more HLA-I / II genes in cells, transfecting cells with one or more nucleic acids for gene editing, culturing the isolated or selected cells, expanding the isolated or selected cells, culturing cells selected for one or more cell surface markers, isolated CD34 expressing γδ TCR. + 1. Culturing Isolated CD34 Cells + The method may include one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or more of the following steps: expanding the cells; culturing the cells under conditions that generate or expand γδ T cells; culturing the cells in an artificial thymic organoid (ATO) system to generate γδ T cells; culturing the cells in serum-free medium; culturing the cells in an ATO system comprising 3D cell aggregates comprising a selected population of stromal cells that express a Notch ligand and serum-free medium. In one embodiment, it is specifically contemplated that one or more steps can be omitted.

[0069] In some embodiments, the present invention provides a method for preparing a population of clonal γδ T cells, comprising: a) isolating CD34 T cells from human peripheral blood cells (PBMCs); + b) introducing one or more nucleic acids encoding a human γδ T cell receptor (TCR); c) introducing an isolated human CD34 + d) eliminating surface expression of one or more HLA-I / II genes in cells; and d) isolating CD34 expressing γδ TCR (e.g., in an artificial thymic organoid system). + Methods exist that involve culturing cells to generate γδ T cells. Typically, the ATO system comprises 3D cell aggregates that contain a selected population of stromal cells that express a Notch ligand and serum-free medium.

[0070] Pluripotent cells that can be used to generate engineered γδ T cells include CD34 +In some embodiments, the method comprises the step of: (i) detecting CD34 cells; (ii) detecting CD34 cells; and (iii) detecting CD34 cells; and (iv) detecting CD34 cells. The cells may be derived from peripheral blood mononuclear cells (PBMCs), bone marrow cells, fetal liver cells, embryonic stem cells, umbilical cord blood cells, induced pluripotent stem cells (iPS cells), or a combination thereof. - Isolating cells or CD34 - and CD34 + In one embodiment, the method further comprises isolating CD34 cells. + This involves further engineering the cells, but not CD34 - The cells may be used to generate γδ T cells. Thus, in some embodiments, CD34 - The cells can then be used or stored for this purpose.

[0071] Certain methods involve introducing CD34 selected in culture medium into cells prior to introducing one or more nucleic acids into the cells. + Culturing the cells includes culturing the selected CD34 +This can include incubating the cells with a medium containing one or more growth factors. In some embodiments, the one or more growth factors include c-kit ligand, flt-3 ligand, and / or human thrombopoietin (TPO). In further embodiments, the medium includes c-kit ligand, flt-3 ligand, and TPO. In some embodiments, the concentration of the one or more growth factors is about 5 ng / ml to about 500 ng / ml for each growth factor or the sum of any and all of these specific growth factors. The concentration of a single growth factor or combination of growth factors in the medium may be about, at least about, or at most about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, The concentration may be 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 410, 420, 425, 430, 440, 441, 450, 460, 470, 475, 480, 490, 500 (or any derivable range) ng / ml or μg / ml or greater.

[0072] In typical embodiments, the nucleic acid may comprise a nucleotide sequence encoding a γ-TCR and / or a δ-TCR, as discussed herein. In specific embodiments, one nucleic acid encodes both the gamma and delta chains of a TCR. In some embodiments, the additional nucleic acid may comprise a nucleic acid sequence encoding an α-TCR and / or a β-TCR polypeptide, and / or one or more iNKT TCR polypeptides. In additional embodiments, the nucleic acid further comprises a nucleic acid sequence encoding a suicide gene product. In some embodiments, the selected CD34 +The nucleic acid molecule introduced into the cell encodes a TCR and a suicide gene product. In another embodiment, the method also includes transfecting a selected CD34 + This involves introducing into the cell a nucleic acid encoding a suicide gene product, where the different nucleic acid molecule encodes a different suicide gene product than the nucleic acid encoding at least one TCR gene.

[0073] As noted above, in some embodiments, the γδ T cells do not express HLA-I and / or HLA-II molecules on the cell surface, which can be achieved by disrupting the expression of genes encoding beta-2-microglobulin (B2M), transactivator (CIITA), or HLA-I and HLA-II molecules. In certain embodiments, the method involves the production of isolated human CD34 T cells. + This method involves eliminating the surface expression of one or more HLA-I / II molecules in a cell. In certain embodiments, eliminating expression can be achieved by gene editing of the genomic DNA of the cell. Some methods include introducing CRISPR and one or more guide RNAs (gRNAs) corresponding to B2M or CIITA into a cell. In certain embodiments, CRISPR or one or more gRNAs are transfected into a cell by electroporation or lipid-mediated transfection. Thus, the method can include introducing CRISPR and one or more gRNAs into a cell by transfecting the cell with a nucleic acid encoding the CRISPR and one or more gRNAs. In some embodiments, different gene editing techniques can be used.

[0074] Similarly, in some embodiments, one or more nucleic acids encoding a TCR receptor are introduced into a cell. This can be done by transfecting or infecting the cell with a recombinant vector, which may or may not be a viral vector as discussed herein. The exogenous nucleic acid can, in some embodiments, be integrated into the genome of the cell.

[0075] In some embodiments, the cells are cultured in cell-free medium. In certain embodiments, the serum-free medium further comprises exogenously added ascorbic acid. In certain embodiments, the method comprises adding ascorbic acid to the medium. In further embodiments, the serum-free medium further comprises one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or all sixteen (or a derivable range therein) of the following exogenously added components: FLT3 ligand (FLT3L), interleukin-7 (IL-7), stem cell factor (SCF), thrombopoietin (TPO), stem cell factor (SCF), IL-2, IL-4, IL-6, IL-15, IL-21, TNF-alpha, TGF-beta, interferon-gamma, interferon-lambda, TSLP, thymopentin, pleotrophin, or midkine. In additional embodiments, the serum-free medium comprises one or more vitamins. In some cases, the serum-free medium comprises one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve (or any range derivable therein) of the following vitamins: biotin, DL-alpha-tocopherol acetate, DL-alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid nicotinamide, pyridoxine, riboflavin, thiamine, inositol, vitamin B12, or a salt thereof. In certain embodiments, the medium comprises, or at least comprises, biotin, DL-alpha-tocopherol acetate, DL-alpha-tocopherol, vitamin A, or a combination or salt thereof. In additional embodiments, the serum-free medium comprises one or more proteins. In some embodiments, the serum-free medium comprises one, two, three, four, five, six or more of the following proteins (or any range derivable therein): albumin or bovine serum albumin (BSA), a fraction of BSA, catalase, insulin, transferrin, superoxide dismutase, or a combination thereof.In other embodiments, the serum-free medium comprises one, two, three, four, five, seven, eight, nine, ten, or eleven of the following compounds: corticosterone, D-galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triiodo-I-thyronine, or a combination thereof. In further embodiments, the serum-free medium comprises B-27® supplement, XenoFree B-27® supplement, GS21™ supplement, or a combination thereof. In additional embodiments, the serum-free medium comprises or further comprises amino acids, simple sugars, and / or inorganic ions. In some embodiments, the serum-free medium comprises one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or thirteen of the following amino acids: arginine, cysteine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine, or a combination thereof. In other embodiments, the serum-free medium comprises one, two, three, four, five, or six of the following inorganic ions: sodium, potassium, calcium, magnesium, nitrogen, or phosphorus, or a combination or salt thereof. In additional embodiments, the serum-free medium comprises one, two, three, four, five, six, or seven of the following elements: molybdenum, vanadium, iron, zinc, selenium, copper, or manganese, or a combination thereof.

