Novel Gene Armoring

Abstract

The present disclosure provides compositions and methods for modifying γδ T cells (e.g., vδ1 and vδ2 T cells) with the IL-15 receptor β subunit (IL-15Rβ), e.g., by transduction with a viral vector, to restore IL-15 responsiveness. Additionally, populations of modified γδ T cells and methods of use thereof are provided.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This PCT application claims the benefit of priority to U.S. Provisional Application No. 63 / 327,230, filed April 4, 2022, U.S. Patent Application No. 63 / 371,860, filed August 18, 2022, and U.S. Provisional Application No. 63 / 482,755, filed February 1, 2023, each of which is incorporated by reference in its entirety.

[0002] Reference to sequence listings submitted electronically via EFS-Web The contents of the electronically submitted sequence listing (Name: 3817_170PC03_Seqlisting_ST26, Size: 39,258 bytes, Creation Date: April 3, 2023) submitted with the application in ASCII text file format are hereby incorporated by reference in their entirety.

[0003] Conventional chimeric antigen receptor (CAR) armoring aims to improve the persistence, proliferation, or other properties of CAR+ cells by having the cells also express IL-15 (either secreted or membrane-bound). Co-expression of IL-15 by CAR+ γδ T cells in the same or separate construct reduces the responsiveness of the cells to IL-15. Thus, novel compositions and methods are needed to reprogram these cells to provide responsiveness to IL-15 in order to adapt γδ T cells to various immunotherapies. [Background technology]

[0004] The growing interest in T cell immunotherapy for cancer has focused on the apparent potential of engineered T cells as therapeutic moieties. Gamma delta (γδ) T cells are a subset of T cells that express a distinct and distinctive γδ T cell receptor (TCR) on their surface. This TCR is composed of one gamma (γ) chain and one delta (δ) chain. Human γδ T cells can be broadly classified into one or two types: peripheral blood resident γδ T cells and non-hematopoietic tissue resident γδ T cells. Most blood resident γδ T cells express the Vδ2 TCR, which is less common in tissue resident γδ T cells, with Vδ1 and / or other Vδ chains being used more frequently.

[0005] IL-15 is a cytokine and T cell growth factor that binds to and signals through a receptor complex consisting of the IL-15 receptor beta subunit (IL-15Rβ, also known as IL-2Rβ), the IL-15 receptor alpha subunit (IL-15Rα), and the common gamma chain. IL-15 is typically secreted by phagocytes after infection and induces the proliferation of certain immune cells (e.g., natural killer cells), which then begin to kill the infected cells. Traditional chimeric antigen receptor (CAR) armoring aims to improve the persistence, proliferation, or other properties of CAR+ cells by having the cells also express IL-15 (either secreted or membrane-bound). Co-expression of IL-15 by CAR+Vδ1 T cells in the same or separate constructs reduces the responsiveness of the cells to IL-15. Thus, novel compositions and methods are needed to reprogram these cells to provide responsiveness to IL-15 in order to adapt gamma delta T cells to various immunotherapies. Summary of the Invention

[0006] In one aspect, the invention features a γδ T cell expressing a recombinant IL-15 receptor β subunit (IL-15Rβ) (e.g., expressing IL-15Rβ without co-expressing a recombinant IL-15 receptor α subunit (IL-15Rα) (e.g., without co-expressing a recombinant IL-15 receptor α subunit (IL-15Rα) where the N-terminus of IL-15Rα, or a fragment thereof, is tethered to the C-terminus of IL-15, or a variant thereof).

[0007] In some embodiments, the γδ T cells further express a recombinant IL-15 receptor α subunit (IL-15Rα) or a fragment thereof.

[0008] In some embodiments, the IL-15Rα or fragment thereof is tethered to IL-15 or a variant thereof. In some embodiments, the C-terminus of the IL-15Rα or fragment thereof is tethered to the N-terminus of IL-15 or a variant thereof. In some embodiments, the N-terminus of the IL-15Rα or fragment thereof is tethered to the C-terminus of IL-15 or a variant thereof.

[0009] In some embodiments, IL-15Rβ and IL-15Rα or fragments thereof are expressed in tandem. IL-15Rβ and IL-15Rα or fragments thereof may include a linker between them. The linker may include a cleavable peptide. The cleavable peptide may be, for example, a self-cleaving peptide, such as a 2A peptide. In some embodiments, the 2A peptide is a foot and mouth disease virus 18 2A (F2A) peptide, an equine rhinitis A virus 2A (E2A) peptide, a porcine teschovirus-1 2A (P2A) peptide, or a thosea asigna virus 2A (T2A) peptide.

[0010] In some embodiments, the γδ T-cell further expresses a chimeric antigen receptor (CAR), which may lack an intracellular signaling domain.

[0011] In some embodiments, the γδ T cells are deleted for the endogenous gene encoding IL-15.

[0012] In some embodiments, the γδ T cells further express secreted IL-15 or a variant thereof.

[0013] In another aspect, the invention features a γδ T cell expressing recombinant IL-15Rβ and recombinant IL-15 or a variant thereof.

[0014] In some embodiments, IL-15Rβ and IL-15 or a variant thereof are expressed in tandem. IL-15Rβ and IL-15 or a variant thereof may include a linker between them. The linker may include a cleavable peptide. The cleavable peptide may be, for example, a self-cleaving peptide, such as a 2A peptide. In some embodiments, the 2A peptide is an F2A peptide, an E2A peptide, a P2A peptide, or a T2A peptide.

[0015] In some embodiments, the γδ T cell further expresses recombinant IL-15Rα or a fragment thereof. The fragment of IL-15Rα may include, for example, a soluble fragment. The soluble fragment may include, for example, a sushi domain. The sushi domain may be linked to an Fc domain. In some embodiments, the IL-15Rα or a fragment thereof is tethered to IL-15 or a variant thereof. In some embodiments, the C-terminus of the IL-15Rα or a fragment thereof is tethered to the N-terminus of the IL-15 or a variant thereof. In some embodiments, the N-terminus of the IL-15Rα or a fragment thereof is tethered to the C-terminus of the IL-15 or a variant thereof.

[0016] In some embodiments, IL-15Rβ and IL-15Rα or fragments thereof are expressed in tandem. For example, IL-15Rβ and IL-15Rα or fragments thereof can include a second linker between them. The second linker can include a cleavable peptide. The cleavable peptide can be, for example, a self-cleaving peptide, such as a 2A peptide. In some embodiments, the 2A peptide is an F2A peptide, an E2A peptide, a P2A peptide, or a T2A peptide.

[0017] In some embodiments, IL-15Rβ, IL-15 or a variant thereof, and recombinant IL-15Rα or a fragment thereof are expressed in tandem.

[0018] In some embodiments, the γδ T-cell further expresses a CAR, which may lack an intracellular signaling domain.

[0019] In some embodiments, the γδ T cells are deleted for the endogenous gene encoding IL-15.

[0020] In another aspect, the invention features an isolated population of γδ T cells, wherein at least 10% (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, or 99%) of the population of γδ T cells express recombinant IL-15Rβ. In some embodiments, at least 50% of the population of γδ T cells express recombinant IL-15Rβ.

[0021] In some embodiments, the isolated population of γδ T cells further expresses recombinant IL-15Rα or a fragment thereof.

[0022] In some embodiments, the IL-15Rα or fragment thereof is tethered to IL-15 or a variant thereof. In some embodiments, the C-terminus of the IL-15Rα or fragment thereof is tethered to the N-terminus of IL-15 or a variant thereof. In some embodiments, the N-terminus of the IL-15Rα or fragment thereof is tethered to the C-terminus of IL-15 or a variant thereof.

[0023] In some embodiments, IL-15Rβ and IL-15Rα or fragments thereof are expressed in tandem. IL-15Rβ and IL-15Rα or fragments thereof may include a linker between them. The linker may include a cleavable peptide. The cleavable peptide may be, for example, a self-cleaving peptide, such as a 2A peptide. In some embodiments, the 2A peptide is an F2A peptide, an E2A peptide, a P2A peptide, or a T2A peptide.

[0024] In some embodiments, the isolated population of γδ T-cells further expresses a CAR, which may lack an intracellular signaling domain.

[0025] In some embodiments, the isolated population of γδ T cells is deleted for the endogenous gene encoding IL-15.

[0026] In some embodiments, the isolated population of γδ T cells further expresses secreted IL-15 or a variant thereof.

[0027] In another aspect, the invention features an isolated population of γδ T cells, wherein at least 10% (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, or 99%) of the population of γδ T cells express recombinant IL-15Rβ and IL-15 or a variant thereof. In some embodiments, at least 50% of the population of γδ T cells express recombinant IL-15Rβ and IL-15 or a variant thereof.

[0028] In some embodiments, IL-15Rβ and IL-15 or a variant thereof are expressed in tandem. For example, the IL-15Rβ and the IL-15 receptor or a variant thereof can include a linker between them. The linker can include a cleavable peptide. The cleavable peptide can be, for example, a self-cleaving peptide, such as a 2A peptide. In some embodiments, the 2A peptide is an F2A peptide, an E2A peptide, a P2A peptide, or a T2A peptide.

[0029] In some embodiments, the isolated population of γδ T cells further expresses recombinant IL-15Rα or a fragment thereof. The fragment of IL-15Rα may include, for example, a soluble fragment. The soluble fragment may include, for example, a sushi domain. The sushi domain may be linked to an Fc domain. In some embodiments, the IL-15Rα or a fragment thereof is tethered to IL-15 or a variant thereof. In some embodiments, the C-terminus of the IL-15Rα or a fragment thereof is tethered to the N-terminus of the IL-15 or a variant thereof. In some embodiments, the N-terminus of the IL-15Rα or a fragment thereof is tethered to the C-terminus of the IL-15 or a variant thereof.

[0030] In some embodiments, IL-15Rβ and IL-15Rα or fragments thereof are expressed in tandem. For example, IL-15Rβ and IL-15Rα or fragments thereof may include a second linker between them. For example, IL-15Rβ and IL-15Rα or fragments thereof may include a second linker between them. The second linker may include a cleavable peptide. The cleavable peptide may be, for example, a self-cleaving peptide, such as a 2A peptide. In some embodiments, the 2A peptide is an F2A peptide, an E2A peptide, a P2A peptide, or a T2A peptide.

[0031] In some embodiments, IL-15Rβ, IL-15 or a variant thereof, and recombinant IL-15Rα or a fragment thereof are expressed in tandem.

[0032] In some embodiments, the isolated population of γδ T-cells further expresses a CAR, which may lack an intracellular signaling domain.

[0033] In some embodiments, the isolated population of γδ T cells is deleted for the endogenous gene encoding IL-15.

[0034] In another aspect, the invention features a method of generating a population of γδ T cells that express recombinant IL-15Rβ, the method includes: (a) providing a population of γδ T cells; and (b) transducing the population of γδ T cells with a polynucleotide encoding IL-15Rβ, thereby generating a population of γδ T cells that express recombinant IL-15Rβ.

[0035] In some embodiments, the polynucleotide comprises an IRES operably linked to IL-15Rβ.

[0036] In some embodiments, the method further comprises transducing the starting population of γδ T cells with a polynucleotide encoding IL-15Rα or a fragment thereof. In some embodiments, the IL-15Rα or a fragment thereof is tethered to IL-15 or a variant thereof. In some embodiments, the C-terminus of the IL-15Rα or a fragment thereof is tethered to the N-terminus of IL-15 or a variant thereof. In some embodiments, the N-terminus of the IL-15Rα or a fragment thereof is tethered to the C-terminus of IL-15 or a variant thereof.

[0037] In some embodiments, the polynucleotide encodes IL-15Rβ and IL-15Rα in tandem. The polynucleotide may include a linker between IL-15Rβ and IL-15Rα. The linker may encode a cleaving peptide or may include an IRES. The cleaving peptide may be, for example, a self-cleaving peptide, such as a 2A peptide. In some embodiments, the 2A peptide is an F2A peptide, an E2A peptide, a P2A peptide, or a T2A peptide.

[0038] In some embodiments, the method further comprises transducing the cell with a polynucleotide encoding a CAR. In some embodiments, the polynucleotide encodes a CAR and an IL-15Rβ in tandem. For example, the polynucleotide can comprise a linker between the CAR and the IL-15Rβ. The linker can encode a cleavable peptide or can comprise an IRES. The cleavable peptide can be, for example, a self-cleaving peptide, such as a 2A peptide. In some embodiments, the 2A peptide is an F2A peptide, an E2A peptide, a P2A peptide, or a T2A peptide.

[0039] In another aspect, the invention features a method of generating a population of γδ T cells that express recombinant IL-15Rβ and IL-15 or a variant thereof, the method includes (a) providing a population of γδ T cells, (b) transducing the population of γδ T cells with a polynucleotide encoding IL-15Rβ, and (c) transducing the population of γδ T cells with a polynucleotide encoding IL-15 or a variant thereof, thereby generating a population of γδ T cells that expresses recombinant IL-15Rβ and IL-15 or a variant thereof.

[0040] In some embodiments, the polynucleotide comprises an IRES operably linked to IL-15Rβ and / or IL-15 or a variant thereof.

[0041] In some embodiments, the polynucleotide encodes IL-15Rβ and IL-15 or a variant thereof in tandem. For example, the polynucleotide may include a linker between IL-15Rβ and IL-15 or a variant thereof. The linker may encode a cleavable peptide or may include an IRES. The cleavable peptide may be, for example, a self-cleaving peptide, such as a 2A peptide. In some embodiments, the 2A peptide is an F2A peptide, an E2A peptide, a P2A peptide, or a T2A peptide.

[0042] In some embodiments, the method further comprises transducing the starting population of γδ T cells with a polynucleotide encoding IL-15Rα or a fragment thereof. The fragment of IL-15Rα may include a soluble fragment. For example, the soluble fragment may include a sushi domain. In some embodiments, the sushi domain is linked to an Fc domain.

[0043] In some embodiments, the IL-15Rα or fragment thereof is tethered to IL-15 or a variant thereof. In some embodiments, the C-terminus of the IL-15Rα or fragment thereof is tethered to the N-terminus of IL-15 or a variant thereof. In some embodiments, the N-terminus of the IL-15Rα or fragment thereof is tethered to the C-terminus of IL-15 or a variant thereof.

[0044] In some embodiments, the polynucleotide encodes IL-15Rβ and IL-15Rα in tandem.

[0045] In some embodiments, the polynucleotide comprises a second linker between IL-15Rβ and IL-15Rα. The second linker may code for a cleavable peptide or may comprise an IRES. The cleavable peptide may be, for example, a self-cleaving peptide, for example, a 2A peptide. In some embodiments, the 2A peptide is an F2A peptide, an E2A peptide, a P2A peptide, or a T2A peptide.

[0046] In some embodiments, the polynucleotide tandemly encodes IL-15Rβ, IL-15 or a variant thereof, and recombinant IL-15Rα or a fragment thereof.

[0047] In some embodiments, the method further comprises transducing the cell with a polynucleotide encoding a CAR. The polynucleotide may encode a CAR and an IL-15Rβ in tandem. For example, the polynucleotide may comprise a linker between the CAR and the IL-15Rβ. The linker may encode a cleavable peptide or may comprise an IRES. The cleavable peptide may be, for example, a self-cleaving peptide, such as a 2A peptide. In some embodiments, the 2A peptide is an F2A peptide, an E2A peptide, a P2A peptide, or a T2A peptide.

[0048] In some embodiments of any of the methods described herein, the method comprises providing a vector comprising polynucleotide.Vector can be, for example, a viral vector.Viral vector can be, for example, a retroviridae virus, adenovirus, parvovirus, coronavirus, rhabdovirus, paramyxovirus, picornavirus, alphavirus, herpesvirus or poxvirus.

[0049] In some embodiments, the parvovirus vector is an AAV vector.

[0050] In some embodiments, the viral vector is a retroviridae viral vector. The retroviridae viral vector can be, for example, a lentiviral vector. The retroviridae viral vector can be, for example, an alpha retroviral vector or a gamma retroviral vector. In some embodiments, the retroviridae viral vector comprises a central polypurine tract, a woodchuck hepatitis virus post-transcriptional regulatory element, a 5'-LTR, an HIV signal sequence, an HIV Psi signal 5'-splice site, a delta-GAG element, a 3'-splice site, and a 3'-self-inactivating LTR.