[0076] In some methods, the cells are cultured in an artificial thymic organoid (ATO) system. The ATO system comprises three-dimensional (3D) cell aggregates, which are aggregates of cells. In certain embodiments, the 3D cell aggregates comprise a selected population of stromal cells that express Notch ligands. In some embodiments, the 3D cell aggregates are created by mixing CD34+ transduced cells with a selected population of stromal cells on a physical matrix or scaffold. In further embodiments, the method comprises: +The transduced cells and stromal cells are centrifuged to form a cell pellet that is placed on a physical matrix or scaffold. In certain embodiments, the stromal cells express intact, partial, or modified Notch ligands, such as DLL1, DLL4, JAG1, JAG2, or a combination thereof. In further embodiments, the Notch ligand is a human Notch ligand. In other embodiments, the Notch ligand is human DLL1.

[0077] The disclosed methods involve detecting at least 1×10 cells that express a marker or may have high or low levels of a particular marker. 2 , 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 , 1×10 18 , 1×10 19 , 1×10 20 , or 1 × 10 21 In some embodiments, a cell population can be generated (e.g., via differentiation and / or expansion steps) comprising at least 1 x 10 cells (or any derivable range therein). The cell population number may be achieved without cell sorting based on marker expression, or without cell sorting based on γδ T cell marker expression, or without cell sorting based on T cell marker expression. In some embodiments, the cell population size may be achieved without cell sorting based on binding of an antigen to a heterologous targeting element, such as a CAR, TCR, BiTE, or other heterologous tumor targeting agent. Furthermore, the achieved cell population can be at least 1 x 10 cells. 2 , 1×10 3 , 1×10 4 , 1×105 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 , 1×10 18 , 1×10 19 , 1×10 20 , or 1 × 10 21 The cell population may comprise 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 days or 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 weeks (or any range derivable therein), where the cells are generated within a period of time, such as, for example, a period of time that is at least, at most, or exactly 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 weeks (or any range derivable therein).

[0078] In some embodiments, the feeder cells used in the methods are CD34 - These CD34 - The cells were CD34 + The cells may be derived from the same population of cells selected for the treatment. In additional embodiments, the cells may be activated. In certain embodiments, the method includes activating γδ T cells. In certain embodiments, the γδ T cells have been activated and expanded with ZOL. To activate and expand the cells, the cells can be incubated or cultured with ZOL. In some embodiments, feeder cells are pulsed with ZOL.

[0079] The cells may be used immediately or stored for future use. In certain embodiments, the cells used to generate γδ T cells are frozen, although in some embodiments, the generated γδ T cells may be frozen. In some aspects, the cells are present in a solution comprising dextrose, one or more electrolytes, albumin, dextran, and DMSO. In other embodiments, the cells are present in a solution that is sterile, non-pyrogenic, and isotonic. In some embodiments, the engineered γδ T cells are derived from hematopoietic stem cells. In some embodiments, the engineered γδ T cells are derived from G-CSF-mobilized CD34 + In some embodiments, the cells are derived from cells from a human patient who does not have cancer. In some embodiments, the cells do not express an endogenous TCR.

[0080] The number of cells produced by a production cycle is about, at least about, or at most about 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 or more (or any derivable range therein), and in some embodiments are engineered γδ T cells. In some cases, the cell population comprises at least about 10 6 ~10 12 engineered γδ T cells. In some embodiments, it is contemplated that populations of cells having these numbers are generated from a single batch of cells and are not the result of pooling batches of cells that were generated separately, i.e., from a single cycle. In some embodiments, the cell population is frozen and then thawed. The cell population may be used to make engineered γδ T cells or may comprise engineered γδ T cells.

[0081] In some embodiments, the method comprises introducing one or more additional nucleic acids into the cell population, which may or may not have been previously frozen and thawed. This use provides one of the advantages of generating pre-made γδ T cells. In certain embodiments, the one or more additional nucleic acids encode one or more therapeutic gene products. Examples of therapeutic gene products include at least the following: 1. antigen recognition molecules, such as CARs (chimeric antigen receptors) and / or TCRs (T cell receptors); 2. costimulatory molecules, such as CD28, 4-1BB, 4-1BBL, CD40, CD40L, and ICOS; and / or 3. cytokines, such as IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-7, IL-9, IL-15, IL-12, IL-17, IL-21, IL-23, IFN-γ, TNF-α, TGF-β, G-CSF, and GM-CSF; and 4. transcription factors, such as T-bet, GATA-3, RORγt, FOXP3, and Bcl-6. Therapeutic antibodies are also included, as well as chimeric antigen receptors, single-chain antibodies, monobodies, humanized antibodies, antibodies, bispecific antibodies, single-chain FV antibodies, or combinations thereof.

[0082] In some embodiments, the method comprises: a) isolating CD34 from human peripheral blood cells (PBMCs); + b) selecting cells; and c) CD34 + c) culturing the cells in a medium containing growth factors such as c-kit ligand, flt-3 ligand, and human thrombopoietin (TPO); and c) selecting CD34 + transducing the cells with a lentiviral vector containing nucleic acid sequences encoding γ-TCR, δ-TCR, thymidine kinase, and a reporter gene product; d) selecting CD34 +e) introducing Cas9 and gRNA against beta-2 microglobulin (B2M) and / or CTIIA into cells to eliminate expression of B2M or CTIIA; f) culturing the transduced cells with an irradiated stromal cell line expressing an exogenous Notch ligand for 2-10 weeks to expand the γδ T cells in 3D aggregate cell culture; f) selecting γδ T cells lacking expression of B2M and / or CTIIA; and g) culturing the selected γδ T cells with irradiated feeder cells.

[0083] In certain embodiments, γδ T cells generated from transduced cells (e.g., HSPCs) are further modified to have one or more characteristics, including making the cells suitable for allogeneic use or more suitable for allogeneic use than if the cells had not been further modified to have one or more characteristics. U The present invention encompasses HSC-γδ T cells. In some embodiments, the HSC-γδ T cells are non-allo-reactive and express exogenous gamma delta TCRs. These cells are useful for "off-the-shelf" cell therapy, eliminating the need for the use of the patient's own γδ T or other cells. Thus, the present method provides a more cost-effective and less labor-intensive cellular immunotherapy.

[0084] In specific embodiments, HSC-γδ T cells are engineered to be HLA-negative to achieve safe and successful allogeneic transplantation without causing graft-versus-host disease (GvHD) and without being rejected by host immune cells (HvG rejection). In certain embodiments, expression of the transgenic γδ TCR gene blocks recombination of endogenous TCRs via allelic exclusion, so allogeneic HSC-γδ T cells do not express endogenous TCRs and do not cause GvHD. In certain embodiments, allogeneic HSC-γδ T cells are engineered to be HLA-negative to achieve safe and successful allogeneic transplantation without causing graft-versus-host disease (GvHD) and without being rejected by host immune cells (HvG rejection). In certain embodiments, expression of the transgenic γδ TCR gene blocks recombination of endogenous TCRs via allelic exclusion, so allogeneic HSC-γδ T cells do not express endogenous TCRs and do not cause GvHD. U HSC-γδ T cells do not express HLA-I and / or HLA-II molecules on their cell surface and do not bind to host CD8 +and CD4 + Resist T cell-mediated allograft depletion and sr39TK immunogen target depletion. Thus, in certain embodiments, engineered γδ T cells do not express surface HLA-I or -II molecules, achieved by disruption of genes encoding proteins associated with HLA-I / II expression, including, but not limited to, beta2-microglobulin (B2M), major histocompatibility complex II transactivator (CIITA), or HLA-I / II molecules. In some cases, HLA-I or HLA-II is not expressed on the surface of γδ T cells because the cells have been engineered by gene editing, which may or may not involve CRISPR-Cas9.

[0085] Where a γδ T cell has been engineered to exhibit one or more characteristics of any type, the γδ T cell may comprise a nucleic acid sequence from a recombinant vector introduced into the cell. The vector may be a non-viral vector, such as a plasmid, or a viral vector, such as a lentivirus, retrovirus, adeno-associated virus (AAV), herpesvirus, or adenovirus.

[0086] The γδ T cells of the present invention may or may not have been exposed to one or more specific conditions before, during, or after their generation. In specific cases, the cells are not or have not been exposed to medium containing animal serum. The cells may be frozen. The cells may be present in a solution containing dextrose, one or more electrolytes, albumin, dextran, and / or DMSO. Any solution in which the cells are present may be a sterile, non-pyrogenic, and isotonic solution. The cells may have been activated and expanded by any suitable method, such as by activation with ZOL.

[0087] Aspects of the present disclosure relate to human cells comprising: i) an exogenous expression or activity inhibitor of one or more of beta2 microglobulin (B2M), CIITA, TRAC, TRBC1, or TRBC2; or ii) a genomic mutation of one or more of beta2 microglobulin (B2M), CIITA, TRAC, TRBC1, or TRBC2. In some embodiments, the cell comprises a genomic mutation. In some embodiments, the genomic mutation comprises a mutation of one or more endogenous genes in the genome of the cell, wherein the one or more endogenous genes comprise B2M, CIITA, TRAC, TRBC1, or TRBC2 genes. In some embodiments, the mutation comprises a loss-of-function mutation. In some embodiments, the inhibitor is an expression inhibitor. In some embodiments, the inhibitor comprises an inhibitory nucleic acid. In some embodiments, the inhibitory nucleic acid comprises one or more of an siRNA, shRNA, miRNA, or antisense molecule. In some embodiments, the cell comprises an activity inhibitor. In some embodiments, after modification, the cells lack any detectable expression of one or more of the B2M, CIITA, TRAC, TRBC1, or TRBC2 proteins. In some embodiments, the cells comprise an inhibitor or genomic mutation of B2M. In some embodiments, the cells comprise an inhibitor or genomic mutation of CIITA. In some embodiments, the cells comprise an inhibitor or genomic mutation of TRAC. In some embodiments, the cells comprise an inhibitor or genomic mutation of TRBC1. In some embodiments, the cells comprise an inhibitor or genomic mutation of TRBC2. In some embodiments, at least 90% of the genomic DNA encoding B2M, CIITA, TRAC, TRBC1, and / or TRBC2 is deleted. In some embodiments, at least or at most 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, or 100% (or any derivable range therein) of the genomic DNA encoding B2M, CIITA, TRAC, TRBC1, and / or TRBC2 is deleted. In other embodiments, deletions, insertions and / or substitutions are made in the genomic DNA. In some embodiments, the cells are the progeny of human stem or progenitor cells.

[0088] Modified to be HLA negative U HSC-γδ T cells can be genetically modified by any suitable method. Genetic mutations of the present disclosure, such as CIITA and / or B2M genes, can be introduced by methods known in the art. In certain embodiments, engineered nucleases can be used to introduce exogenous nucleic acid sequences for genetic modification of any cell mentioned herein. Genome editing, or genome editing with engineered nucleases (GEEN), is a type of genetic engineering in which DNA is inserted, replaced, or removed from the genome using artificially engineered nucleases, or "molecular scissors." The nucleases create specific double-strand breaks (DSBs) at desired locations within the genome, and the cell's endogenous machinery is used to repair the induced breaks through the natural processes of homologous recombination (HR) and non-homologous end joining (NHEJ). Non-limiting examples of engineered nucleases include zinc finger nucleases (ZFNs), TAL effector nucleases (TALENs), CRISPR / Cas9 systems, and engineered meganucleases and engineered homing endonucleases. Any engineered nuclease known in the art can be used in certain embodiments of the methods and compositions.

[0089] When engineered γδ T cells contain one or more suicide genes for subsequent depletion as needed, the suicide genes may be of any suitable type. The γδ T cells of the present disclosure may express a suicide gene product, which may be, for example, enzyme-based. Examples of suicide gene products include herpes simplex virus thymidine kinase (HSV-TK), purine nucleoside phosphorylase (PNP), cytosine deaminase (CD), carboxypeptidase G2, cytochrome P450, linamarase, beta-lactamase, nitroreductase (NTR), carboxypeptidase A, or inducible caspase 9. Thus, in specific cases, the suicide gene may encode thymidine kinase (TK). In specific cases, the TK gene is a viral TK gene, such as the herpes simplex virus TK gene. In certain embodiments, the suicide gene product is activated by a substrate such as ganciclovir, penciclovir, or a derivative thereof.

[0090] In some embodiments, the engineered γδ T cells can be imaged or otherwise detected. In certain cases, the cells contain an exogenous nucleic acid encoding a polypeptide with a substrate that can be labeled for imaging, and the imaging may be fluorescent, radioactive, colorimetric, etc. In specific cases, the cells are detected by positron emission tomography. In at least some cases, the cells express the sr39TK gene, a positron emission tomography (PET) reporter / thymidine kinase gene, allowing these genetically modified cells to be tracked by PET imaging and eliminated by the sr39TK suicide gene function.

[0091] A population of engineered γδ T cells is encompassed by the present disclosure. In certain embodiments, the γδ T clonal cells comprise an exogenous nucleic acid encoding a γδ T cell receptor and lack surface expression of one or more HLA-I or HLA-II molecules. The γδ T cells may comprise an exogenous nucleic acid encoding a suicide gene, including an enzyme-based suicide gene such as thymidine kinase (TK). The TK gene may be a viral TK gene, such as the herpes simplex virus TK gene. In cells of the population, the suicide gene can be activated by a substrate, such as ganciclovir, penciclovir, or a derivative thereof. The cells may comprise an exogenous nucleic acid encoding a polypeptide having a substrate that can be labeled for imaging; in some cases, the suicide gene product is a polypeptide having a substrate that can be labeled for imaging. In certain embodiments, the suicide gene is sr39TK. In certain cases of γδ T cell populations, the γδ T cells comprise nucleic acid sequences from a recombinant vector, such as a viral vector (including at least a lentivirus, retrovirus, adeno-associated virus (AAV), herpesvirus, or adenovirus), that has been introduced into the cells.

[0092] In certain embodiments, the cells of the γδ T cell population may or may not have been exposed to one or more specific conditions, or may be exposed to one or more specific conditions. In certain cases, for example, the cells of the population are not or have not been exposed to a medium containing animal serum. The cells of the population may or may not be frozen. In some cases, the cells of the population are present in a solution comprising dextrose, one or more electrolytes, albumin, dextran, and / or DMSO. The solution may comprise dextrose, one or more electrolytes, albumin, dextran, and DMSO. The cells may be present in a solution that is sterile, non-pyrogenic, and isotonic. In specific cases, the γδ T cells are activated, such as activated with ZOL. In certain aspects, the cell population comprises at least about 10 2 ~10 6 The cell population optionally comprises at least about 10 clonal cells. 6~10 12 The total number of cells may be 100.

[0093] In certain embodiments, there is a T cell population comprising clonal γδ T cells comprising a gamma delta (γδ) T cell receptor and one or more exogenous nucleic acids encoding thymidine kinase suicide, wherein the clonal γδ T cells have been engineered not to express functional beta 2 microglobulin (B2M), major histocompatibility complex class II transactivator (CIITA), and / or HLA-I and HLA-II molecules, and the cell population comprises at least about 10 total cells. 6 ~10 12 At least about 10 2 ~10 6 Optionally, the cells are frozen in solution.