[0051] In some embodiments, the viral vector is a pseudotyped viral vector. The pseudotyped viral vector may be, for example, a pseudotyped adenovirus, a pseudotyped parvovirus, a pseudotyped coronavirus, a pseudotyped rhabdovirus, a pseudotyped paramyxovirus, a pseudotyped picornavirus, a pseudotyped alphavirus, a pseudotyped herpesvirus, a pseudotyped poxvirus, and a pseudotyped retroviridae virus. In some embodiments, the pseudotyped viral vector is a lentiviral vector. In some embodiments, the pseudotyped viral vector comprises one or more envelope proteins from a virus selected from vesicular stomatitis virus VSV, RD114 virus, MLV, FeLV, VEE, HFV, WDSV, SFV, rabies virus, ALV, BIV, BLV, EBV, CAEV, SNV, ChTLV, STLV, MPMV, SMRV, RAV, FuSV, MH2, AEV, AMV, avian sarcoma virus CT10, and EIAV. For example, in some embodiments, the pseudotyped viral vector comprises a VSV-G envelope protein.

[0052] In some embodiments of any of the methods described herein, the method further comprises culturing the starting population of γδ T cells in the presence of exogenous IL-2 and / or IL-15.

[0053] In some embodiments, at least 10% (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, or 99%) of the population of γδ T cells express IL-15Rβ. In some embodiments, at least 50% of the population of γδ T cells express IL-15Rβ.

[0054] In another aspect, the invention features a method of expanding γδ T cells, the method including (a) providing a population of γδ T cells that express IL-15Rβ, and (b) culturing the population of γδ T cells in the presence of exogenous IL-2 and / or IL-15 or a variant thereof.

[0055] In some embodiments, the population of γδ T cells further expresses recombinant IL-15Rα or a fragment thereof. The IL-15Rα or a fragment thereof may be tethered to IL-15 or a variant thereof. In some embodiments, the C-terminus of the IL-15Rα or a fragment thereof is tethered to the N-terminus of IL-15 or a variant thereof. In some embodiments, the N-terminus of the IL-15Rα or a fragment thereof is tethered to the C-terminus of IL-15 or a variant thereof.

[0056] In another aspect, the invention features a method of expanding γδ T cells, the method includes (a) providing a population of γδ T cells that express IL-15Rβ and IL-15 or a variant thereof, and (b) culturing the γδ T cells, where the IL-15 or variant thereof expands the population of γδ T cells.

[0057] In some embodiments, the method further comprises culturing the population of γδ T cells in the presence of exogenous IL-2.

[0058] In some embodiments, the population of γδ T cells further expresses recombinant IL-15Rα or a fragment thereof. The fragment of IL-15Rα can include, for example, a soluble fragment. The soluble fragment can include, for example, a sushi domain. In some embodiments, the sushi domain is linked to an Fc domain.

[0059] In some embodiments, IL-15Rα or a fragment thereof is tethered to IL-15 or a variant thereof. In some embodiments, the C-terminus of IL-15Rα or a fragment thereof is tethered to the N-terminus of IL-15 or a variant thereof. In some embodiments, the N-terminus of IL-15Rα or a fragment thereof is tethered to the C-terminus of IL-15 or a variant thereof. As used herein, "mbIL-15" refers to membrane-bound IL-15Rα tethered to IL-15 (e.g., where the C-terminus of IL-15Rα is tethered to the N-terminus of IL-15). [Brief description of the drawings]

[0060] [Figure 1] Schematic diagram of conventional and novel IL-15 armoring constructs. A shows a diagram of the armoring strategies evaluated in the present invention. Conventional armoring constructs used in the context of αβT cells, NK cells, and iNKT cells utilize secreted (sIL-15) or membrane-tethered IL-15 (mbIL-15) constructs. Novel armoring strategies are constructed with the introduction of a common β chain (also known as IL-2Rβ, IL-15Rβ, or CD122) alone or in combination with wild-type or IL-15-tethered IL-15Rα (CD215) chains. B shows the domain structure of conventional armoring CAR encoding gamma retroviral vectors. C shows the domain structure of novel armoring CAR encoding gamma retroviral vectors. [Diagram 2] Graphs evaluating the effect of conventional armoring strategies on V51 T cell proliferation and phenotype. A shows fold expansion of CAR-modified non-armored (muCAR19) and conventional armored (muCAR19.sIL-15 and muCAR19-mbIL-15) V51 T cells. B shows analysis of surface expression of CAR, IL-15, and IL-15Rα by flow cytometry in V51 T cells modified with mbIL-15 armored CAR. C shows analysis of IL-15 production by ELISA in non-armored (muCAR19) and secreted IL-15 armored (muCAR19.sIL-15) CAR-modified V51 T cells. [Diagram 3]Graphs evaluating the effect of conventional armoring strategies on V51 T cell cytotoxicity, viability, and repeated antigen stimulation-induced proliferation. A shows a comparison of the cytotoxic activity of non-armored (muCAR19) and conventional armored V51 T cells against the NALM-6 acute lymphoblastic leukemia cell line. B shows analysis of survival of non-armored and conventional armored CAR-modified V51 T cells by flow cytometry. C shows fold expansion of CAR-modified non-armored and conventional armored V51 T cells during three repeated antigen stimulations with CD19+NALM-6 cells. [Figure 4] Graph assessing the effect of armouring of mbIL-15 on the fold expansion of CAR-modified V51 T cells. The graph shows a comparison of the fold expansion of non-armoured and mbIL-15 armoured V51 CAR-T cells in the presence of high and low concentrations of IL-15. [Diagram 5] Graph showing analysis of IL-15 receptor chain component expression using LEGENDScreen. Heatmap shows percentage of V51 T cells expressing IL-15Rα (CD215), IL-15Rβ (CD122, also known as IL-2Rβ) and common gamma chain (CD123, γc) among naive V51 T cells. [Figure 6] Graphs evaluating the effect of IL-15Rβ chain transfer in the presence of mbIL-15 or IL-15Rα chain. A shows a comparison of fold expansion of non-armored (muCAR19), mbIL-15 armored (muCAR19.α.15), mbIL-15 and IL-15β chain armored (muCAR19.α.15.β) and IL-15Rα and β armored (muCAR19.α.β) CAR modified Vδ1 T cells. B shows fold expansion of α.15, α.β, or α.15.β chain expressing Vδ1 T cells in the presence of low and high concentrations of IL-15. [Figure 7]Graphs assessing the effect of IL-15Rβ chain transfer on V51 T cell cytotoxicity, viability, and repeated antigen stimulation-induced proliferation. A shows a comparison of cytotoxic activity of conventional armoring (mbIL-15, α.15), mbIL-15 and IL-15β chain armoring (muCAR19.α.15.β), and IL-15Rα and β armoring (muCAR19.α.β) CAR-modified V51 T cells against the NALM-6 acute lymphoblastic leukemia cell line. B shows the effect of armoring on fold expansion, enrichment, and target (NALM-6) clearance of CAR-modified V51 T cells during repeated antigen stimulation with CD19+NALM-6 cells in the absence and presence of exogenous IL-15 (C). [Figure 8] Graph showing analysis of CAR and IL-15R chain expression 3 days after transduction. (A) Comparison of CAR expression in V51 T cells armoured with novel armouring constructs 3 days after transduction. (B) Comparison of IL-15Rα, IL-15Rβ, common gamma chain and tethered IL-15 expression in armoured CAR-modified V51 T cells. [Figure 9] Graphs assessing the effect of IL15Rβ chain transfer alone or in combination with wild type or IL-15 tethered IL-15Rα chain on V51 T cell proliferation. (A) shows a comparison of fold expansion of IL-15Rβ (muCAR19.β), mbIL-15 and IL-15Rβ (muCAR19.α.15.β), and IL-15Rα and β (muCAR19.α.β) armoured CAR modified V51 T cells. (B) shows fold expansion of CAR modified V51 T cells armoured with novel constructs. [Figure 10A] Graph showing phenotypic analysis of frozen-thawed Armouring CAR-modified V51 T cell preparations. The figure shows analysis of CAR expression in Armouring V51 T cells. [Figure 10B] Graph showing phenotypic analysis of frozen-thawed Armouring CAR-modified V51 T cell preparations. The figure shows a comparison of IL-15Rα, IL-15Rβ, common gamma chain and tethered IL-15 expression in Armouring CAR-modified V51 T cells. [Figure 10C] Graph showing phenotypic analysis of frozen-thawed Armouring CAR-modified V51 T cell preparations. The figure shows a comparison of CAR expression in Armouring V51 T cells grown in the presence of high and low concentrations of IL-15 3 days after transduction (day 7) and on the day of harvest (day 14). [Figure 10D] Graph showing phenotypic analysis of frozen-thawed Armoring CAR-modified V51 T cell preparations. The figure shows flow cytometry analysis of immune cell activation and inhibitory marker expression in CAR+ve and CAR-ve V51 T cell fractions. [Figure 11] Graph showing cytotoxic activity of freshly thawed, armored CAR-modified V51 T cell preparations. Cytotoxic activity of armored cells is shown in the absence (A) and presence (B) of IL-15. Cytotoxicity of CD19-targeted CAR-modified cells against antigen-negative targets (C) is shown against AML targets MV4-11 on the left and MOLM-13 on the right. D shows cytotoxicity of CD123-targeted CAR-modified cells non-armored (CAR123) or armored with mbIL-15 and IL-15Rβ (CAR123.α.15.β). E shows cytotoxicity of mesothelin-targeted CAR-modified cells non-armored (Meso-CAR) or armored with mbIL-15 and IL-15Rβ (Meso-CAR.α.15.β). [Figure 12] Graph showing in vitro viability of freshly thawed Armouring CAR-modified V51 T cell preparations in the absence of exogenous IL-15. A shows the fold expansion of Armouring CAR-modified V51 T cells. B shows the relative fold change in number of non-transduced (CAR-ve) V51 T cells. C shows enrichment of CAR-modified V51 T cells. D shows the percentage of viable lymphocytes over the assay period. [Figure 13] Graphs assessing the effect of repeated antigen exposure on the proliferation, enrichment, and target cell clearance of Armoring CAR-modified Vδ1 T cells. A-C show repeated antigen stimulation with NALM-6 cells in the presence of supraphysiological concentrations (10 ng / mL) and D-F show physiological IL-15 concentrations (70 pg / mL). [Figure 14A] Schematic and results evaluating the in vivo performance of Armouring CAR-modified V51 T cells in NSG and NOG-IL15 mice challenged with NALM6 tumor cells. The figure shows the in vivo study design and experimental study classification. [Figure 14B] Schematic and results evaluating the in vivo performance of Armoring CAR-modified V51 T cells in NSG and NOG-IL15 mice challenged with NALM6 tumor cells. The figure shows in vivo efficacy in NSG mice in the presence of exogenous IL-15. [Figure 14C] Schematic and results evaluating the in vivo performance of Armoring CAR-modified V51 T cells in NSG and NOG-IL15 mice challenged with NALM6 tumor cells. The figure shows in vivo efficacy in NSG mice in the absence of exogenous IL-15. [Figure 14D] Schematic and results evaluating the in vivo performance of Armouring CAR-modified V51 T cells in NSG and NOG-IL15 mice challenged with NALM6 tumor cells. The figure shows in vivo efficacy in NOG-hIL15 mice. [Figure 14E] Schematic and results evaluating the in vivo performance of Armoring CAR-modified V51 T cells in NSG and NOG-IL15 mice challenged with NALM6 tumor cells. The figure shows the in vivo persistence of CD19 CAR+ and CAR-V51 T cells in the bone marrow of NSG mice 3 days post-administration. [Figure 14F] Schematic and results evaluating the in vivo performance of Armoring CAR-modified V51 T cells in NSG and NOG-IL15 mice challenged with NALM6 tumor cells. The figure compares the percentage of V51 CD19 CAR+ in the formulation upon freeze-thawing with cells harvested from the bone marrow of NSG mice 3 days after dosing. [Figure 14G]Schematic and results evaluating the in vivo performance of Armoring CAR-modified V51 T cells in NSG and NOG-IL15 mice challenged with NALM6 tumor cells. The figure shows in vivo efficacy in NSG mice in the presence of exogenous IL-15 through to the end of the study on days 20 / 21. [Figure 14H] Schematic and results evaluating the in vivo performance of Armoring CAR-modified V51 T cells in NSG and NOG-IL15 mice challenged with NALM6 tumor cells. The figure shows in vivo efficacy in NSG mice in the absence of exogenous IL-15 through the end of the study on days 20 / 21. [Figure 14I] Schematic and results evaluating the in vivo performance of Armoring CAR-modified V51 T cells in NSG and NOG-IL15 mice challenged with NALM6 tumor cells. The figure shows in vivo efficacy in NOG-hIL15 mice through the end of the study on days 20 / 21. [Figure 14J] Schematic and results evaluating the in vivo performance of Armoring CAR-modified V51 T cells in NSG and NOG-IL15 mice challenged with NALM6 tumor cells. The figure compares the percentage of V51 CD19 CAR+ of the formulation upon freeze-thaw with cells harvested from the bone marrow of NSG mice 3 and 14 days after dosing. [Figure 14K] Schematic and results evaluating the in vivo performance of Armoring CAR-modified V51 T cells in NSG and NOG-IL15 mice challenged with NALM6 tumor cells. The figure compares the numbers of V51 CD19 CAR+ and CAR- cells recovered from bone marrow samples taken 14 days after exogenous IL-15 treatment from NSG mice. [Figure 14L] Schematic and results evaluating the in vivo performance of Armoring CAR-modified V51 T cells in NSG and NOG-IL15 mice challenged with NALM6 tumor cells. The figure compares the numbers of V51 CD19 CAR+ and CAR- cells recovered from bone marrow samples taken 14 days post-treatment from NSG mice that did not receive exogenous IL-15. [Figure 14M] Schematic and results evaluating the in vivo performance of Armoring CAR-modified V51 T cells in NSG and NOG-IL15 mice challenged with NALM6 tumor cells. The figure compares the number of NALM-6 tumor cells recovered from bone marrow samples taken 14 days post-treatment from NSG mice that did not receive exogenous IL-15. [Figure 15A] Graph assessing the in vitro viability and effect of repeated antigen exposure on two different mesothelin-targeting CAR (YP218 or P4) modified V51 T that are non-armored, mbIL-15 and IL-15Rβ armored (CAR.α.15.β), or IL-15Rβ armored (CAR.β). The figure shows the viability of non-armored and α.15β armored CAR T cells. [Figure 15B] Graph assessing in vitro viability and effect of repeated antigen exposure on two different mesothelin-targeting CAR (YP218 or P4) modified V51 T that are non-armored, mbIL-15 and IL-15Rβ armored (CAR.α.15.β), or IL-15Rβ armored (CAR.β). The figure shows tumor confluence measured by following GFP fluorescence over time after treatment with different Meso-CAR T cell products. [Figure 15C] Graph assessing the in vitro viability and effect of repeated antigen exposure on two different mesothelin-targeting CAR (YP218 or P4) modified V51 T that are non-armored, mbIL-15 and IL-15Rβ armored (CAR.α.15.β), or IL-15Rβ armored (CAR.β). The figure shows the fold expansion of CAR T cells following a repeated antigen stimulation assay. [Figure 15D]Graph assessing in vitro viability and effect of repeated antigen exposure on two different mesothelin-targeting CAR (YP218 or P4) modified V51 T that are non-armored, mbIL-15 and IL-15Rβ armored (CAR.α.15.β), or IL-15Rβ armored (CAR.β). The figure shows tumor confluence measured by following GFP fluorescence over time after treatment with different Meso-CAR T cell products. [Figure 15E] Graph assessing the in vitro viability and effect of repeated antigen exposure on two different mesothelin-targeting CAR (YP218 or P4) modified V51 T that are non-armored, mbIL-15 and IL-15Rβ armored (CAR.α.15.β), or IL-15Rβ armored (CAR.β). The figure shows the fold expansion of CAR T cells following a repeated antigen stimulation assay. [Figure 16] Schematic diagram and results evaluating the in vivo performance of non-armored, mbIL-15 and IL-15Rβ armored, and IL-15Rβ armored MesoCAR modified Vδ1 T cells (P4 CAR, P4 CAR.α.15.β, and P4 CAR.β, respectively) in a disseminated A549 tumor model. The A549 cell line is engineered to express mesothelin (A549-Meso). A shows a schematic diagram of the model used. B shows the in vivo efficacy of the products tested. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0061] The present invention provides compositions and methods for modifying γδ T cells (e.g., vδ1 and vδ2 T cells) to respond to IL-15, a cytokine and T cell growth factor that binds to and signals through a receptor complex consisting of the IL-2 receptor β subunit (IL-15Rβ), the IL-15 receptor α subunit (IL-15Rα), and the common γ chain.