[0094] In certain embodiments, U HSC-γδ T cells and / or their precursors U At any stage in the process of generating HSC-γδ T cells, cells can be specifically constructed and / or cultured in a particular medium (whether or not present in an in vitro ATO culture system), and the cells can be constructed so that they are suitable for delivery to a recipient without adverse effects.

[0095] In certain embodiments, the medium can be prepared using any of media used to culture animal cells, such as AIMV, X-VIVO-15, NeuroBasal, EGM2, TeSR, BME, BGJb, CMRL1066, Glasgow MEM, Improved MEM Zinc Option, IMDM, Medium 199, Eagle MEM, αMEM, DMEM, Ham, RPMI-1640, and Fischer's medium, as well as any combination thereof, as its basal medium, but is not limited thereto as long as it can be used to culture animal cells. In particular, the medium may be xeno-free or chemically defined.

[0096] The medium may be serum-containing or serum-free, or xeno-free. To prevent contamination with components derived from different animals, the serum may be derived from the same animal as the stem cells. Serum-free medium refers to a medium that does not contain untreated or unpurified serum, and therefore may include a medium containing purified blood-derived components or animal tissue-derived components (such as growth factors).

[0097] The medium may or may not contain a serum replacement. Serum replacements include materials that suitably contain albumin (e.g., lipid-rich albumin, bovine albumin, albumin substitutes such as recombinant or humanized albumin, plant starch, dextran, and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thioglycerol, or equivalents thereof. Serum replacements can be prepared, for example, by the methods disclosed in International Publication No. 98 / 30679 (incorporated herein in its entirety). Alternatively, for greater convenience, any commercially available material can be used. Commercially available materials include knockout serum replacement (KSR), chemically-defined lipid concentrated (Gibco), and Glutamax (Gibco).

[0098] In further embodiments, the medium may be a serum-free medium suitable for cell development. For example, the medium may contain B-27® supplement, Xenofree B-27® supplement (available on the World Wide Web at thermofisher.com / us / en / home / technical-resources / media-formulation.250.html), NS21 supplement (Chen et al., J Neurosci Methods, 2008 Jun 30;171(2):239-247, incorporated herein in its entirety), GS21™ supplement (available on the World Wide Web at amsbio.com / B-27.aspx), or a combination thereof, at a concentration effective to generate T cells from the 3D cell aggregates.

[0099] Cells expressing polypeptides comprising the amino acid sequences set forth in Table 1 (SEQ ID NO: 1-SEQ ID NO: 52) and / or other γδ T cells can be generated by any suitable method. The one or more methods may utilize one or more sequential steps for one or more modifications to the cells, and / or may utilize one or more simultaneous steps for one or more modifications to the cells. In certain embodiments, a starting source of cells is modified to function as γδ T cells, followed by one or more steps that confer one or more additional characteristics to the cells, e.g., the ability to be imaged, and / or the ability to be selectively killed, and / or the ability to be used in an allogeneic system. In certain embodiments, U At least part of the process for generating HSC-γδ T cells is carried out in a specific in vitro culture system.An example of a specific in vitro culture system is that which allows the differentiation of specific cells with high efficiency and high yield.In certain embodiments, the in vitro culture system is an artificial thymic organoid (ATO) system.

[0100] In specific cases, UHSC-γδ T cells can be generated by: 1) genetic modification of donor HSCs to express γδ TCR (e.g., via a lentiviral vector) and eliminate expression of HLA-I / II molecules (e.g., via CRISPR / Cas9-based gene editing), 2) in vitro differentiation into γδ T cells via ATO culture, 3) in vitro purification and expansion of γδ T cells, and 4) construction and cryopreservation and / or use.

[0101] Certain embodiments of the present disclosure are directed to a method for preparing a population of clonal gamma delta (γδ) T cells, comprising: a) isolating CD34 T cells from human peripheral blood cells (PBMCs); + The present invention provides a method for generating γδ T cells by culturing isolated human CD34+ cells expressing γδ TCRs in an artificial thymic organoid (ATO) system, comprising: selecting γδ T cells; b) introducing one or more nucleic acids encoding a human γδ T cell receptor (TCR); c) eliminating expression of one or more HLA-I / II genes in the isolated human CD34+ cells; and d) culturing the isolated CD34+ cells expressing γδ TCRs in an artificial thymic organoid (ATO) system to generate γδ T cells, wherein the ATO system comprises 3D cell aggregates comprising a selected population of stromal cells expressing a Notch ligand and serum-free medium. - This may further include isolating the cells. In alternative embodiments, other culture systems than the ATO system are used, such as 2D culture systems or other forms of 3D culture systems (e.g., FTOC-like cultures, matrigel-supported cultures).

[0102] Certain aspects of the present disclosure relate to novel three-dimensional cell culture systems for generating γδ T cells from less differentiated cells, such as embryonic stem cells, pluripotent stem cells, hematopoietic stem or progenitor cells, induced pluripotent stem (iPS) cells, or stem or progenitor cells. Stem cells of any type can be cultured, for example, from at least fetal liver, umbilical cord blood, and peripheral blood CD34 cells. + They can be utilized from a variety of sources, including cells (either G-CSF mobilized or non-G-CSF mobilized).

[0103] In certain embodiments, the system comprises using a serum-free medium. In certain aspects, the system uses a serum-free medium suitable for cell development for the culture of three-dimensional cell aggregates. Such a system comprises a sufficient amount of U In an embodiment of the present disclosure, the 3D cell aggregates are generated from stem or progenitor cells. U From HSC-γδ T cells or precursors U The cells are cultured in serum-free medium containing insulin for a period sufficient for in vitro differentiation into HSC-γδ T cells.

[0104] Embodiments of cell culture compositions include ATO 3D cultures, which use highly standardized serum-free components and stromal cell lines to promote robust and highly reproducible T cell differentiation from human HSCs. In certain embodiments, cell differentiation in ATO closely mimics endogenous thymocyte formation and, in contrast to monolayer co-cultures, provides functionally robust T cell differentiation. U Certain aspects of the 3D culture composition use serum-free conditions, avoid the use of human thymus tissue or proprietary scaffold materials, and allow for the efficient positive selection of HSC-γδ T cells from source cells. U Promotes positive selection and robust generation of HSC-γδ T cells.

[0105] The cells produced by the preparation method can be frozen. The cells can be in a solution containing dextrose, one or more electrolytes, albumin, dextran, and DMSO. The solution can be sterile, non-pyrogenic, and isotonic.

[0106] Genetic modifications can also be introduced into specific components to generate antigen-specific T cells and model positive and negative selection. Examples of these modifications include transducing HSCs with lentiviral vectors encoding antigen-specific T cell receptors (TCRs) or chimeric antigen receptors (CARs) to generate antigen-specific, allelically excluded naive T cells, and transducing HSCs with genes to direct lineage commitment to specific lymphoid cells. For example, transducing HSCs with gamma delta (γδ)-related TCRs to generate functional γδ T cells in ATO, transducing ATO stromal cell lines (e.g., MS5-hDLL1) with human MHC genes (e.g., human CD1d genes) to enhance the positive selection and maturation of both TCR-engineered and non-TCR-engineered T cells in ATO, and / or transducing ATO stromal cell lines with antigens plus costimulatory molecules or cytokines to enhance the positive selection of CAR T cells in ATO.