[0062] Traditional chimeric antigen receptor (CAR) armoring aims to improve the persistence, proliferation, or other properties of CAR+ cells by also expressing IL-15 (either secreted or membrane-bound) in the cells. Co-expression of exogenous IL-15 by the CAR+ cells in the same or separate construct reduces the responsiveness of the cells to IL-15. The present invention introduces a positive IL-15 signaling loop and boosts the responsiveness of CAR+ cells to IL-15 by co-expressing IL-15 receptor β and / or α subunits, which appear to be restricted to some cells, including γδ T cells. This can improve the sensitivity of CAR+ cells even in the absence of an armoring construct. The described strategy can be utilized alone or in combination with CARs to enhance the persistence and proliferation of γδ T cells.

[0063] Conventional approaches to modify the IL-15 signaling loop in NK cells and αβ T cells include transduction with constructs encoding IL-15Rα. In both cases, NK cells or αβ T cells expressing IL-15Rα and treated with IL-15 increase cell proliferation, viability, and toxicity. In contrast, γδ T cells modified with IL-15Rα show reduced proliferation and only a moderate effect on viability. The present invention is based on the unexpected discovery that γδ T cells can be modified with recombinant IL-15Rβ to effectively introduce a positive IL-15 signaling loop. This was unexpected, since it was expected that IL-15Rα was required for IL-15 responsiveness.

[0064] The compositions and methods described herein feature γδ T cells and populations thereof engineered to express recombinant IL-15Rβ. These IL-15Rβ engineered γδ T cells provide efficient proliferation and survival in the presence of IL-15, arming them with substantial cytotoxicity for a variety of therapeutic approaches.

[0065] I. Definition It is understood that aspects and embodiments of the invention described herein include aspects and embodiments "comprising," "consisting of," and "consisting essentially of." As used herein, the singular forms "a," "an," and "the" include plural referents unless otherwise indicated.

[0066] The term "about" as used herein refers to the normal error range of the respective value, which is readily known to one of ordinary skill in the art. Reference herein to a value or parameter of "about" includes (and describes) embodiments directed to the value or parameter itself. In some instances, "about" encompasses a variation of +20%, in some instances +10%, in some instances +5%, in some instances +1%, or in some instances +0.1% from the particular value, where such variations are appropriate for carrying out the disclosed methods.

[0067] As used herein, the term "engineered γδ T-cell" refers to a γδ T-cell that expresses a transgene (i.e., a gene that has been transduced into the engineered γδ T-cell or its parent cell).

[0068] As used herein, the term "primed γδ T cells" refers to a starting population (e.g., an endogenous population of γδ T cells) that has been affected by culture conditions. In some examples, primed γδ T cells have different functional viral entry receptor properties compared to their unprimed counterparts prior to experiencing the culture conditions. In some embodiments, the population of primed γδ T cells is an expanded population of γδ T cells.

[0069] As used herein, an "expanded population of γδ cells" refers to a population of hematopoietic cells comprising γδ T cells cultured under conditions and for a period of time that induces proliferation of γδ cells, i.e., an increase in the number of γδ cells. Similarly, as used herein, an "expanded population of V51 T cells" refers to a population of hematopoietic cells comprising V51 T cells cultured under conditions and for a period of time that induces proliferation of V51 T cells, i.e., an increase in the number of V51 T cells. Similarly, as used herein, an "expanded population of V52 T cells" refers to a population of hematopoietic cells comprising V52 T cells cultured under conditions and for a period of time that induces proliferation of V52 T cells, i.e., an increase in the number of V52 T cells.

[0070] As used herein, a "population" of γδ T cells refers to a group of three or more γδ T cells (e.g., at least 10, at least 10, at least 10, at least 10, at least 10, at least 10, at least 10, at least 10, at least 10, at least 10, or at least 10). A population of a particular cell type (e.g., a population of endogenous γδ T cells, a population of primed γδ T cells, or a population of engineered γδ T cells) refers to cells of that type and not to different types of cells within a broader population. For example, if 10% of the cells in a starting population of 10 T cells are γδ T cells, then the starting population of γδ T cells is 10.

[0071] As used herein, "armor protein" refers to a protein encoded by a transgene that, when expressed by a γδ T cell (e.g., a γδ T cell expressing a CAR), increases the persistence of the γδ T cell, increases the proliferation of the γδ T cell, or increases the cytotoxicity of the γδ T cell against a target cell, e.g., via signaling (e.g., cytokine signaling) to improve cell persistence, cell viability, activation, and other desirable properties. Armor proteins can be membrane-bound or soluble proteins. For example, armor proteins include membrane-bound receptors (e.g., IL-15R, e.g., IL-15Rβ and IL-15Rα, αβTCR, natural cytotoxicity receptors (e.g., NKp30, NKp44, or NKp46), cytokine receptors (e.g., IL-12 receptor), and / or chemokine receptors (e.g., CCR2 receptor), and / or membrane-bound ligands or cytokines (e.g., membrane-bound IL-15, membrane-bound IL-7, Armor proteins include membrane-bound CD40L, membrane-bound 4-1BB, membrane-bound 4-1BBL, membrane-bound CCL19. Additionally or alternatively, armor proteins can be soluble proteins such as soluble ligands or cytokines (e.g., soluble IL-15, soluble IL-7, soluble IL-12, soluble CD40L, soluble 4-1BBL, and / or soluble CCL19). In some embodiments, armor proteins are not antigen-specific.

[0072] As used herein, "IL-15" refers to natural or recombinant IL-15 or variants thereof (e.g., muteins, analogs, subunits, receptor complexes, fragments, isoforms, and peptidomimetics thereof) having at least 70% (e.g., at least 80%, 85%, 90%, 95%, 97%, 99%, e.g., at least 85%) sequence identity over the full length that acts as an agonist of one or more IL-15 receptor (IL-15R) subunits. IL-15, like IL-2, is a known T cell growth factor and can support the proliferation of CTLL-2, an IL-2-dependent cell line. IL-15 was first reported by Grabstein et al. as a 114 amino acid mature protein (Science 264.5161:965-969,1994). As used herein, the term "IL-15" refers to native or recombinant IL-15 and its muteins, analogs, subunits, or complexes thereof (e.g., receptor complexes, e.g., sushi peptides, as described in PCT Publication No. WO2007 / 046006), each of which can stimulate proliferation of CTLL-2 cells. In a CTLL-2 proliferation assay, supernatants from cells transfected with recombinantly expressed in-frame fusions of precursor and mature forms of IL-15 can induce proliferation of CTLL-2 cells.

[0073] Human IL-15 can be obtained by conventional procedures such as the polymerase chain reaction (PCR) or the method described by Grabstein et al. (Science 264.5161:965-969, 1994). Human IL-15 cDNA was deposited with the ATCC on February 19, 1993 and assigned accession number 69245.

[0074] The amino acid sequence of human IL-15 (gene ID 3600) is found in Genbank under the accession locators NP000576.1 GI:10835153 (isoform 1) and NP_751915.1 GI:26787986 (isoform 2). The mouse (Mus musculus) IL-15 amino acid sequence (gene ID 16168) is found in Genbank as the accession locator NP_001241676.1 GI:363000984.

[0075] IL-15 may also refer to IL-15 from various mammalian species, including, for example, human, monkey, cow, pig, horse, and mouse. As referred to herein, IL-15 "mutant protein" is a polypeptide having an amino acid sequence that differs from a natural mammalian IL-15 polypeptide due to one or more amino acid deletions, insertions, and / or substitutions. Variants may include conservative substitution sequences, meaning that a given amino acid residue is replaced by a residue with similar physiochemical properties. Examples of conservative substitutions include the substitution of one aliphatic residue for another aliphatic residue, such as Ile, Val, Leu, or Ala for each other, or the substitution of one polar residue for another, such as Lys for Arg, Glu for Asp, or Gln for Asn. Other such conservative substitutions are well known, such as the substitution of whole regions with similar hydrophobic properties. Natural IL-15 variants are also encompassed by the present invention. Examples of such variants are proteins resulting from alternate mRNA splicing events or from proteolytic cleavage of the IL-15 protein, in which case IL-15 binding properties are retained. Alternative splicing of the mRNA can result in a truncated but biologically active IL-15 protein. Mutations resulting from proteolysis include, for example, differences in the N-terminus or C-terminus upon expression in different types of host cells due to proteolytic removal of one or more terminal amino acids (generally 1-10 amino acids) of the IL-15 protein. In some embodiments, the termini of the protein can be modified with chemical groups, such as, for example, polyethylene glycol, to alter its physical properties (Yang et al. Cancer 76:687-694,1995). In some embodiments, the termini or interiors of the protein can be modified with additional amino acids (Clark-Lewis et al. PNAS 90:3574-3577,1993).

[0076] As used herein, "IL-2 receptor beta subunit," "IL-2Rβ," "IL-15 receptor beta subunit," and "IL-15Rβ," used interchangeably herein, refer to natural or recombinant IL-15Rβ or variants thereof having at least 70% (e.g., at least 80%, 85%, 90%, 95%, 97%, 99%, e.g., at least 85%) sequence identity over isometric portions that act as receptors for one or more of IL-2 or IL-15. IL-15Rβ includes full length and fragments thereof. IL-2Rβ, referred to herein as IL-15Rβ, is a known T cell growth factor receptor that can support the proliferation of CTLL-2, an IL-2-dependent cell line. IL-2Rβ is a mature protein of 551 amino acids. As used herein, the term "IL-15Rβ" refers to natural or recombinant IL-2Rβ, and muteins, analogs, subunits, or complexes thereof.

[0077] The amino acid sequence of human IL-2Rβ (gene ID 3560) is found in Genbank under the accession locators NP_000869, NP_001333151, and NP_001333152. The mouse (Mus musculus) IL-2Rβ amino acid sequence (gene ID 16185) is found in Genbank under the accession locator NP_032394.

[0078] IL-15Rβ may also refer to IL-15Rβ derived from various mammalian species, including, for example, human, monkey, bovine, porcine, equine, and murine. As referred to herein, an IL-15Rβ "mutant protein" is a polypeptide having an amino acid sequence that differs from a natural mammalian IL-15Rβ polypeptide due to one or more amino acid deletions, insertions, and / or substitutions. A variant may include a conservative substitution sequence, meaning that a given amino acid residue is replaced by a residue with similar physiochemical properties. Examples of conservative substitutions include the substitution of one aliphatic residue for another aliphatic residue, such as Ile, Val, Leu, or Ala for each other, or the substitution of one polar residue for another, such as Lys for Arg, Glu for Asp, or Gln for Asn. Other such conservative substitutions are well known, such as the substitution of whole regions with similar hydrophobic properties. Natural IL-15Rβ variants are also encompassed by the present invention. Examples of such variants are proteins resulting from alternate mRNA splicing events or from proteolytic cleavage of the IL-15Rβ protein, in which case IL-15Rβ binding properties are retained. Alternative splicing of the mRNA can result in a truncated but biologically active IL-15Rβ protein. Mutations resulting from proteolysis include, for example, differences in the N-terminus or C-terminus upon expression in different types of host cells due to proteolytic removal of one or more terminal amino acids (generally 1-10 amino acids) of the IL-15Rβ protein.

[0079] As used herein, "IL-15 receptor alpha subunit" and "IL-15Rα" refer to natural or recombinant IL-15Rα or variants thereof (e.g., muteins, analogs, fragments, isoforms, and peptidomimetics thereof) that act as a receptor for IL-15. IL-15Rα includes full-length and fragments thereof. IL-15Rα is a known T cell growth factor receptor. IL-15Rα is a mature protein of 267 amino acids. As used herein, the term "IL-15Rα" refers to natural or recombinant IL-15Rα, and muteins, analogs, subunits, or complexes thereof. Fragments of IL-15Rα include sushi peptides, for example, as described in PCT Publication No. WO2007 / 046006. Soluble fragments (e.g., sushi peptides, e.g., fragments containing residues 31-95 of IL-15Rα, or variants having at least 70% (e.g., at least 80%, 85%, 90%, 95%, 97%, 99%, e.g., at least 85%) sequence identity over the entire length) also include muteins, analogs, fragments, isoforms, and peptidomimetics thereof. In some embodiments, the soluble fragment is linked to an Fc domain (e.g., Fc-sushi or sushi-Fc).

[0080] The amino acid sequence of human IL-15Rα (gene ID 3601) is found in Genbank under the accession locators NP_001230468, NP_001243694, NP_002180, NP_751950, and NP_001338024. The mouse (Mus musculus) IL-15Rα amino acid sequence (gene ID 16169) is found in Genbank as the accession locator NP_001258426.

[0081] IL-15Rα may also refer to IL-15Rα derived from various mammalian species, including, for example, human, monkey, cow, pig, horse, and mouse. As referred to herein, an IL-15Rα "mutant protein" is a polypeptide having an amino acid sequence that differs from a natural mammalian IL-15Rα polypeptide due to one or more amino acid deletions, insertions, and / or substitutions. A variant may include a conservative substitution sequence, meaning that a given amino acid residue is replaced by a residue with similar physiochemical properties. Examples of conservative substitutions include the substitution of one aliphatic residue for another aliphatic residue, such as Ile, Val, Leu, or Ala for each other, or the substitution of one polar residue for another polar residue, such as Lys for Arg, Glu for Asp, or Gln for Asn. Other such conservative substitutions are well known, such as the substitution of whole regions with similar hydrophobic properties. Natural IL-15Rα variants are also encompassed by the present invention. Examples of such variants are proteins resulting from alternate mRNA splicing events or from proteolytic cleavage of the IL-15Rα protein, in which case IL-15Rα binding properties are retained. Alternative splicing of the mRNA can result in a truncated but biologically active IL-15Rα protein. Mutations resulting from proteolysis include, for example, differences in the N-terminus or C-terminus upon expression in different types of host cells due to proteolytic removal of one or more terminal amino acids (generally 1-10 amino acids) of the IL-15Rα protein. In some embodiments, the termini of the protein can be modified with chemical groups, such as, for example, polyethylene glycol, to alter its physical properties (Yang et al. Cancer 76:687-694,1995). In some embodiments, the termini or interiors of the protein can be modified with additional amino acids (Clark-Lewis et al. PNAS 90:3574-3577,1993).

[0082] As used herein, "internal ribosome entry site" or "IRES" refers to an RNA element that allows translation initiation in a 5' cap-independent manner. In some embodiments, the polyribonucleotide described herein comprises one or more internal ribosome entry site (IRES) elements. In some embodiments, the IRES is operably linked to one or more transgene sequences (e.g., each IRES is operably linked to one or more expression sequences). In an embodiment, the IRES is located between a heterologous promoter and the 5' end of a coding sequence. The IRES may comprise an RNA sequence that can bind to a eukaryotic ribosome. In some embodiments, the IRES element is at least about 5 nt, at least about 8 nt, at least about 9 nt, at least about 10 nt, at least about 15 nt, at least about 20 nt, at least about 25 nt, at least about 30 nt, at least about 40 nt, at least about 50 nt, at least about 100 nt, at least about 200 nt, at least about 250 nt, at least about 350 nt, or at least about 500 nt.

[0083] As used herein, the phrase "in an amount effective to" refers to an amount that induces a detectable result (e.g., a number of cells having a statistically significant, e.g., p<0.05, increased number compared to the starting population).

[0084] As used herein, an "expanded population of γδ cells" refers to a population of hematopoietic cells comprising γδ T cells cultured under conditions and for a period of time that induces proliferation of γδ cells, i.e., an increase in the number of γδ cells. Similarly, as used herein, an "expanded population of V51 T cells" refers to a population of hematopoietic cells comprising V51 T cells cultured under conditions and for a period of time that induces proliferation of V51 T cells, i.e., an increase in the number of V51 T cells. Similarly, as used herein, an "expanded population of V52 T cells" refers to a population of hematopoietic cells comprising V52 T cells cultured under conditions and for a period of time that induces proliferation of V52 T cells, i.e., an increase in the number of V52 T cells.

[0085] The term "marker," as used herein, refers to a DNA, RNA, protein, carbohydrate, glycolipid, or cell-based molecular marker, the expression or presence of which in a patient sample can be detected by standard methods (or methods disclosed herein).

[0086] A cell or cell population that "expresses" a marker of interest is one in which the mRNA encoding the protein, or the protein itself (including fragments thereof), is determined to be present in the cell or population. Expression of a marker can be detected by various means. For example, in some embodiments, expression of a marker refers to the surface density of the marker on the cells. For example, mean fluorescence intensity (MFI), used as a readout in flow cytometry, represents the density of the marker on a population of cells. One skilled in the art will appreciate that MFI values ​​depend on the staining parameters (e.g., concentration, duration, and temperature) as well as the fluorochrome composition. However, MFI can be quantitative when considered in the context of appropriate controls. For example, a population of cells can be said to express a marker if the MFI of an antibody against that marker is significantly higher than the MFI of an appropriate isotype control antibody in the same population of cells stained under comparable conditions. Additionally or alternatively, a population of cells can be said to express a marker on a cell-by-cell basis using positive and negative gates according to conventional flow cytometry analysis methods (e.g., by setting gates according to isotype or "fluorescence minus one (FMO) controls). According to this metric, a population is said to "express" a marker if the number of cells detected as positive for the marker is significantly higher than background (e.g., by gating on an isotype control).

[0087] As used herein, "functional expression of VSV-G entry receptor" refers to a level of expression of VSV-G entry receptor sufficient to mediate detectable VSV-G entry in at least 5% of a target cell population, as measured by a β-lactamase-Vpr (BlaM-VpR)-based assay. See, e.g., Cavrois et al., Nat Biotechnol. 11:1151-1154, 2002. Conversely, in a population of cells that is "deficient in functional expression of VSV-G entry receptor," more than 95% of the cell population lacks a sufficient level of expression of VSV-G entry receptor to mediate detectable VSV-G entry, as measured by a BlaM-VpR-based assay.

[0088] As used herein, when expression of a population is described as a percentage of positive cells and that percentage is compared to the corresponding percentage of positive cells in a reference population, the difference in percentage is the percentage of the parent population of each population. For example, if a marker is expressed in 10% of the cells in population A and the same marker is expressed in 1% of the cells in population B, population A is said to have a 9% higher frequency of marker positivity compared to population B (i.e., 10% - 1%, not 10% ÷ 1%). When multiplying the frequency by the number of cells in the parent populations, the difference in absolute number of cells is calculated. In the above example, if there are 100 cells in population A and 10 cells in population B, population A has 100 times the number of cells relative to population B (i.e., (10% x 100) ÷ (1% x 10)).

[0089] The expression level of the marker may be a nucleic acid expression level (e.g., a DNA expression level or an RNA expression level, e.g., an mRNA expression level). Any suitable method of determining the nucleic acid expression level may be used. In some embodiments, the nucleic acid expression level is determined using qPCR, rtPCR, RNA-seq, multiplex qPCR or RT-qPCR, microarray analysis, sequential analysis of gene expression (SAGE), MASSARRAY® technology, in situ hybridization (e.g., FISH), or a combination thereof.

[0090] As used herein, a "reference population" of cells refers to a population of cells corresponding to a cell of interest, against which the phenotype of the cell of interest is measured. For example, the expression level of a marker on an isolated population of non-hematopoietic tissue-derived γδ cells may be compared to the expression level of the same marker on hematopoietic tissue-derived γδ T cells (e.g., blood-resident γδ cells, e.g., blood-resident γδ cells from the same or different donor), or on non-hematopoietic tissue-derived γδ T cells expanded under different conditions (e.g., in the presence of substantial TCR activation, in the presence of an exogenous TCR activator (e.g., anti-CD3), or in substantial contact with stromal cells (e.g., fibroblasts)). A population may also be compared to itself in an initial state. For example, the reference population can be a cell population that is isolated before its expansion. In this case, the expanded population is compared to its own composition before the expansion step, i.e., in this case, its past composition is the reference population.

[0091] As used herein, the term "chimeric antigen receptor" or alternatively "CAR" refers to a recombinant polypeptide construct that includes an extracellular antigen-binding domain, a transmembrane domain, and optionally an intracellular domain that propagates an activation signal and / or a costimulatory signal to activate the cell. In some embodiments, the CAR includes an optional leader sequence at the N-terminus of the CAR fusion protein. In some embodiments, the CAR lacks the intracellular (e.g., signaling) domain.

[0092] As used herein, the term "tether" or "tethered", when present, refers to a polypeptide that connects two or more polypeptides (e.g., IL-15Rα or a fragment thereof (e.g., the sushi domain) and IL-15 or a variant thereof). In general, a tethering factor can be, for example, about 1-100 (e.g., 5-50, 5-30, 5-20, e.g., 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, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100) amino acids in length. In some embodiments, the tethering factor is highly flexible and can contain many glycine (G) and / or serine (S) residues, which can be present in the form of GS repeats. In some embodiments, the tethering agent may link the C-terminus of a first protein to the N-terminus of a second protein. In other embodiments, the tethering agent may join the C-terminus of a second protein to the N-terminus of a first protein.

[0093] As used herein, the term "percentage of identity" refers to the percentage of amino acid residues in a candidate sequence, e.g., an IL-15Rβ variant, that are identical to the amino acid residues in a reference sequence, e.g., a wild-type IL-15Rβ polypeptide, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage of identity (i.e., for optimal alignment, gaps can be introduced in one or both of the candidate and reference sequences, and non-homologous sequences can be ignored for comparison purposes). Alignment for purposes of determining percentage of identity can be achieved in a variety of ways that are within the skill of the art, e.g., using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared. In some embodiments, the percentage of amino acid sequence identity of a given candidate sequence (alternatively expressed as a given candidate sequence having or containing a specific percentage of amino acid sequence identity to a given reference sequence) to a given reference sequence is calculated as follows:

[0094] 100 x (A / B ratio)

[0095] where A is the number of amino acid residues scored as identical in the alignment of the candidate sequence with the reference sequence, and B is the total number of amino acid residues in the reference sequence. In some embodiments where the length of the candidate sequence is not equal to the length of the reference sequence, the percentage of amino acid sequence identity of the candidate sequence to the reference sequence will not be equal to the percentage of amino acid sequence identity of the reference sequence to the candidate sequence.

[0096] As used herein, "2A peptide" refers to a class of 18-22 amino acids long peptides that share a core sequence motif of DxExNPGP, where X is any amino acid. The 2A peptide may be the foot and mouth disease virus 18 2A (F2A) peptide, the equine rhinitis A virus 2A (E2A) peptide, the porcine teschovirus-1 2A (P2A) peptide, or the thosea asigna virus 2A (T2A) peptide.

[0097] "Immune response," as understood in the art, generally refers to a biological response in a vertebrate to foreign agents or abnormalities (e.g., cancerous cells) that protects the organism from such agents and the diseases they cause. The immune response is mediated by the action of one or more cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and soluble macromolecules produced by these cells or the liver, including antibodies, cytokines, and complement, which results in the selective targeting, binding, damage, destruction, and / or elimination from the vertebrate body of invading pathogens, pathogen-infected cells or tissues, cancerous or other abnormal cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues. An immune response can include, for example, the activation or inhibition of T cells (e.g., effector T cells, Th cells, CD4+ cells, CD8+ T cells, or Treg cells), or the activation or inhibition of any other cell of the immune system (e.g., NK cells).

[0098] "Immunotherapy" refers to the treatment of a subject suffering from or at risk of recurrence of a disease by methods involving inducing, enhancing, suppressing, or modifying the immune system or immune response.

[0099] As used herein, the terms "treat", "treatment", or "treatment of", when used in reference to treating a disease or condition, e.g., cancer, in a subject, refer to reducing the pathology of the disease, reducing or eliminating symptoms of the disease, promoting improved survival, and / or reducing discomfort. For example, treatment refers to the ability of a therapeutic when administered to a subject to reduce one or more symptoms, signs, or pathogenesis of a disease. Treatment also refers to alleviating or reducing one or more clinical symptoms and / or inhibiting or slowing the progression of a condition and / or preventing or delaying the onset of a disease or condition.

[0100] As used herein, "cancer" refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division can lead to the formation of malignant tumors or malignant cells that can invade adjacent tissues and metastasize to distant parts of the body via the lymphatic system or bloodstream.

[0101] As used herein, the term "effective amount" or "therapeutically effective amount" of an administered therapeutic agent, such as immune cells comprising a polynucleotide encoding a CAR, is an amount sufficient to carry out a specifically stated or intended purpose, such as treating cancer or treating cancer. An "effective amount" can be empirically determined in a routine manner in relation to the stated purpose.

[0102] As used herein, the terms "subject," "individual," or "patient" refer to any subject for whom diagnosis, prognosis, or treatment is desired, particularly mammalian subjects. Mammalian subjects include, for example, humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, bears, etc.

[0103] As used herein, the terms "ug" and "uM" are used interchangeably with "μg" and "μM", respectively.

[0104] The various aspects described herein are described in further detail in the following subsections.

[0105] II. Engineered γδ T Cells II.A. IL-15Rβ-expressing γδ T cells The present invention features engineered γδ T cells expressing recombinant IL-15Rβ. Such engineered cells have enhanced responsiveness to IL-15 without further expression of additional IL15 receptor complex components. In some embodiments, the γδ T cells are deleted for the endogenous gene encoding IL-15. Such engineered cells exhibit enhanced sensitivity to exogenous IL-15 with respect to signal transduction.

[0106] In another embodiment, described herein is a γδ T cell expressing recombinant IL-15Rβ and recombinant IL-15 or a variant thereof. In some embodiments, the recombinant IL-15 is a secreted IL-15 or a variant thereof or a tethered IL-15 or a variant thereof. Such modified cells exhibit a continuous IL-15 signal and are not dependent on exogenous IL-15 for signaling.

[0107] In some embodiments, the γδ T cells expressing recombinant IL-15Rβ further express recombinant IL-15Rα or a fragment thereof (e.g., a soluble fragment, e.g., sushi domain). The IL-15Rα or a fragment thereof may be tethered to IL-15 or a variant thereof. In some embodiments, the C-terminus of the IL-15Rα or a fragment thereof is tethered to the N-terminus of IL-15 or a variant thereof. In some embodiments, the N-terminus of the IL-15Rα or a fragment thereof is tethered to the C-terminus of IL-15 or a variant thereof. Such modified cells have additional responsiveness as shown herein.

[0108] The various expression transgenes described herein may be expressed in tandem, for example, within a single construct. For example, in some embodiments, IL-15Rβ and IL-15Rα or fragments thereof are expressed in tandem. In some embodiments, IL-15Rβ and IL-15 or a variant thereof are expressed in tandem. In some embodiments, IL-15Rβ and IL-15Rα or fragments thereof are expressed in tandem. In some embodiments, IL-15Rβ, IL-15 or a variant thereof, and recombinant IL-15Rα or fragments thereof are expressed in tandem.

[0109] The one or more transgenes may include a linker between them. The IL-15Rβ and IL-15Rα or fragments thereof may include a linker between them. The linker may include a cleavable peptide. The cleavable peptide may be, for example, a self-cleaving peptide, for example, a 2A peptide. In some embodiments, the 2A peptide is a foot and mouth disease virus 18 2A (F2A) peptide, an equine rhinitis A virus 2A (E2A) peptide, a porcine teschovirus-1 2A (P2A) peptide, or a thosea asigna virus 2A (T2A) peptide. The IL-15Rβ and IL-15 or variants thereof may include a linker between them. The linker may include a cleavable peptide. The cleavable peptide may be, for example, a self-cleaving peptide, for example, a 2A peptide. In some embodiments, the 2A peptide is a F2A peptide, an E2A peptide, a P2A peptide, or a T2A peptide. 2A peptides are used to link two or more transgenes and allow the translated polypeptide to self-cleave into individual polypeptide chains (e.g., IL-15Rβ and IL-15Rα). Thus, in some embodiments, the transgenes encode a 2A peptide between a first transgene and a second transgene, optionally with flexible linkers (e.g., GSG linkers) on either side of the 2A peptide. The transgenes may further include one or more engineered cleavage sequences, such as a furin cleavage sequence, to remove 2A peptide residues bound to the peptide. Exemplary 2A peptides are described, for example, in Chng et al. MAbs 7:403-412,2015, and Lin et al. Front. Plant Sci.9:1379,2018, the disclosures of which are incorporated herein by reference in their entireties.

[0110] In some embodiments, IL-15Rα or a fragment thereof is tethered to IL-15 or a variant thereof. The tethering factor can be a peptide linker of any suitable length, for example, having 1-100 (e.g., 5-50, 5-30, 5-20, e.g., 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, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100) amino acid residues. The linker can be a flexible linker, for example, comprising one or more glycines or serines.

[0111] Examples of armoring proteins are shown in Table 1.

[0112] [Table 1]

[0113] In some embodiments, the IL-15Rα polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the IL-15Rα polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the IL-15Rα polypeptide comprises a signal peptide sequence.

[0114] In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the IL-15 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the IL-15 polypeptide comprises a signal peptide sequence.

[0115] In some embodiments, the IL-15Rα polypeptide is tethered to the IL-15 polypeptide by a linker. For example, in such cases, the IL-15Rα polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:3. In some embodiments, the IL-15Rα polypeptide comprises an amino acid sequence set forth in SEQ ID NO:3, and the IL-15 polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:2. In some embodiments, the IL-15 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2. In some embodiments, the linker comprises one or more peptide bonds. In some embodiments, the linker comprises a peptide linker. In some embodiments, the peptide linker is a flexible linker. In some embodiments, the linker is a rigid linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker is a Gly-Ser linker. In some embodiments, the linker comprises one or more repeats of the sequence GGGS, GGGS (SEQ ID NO:6), or a combination thereof. In some embodiments, the IL-15Rα polypeptide is tethered to the IL-15 polypeptide by a linker comprising the amino acid sequence set forth in SEQ ID NO:7. In some embodiments, the IL-15Rα polypeptide is tethered to the IL-15 polypeptide by a linker comprising the amino acid sequence set forth in SEQ ID NO:8. In some embodiments, the IL-15Rα polypeptide is tethered to the IL-15 polypeptide by a linker comprising the amino acid sequence set forth in SEQ ID NO:9. In some embodiments, the IL-15Rα polypeptide is tethered to the IL-15 polypeptide by a linker comprising the amino acid sequence set forth in SEQ ID NO:10.In some embodiments, the IL-15Rα polypeptide is tethered to the IL-15 polypeptide by a linker comprising the amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, the IL-15Rα polypeptide is tethered to the IL-15 polypeptide by a linker comprising the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the IL-15Rα polypeptide is tethered to the IL-15 polypeptide by a linker comprising the amino acid sequence set forth in SEQ ID NO: 13.

[0116] In some embodiments, a construct comprising an IL-15Rα polypeptide tethered to an IL-15 polypeptide is arranged N-terminally to C-terminally in the following order: (i) the N-terminus, (ii) the IL-15 polypeptide, (iii) a polypeptide comprising a peptide linker disclosed herein (e.g., GGGS, GGGS (SEQ ID NO:6), or a combination thereof, (iv) the IL-15Rα polypeptide, (v) the C-terminus. In some embodiments, a construct comprising an IL-15Rα polypeptide tethered to an IL-15 polypeptide is arranged N-terminally to C-terminally in the following order: (i) the N-terminus, (ii) an IL-15 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:2, (iii) a polypeptide comprising a peptide linker disclosed herein (e.g., GGGS, GGGS (SEQ ID NO:6), or a combination thereof, (iv) an IL-15Rα polypeptide comprising the amino acid sequence set forth in SEQ ID NO:3, (v) the C-terminus.