[0107] CD34 from human peripheral blood cells (PBMCs) to generate engineered γδ T cells + Cells can be modified by introducing specific exogenous genes and knocking out specific endogenous genes. This method involves introducing CD34 selected in culture medium prior to introducing one or more nucleic acids into the cells. + The cell line may further comprise culturing the cells. The culturing may comprise culturing CD34 cells selected in a medium containing one or more growth factors. + This can include incubating the cells with, optionally, one or more growth factors, which can include, for example, c-kit ligand, flt-3 ligand, and / or human thrombopoietin (TPO), at a particular concentration, e.g., from about 5 ng / ml to about 500 ng / ml.

[0108] In certain methods, the nucleic acid introduced into the cells is one or more nucleic acids comprising nucleic acid sequences encoding the γ-TCR and δ-TCR (e.g., SEQ ID NO: 1-52). The method may also include introducing a nucleic acid encoding a suicide gene into the selected cells. In certain embodiments, one nucleic acid encodes both the γ-TCR and the δ-TCR, or one nucleic acid encodes the γ-TCR, the δ-TCR, and the suicide gene. The suicide gene may be enzyme-based, such as thymidine kinase (TK), including viral TK genes, such as those derived from the herpes simplex virus TK gene. The suicide gene may be activated by a substrate, such as ganciclovir, penciclovir, or a derivative thereof. The cells may be engineered to contain an exogenous nucleic acid encoding a polypeptide bearing a substrate that can be labeled for imaging. In some cases, the suicide gene product is a polypeptide bearing a substrate that can be labeled for imaging, such as sr39TK.

[0109] In producing engineered γδ T cells, the cells may be present in certain serum-free media, including those containing exogenously added ascorbic acid. In certain embodiments, the serum-free media further contains exogenously added FLT3 ligand (FLT3L), interleukin 7 (IL-7), stem cell factor (SCF), thrombopoietin (TPO), IL-2, IL-4, IL-6, IL-15, IL-21, TNF-alpha, TGF-beta, interferon-gamma, interferon-lambda, TSLP, thymopentin, pleotrophin, or midkine, or a combination thereof. The serum-free medium may further contain vitamins, including biotin, DL-alpha tocopherol acetate, DL-alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid nicotinamide, pyridoxine, riboflavin, thiamine, inositol, vitamin B12, or combinations thereof or salts thereof. The serum-free medium may further contain one or more exogenously added (or non-added) proteins, such as albumin or bovine serum albumin, a fraction of BSA, catalase, insulin, transferrin, superoxide dismutase, or combinations thereof. The serum-free medium may further contain corticosterone, D-galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triiodo-I-thyronine, or combinations thereof. The serum-free medium may contain B-27® supplement, Xenofree B-27® supplement, GS21™ supplement, or a combination thereof. Amino acids (including arginine, cysteine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine, or a combination thereof), simple sugars, and / or inorganic ions (including, for example, sodium, potassium, calcium, magnesium, nitrogen, or phosphorus, or a combination or salt thereof) may be present in the serum-free medium.The serum-free medium may further comprise molybdenum, vanadium, iron, zinc, selenium, copper, or manganese, or a combination thereof.

[0110] Further aspects and embodiments of the invention are described in the following sections. [Example]

[0111] Human Vγ9Vδ2 TCR clones, sequences, and gene delivery vectors The human Vy9V52 TCR (referred to herein as γδ TCR) was cloned from γδ T cells derived from peripheral blood mononuclear cells (PBMCs) of a healthy donor (PBMC-γδ T). Exemplary embodiments of the methods disclosed herein, as well as γδ TCR sequences (e.g., amino acid sequences and / or gene coding sequences) and exemplary γδ TCR gene delivery vectors, are discussed below.

[0112] method Human γδ T cells are derived from stem and progenitor cells (e.g., CD34 + They can be generated by γδ TCR genetic engineering of HSCs, ESCs, iPSCs) followed by differentiation (in vivo or ex vivo) into transgenic γδ T cells.

[0113] HSCs can be isolated directly from umbilical cord blood or G-CSF-mobilized peripheral blood (CB-HSCs or PBSCs) or can be derived from embryonic or induced pluripotent stem cells (ES-HSCs or iPS-HSCs) containing human CD34 + Refers to hematopoietic progenitor and stem cells. HSCs can be genetically engineered via vector-dependent or vector-independent gene delivery methods or other gene editing methods (e.g., CRISPR, TALEN, zinc finger, etc.).

[0114] In addition to the antigen specificity conferred by the monoclonal transgenic γδ TCR, HSC-γδ T cells can be further engineered to express additional targeting molecules to enhance their disease-targeting capabilities. Such targeting molecules may be chimeric antigen receptors (CARs), natural or synthetic receptors / ligands, etc. The resulting CAR-γδ T cells can then be utilized for off-the-shelf disease-targeting cell therapy.

[0115] In addition to the antigen specificity conferred by the monoclonal transgenic TCR, HSC-γδ T cells can be further engineered to express additional targeting molecules to enhance their disease-targeting capabilities. Such targeting molecules can be chimeric antigen receptors (CARs), natural or synthetic receptors / ligands, etc. The resulting CAR-γδ T cells can then be utilized for off-the-shelf disease-targeting cell therapy.

[0116] HSC-γδ T cells and derivatives can also be further engineered to overexpress genes encoding T cell stimulatory factors or to disrupt genes encoding T cell inhibitory factors, resulting in functionally enhanced HSC-γδ T cells and derivatives.

[0117] In vivo generation of HSC-engineered γδT (HSC-γδT) cells for HSC adoptive therapy A γδ TCR engineered HSC adoptive transfer method is disclosed that can generate HSC-γδ T cells in vivo, cells that can potentially provide patients with a lifelong supply of disease-targeting engineered HSC-γδ T cells.

[0118] The procedure involves 1) transfecting human CD34 cells to express the selected γδ TCR gene. + 1) genetic modification of hematopoietic stem cells (HSCs); 2) adoptive transfer of γδ TCR-engineered HSCs into the patient; 3) in vivo generation of HSC-γδ T cells; and 4) due to the self-renewing lifespan of HSCs, the method can potentially protect the patient with a lifelong supply of HSC-γδ T cells.

[0119] Allogeneic HSC-engineered γδT (γδT) for off-the-shelf cell therapy Allo Ex vivo generation of HSC-γδT cells For off-the-shelf cell therapy applications Allo An ex vivo differentiation culture method for generating HSC-γδ T cells is disclosed.

[0120] Feeder-dependent culture The procedure involves 1) transfecting human CD34 cells to express the selected γδ TCR gene. + 3) Genetic modification of hematopoietic stem cells (HSCs), and 3) using feeder cells Allo 3) ex vivo generation of HSC-γδ T cells (e.g., artificial thymic organoid culture), and 3) differentiated Allo This involves ex vivo expansion of HSC-γδ T cells.

[0121] Feeder-free culture The generation procedure involves 1) transfecting human CD34 cells to express the selected TCR gene; + Genetic modification of hematopoietic stem cells (HSCs), 2) feeder cell-free Allo 3) ex vivo generation of HSC-γδ T cells; Allo This involves ex vivo expansion of HSC-γδ T cells.

[0122] Purpose Engineered γδ T cells can be used to target multiple diseases, including cancer and infectious diseases.

[0123] γδ T cell therapy for cancer Proof-of-principle data are provided for treating a large collection of human cancers, including hematological cancers (eg, multiple myeloma) and solid tumors (eg, ovarian, melanoma, prostate, breast, and lung cancer).

[0124] γδ T cell therapy for infectious diseases Proof-of-principle data will be provided to target COVID-19.