[0117] In some embodiments, the Armor protein is expressed as a single polypeptide. In some embodiments, the Armor protein is expressed as a single polypeptide comprising (i) an IL-2Rβ polypeptide, (ii) a linker, and (iii) an IL-15Rα polypeptide tethered to an IL-15 polypeptide. In some embodiments, the Armor protein is expressed as a single polypeptide comprising (i) an IL-2Rβ polypeptide, (ii) a cleavable linker, and (iii) an IL-15Rα polypeptide tethered to an IL-15 polypeptide. In some embodiments, the cleavable linker comprises a P2A sequence. In some embodiments, the linker comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:4. In some embodiments, the armor protein is expressed as a single polypeptide comprising (i) an IL-2Rβ polypeptide, (ii) a linker comprising the amino acid sequence set forth in SEQ ID NO:4, and (iii) an IL-15Rα polypeptide tethered to an IL-15 polypeptide.

[0118] In some embodiments, the Armor protein comprises (i) an IL-2Rβ polypeptide comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:1; (ii) a linker comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:4; and (iii) an IL-15 polypeptide tethered thereto. and wherein the IL-15Rα polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:3, and the IL-15 polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:2.

[0119] In some aspects, the armor protein is expressed as a single polypeptide comprising (i) an IL-2Rβ polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1, (ii) a linker comprising the amino acid sequence set forth in SEQ ID NO:4, and (iii) an IL-15Rα polypeptide tethered to an IL-15 polypeptide, where the IL-15Rα polypeptide comprises the amino acid sequence set forth in SEQ ID NO:3 and the IL-15 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2.

[0120] II.B.γδT cells γδ T cells are a subset of T cells that express a distinct and characteristic γδ T cell receptor (TCR) on their surface. This TCR is composed of one gamma (γ) chain and one delta (δ) chain. Human γδ T cells can be broadly classified into one or two types: peripheral blood resident γδ T cells and non-hematopoietic tissue resident γδ T cells. Most blood resident γδ T cells express the Vδ2 TCR, which is less common in tissue resident γδ T cells, with Vδ1 and / or other Vδ chains being used more frequently. The present invention provides γδ T cells transduced with a viral vector encoding a desired transgene as described herein.

[0121] In some embodiments, suitable γδ T-cells for use as a source for engineered γδ T-cells described herein include V51 cells, V52 cells, V53 cells, V55 cells, and V58 cells. In some embodiments, the population of engineered γδ T-cells is derived from a population of V51 cells or V52 cells. In some examples, the population of engineered γδ T-cells is derived from a population of non-V51 / V52 T cells. In some examples, the population of engineered γδ T-cells is derived from a mixed population of V51 and V52 cells.

[0122] The γδ T cells described herein (e.g., endogenous γδ T cells or primed γδ T cells) may lack the vesicular stomatitis virus G glycoprotein (VSV-G) entry receptor (e.g., LDL). The γδ T cells (e.g., endogenous γδ T cells or primed γδ T cells) may express ASCT-1 and / or ASCT-2. Expression of ASCT-1 and / or ASCT-2 may allow transduction with betaretrovirus pseudotyped vectors (e.g., BaEV and RD114). Lack of expression of VSV-G may prevent transduction with VSV-G pseudotyped vectors.

[0123] In one aspect, the invention relates to a method and / or method for the treatment of a cell-associated disease, comprising administering to a patient a therapeutically effective amount of a cell-associated disease, the method ... For example, the invention provides a population of γδ T cells engineered to express one or more transgenes, which may encode a marker (e.g., soluble IL-15, soluble IL-7, soluble IL-12, soluble CD40L, soluble 4-1BBL, and / or soluble CCL19), a selection marker (e.g., a reporter gene), or a suicide gene. In some examples, the invention provides a population of γδ T cells engineered to express IL-15Rβ, a CAR, and one or more additional transgene-encoded proteins (e.g., armor proteins). In some embodiments, the one or more transgenes are codon-optimized.

[0124] In some embodiments, a γδ T cell is transduced with a viral vector encoding a transgene (e.g., IL-15Rβ and / or a CAR). In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is a lentiviral vector. In some such embodiments, the cell may stably express the transgene. In some embodiments, the cell may transiently express the transgene.

[0125] In one aspect, the invention features a cell population (e.g., an isolated cell population) of engineered γδ T-cells (e.g., at least 10, 10, 10, 10, 10, 10, 10, 10, 10, or 10 cells), where at least 3% (e.g., at least 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 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%, 100%, 101%, 102%, 103%, 104%, 105%, 10 3%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or substantially all) of the γδ T cells expressing the transgene (e.g., IL-15Rβ and / or CAR and / or one or more additional proteins).

[0126] II.C. Chimeric antigen receptors Some embodiments of the present disclosure relate to a population of γδ T cells (e.g., a population of Vd1+ γδ cells derived from blood or skin) comprising γδ T cells comprising a heterologous nucleic acid encoding a CAR, where the CAR comprises a binding domain that specifically binds human mesothelin. In some embodiments, the CAR comprises an antigen binding domain that specifically binds human mesothelin (e.g., the antigen binding domain comprises a sequence of any of SEQ ID NOs: 1-8). In some embodiments, the cells may further comprise a 4-1BB and a CD3ζ domain. In still further embodiments, the cells relate to a population of γδ T cells (e.g., a population of Vd1+ γδ cells derived from blood or skin) comprising γδ T cells comprising a heterologous nucleic acid encoding a CAR, where the CAR comprises an antigen binding domain that specifically binds human mesothelin (e.g., the antigen binding domain comprises a sequence of any of SEQ ID NOs: 1-8), and the γδ T cells further comprise a CD8 transmembrane domain. In yet a further aspect, the cell relates to a population of γδ T cells (e.g., a population of Vd1+ γδ cells derived from blood or skin) comprising γδ T cells comprising a heterologous nucleic acid encoding a CAR, where the CAR comprises an antigen binding domain that specifically binds human mesothelin (e.g., the antigen binding domain comprises a sequence of any of SEQ ID NOs: 1-8), and the γδ T cells further comprise (a) IL-15Rβ, or (b) (i) IL-15Rα tethered to IL-15 or a variant thereof, and (ii) IL-15Rβ. In a further aspect, a γδ T-cell as described herein comprising a heterologous nucleic acid encoding a CAR (wherein the CAR comprises an antigen binding domain that specifically binds human mesothelin (e.g., the antigen binding domain comprises any of SEQ ID NOs: 1-8)) can further comprise 4-1BB and a CD3ζ domain and / or a CD8 domain, and (a) IL-15Rβ, or (b) (i) IL-15Rα tethered to IL-15 or a variant thereof, and (ii) IL-15Rβ.

[0127] In some aspects, an antigen binding domain that specifically binds human mesothelin comprises a variable heavy chain (VH) complementarity determining region 1 (CDR1), a VH-CDR2, a VH-CDR3, a variable light chain (VL) CDR-1, a VL-CDR2, and a VL-CDR3. In some aspects, VH-CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 16. In some aspects, VH-CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15. In some aspects, VH-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 14. In some aspects, VL-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 17. In some aspects, VL-CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 18. In some aspects, VL-CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 19.

[0128] In some embodiments, a γδ T-cell (e.g., of a population of blood- or skin-derived Vd1+ γδ cells) comprises a heterologous nucleic acid molecule encoding a CAR, wherein the CAR comprises an antigen-binding domain that specifically binds human mesothelin, and wherein the CAR comprises a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 19.

[0129] In some embodiments, a γδ T-cell (e.g., of a population of blood- or skin-derived Vd1+ γδ cells) comprises a heterologous nucleic acid molecule encoding a CAR, wherein the CAR comprises an antigen-binding domain that specifically binds human mesothelin, and the CAR comprises (i) a VH comprising an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:20, (ii) a VL comprising an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:21, or (iii) both (i) and (ii).

[0130] In some embodiments, a γδ T cell (e.g., of a population of blood- or skin-derived Vd1+ γδ cells) comprises a heterologous nucleic acid molecule encoding a CAR, wherein the CAR comprises an antigen-binding domain that specifically binds human mesothelin, and the CAR comprises (i) a VH comprising the amino acid sequence set forth in SEQ ID NO: 20, (ii) a VL comprising the amino acid sequence set forth in SEQ ID NO: 21, or (iii) both (i) and (ii).

[0131] In some embodiments, the antigen binding domain that specifically binds to human mesothelin comprises the P4 antigen binding domain (Table 2).

[0132] In some aspects, an antigen binding domain that specifically binds human mesothelin comprises a variable heavy chain (VH) complementarity determining region 1 (CDR1), a VH-CDR2, a VH-CDR3, a variable light chain (VL) CDR-1, a VL-CDR2, and a VL-CDR3. In some aspects, the VH-CDR3 comprises the amino acid sequence set forth in SEQ ID NO:24. In some aspects, the VH-CDR2 comprises the amino acid sequence set forth in SEQ ID NO:23. In some aspects, the VH-CDR1 comprises the amino acid sequence set forth in SEQ ID NO:22. In some aspects, the VL-CDR1 comprises the amino acid sequence set forth in SEQ ID NO:25. In some aspects, the VL-CDR2 comprises the amino acid sequence set forth in SEQ ID NO:26. In some aspects, the VL-CDR3 comprises the amino acid sequence set forth in SEQ ID NO:27.

[0133] In some embodiments, a γδ T-cell (e.g., of a population of blood- or skin-derived Vd1+ γδ cells) comprises a heterologous nucleic acid molecule encoding a CAR, wherein the CAR comprises an antigen-binding domain that specifically binds human mesothelin, and wherein the CAR comprises a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 22, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 23, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 24, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27.

[0134] In some embodiments, a γδ T-cell (e.g., of a population of blood- or skin-derived Vd1+ γδ cells) comprises a heterologous nucleic acid molecule encoding a CAR, wherein the CAR comprises an antigen-binding domain that specifically binds human mesothelin, and the CAR comprises (i) a VH comprising an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:28, (ii) a VL comprising an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:29, or (iii) both (i) and (ii).

[0135] In some embodiments, a γδ T cell (e.g., of a population of blood- or skin-derived Vd1+ γδ cells) comprises a heterologous nucleic acid molecule encoding a CAR, wherein the CAR comprises an antigen-binding domain that specifically binds human mesothelin, and the CAR comprises (i) a VH comprising the amino acid sequence set forth in SEQ ID NO: 28, (ii) a VL comprising the amino acid sequence set forth in SEQ ID NO: 29, or (iii) both (i) and (ii).

[0136] [Table 2]

[0137] In some embodiments, the CAR further comprises a hinge region between the antigen binding domain and the transmembrane domain. In some embodiments, the hinge is derived from an immunoglobulin (e.g., derived from a hinge region or loop region). In certain embodiments, these hinges include, for example, an IgA1, IgA2, IgG1, IgG2, IgG3, IgG4, IgD, IgE, or IgM hinge region, a fragment thereof (alone or capped with additional sequences, e.g., sequences of the CH1 or CH2 regions), or a combination of fragments derived from an IgA1, IgA2, IgG1, IgG2, IgG3, IgG4, IgD, IgE, or IgM hinge region. In some embodiments, the hinge comprises, for example, a constant domain loop region of IgA1, IgA2, IgG1, IgG2, IgG3, IgG4, IgD, IgE, or IgM, a fragment thereof (either alone or capped with additional sequence, e.g., from an adjacent β-strand), or a combination of fragments from the loop region of IgA1, IgA2, IgG1, IgG2, IgG3, IgG4, IgD, IgE, or IgM. In some embodiments, the hinges of the present disclosure comprise sequences from a hinge region, sequences from a loop region, or a combination thereof.

[0138] In some embodiments, the hinge region is selected from a CD8 hinge, a CD28 hinge, and an immunoglobulin hinge. In some embodiments, the hinge comprises a CD28 hinge. In some embodiments, the CAR comprises a hinge comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 30 (Table 3). In some embodiments, the CAR comprises a hinge comprising the amino acid sequence set forth in SEQ ID NO: 30.

[0139] [Table 3]

[0140] Thus, in some aspects, the disclosure provides a CAR (or a polynucleotide encoding a CAR), comprising (i) an antigen binding domain that specifically binds mesothelin (e.g., an anti-mesothelin scFv), (ii) a hinge region that comprises the hinge region of CD28, (iii) a transmembrane domain, and (iv) an intracellular domain. In some aspects, the disclosure provides a CAR (or a polynucleotide encoding a CAR), comprising (i) an antigen binding domain that specifically binds mesothelin (e.g., an anti-mesothelin scFv), (ii) a hinge region that comprises the amino acid sequence set forth in SEQ ID NO: 30, (iii) a transmembrane domain, and (iv) an intracellular domain.

[0141] In some embodiments, the hinge comprises a CD8 hinge. In some embodiments, the CAR comprises a hinge comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 32 (Table 3). In some embodiments, the CAR comprises a hinge comprising the amino acid sequence set forth in SEQ ID NO: 32.

[0142] Thus, in some aspects, the disclosure provides a CAR (or a polynucleotide encoding a CAR), comprising (i) an antigen binding domain that specifically binds mesothelin (e.g., an anti-mesothelin scFv), (ii) a hinge region that comprises the hinge region of CD8, (iii) a transmembrane domain, and (iv) an intracellular domain. In some aspects, the disclosure provides a CAR (or a polynucleotide encoding a CAR), comprising (i) an antigen binding domain that specifically binds mesothelin (e.g., an anti-mesothelin scFv), (ii) a hinge region that comprises the amino acid sequence set forth in SEQ ID NO: 32, (iii) a transmembrane domain, and (iv) an intracellular domain.

[0143] In some embodiments, the CAR further comprises a transmembrane domain. The transmembrane domain can be derived from either natural or recombinant sources. If the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. In some embodiments, the transmembrane domain can transmit a signal to the intracellular domain(s) whenever the CAR of the present disclosure binds to a target.

[0144] In some embodiments, the transmembrane domain is selected from the group consisting of, for example, CD8, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2Rβ, IL2Rγ, IL7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD1 1b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKG2D, NKG2C, or at least the transmembrane region(s) of CD19.

[0145] In some embodiments, the TM domain is derived from CD8, CD2, CD4, CD28, CD45, PD1, CD152, or any combination thereof. In some embodiments, the TM domain is derived from CD28. In some embodiments, the TM domain comprises an amino acid sequence having at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:31. In certain embodiments, the TM domain comprises the amino acid sequence set forth in SEQ ID NO:31.

[0146] Thus, in some aspects, the disclosure provides a CAR (or a polynucleotide encoding a CAR), comprising (i) an antigen binding domain that specifically binds mesothelin (e.g., an anti-mesothelin scFv), (ii) a hinge region, (iii) a CD28 transmembrane domain, and (iv) an intracellular domain. In some aspects, the disclosure provides a CAR (or a polynucleotide encoding a CAR), comprising (i) an antigen binding domain that specifically binds mesothelin (e.g., an anti-mesothelin scFv), (ii) a hinge region, (iii) a transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO:31, and (iv) an intracellular domain.

[0147] In some embodiments, the TM domain is derived from CD8. In some embodiments, the TM domain comprises an amino acid sequence having at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 33. In certain embodiments, the TM domain comprises the amino acid sequence set forth in SEQ ID NO: 33.

[0148] Thus, in some aspects, the disclosure provides a CAR (or a polynucleotide encoding a CAR), comprising (i) an antigen binding domain that specifically binds mesothelin (e.g., an anti-mesothelin scFv), (ii) a hinge region, (iii) a CD8 transmembrane domain, and (iv) an intracellular domain. In some aspects, the disclosure provides a CAR (or a polynucleotide encoding a CAR), comprising (i) an antigen binding domain that specifically binds mesothelin (e.g., an anti-mesothelin scFv), (ii) a hinge region, (iii) a transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO: 33, and (iv) an intracellular domain.