[0125] Allo Detailed description of the culture method for HSC-γδ T cells In vivo generation of HSC-γδ T cells Human CD34 + HSCs were cultured in X-VIVO15 serum-free hematopoietic cell medium containing recombinant human Flt3 ligand, SCF, TPO, and IL-3 on retronectin-coated, tissue-culture-untreated plates for up to 48 hours. Viral transduction was performed for 24 hours by adding concentrated lentivectors directly to the culture medium. Approximately 48 hours later, CD34+ cells were harvested and transduced into 270-rad total-body-irradiated NOD.Cg-Prkdc cells. scid Il2rg tm1Wjl / SzJ(NSG) mice were intravenously (iv). One to two fragments of human fetal or postnatal thymus were transplanted under the kidney capsule of each recipient NSG mouse.

[0126] Allo Feeder-dependent ex vivo generation of HSC-γδ T cells Phase 1: Allo Differentiation of HSC-γδ T cells Fresh or frozen / thawed CD34 + HSCs are cultured in stem cell culture medium (a base medium supplemented with a cytokine cocktail including IL-3, IL-7, IL-6, SCF, EPO, TPO, FLT3L, etc.) in retronectin-coated flasks for 12–72 h, followed by the addition of TCR gene delivery vectors and further culture for 12–48 h. TCR gene-modified HSCs are then cultured in feeder-dependent cultures (e.g., artificial thymus organoid cultures) for 4–10 weeks. Allo Differentiation into HSC-γδ T cells. Artificial thymic organoids (ATOs) were generated according to a previously established protocol (Seet et al., Cell Stem Cell. 2019 Mar 7;24(3):376-389).

[0127] Phase 2: Allo Expansion of HSC-γδ T cells At stage 2, differentiated AlloHSC-γδ T cells are stimulated with TCR cognate antigens (proteins, peptides, lipids, fluorophores, small molecules, etc.) or nonspecific TCR stimulatory reagents (anti-CD3 / anti-CD28 antibodies or antibody-coated beads, concanavalin A, PMA / ionomycin, etc.) and expanded in T cell culture medium for up to 1 month. Cultures can be supplemented with cytokines (IL-2, IL-7, IL-15, etc.) that respond to T cell responses.

[0128] Allo HSC-γδT cell derivative In some embodiments, Allo HSC-γδ T cells can be further engineered to express additional transgenes. In one embodiment, such transgenes encode disease-targeting molecules such as chimeric antigen receptors (CARs), T cell receptors (TCRs), and other natural or synthetic receptors / ligands. In another embodiment, such transgenes can encode T cell regulatory proteins such as IL-2, IL-7, IL-15, IFN-γ, TNF-α, CD28, 4-1BB, OX40, ICOS, and FOXP3. At various culture stages, transgenes can be engineered to express the T cells after expansion. Allo HSC-γδ T cells or their progenitors (HSCs, newly differentiated Allo HSC-γδT cells, proliferating Allo It can be introduced into HSC-γδT cells.

[0129] In some embodiments, Allo HSC-γδ T cells can be further engineered to disrupt selected genes using gene editing tools (CRISPR, TALEN, zinc finger, etc.). In one embodiment, the disrupted gene encodes a T cell immune checkpoint inhibitor (PD-1, CTLA-4, TIM-3, LAG-3, etc.). Deletion of these negative regulatory genes results in: Allo This may enhance the disease-fighting capacity of HSC-γδ T cells and result in resistance to disease-induced anergy and tolerance.

[0130] AlloFeeder-free ex vivo generation of HSC-γδ T cells Phase 1: Allo Differentiation of HSC-γδ T cells Fresh or frozen / thawed CD34 + HSCs are cultured in stem cell culture medium (a base medium supplemented with a cytokine cocktail including IL-3, IL-7, IL-6, SCF, EPO, TPO, FLT3L, etc.) in retronectin-coated flasks for 12–72 hours, followed by the addition of the TCR gene delivery vector and further culture for 12–48 hours.

[0131] TCR gene-modified HSCs were then cultured in differentiation medium without feeders for 4–10 weeks. Allo Differentiation into HSC-γδ T cells. Allo Coat with HSC-γδT culture coating materials (DLL-1 / 4, VCAM-1 / 5, retronectin, etc.). + HSCs are suspended in proliferation medium (a base medium containing serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, SCF, TPO, IL-3, IL-6, Flt3 ligand, human LDL, UM171, and additives), seeded into the coated wells of a plate, and cultured for 3-7 days. The proliferation medium is renewed every 3-4 days. The cells are then harvested and suspended in maturation medium (a base medium containing serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, SCF, IL-3, IL-6, IL-7, IL-15, Flt3 ligand, ascorbic acid, and additives). The maturation medium is renewed once or twice a week.

[0132] Phase 2: Allo Expansion of HSC-γδ T cells differentiated AlloHSC-γδ T cells are stimulated with TCR alloantigens (proteins, peptides, lipids, fluorophore antigens, small molecules, etc.) or nonspecific TCR stimulatory reagents (anti-CD3 / anti-CD28 antibodies or antibody-coated beads, concanavalin A, PMA / ionomycin, and artificial APCs) and grown in T cell culture medium for up to 1 month. Cultures can be supplemented with cytokines (IL-2, IL-7, IL-15, etc.) that respond to T cell responses.

[0133] Allo HSC-γδT cell derivative In some embodiments, Allo HSC-γδ T cells can be further engineered to express additional transgenes. In one embodiment, such transgenes encode disease-targeting molecules such as chimeric antigen receptors (CARs), T cell receptors (TCRs), and other natural or synthetic receptors / ligands. In another embodiment, such transgenes can encode T cell regulatory proteins such as IL-2, IL-7, IL-15, IFN-γ, TNF-α, CD28, 4-1BB, OX40, ICOS, and FOXP3. At various culture stages, transgenes can be engineered to express the T cells after expansion. Allo HSC-γδ T cells or their progenitors (HSCs, newly differentiated Allo HSC-γδT cells, proliferating Allo It can be introduced into HSC-γδT cells.

[0134] In some embodiments, Allo HSC-γδ T cells can be further engineered to disrupt selected genes using gene editing tools (CRISPR, TALEN, zinc finger, etc.). In one embodiment, the disrupted gene encodes a T cell immune checkpoint inhibitor (PD-1, CTLA-4, TIM-3, LAG-3, etc.). Deletion of these negative regulatory genes results in: Allo This may enhance the disease-fighting capacity of HSC-γδ T cells and result in resistance to disease-induced anergy and tolerance.

[0135] In some embodiments, AlloHSC-γδ T cells or enhanced Allo HSC-γδ T cells can be further engineered to make them suitable for allogeneic adoptive transfer, thereby serving as a ready-made cell product. In one embodiment, genes encoding MHC molecules or MHC expression / display regulatory molecules (such as MHC molecules, B2M, CIITA (class II transcription activator-regulated induction of MHC class II mRNA expression)) are inserted into the HSC-γδ T cells. Allo The lack of MHC molecule expression on HSC-γδ T cells renders the cells resistant to allogeneic host T cell-mediated depletion. Allo HSC-γδ T cells are further engineered to overexpress the HLA-E gene, which confers resistance to host NK cell-mediated depletion.

[0136] Allo HSC-γδ T cells and derivatives can be used fresh or cryopreserved. Allo Various intermediate cell products produced during HSC-γδ T cell culture can be paused for cryopreservation, stored, and retrieved for continued production.

[0137] Novel Features and Advantages Using feeder-dependent cultures (e.g., ATO cultures) Allo Compared to methods for generating HSC-γδ T cells, the present invention provides an in vitro differentiation method that does not require feeder cells. This novel method significantly improves the process for scale-up production and GMP-compliant manufacturing of therapeutic cells for human use.