[0149] In some embodiments, the CAR further comprises a costimulatory domain. In some embodiments, the costimulatory domain comprises a costimulatory domain of 4-1BB / CD137, interleukin-2 receptor (IL-2R), interleukin-12 receptor (IL-12R), IL-7, IL-21, IL-23, IL-15, CD2, CD3, CD4, CD7, CD8, CD27, CD28, CD30, CD40, ICOS, lymphocyte function-associated antigen 1 (LFA-1), LIGHT, NKG2C, OX40, DAP10, B7-H3, Lck binding-deficient CD28 (ICA), BTLA, GITR, HVEM, LFA-1, LIGHT, NKG2C, PD-1, TILR2, TILR4, TILR7, TILR9, Fc receptor gamma chain, Fc receptor epsilon chain, a ligand that specifically binds CD83, or any combination thereof. In some embodiments, the CAR comprises a 4-1BB costimulatory domain.

[0150] In some embodiments, the costimulatory domain comprises an amino acid sequence having at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 34. In certain embodiments, the costimulatory domain comprises the amino acid sequence set forth in SEQ ID NO:34.

[0151] Thus, in some aspects, the disclosure provides a CAR (or a polynucleotide encoding a CAR), comprising (i) an antigen binding domain that specifically binds mesothelin (e.g., an anti-mesothelin scFv), (ii) a hinge region, (iii) a transmembrane domain, (vi) a costimulatory domain, and (v) an intracellular signaling domain. In some aspects, the disclosure provides a CAR (or a polynucleotide encoding a CAR), comprising (i) an antigen binding domain that specifically binds mesothelin (e.g., an anti-mesothelin scFv), (ii) a hinge region, (iii) a transmembrane domain, (vi) a 4-1BB costimulatory domain, and (v) an intracellular signaling domain. In some aspects, the disclosure provides a CAR (or a polynucleotide encoding a CAR), the CAR comprising (i) an antigen binding domain that specifically binds mesothelin (e.g., an anti-mesothelin scFv), (ii) a hinge region, (iii) a transmembrane domain, (iv) a costimulatory domain comprising the amino acid sequence set forth in SEQ ID NO: 34, and (v) an intracellular signaling domain.

[0152] In some embodiments, the CAR further comprises an intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a CD3zeta activation domain, a CD3delta activation domain, a CD3epsilon activation domain, a CD3etta activation domain, a CD79A activation domain, a DAP12 activation domain, a FCER1G activation domain, a DAP10 / CD28 activation domain, a ZAP70 activation domain, or any combination thereof. In some embodiments, the intracellular signaling domain comprises a CD3zeta activation domain.

[0153] In some embodiments, the intracellular signaling domain comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 35. In certain embodiments, the intracellular signaling domain comprises the sequence set forth in SEQ ID NO:35.

[0154] Thus, in some aspects, the disclosure provides a CAR (or a polynucleotide encoding a CAR), comprising (i) an antigen binding domain that specifically binds mesothelin (e.g., an anti-mesothelin scFv), (ii) a hinge region, (iii) a transmembrane domain, (vi) a costimulatory domain, and (v) an intracellular signaling domain comprising a CD3ζ activation domain. In some aspects, the disclosure provides a CAR (or a polynucleotide encoding a CAR), comprising (i) an antigen binding domain that specifically binds mesothelin (e.g., an anti-mesothelin scFv), (ii) a hinge region, (iii) a transmembrane domain, (iv) a costimulatory domain, and (v) an intracellular signaling domain comprising the amino acid sequence set forth in SEQ ID NO:35.

[0155] II.D. Armoring CAR-γδ T cells In some embodiments, an engineered γδ T-cell (e.g., a Vd1+ cell) comprises: (A) (i) an antigen-binding domain that specifically binds to human mesothelin (the antigen-binding domain comprises a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 19); (ii) a hinge region comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 30; (iii) a hinge region comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 31; (B) a CAR comprising an amino acid sequence having sequence identity to the sequence set forth in SEQ ID NO:34; (iv) a costimulatory domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:34; and (v) an intracellular signaling domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:35; and (B) an armor protein comprising an IL-2Rβ polypeptide comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the engineered γδ T-cell does not comprise an IL-15Rα polypeptide or an IL-15 polypeptide.

[0156] In some embodiments, an engineered γδ T-cell (e.g., a Vd1+ cell) comprises: (A)(i) an antigen-binding domain that specifically binds to human mesothelin (the antigen-binding domain comprises a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 19); (ii) a hinge region comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:30; (iii) a transmembrane domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:31; (iv) a transmembrane domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:34; (B) a CAR comprising (i) an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1; and (v) an intracellular signaling domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 35. (ii) an IL-2Rβ polypeptide comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:4;or a linker comprising an amino acid sequence having at least about 99% sequence identity, and (iii) an armor protein comprising an IL-15Rα polypeptide tethered to an IL-15 polypeptide, wherein the IL-15Rα polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:3, and the IL-15 polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:2.

[0157] In some embodiments, an engineered γδ T-cell (e.g., a Vd1+ cell) comprises: (A)(i) an antigen-binding domain that specifically binds human mesothelin (the antigen-binding domain comprises a VH comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:20, and a VH comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:21); (ii) a hinge region comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:30; and (iii) an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:31. (B) a transmembrane domain comprising: (iv) a costimulatory domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 34; and (v) an intracellular signaling domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 35; and (i) an IL-2Rβ polypeptide comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:1; (ii) an IL-2Rβ polypeptide comprising at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%,a linker comprising an amino acid sequence having at least about 97%, at least about 98%, or at least about 99% sequence identity; and (iii) an armor protein comprising an IL-15Rα polypeptide tethered to an IL-15 polypeptide, wherein the IL-15Rα polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:3, and the IL-15 polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:2.

[0158] In some embodiments, an engineered γδ T-cell (e.g., a Vd1+ cell) comprises: (A) (i) an antigen-binding domain that specifically binds to human mesothelin (the antigen-binding domain comprises a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 19); (ii) a hinge region comprising the amino acid sequence set forth in SEQ ID NO: 30; (iii) a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 31; (B) a CAR comprising (i) an IL-2Rβ polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1, (ii) a linker comprising the amino acid sequence set forth in SEQ ID NO:4, and (iii) an IL-15Rα polypeptide tethered to an IL-15 polypeptide, wherein the IL-15Rα polypeptide comprises the amino acid sequence set forth in SEQ ID NO:3 and the IL-15 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2.

[0159] In some aspects, an engineered γδ T (e.g., a Vd1+ cell) cell comprises: (A) a CAR comprising (i) an antigen binding domain that specifically binds human mesothelin (the antigen binding domain comprises a VH comprising the amino acid sequence set forth in SEQ ID NO:20, a VL comprising the amino acid sequence set forth in SEQ ID NO:21), (ii) a hinge region comprising the amino acid sequence set forth in SEQ ID NO:30, (iii) a transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO:31, (iv) a costimulatory domain comprising the amino acid sequence set forth in SEQ ID NO:34, and (v) an intracellular signaling domain comprising the amino acid sequence set forth in SEQ ID NO:35; and (B) an armor protein comprising (i) an IL-2Rβ polypeptide comprising the amino acid sequence set forth in SEQ ID NO:15, (ii) a linker comprising the amino acid sequence set forth in SEQ ID NO:4, and (iii) an IL-15Rα polypeptide tethered to an IL-15 polypeptide, wherein the IL-15Rα polypeptide comprises the amino acid sequence set forth in SEQ ID NO:3, and the IL-15 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2.

[0160] In some embodiments, an engineered γδ T (e.g., a Vd1+ cell) cell comprises: (A) (i) an antigen-binding domain that specifically binds to human mesothelin (the antigen-binding domain comprises a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 19); (ii) a hinge region comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 32; (iii) a hinge region comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 33; (B) a CAR comprising an amino acid sequence having sequence identity to the sequence set forth in SEQ ID NO:34; (iv) a costimulatory domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:34; and (v) an intracellular signaling domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:35; and (B) an armor protein comprising an IL-2Rβ polypeptide comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the engineered γδ T-cell does not comprise an IL-15Rα polypeptide or an IL-15 polypeptide.

[0161] In some embodiments, an engineered γδ T-cell (e.g., a Vd1+ cell) comprises: (A)(i) an antigen-binding domain that specifically binds to human mesothelin (the antigen-binding domain comprises a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 19); (ii) a hinge region comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:32; (iii) a transmembrane domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:33; (iv) a transmembrane domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:34; (B) a CAR comprising (i) an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1; and (v) an intracellular signaling domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 35. (ii) an IL-2Rβ polypeptide comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:4;or a linker comprising an amino acid sequence having at least about 99% sequence identity, and (iii) an armor protein comprising an IL-15Rα polypeptide tethered to an IL-15 polypeptide, wherein the IL-15Rα polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:3, and the IL-15 polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:2.

[0162] In some embodiments, an engineered γδ T-cell (e.g., a Vd1+ cell) comprises: (A)(i) an antigen-binding domain that specifically binds human mesothelin (the antigen-binding domain comprises a VH comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:20, and a VH comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:21); (ii) a hinge region comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:32; and (iii) an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:33. (B) a transmembrane domain comprising: (iv) a costimulatory domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 34; and (v) an intracellular signaling domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 35; and (i) an IL-2Rβ polypeptide comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:1; (ii) an IL-2Rβ polypeptide comprising at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%,a linker comprising an amino acid sequence having at least about 97%, at least about 98%, or at least about 99% sequence identity; and (iii) an armor protein comprising an IL-15Rα polypeptide tethered to an IL-15 polypeptide, wherein the IL-15Rα polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:3, and the IL-15 polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:2.

[0163] In some embodiments, an engineered γδ T-cell (e.g., a Vd1+ cell) comprises: (A) (i) an antigen-binding domain that specifically binds to human mesothelin (the antigen-binding domain comprises a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 19); (ii) a hinge region comprising the amino acid sequence set forth in SEQ ID NO: 32; (iii) a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 33; (B) a CAR comprising (i) an IL-2Rβ polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1, (ii) a linker comprising the amino acid sequence set forth in SEQ ID NO:4, and (iii) an IL-15Rα polypeptide tethered to an IL-15 polypeptide, wherein the IL-15Rα polypeptide comprises the amino acid sequence set forth in SEQ ID NO:3 and the IL-15 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2.

[0164] In some aspects, an engineered γδ T-cell (e.g., a Vd1+ cell) comprises: (A) a CAR comprising (i) an antigen binding domain that specifically binds human mesothelin (the antigen binding domain comprises a VH comprising the amino acid sequence set forth in SEQ ID NO:20, a VL comprising the amino acid sequence set forth in SEQ ID NO:21), (ii) a hinge region comprising the amino acid sequence set forth in SEQ ID NO:32, (iii) a transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO:33, (iv) a costimulatory domain comprising the amino acid sequence set forth in SEQ ID NO:34, and (v) an intracellular signaling domain comprising the amino acid sequence set forth in SEQ ID NO:35; and (B) an armor protein comprising (i) an IL-2Rβ polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1, (ii) a linker comprising the amino acid sequence set forth in SEQ ID NO:4, and (iii) an IL-15Rα polypeptide tethered to an IL-15 polypeptide, wherein the IL-15Rα polypeptide comprises the amino acid sequence set forth in SEQ ID NO:3, and the IL-15 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2.

[0165] In some embodiments, an engineered γδ T-cell (e.g., a Vd1+ cell) comprises: (A) (i) an antigen-binding domain that specifically binds to human mesothelin (the antigen-binding domain comprises a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 22, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 23, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 24, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27); (ii) a hinge region comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 30; (iii) a hinge region comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 31; (B) a CAR comprising an amino acid sequence having sequence identity to the sequence set forth in SEQ ID NO:34; (iv) a costimulatory domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:34; and (v) an intracellular signaling domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:35; and (B) an armor protein comprising an IL-2Rβ polypeptide comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the engineered γδ T-cell does not comprise an IL-15Rα polypeptide or an IL-15 polypeptide.

[0166] In some embodiments, an engineered γδ T-cell (e.g., a Vd1+ cell) comprises: (A)(i) an antigen-binding domain that specifically binds to human mesothelin (the antigen-binding domain comprises a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 22, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 23, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 24, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27); (ii) a hinge region comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:30; (iii) a transmembrane domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:31; (iv) a transmembrane domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:34; (B) a CAR comprising (i) an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1; and (v) an intracellular signaling domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 35. (ii) an IL-2Rβ polypeptide comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:4;or a linker comprising an amino acid sequence having at least about 99% sequence identity, and (iii) an armor protein comprising an IL-15Rα polypeptide tethered to an IL-15 polypeptide, wherein the IL-15Rα polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:3, and the IL-15 polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:2.

[0167] In some embodiments, an engineered γδ T-cell (e.g., a Vd1+ cell) comprises: (A)(i) an antigen-binding domain that specifically binds human mesothelin (the antigen-binding domain comprises a VH comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:28, and an antigen-binding domain that comprises at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:29); (ii) a hinge region comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:30; and (iii) an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:31. (B) a transmembrane domain comprising: (iv) a costimulatory domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 34; and (v) an intracellular signaling domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 35; and (i) an IL-2Rβ polypeptide comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:1; (ii) an IL-2Rβ polypeptide comprising at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%,a linker comprising an amino acid sequence having at least about 97%, at least about 98%, or at least about 99% sequence identity; and (iii) an armor protein comprising an IL-15Rα polypeptide tethered to an IL-15 polypeptide, wherein the IL-15Rα polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:3, and the IL-15 polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:2.

[0168] In some embodiments, an engineered γδ T-cell (e.g., a Vd1+ cell) comprises: (A) (i) an antigen-binding domain that specifically binds to human mesothelin (the antigen-binding domain comprises a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 22, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 23, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 24, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27); (ii) a hinge region comprising the amino acid sequence set forth in SEQ ID NO: 30; (iii) a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 31; (B) a CAR comprising (i) an IL-2Rβ polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1, (ii) a linker comprising the amino acid sequence set forth in SEQ ID NO:4, and (iii) an IL-15Rα polypeptide tethered to an IL-15 polypeptide, wherein the IL-15Rα polypeptide comprises the amino acid sequence set forth in SEQ ID NO:3 and the IL-15 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2.

[0169] In some aspects, an engineered γδ T-cell (e.g., a Vd1+ cell) comprises: (A) a CAR comprising (i) an antigen binding domain that specifically binds human mesothelin (the antigen binding domain comprises a VH comprising the amino acid sequence set forth in SEQ ID NO:28, a VL comprising the amino acid sequence set forth in SEQ ID NO:29), (ii) a hinge region comprising the amino acid sequence set forth in SEQ ID NO:30, (iii) a transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO:31, (iv) a costimulatory domain comprising the amino acid sequence set forth in SEQ ID NO:34, and (v) an intracellular signaling domain comprising the amino acid sequence set forth in SEQ ID NO:35; and (B) an armor protein comprising (i) an IL-2Rβ polypeptide comprising the amino acid sequence set forth in SEQ ID NO:15, (ii) a linker comprising the amino acid sequence set forth in SEQ ID NO:4, and (iii) an IL-15Rα polypeptide tethered to an IL-15 polypeptide, wherein the IL-15Rα polypeptide comprises the amino acid sequence set forth in SEQ ID NO:3, and the IL-15 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2.

[0170] In some embodiments, an engineered γδ T-cell (e.g., a Vd1+ cell) comprises: (A) (i) an antigen-binding domain that specifically binds to human mesothelin (the antigen-binding domain comprises a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 22, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 23, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 24, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27); (ii) a hinge region comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 32; (iii) a hinge region comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 33; (B) a CAR comprising an amino acid sequence having sequence identity to the sequence set forth in SEQ ID NO:34; (iv) a costimulatory domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:34; and (v) an intracellular signaling domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:35; and (B) an armor protein comprising an IL-2Rβ polypeptide comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the engineered γδ T-cell does not comprise an IL-15Rα polypeptide or an IL-15 polypeptide.

[0171] In some embodiments, an engineered γδ T-cell (e.g., a Vd1+ cell) comprises: (A)(i) an antigen-binding domain that specifically binds to human mesothelin (the antigen-binding domain comprises a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 22, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 23, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 24, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27); (ii) a hinge region comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:32; (iii) a transmembrane domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:33; (iv) a transmembrane domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:34; (B) a CAR comprising (i) an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1; and (v) an intracellular signaling domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 35. (ii) an IL-2Rβ polypeptide comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:4;or a linker comprising an amino acid sequence having at least about 99% sequence identity, and (iii) an armor protein comprising an IL-15Rα polypeptide tethered to an IL-15 polypeptide, wherein the IL-15Rα polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:3, and the IL-15 polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:2.