[0138] It is a cell product Allo HSC-γδ T cells exhibit a phenotype / functionality that is distinct from that of their natural counterparts T cells as well as their counterparts T cells generated using other ex vivo culture methods (e.g., ATO culture methods); Allo making HSC-γδ T cells a unique cell product.

[0139] AlloUnique features of HSC-γδ T cell differentiation cultures include the following: 1) It is ex vivo and feeder-free. 2) It does not support TCRV / D / J recombination, so there are no randomly rearranged endogenous TCRs and therefore no GvHD risk. 3) Transgenic Allo Supports synchronized differentiation of HSC-γδ T cells, thereby eliminating the presence of undifferentiated progenitor cells and other lineages of bystander immune cells. 4) As a result, Allo The HSC-γδ T cell product contains a homogenous and pure population of monoclonal TCR-engineered T cells, free of escaped random T cells, free of other immune cell lineages, and free of undifferentiated progenitor cells. Therefore, no purification steps are required. 5) High yield. Approximately 10 13 pieces Allo HSC-γδ T cells (10,000–100,000 doses) can be generated from PBSCs of healthy donors, with approximately 10 13 pieces Allo HSC-γδ T cells (10,000-100,000 doses) can be generated from CB HSCs of healthy donors. 6) Allo Intrinsic phenotype of HSC-γδ T cells - transgenic TCR + endogenous TCR - CD3 + . (Note: Allo These unique characteristics of HSC-γδ T cell differentiation cultures distinguish them from other methods of generating ready-made T cell products, including healthy donor PBMC-based T cell cultures, ATO cultures, etc.

[0140] Proof of principle Proof-of-principle research was conducted, Allo This shows the successful generation of HSC-γδT cells. Allo BCAR-γδT cell product), and interleukin-15 (IL-15) ( Allo15 BCAR-γδ T cell product) for further expression AlloFurther engineering of CAR-γδ T cells also proved successful, and pilot CMC, pharmacology, efficacy, and safety studies were performed analyzing these cell products.

[0141] Table 1: Amino acid sequences of the cloned γδ TCR CDR3 region Human γδ TCR genes were cloned using a single-cell RT-PCR approach (see, e.g., Figure 1). Briefly, human γδ T cells were expanded from healthy donor peripheral blood mononuclear cells (PBMCs) and sorted using flow cytometry based on a stringent combination of surface markers gated as hCD3+Vγ9+Vδ2+ (Figures 1A and 1B). Single cells were sorted directly onto PCR plates containing cell lysis buffer and then subjected to TCR cloning using one-step RT-PCR, followed by Sanger sequencing analysis (Figure 1A). As shown below, more than 25 pairs of γδ TCR γ9 and δ2 chain genes were identified. [Table 1-1] [Table 1-2]

[0142] Exemplary Vector Sequences pMNDW-G115 DNA SEQ ID NO: [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0143] pMNDW-γδ1 DNA SEQ ID NO: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0144] All publications mentioned herein (e.g., PCT Published International Application Nos. PCT / US19 / 36786 and PCT / US2020 / 037486, U.S. Patent Application No. 15 / 320,037, and Zarin et al., Cell Immunol., 2015 Jul;296(1):70-5.doi:10.1016 / j.cellimm.2015.03.007.Epub 2015, such as those listed above) are incorporated by reference to disclose and describe the aspects, methods and / or materials related to the cited publications. Many of the techniques and procedures described or referenced herein are well understood and commonly used by those skilled in the art.

[0145] Unless otherwise defined, all technical terms, notations, and other scientific or technical terms used herein are intended to have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. In some cases, terms having a commonly understood meaning are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference from what is commonly understood in the art. In certain embodiments, for example, the following are provided: (Item 1) An engineered cell is a cell that has been genetically modified to contain at least one exogenous gamma delta T cell receptor (γδ TCR) nucleic acid molecule. (Item 2) 2. The engineered cell of item 1, wherein the cell is a pluripotent stem cell, a hematopoietic stem cell, a hematopoietic progenitor cell, or an immune cell. (Item 3) 2. The engineered cell of item 1, wherein the cell is a human cell. (Item 4) 2. The engineered cell of paragraph 1, wherein the γδ TCR nucleic acid molecule is a clone of the T cell receptor of a γδ T cell or has a sequence that is modified from the sequence of the T cell receptor of the γδ T cell. (Item 5) 2. The engineered cell of claim 1, wherein the γδ TCR nucleic acid molecule is a clone of the T cell receptor of a human γδ T cell or has a sequence that is modified from the sequence of the T cell receptor of the human γδ T cell. (Item 6) 2. The engineered cell of paragraph 1, wherein the γδ TCR nucleic acid molecule comprises a nucleic acid sequence obtained from a human γδ T cell receptor. (Item 7) 2. The engineered cell of item 1, wherein the engineered cell lacks an exogenous oncogene. (Item 8) 2. The engineered cell of item 1, wherein the gamma delta T cell receptor nucleic acid molecule encodes at least one amino acid sequence set forth in SEQ ID NO: 1 to SEQ ID NO: 52. (Item 9) A composition comprising engineered cells transduced with at least one polynucleotide encoding a T cell receptor gamma chain polypeptide and / or a T cell receptor delta chain polypeptide, wherein the T cell receptor gamma chain polypeptide and / or the T cell receptor delta chain polypeptide comprises at least one amino acid sequence set forth in SEQ ID NO:1 to SEQ ID NO:52. (Item 10) 1. A method for generating an engineered functional gamma delta T cell comprising at least one exogenous nucleic acid molecule encoding a T cell receptor gamma chain polypeptide and / or a T cell receptor delta chain polypeptide, comprising: transducing human hematopoietic stem / progenitor cells with at least one exogenous nucleic acid molecule encoding a T cell receptor gamma chain polypeptide and a T cell receptor delta chain polypeptide, such that the human pluripotent cells transduced with the at least one exogenous nucleic acid molecule express a T cell receptor comprising the gamma chain polypeptide and the delta chain polypeptide encoded by the at least one exogenous nucleic acid molecule; differentiating said human hematopoietic stem / progenitor cells to produce said engineered functional gamma delta T cells; A method comprising: (Item 11) The method comprises: (a) differentiating the transduced human hematopoietic stem / progenitor cells in a first in vitro culture; (b) expanding the differentiated cells of (a) in a second in vitro culture; and Item 11. The method of item 10, further comprising: (Item 12) the hematopoietic stem / progenitor cells are cultured in feeder cell-free medium, and / or 12. The method of claim 11, wherein the hematopoietic stem / progenitor cells are cultured in a medium comprising one or more of IL-3, IL-7, IL-6, SCF, MCP-4, EPO, TPO, FLT3L and / or retronectin. (Item 13) 13. The method of claim 12, further comprising expanding in vitro the cells transduced with the nucleic acid molecule encoding a T cell receptor gamma chain polypeptide and / or a T cell receptor delta chain polypeptide. (Item 14) 11. The method of claim 10, further comprising transplanting the hematopoietic stem / progenitor cells transduced with the nucleic acid molecule encoding the T cell receptor gamma chain polypeptide and / or the T cell receptor delta chain polypeptide into a subject to generate a clonal population of the engineered cells in vivo. (Item 15) the engineered functional gamma delta T cells are HLA-I low / negative, HLA-II low / negative, HLA-E positive, and 11. The method of claim 10, comprising a gene expression profile characterized by at least one of expressing an immunomodulatory gene and / or a suicide gene. (Item 16) the exogenous nucleic acid molecule is contained in a lentiviral expression vector; and / or 11. The method of claim 10, wherein the method further comprises contacting the transduced cells with an agent selected to promote growth and / or differentiation. (Item 17) 17. The method of claim 16, further comprising co-culturing the transduced cells with peripheral blood mononuclear cells, antigen-presenting cells, or artificial antigen-presenting cells. (Item 18) The hematopoietic stem / progenitor cells are CD34 + 11. The method of item 10, comprising hematopoietic stem or progenitor cells. (Item 19) Item 11. The method according to Item 10, wherein the T cell receptor gamma chain polypeptide and / or the T cell receptor delta chain polypeptide comprises at least one amino acid sequence set forth in SEQ ID NO: 1 to SEQ ID NO: 52. (Item 20) 20. An engineered functional gamma delta T cell produced by the method of any one of items 10 to 19. (Item 21) 21. A method of treating a subject in need of gamma delta T cells, comprising administering to said subject the cells of items 1 to 9 or 20. (Item 22) the gamma delta T cells are CD34 + 22. The method of claim 21, wherein the hematopoietic stem or progenitor cells are produced by transducing at least one exogenous nucleic acid molecule encoding a T cell receptor gamma chain polypeptide, a T cell receptor delta chain polypeptide, IL-15, and a suicide gene. (Item 23) 23. The method according to Item 22, wherein the gamma delta T cells comprise at least one amino acid sequence set forth in SEQ ID NO: 1 to SEQ ID NO: 52. (Item 24) a subject in need of said gamma delta T cells has been diagnosed with cancer; or the subject in need of the gamma delta T cells has been diagnosed with a viral infection, a fungal infection, or a protozoal infection. Item 21. The method according to item 21. (Item 25) 22. The method of claim 21, wherein the T cell receptor gamma chain polypeptide and the T cell receptor delta chain polypeptide are selected from γδ T cell receptors, which are observed to target cancer cells or cells infected with viruses, fungi, or protozoa.