[0172] In some embodiments, an engineered γδ T-cell (e.g., a Vd1+ cell) comprises: (A)(i) an antigen-binding domain that specifically binds human mesothelin (the antigen-binding domain comprises a VH comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:28, and an antigen-binding domain that comprises at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:29); (ii) a hinge region comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:32; and (iii) an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:33. (B) a transmembrane domain comprising: (iv) a costimulatory domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 34; and (v) an intracellular signaling domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 35; and (i) an IL-2Rβ polypeptide comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:1; (ii) an IL-2Rβ polypeptide comprising at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%,a linker comprising an amino acid sequence having at least about 97%, at least about 98%, or at least about 99% sequence identity; and (iii) an armor protein comprising an IL-15Rα polypeptide tethered to an IL-15 polypeptide, wherein the IL-15Rα polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:3, and the IL-15 polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:2.

[0173] In some embodiments, an engineered γδ T-cell (e.g., a Vd1+ cell) comprises: (A) (i) an antigen-binding domain that specifically binds to human mesothelin (the antigen-binding domain comprises a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 22, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 23, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 24, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27); (ii) a hinge region comprising the amino acid sequence set forth in SEQ ID NO: 32; (iii) a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 33; (B) a CAR comprising (i) an IL-2Rβ polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1, (ii) a linker comprising the amino acid sequence set forth in SEQ ID NO:4, and (iii) an IL-15Rα polypeptide tethered to an IL-15 polypeptide, wherein the IL-15Rα polypeptide comprises the amino acid sequence set forth in SEQ ID NO:3 and the IL-15 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2.

[0174] In some aspects, an engineered γδ T-cell (e.g., a Vd1+ cell) comprises: (A) a CAR comprising (i) an antigen binding domain that specifically binds human mesothelin (the antigen binding domain comprises a VH comprising the amino acid sequence set forth in SEQ ID NO:28, a VL comprising the amino acid sequence set forth in SEQ ID NO:29), (ii) a hinge region comprising the amino acid sequence set forth in SEQ ID NO:32, (iii) a transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO:33, (iv) a costimulatory domain comprising the amino acid sequence set forth in SEQ ID NO:34, and (v) an intracellular signaling domain comprising the amino acid sequence set forth in SEQ ID NO:35; and (B) an armor protein comprising (i) an IL-2Rβ polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1, (ii) a linker comprising the amino acid sequence set forth in SEQ ID NO:4, and (iii) an IL-15Rα polypeptide tethered to an IL-15 polypeptide, wherein the IL-15Rα polypeptide comprises the amino acid sequence set forth in SEQ ID NO:3, and the IL-15 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2.

[0175] III. Transduction Methods In one aspect, the invention provides a method of generating a population of engineered γδ T cells by transducing a population of γδ T cells (e.g. V51 T cells, V52 T cells, and / or non-V51 / V52 T cells) with a polynucleotide, such as a viral vector, such as a retroviral vector. The method comprises providing a population of γδ T cells and transducing the population of γδ T cells with a polynucleotide encoding IL-15Rβ, thereby generating a population of γδ T cells expressing recombinant IL-15Rβ. The method may further comprise transducing the starting population of γδ T cells with a polynucleotide encoding an IL-15 receptor α subunit or a fragment thereof. In other embodiments, the method comprises generating a population of γδ T cells expressing recombinant IL-15Rβ and IL-15 or a variant thereof. The method includes providing a population of γδ T cells, transducing the population of γδ T cells with a polynucleotide encoding IL-15Rβ, and transducing the population of γδ T cells with a polynucleotide encoding IL-15 or a variant thereof, thereby generating a population of γδ T cells expressing recombinant IL-15Rβ and IL-15 or a variant thereof. The method may include transducing the starting population of γδ T cells with a polynucleotide encoding an IL-15 receptor α subunit or a fragment thereof. The method described herein may further include transducing the cells with a polynucleotide encoding a CAR.

[0176] In another aspect, the invention provides a method of generating a population of engineered γδ T-cells by providing a starting population of γδ T-cells, priming the γδ T-cells in the absence of a viral vector, and culturing the population of primed γδ T-cells in the presence of an amount of viral vector effective to transduce at least 3% (e.g., at least 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or substantially all) of the primed γδ T-cells. In some embodiments, the population of primed γδ T cells is cultured in the presence of an amount of a viral vector effective to transduce at least 5% of the primed γδ T cells. In some embodiments, the population of primed γδ T cells is cultured in the presence of an amount of a viral vector effective to transduce at least 20% of the primed γδ T cells.

[0177] Primed γδ T cells may be obtained by culturing the starting population of γδ T cells in the absence of a viral vector. For example, the starting population of γδ T cells may be cultured for a first culture period of at least 1 hour (e.g., at least 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, or more, e.g., about 1 hour to about 14 days, about 6 hours to about 14 days, about 1 day to about 14 days, about 2 days to about 14 days, about 5 days to about 14 days, about 7 days to about 14 days, about 5 days to about 10 days, about 5 days to about 7 days, or about 7 days to about 10 days). When primed γδ T cells are obtained, for example after culturing the cells in the absence of a viral vector, the primed γδ T cells may be further cultured for a second culture period of at least 1 day (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days, or more, e.g., about 1 to about 14 days, about 2 to about 14 days, about 5 to about 14 days, about 7 to about 14 days, about 5 to about 10 days, about 5 to about 7 days, or about 7 to about 10 days). The second culture period may be about 1 to about 14 days (e.g., about 3 to about 14 days, about 3 to about 12 days, about 4 to about 1 day, about 5 to about 10 days, or about 5 to about 7 days).

[0178] In some embodiments, the viral vector is cultured with the primed γδ T cells at a multiplicity of infection (MOI) of about 10 or less, such as about 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, or 0.25 or less. In some embodiments, the viral vector is cultured with the primed γδ T cells at a multiplicity of infection (MOI) of about 5 or less. In some embodiments, the viral vector is cultured with the primed γδ T cells at a multiplicity of infection (MOI) of about 4 or less. In some embodiments, the viral vector is cultured with the primed γδ T cells at a multiplicity of infection (MOI) of about 3 or less. In some embodiments, the viral vector is cultured with the primed γδ T cells at a multiplicity of infection (MOI) of about 2 or less. In some embodiments, the viral vector is cultured with the primed γδ T cells at a multiplicity of infection (MOI) of about 1 or less. In some embodiments, the viral vector is cultured with the primed γδ T cells at a multiplicity of infection (MOI) of about 0.5 or less. In some embodiments, the viral vectors are cultured with primed γδ T cells at a multiplicity of infection (MOI) of about 0.25 or less. In some embodiments, the viral vectors are cultured with primed γδ T cells at a multiplicity of infection (MOI) of about 0.25 to about 10 (e.g., about 0.5 to about 10, about 1 to about 10, or about 1 to about 5).

[0179] In some embodiments, transduction of γδ T cells includes the use of a transduction enhancer to enhance transduction efficiency. Suitable transduction enhancers include, for example, vectorfusin, spermid, and / or retronectin. The method may include contacting the γδ T cells with the transduction enhancer during culture. In some embodiments, the method further includes contacting the cells with nevirapine. In some embodiments, transduction of γδ T cells includes adding IL-15 to the culture medium, which can increase γδ T cell expression of ASCT-2, a viral entry receptor for betaretroviral pseudotyped viral vectors.

[0180] IV. Viral Vectors The compositions and methods described herein include the use of viral vectors for efficient transduction of γδ T cells. Viral genomes provide a rich source of vectors that can be used to efficiently deliver exogenous genes to mammalian cells. Viral genomes are particularly useful vectors for gene delivery because polynucleotides contained within such genomes are usually integrated into the nuclear genome of mammalian cells by generalized or specialized transduction. These processes occur as part of the natural viral replication cycle and do not require the addition of proteins or reagents to induce gene integration. Examples of viral vectors include retroviruses (e.g., Retroviridae viral vectors). Examples of retroviruses are avian leukosis sarcoma, avian C virus, mammalian C, B, D virus, oncoretrovirus, HTLV-BLV group, lentivirus, alpharetrovirus, betaretrovirus, gammaretrovirus, spumavirus (Coffin, JM, Retroviridae: The viruses and their replication, Virology, Third Edition (Lippincott-Raven, Philadelphia, (1996))). Other examples are murine leukemia virus (MLV), murine sarcoma virus, mouse mammalian tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus (BaEV), gibbon ape leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus, and lentiviruses.

[0181] IV.A. Retroviral Vectors In some examples, the viral vector used in the methods and compositions described herein is a retroviral vector. One type of retroviral vector that can be used in the methods and compositions described herein is a lentiviral vector. A subset of retroviruses, lentiviral vectors (LVs), can transduce a wide range of dividing and non-dividing cell types with high efficiency, resulting in stable, long-term expression of transgenes. An overview of the optimization strategy for packaging and transducing LVs is provided in Deleda, The Journal of Gene Medicine 6:S125, 2004, the disclosure of which is incorporated herein by reference.

[0182] The use of lentivirus-based gene transfer techniques relies on the in vitro production of recombinant lentivirus particles carrying a highly deleted viral genome that houses the transgene of interest. In particular, recombinant lentiviruses are recovered by trans co-expression in permissive cell lines of (1) a packaging construct, i.e., a vector expressing the Gag-Pol precursor together with Rev (alternatively expressed in trans), (2) a vector expressing an envelope protein, generally of heterologous nature, and (3) a transfer vector consisting of a viral cDNA lacking all open reading frames but maintaining sequences necessary for replication, encapsidation and expression, into which the sequences to be expressed have been inserted.

[0183] The LV used in the methods and compositions described herein may include one or more of the following: 5'-long terminal repeat (LTR), HIV signal sequence, HIV Psi signal 5'-splice site (SD), delta-GAG element, Rev-responsive element (RRE), 3'-splice site (SA), elongation factor (EF) 1 alpha promoter, and 3'-self-inactivating LTR (SIN-LTR). The lentiviral vector may optionally include a central polypurine tract (cPPT) and a woodchuck hepatitis virus posttranscriptional control element (WPRE), which are described in US 6,136,597, the disclosure of which is incorporated herein by reference as it relates to the WPRE. The lentiviral vector may further include a pHR' backbone, which may include, for example, the following:

[0184] Lentigen LV, described in Lu et al., Journal of Gene Medicine 6:963, 2004, may be used to express DNA molecules and / or transduce cells. The LV used in the methods and compositions described herein may be a 5'-long terminal repeat (LTR), an HIV signal sequence, an HIV Psi signal 5'-splice site (SD), a delta-GAG element, a Rev-responsive element (RRE), a 3'-splice site (SA), an elongation factor (EF) 1 alpha promoter, and a 3'-self-inactivating LTR (SIN-LTR). Optionally, it will be readily apparent to one of skill in the art that one or more of these regions may be replaced with another region that performs a similar function.

[0185] Enhancer elements can be used to increase expression of modified DNA molecules or to increase lentiviral integration efficiency. LVs used in the methods and compositions described herein can include a nef sequence. LVs used in the methods and compositions described herein can include a cPPT sequence that enhances vector integration. The cPPT acts as a second origin of (+)strand DNA synthesis, introducing a partial strand duplication in the middle of the native HIV genome. Introduction of the cPPT sequence into the transfer vector backbone strongly increased nuclear transport and increased the total amount of genome integrated into the DNA of the target cell. LVs used in the methods and compositions described herein can include a woodchuck posttranscriptional regulatory element (WPRE). The WPRE acts at the transcriptional level by facilitating nuclear transport of transcripts and / or increasing the efficiency of polyadenylation of the nascent transcript, thereby increasing the total amount of mRNA in the cell. Addition of the WPRE to the LV substantially increases the level of expression of transgenes from several different promoters, both in vitro and in vivo. The LV used in the methods and compositions described herein may contain both a cPPT sequence and a WPRE sequence. The vector may also contain an IRES sequence, which allows expression of multiple polypeptides from a single promoter.

[0186] In addition to IRES sequences, other elements that allow for the expression of multiple polypeptides are also useful. The vectors used in the methods and compositions described herein may include multiple promoters that allow for the expression of multiple polypeptides. The vectors used in the methods and compositions described herein may include protein cleavage sites that allow for the expression of multiple polypeptides. Examples of protein cleavage sites that allow for the expression of multiple polypeptides are described in Klump et al., Gene Ther.;8:811, 2001, Osborn et al., Molecular Therapy 12:569, 2005, Szymczak and Vignali, Expert Opin. Biol. Ther.5:627, 2005, and Szymczak et al., Nat. Biotechnol.22:589, 2004, the disclosures of which are incorporated herein by reference as they relate to protein cleavage sites that allow for the expression of multiple polypeptides. It will be readily apparent to one of skill in the art that other elements that allow for the expression of multiple polypeptides identified in the future will be useful and may be utilized in vectors suitable for use in the compositions and methods described herein.

[0187] Other retroviral vectors (e.g., retroviral backbones) that may be used in conjunction with the compositions and methods described herein include gamma retroviral vectors. Exemplary gamma retroviral vectors are or are derived from chick syncytial virus, feline leukemia virus, Finkel-Biskis-Jinkins murine sarcoma virus, Gardner-Arnstein feline sarcoma virus, gibbon ape leukemia virus, guinea pig type C oncovirus, Hardy-Zuckerman feline sarcoma virus, Harvey murine sarcoma virus, Kirsten murine sarcoma virus, koala retrovirus, Moloney murine sarcoma virus, murine leukemia virus, porcine type C oncovirus, reticuloendotheliosis virus, Snyder-Scielen feline sarcoma virus, Troeger duck spleen necrosis virus, Viper retrovirus, and woolly monkey sarcoma virus.

[0188] In certain embodiments, the viral vector backbone is derived from lentivirus (LV). In certain embodiments, the viral vector backbone is derived from third generation self-inactivating (SIN) lentivirus vector (LV) (e.g., HIV, SIV, or EIAV). In certain embodiments, the viral vector backbone is derived from non-self-inactivating LV (e.g., ).

[0189] Other retroviral vectors (e.g., retroviral backbones) that may be used in conjunction with the compositions and methods described herein include alpharetroviral vectors. Exemplary alpharetroviral vectors are or are derived from the avian carcinoma Mill Hill Virus 2, avian leukosis virus, avian myeloblastosis virus, avian myelocytoma virus 29, avian sarcoma virus ct10, Fujinami sarcoma virus, Rous sarcoma virus, ur2 sarcoma virus, and y73 sarcoma virus.

[0190] In some embodiments, the viral vector comprises: (A) (i) an antigen-binding domain that specifically binds to human mesothelin (the antigen-binding domain comprises a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 19); (ii) a hinge region comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 30; and (iii) an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 31. (B) a CAR comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:34; (iv) a costimulatory domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:34; and (v) an intracellular signaling domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:35; and (B) a polynucleotide encoding an armor protein comprising an IL-2Rβ polypeptide comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the engineered γδ T-cell does not comprise an IL-15Rα polypeptide or an IL-15 polypeptide.

[0191] In some embodiments, the viral vector comprises: (A) (i) an antigen-binding domain that specifically binds to human mesothelin (the antigen-binding domain comprises a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 19); (iii) a transmembrane domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:31; (iv) a transmembrane domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:34; 35; and (B) a CAR comprising (i) an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:1; and (ii) an intracellular signaling domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:1. (ii) an IL-2Rβ polypeptide comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:4;and (iii) a polynucleotide encoding an armor protein comprising an IL-15Rα polypeptide tethered to an IL-15 polypeptide, wherein the IL-15Rα polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:3, and the IL-15 polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:2.