Claims

1. 1. An engineered cell, which is a cell genetically modified to contain at least one exogenous gamma delta T cell receptor (γδ TCR) nucleic acid molecule, wherein the gamma delta T cell receptor nucleic acid molecule encodes a T cell receptor gamma chain polypeptide and a T cell receptor delta chain polypeptide, and wherein the T cell receptor gamma chain polypeptide and the T cell receptor delta chain polypeptide comprise the amino acid sequences set forth in SEQ ID NO:3 and SEQ ID NO:4, respectively.

2. A composition comprising engineered cells transduced with at least one polynucleotide encoding a T cell receptor gamma chain polypeptide and a T cell receptor delta chain polypeptide, wherein the T cell receptor gamma chain polypeptide and the T cell receptor delta chain polypeptide comprise the amino acid sequences set forth in SEQ ID NO:3 and SEQ ID NO:4, respectively.

3. 1. An ex vivo method for generating engineered functional gamma delta T cells, said method comprising: transducing human hematopoietic stem / progenitor cells with at least one exogenous nucleic acid molecule encoding a T cell receptor gamma chain polypeptide and a T cell receptor delta chain polypeptide, such that the human hematopoietic stem / progenitor cells transduced with the at least one exogenous nucleic acid molecule express T cell receptors comprising the gamma chain polypeptide and the delta chain polypeptide encoded by the at least one exogenous nucleic acid molecule, wherein the T cell receptor gamma chain polypeptide and the T cell receptor delta chain polypeptide comprise the amino acid sequences set forth in SEQ ID NO:3 and SEQ ID NO:4, respectively; differentiating said human hematopoietic stem / progenitor cells to produce said engineered functional gamma delta T cells; Including, the hematopoietic stem / progenitor cells are cultured in a feeder cell-free medium; The method wherein the hematopoietic stem / progenitor cells are cultured in a medium comprising one or more of IL-3, IL-7, IL-6, SCF, MCP-4, EPO, TPO, FLT3L and RetroNectin™.

4. The method comprises: (a) differentiating the transduced human hematopoietic stem / progenitor cells in a first in vitro culture; and (b) expanding the differentiated cells of (a) in a second in vitro culture; and The method of claim 3, comprising:

5. 4. The method of claim 3, wherein the method further comprises expanding in vitro the cells transduced with the nucleic acid molecules encoding T cell receptor gamma chain polypeptides and T cell receptor delta chain polypeptides.

6. A composition for use in a method of making engineered functional gamma delta T cells, said composition comprising at least one exogenous nucleic acid molecule encoding a T cell receptor gamma chain polypeptide and a T cell receptor delta chain polypeptide, wherein said T cell receptor gamma chain polypeptide and said T cell receptor delta chain polypeptide comprise the amino acid sequences set forth in SEQ ID NO:3 and SEQ ID NO:4, respectively, said method comprising: transducing human hematopoietic stem / progenitor cells with at least one exogenous nucleic acid molecule encoding a T cell receptor gamma chain polypeptide and a T cell receptor delta chain polypeptide, such that human hematopoietic stem / progenitor cells transduced with said at least one exogenous nucleic acid molecule express a T cell receptor comprising the gamma chain polypeptide and the delta chain polypeptide encoded by said at least one exogenous nucleic acid molecule; differentiating said human hematopoietic stem / progenitor cells to generate said engineered functional gamma delta T cells; wherein the method further comprises transplanting the hematopoietic stem / progenitor cells transduced with the nucleic acid molecules encoding the T cell receptor gamma chain polypeptide and the T cell receptor delta chain polypeptide into a subject to generate a clonal population of the engineered cells in vivo. composition.

7. the engineered functional gamma delta T cells are HLA-I negative, HLA-II negative, HLA-E positive, and Expressing immunomodulatory and / or suicide genes 4. The method of claim 3, comprising a gene expression profile characterized by at least one of:

8. the exogenous nucleic acid molecule is contained in a lentiviral expression vector; and / or 4. The method of claim 3, wherein the method further comprises contacting the transduced cells with an agent selected to promote growth and / or differentiation.

9. 9. The method of claim 8, wherein the method further comprises co-culturing the transduced cells with peripheral blood mononuclear cells, antigen-presenting cells, or artificial antigen-presenting cells.

10. The hematopoietic stem / progenitor cells are CD34 ＋ The method of claim 3 , comprising hematopoietic stem or progenitor cells.

11. 10. A composition comprising the engineered cells of claim 1 for treating a subject in need of gamma delta T cells, wherein the engineered cells are gamma delta T cells.

12. 10. A composition comprising the engineered cells of claim 1 for treating a subject in need of gamma delta T cells, wherein the engineered cells are gamma delta T cells, and the gamma delta T cells are CD34 ＋ A composition produced by transducing hematopoietic stem or progenitor cells with at least one exogenous nucleic acid molecule encoding a T cell receptor gamma chain polypeptide, a T cell receptor delta chain polypeptide, IL-15, and a suicide gene.

13. The composition of claim 12, wherein the gamma delta T cells comprise a T cell receptor gamma chain polypeptide and a T cell receptor delta chain polypeptide, and the T cell receptor gamma chain polypeptide and the T cell receptor delta chain polypeptide comprise the amino acid sequences set forth in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

14. a subject in need of said gamma delta T cells has been diagnosed with cancer; or the subject in need of the gamma delta T cells has been diagnosed with a viral infection, a fungal infection, or a protozoal infection; The composition of claim 11.

15. 12. The composition of claim 11, wherein the T cell receptor gamma chain polypeptide and the T cell receptor delta chain polypeptide are selected from γδ T cell receptors that are observed to target cancer cells or cells infected with a virus, a fungus, or a protozoan.

Images