[0192] In some embodiments, the viral vector comprises: (A) (i) an antigen-binding domain that specifically binds human mesothelin (the antigen-binding domain is an antigen-binding domain that comprises a VH comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:20, and an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:21); (ii) a hinge region comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:30; (iii) a transmembrane domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:31; (v) a costimulatory domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 34, and (v) an intracellular signaling domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 35, and (B)(i) a CAR comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 1. (ii) an IL-2Rβ polypeptide comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:4;a linker comprising an amino acid sequence having at least about 98%, or at least about 99%, sequence identity; and (iii) a polynucleotide encoding an armor protein comprising an IL-15Rα polypeptide tethered to an IL-15 polypeptide, wherein the IL-15Rα polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:3, and the IL-15 polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:2.

[0193] In some embodiments, the viral vector comprises: (A) (i) an antigen-binding domain that specifically binds to human mesothelin (the antigen-binding domain comprises a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 19); (ii) a hinge region comprising the amino acid sequence set forth in SEQ ID NO: 30; and (iii) a membrane domain comprising the amino acid sequence set forth in SEQ ID NO: 31. (B) a polynucleotide encoding an armor protein comprising: (i) an IL-2Rβ polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1; (ii) a linker comprising the amino acid sequence set forth in SEQ ID NO:4; and (iii) an IL-15Rα polypeptide tethered to an IL-15 polypeptide, wherein the IL-15Rα polypeptide comprises the amino acid sequence set forth in SEQ ID NO:3 and the IL-15 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2.

[0194] In some aspects, the viral vector comprises: (A) a CAR comprising (i) an antigen binding domain that specifically binds human mesothelin (the antigen binding domain comprises a VH comprising the amino acid sequence set forth in SEQ ID NO:20, a VL comprising the amino acid sequence set forth in SEQ ID NO:21), (ii) a hinge region comprising the amino acid sequence set forth in SEQ ID NO:30, (iii) a transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO:31, (iv) a costimulatory domain comprising the amino acid sequence set forth in SEQ ID NO:34, and (v) an intracellular signaling domain comprising the amino acid sequence set forth in SEQ ID NO:35; and (B) a polynucleotide encoding an armor protein comprising (i) an IL-2Rβ polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1, (ii) a linker comprising the amino acid sequence set forth in SEQ ID NO:4, and (iii) an IL-15Rα polypeptide tethered to an IL-15 polypeptide, wherein the IL-15Rα polypeptide comprises the amino acid sequence set forth in SEQ ID NO:3, and the IL-15 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2.

[0195] In some embodiments, (A) a portion of the polynucleotide encoding a CAR and (B) a portion of the polynucleotide encoding an Armor protein are connected by a portion of the polynucleotide encoding a linker. In some embodiments, the linker comprises a peptide bond. In some embodiments, the linker comprises one or more amino acids. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker comprises a T2A linker. In some embodiments, the linker comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:5. In some embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO:5.

[0196] In some embodiments, the viral vector comprises: (A) (i) an antigen-binding domain that specifically binds to human mesothelin (the antigen-binding domain comprises a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 19); (ii) a hinge region comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 32; and (iii) an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 33. (B) a CAR comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:34; (iv) a costimulatory domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:34; and (v) an intracellular signaling domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:35; and (B) a polynucleotide encoding an armor protein comprising an IL-2Rβ polypeptide comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the engineered γδ T-cell does not comprise an IL-15Rα polypeptide or an IL-15 polypeptide.

[0197] In some embodiments, the viral vector comprises: (A) (i) an antigen-binding domain that specifically binds to human mesothelin (the antigen-binding domain comprises a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 19); (iii) a transmembrane domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:33; (iv) a transmembrane domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:34; 35; and (B) a CAR comprising (i) an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:1; and (ii) an intracellular signaling domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:1. (ii) an IL-2Rβ polypeptide comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:4;and (iii) a polynucleotide encoding an armor protein comprising an IL-15Rα polypeptide tethered to an IL-15 polypeptide, wherein the IL-15Rα polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:3, and the IL-15 polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:2.

[0198] In some embodiments, the viral vector comprises: (A) (i) an antigen-binding domain that specifically binds human mesothelin (the antigen-binding domain is an antigen-binding domain that comprises a VH comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:20, and an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:21); (ii) a hinge region comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 32; (iii) a transmembrane domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 33; (v) a costimulatory domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 34, and (v) an intracellular signaling domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 35, and (B)(i) a CAR comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 1. (ii) an IL-2Rβ polypeptide comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:4;a linker comprising an amino acid sequence having at least about 98%, or at least about 99%, sequence identity; and (iii) a polynucleotide encoding an armor protein comprising an IL-15Rα polypeptide tethered to an IL-15 polypeptide, wherein the IL-15Rα polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:3, and the IL-15 polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:2.

[0199] In some aspects, the viral vector comprises: (A) (i) an antigen-binding domain that specifically binds to human mesothelin (the antigen-binding domain comprises a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 19); (ii) a hinge region comprising the amino acid sequence set forth in SEQ ID NO: 32; and (iii) a membrane domain comprising the amino acid sequence set forth in SEQ ID NO: 33. (B) a polynucleotide encoding an armor protein comprising: (i) an IL-2Rβ polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1; (ii) a linker comprising the amino acid sequence set forth in SEQ ID NO:4; and (iii) an IL-15Rα polypeptide tethered to an IL-15 polypeptide, wherein the IL-15Rα polypeptide comprises the amino acid sequence set forth in SEQ ID NO:3 and the IL-15 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2.

[0200] In some aspects, the viral vector comprises: (A) a CAR comprising (i) an antigen binding domain that specifically binds human mesothelin (the antigen binding domain comprises a VH comprising the amino acid sequence set forth in SEQ ID NO:20, a VL comprising the amino acid sequence set forth in SEQ ID NO:21), (ii) a hinge region comprising the amino acid sequence set forth in SEQ ID NO:32, (iii) a transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO:33, (iv) a costimulatory domain comprising the amino acid sequence set forth in SEQ ID NO:34, and (v) an intracellular signaling domain comprising the amino acid sequence set forth in SEQ ID NO:35; and (B) a polynucleotide encoding an armor protein comprising (i) an IL-2Rβ polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1, (ii) a linker comprising the amino acid sequence set forth in SEQ ID NO:4, and (iii) an IL-15Rα polypeptide tethered to an IL-15 polypeptide, wherein the IL-15Rα polypeptide comprises the amino acid sequence set forth in SEQ ID NO:3, and the IL-15 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2.

[0201] In some embodiments, (A) a portion of the polynucleotide encoding a CAR and (B) a portion of the polynucleotide encoding an Armor protein are connected by a portion of the polynucleotide encoding a linker. In some embodiments, the linker comprises a peptide bond. In some embodiments, the linker comprises one or more amino acids. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker comprises a T2A linker. In some embodiments, the linker comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:5. In some embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO:5.

[0202] In some embodiments, the viral vector comprises: (A) (i) an antigen-binding domain that specifically binds to human mesothelin (the antigen-binding domain comprises a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 22, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 23, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 24, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27); (ii) a hinge region comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 30; and (iii) an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 31. (B) a CAR comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:34; (iv) a costimulatory domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:34; and (v) an intracellular signaling domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:35; and (B) a polynucleotide encoding an armor protein comprising an IL-2Rβ polypeptide comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the engineered γδ T-cell does not comprise an IL-15Rα polypeptide or an IL-15 polypeptide.

[0203] In some embodiments, the viral vector comprises: (A) (i) an antigen-binding domain that specifically binds to human mesothelin (the antigen-binding domain comprises a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 22, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 23, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 24, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27); (iii) a transmembrane domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:31; (iv) a transmembrane domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:34; 35; and (B) a CAR comprising (i) an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:1; and (ii) an intracellular signaling domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:1. (ii) an IL-2Rβ polypeptide comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:4;and (iii) a polynucleotide encoding an armor protein comprising an IL-15Rα polypeptide tethered to an IL-15 polypeptide, wherein the IL-15Rα polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:3, and the IL-15 polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:2.

[0204] In some embodiments, the viral vector comprises: (A) (i) an antigen-binding domain that specifically binds human mesothelin (the antigen-binding domain is an antigen-binding domain that comprises a VH comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:28, and an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:29). (ii) a hinge region comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:30; (iii) a transmembrane domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:31; (v) a costimulatory domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 34, and (v) an intracellular signaling domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 35, and (B)(i) a CAR comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 1. (ii) an IL-2Rβ polypeptide comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:4;a linker comprising an amino acid sequence having at least about 98%, or at least about 99%, sequence identity; and (iii) a polynucleotide encoding an armor protein comprising an IL-15Rα polypeptide tethered to an IL-15 polypeptide, wherein the IL-15Rα polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:3, and the IL-15 polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:2.

[0205] In some embodiments, the viral vector comprises: (A) (i) an antigen-binding domain that specifically binds to human mesothelin (the antigen-binding domain comprises a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 22, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 23, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 24, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27); (ii) a hinge region comprising the amino acid sequence set forth in SEQ ID NO: 30; and (iii) a membrane domain comprising the amino acid sequence set forth in SEQ ID NO: 31. (B) a polynucleotide encoding an armor protein comprising: (i) an IL-2Rβ polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1; (ii) a linker comprising the amino acid sequence set forth in SEQ ID NO:4; and (iii) an IL-15Rα polypeptide tethered to an IL-15 polypeptide, wherein the IL-15Rα polypeptide comprises the amino acid sequence set forth in SEQ ID NO:3 and the IL-15 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2.

[0206] In some aspects, the viral vector comprises: (A) a CAR comprising (i) an antigen binding domain that specifically binds human mesothelin (the antigen binding domain comprises a VH comprising the amino acid sequence set forth in SEQ ID NO:28, a VL comprising the amino acid sequence set forth in SEQ ID NO:29), (ii) a hinge region comprising the amino acid sequence set forth in SEQ ID NO:30, (iii) a transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO:31, (iv) a costimulatory domain comprising the amino acid sequence set forth in SEQ ID NO:34, and (v) an intracellular signaling domain comprising the amino acid sequence set forth in SEQ ID NO:35; and (B) a polynucleotide encoding an armor protein comprising (i) an IL-2Rβ polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1, (ii) a linker comprising the amino acid sequence set forth in SEQ ID NO:4, and (iii) an IL-15Rα polypeptide tethered to an IL-15 polypeptide, wherein the IL-15Rα polypeptide comprises the amino acid sequence set forth in SEQ ID NO:3, and the IL-15 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2.

[0207] In some embodiments, (A) a portion of the polynucleotide encoding a CAR and (B) a portion of the polynucleotide encoding an Armor protein are connected by a portion of the polynucleotide encoding a linker. In some embodiments, the linker comprises a peptide bond. In some embodiments, the linker comprises one or more amino acids. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker comprises a T2A linker. In some embodiments, the linker comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:5. In some embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO:5.

[0208] In some embodiments, the viral vector comprises: (A) (i) an antigen-binding domain that specifically binds human mesothelin (the antigen-binding domain comprises a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 22, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 23, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 24, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27); (ii) a hinge region comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 32; and (iii) an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 33. (B) a CAR comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:34; (iv) a costimulatory domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:34; and (v) an intracellular signaling domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:35; and (B) a polynucleotide encoding an armor protein comprising an IL-2Rβ polypeptide comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the engineered γδ T-cell does not comprise an IL-15Rα polypeptide or an IL-15 polypeptide.

[0209] In some embodiments, the viral vector comprises: (A) (i) an antigen-binding domain that specifically binds to human mesothelin (the antigen-binding domain comprises a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 22, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 23, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 24, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27); (iii) a transmembrane domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:33; (iv) a transmembrane domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:34; 35; and (B) a CAR comprising (i) an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:2; and (ii) an intracellular signaling domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:35. (ii) an IL-2Rβ polypeptide comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:4;and (iii) a polynucleotide encoding an armor protein comprising an IL-15Rα polypeptide tethered to an IL-15 polypeptide, wherein the IL-15Rα polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:3, and the IL-15 polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:2.

[0210] In some embodiments, the viral vector comprises: (A) (i) an antigen-binding domain that specifically binds human mesothelin (the antigen-binding domain is an antigen-binding domain that comprises a VH comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:28, and an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:29). (ii) a hinge region comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 32; (iii) a transmembrane domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 33; (v) a costimulatory domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 34, and (v) an intracellular signaling domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 35, and (B)(i) a CAR comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 1. (ii) an IL-2Rβ polypeptide comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:4;a linker comprising an amino acid sequence having at least about 98%, or at least about 99%, sequence identity; and (iii) a polynucleotide encoding an armor protein comprising an IL-15Rα polypeptide tethered to an IL-15 polypeptide, wherein the IL-15Rα polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:3, and the IL-15 polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:2.

[0211] In some embodiments, the viral vector comprises: (A) (i) an antigen-binding domain that specifically binds to human mesothelin (the antigen-binding domain comprises a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 22, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 23, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 24, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27); (ii) a hinge region comprising the amino acid sequence set forth in SEQ ID NO: 32; and (iii) a membrane domain comprising the amino acid sequence set forth in SEQ ID NO: 33. (B) a polynucleotide encoding an armor protein comprising: (i) an IL-2Rβ polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1; (ii) a linker comprising the amino acid sequence set forth in SEQ ID NO:4; and (iii) an IL-15Rα polypeptide tethered to an IL-15 polypeptide, wherein the IL-15Rα polypeptide comprises the amino acid sequence set forth in SEQ ID NO:3 and the IL-15 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2.

[0212] In some aspects, the viral vector comprises: (A) a CAR comprising (i) an antigen binding domain that specifically binds human mesothelin (the antigen binding domain comprises a VH comprising the amino acid sequence set forth in SEQ ID NO:28, a VL comprising the amino acid sequence set forth in SEQ ID NO:29), (ii) a hinge region comprising the amino acid sequence set forth in SEQ ID NO:32, (iii) a transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO:33, (iv) a costimulatory domain comprising the amino acid sequence set forth in SEQ ID NO:34, and (v) an intracellular signaling domain comprising the amino acid sequence set forth in SEQ ID NO:35; and (B) a polynucleotide encoding an armor protein comprising (i) an IL-2Rβ polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1, (ii) a linker comprising the amino acid sequence set forth in SEQ ID NO:4, and (iii) an IL-15Rα polypeptide tethered to an IL-15 polypeptide, wherein the IL-15Rα polypeptide comprises the amino acid sequence set forth in SEQ ID NO:3, and the IL-15 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2.

[0213] In some embodiments, (A) a portion of the polynucleotide encoding a CAR and (B) a portion of the polynucleotide encoding an Armor protein are connected by a portion of the polynucleotide encoding a linker. In some embodiments, the linker comprises a peptide bond. In some embodiments, the linker comprises one or more amino acids. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker comprises a T2A linker. In some embodiments, the linker comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:5. In some embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO:5.

[0214] V. Methods for obtaining and expanding γδ T cells The engineered γδ T-cells can grow in the presence of cytokines such as IL-15, IL-2, or in the absence of exogenous IL-2 or IL-15. In some embodiments, the cells are grown in the presence of exogenous IL-15. In some embodiments, the cells express secreted IL-15 and do not require exogenous IL-15 for growth. In some embodiments, the method comprises growing a population of γδ T-cells in the presence of exogenous IL-2.

[0215] In one aspect, the invention features a method of expanding γδ T cells by providing a population of γδ T cells that express IL-15Rβ and culturing the population of γδ T cells in the presence of exogenous IL-2 and / or IL-15 or a variant thereof.

[0216] In another aspect, the invention features a method of expanding γδ T cells by providing a population of γδ T cells expressing IL-15Rβ and IL-15 or a variant thereof and culturing the γδ T cells, where the IL-15 or a variant thereof expands the population of γδ T cells.

[0217] The engineered γδ T-cells of the invention may be derived from any suitable autologous or allogeneic γδ T-cells or populations thereof. In some embodiments, suitable γδ T-cells for use as a source for engineered γδ T-cells described herein include Vδ1, Vδ2, Vδ3, Vδ5, and Vδ8 cells. In some embodiments, the population of engineered γδ T-cells is derived from a population of Vδ1 or Vδ2 cells.

[0218] For example, provided herein are methods for isolating and expanding V51 cells from non-hematopoietic tissues, such as skin or gut. In other embodiments, suitable γδ T cells may be derived from blood (e.g., peripheral blood). Methods for isolating and expanding V51 cells from blood include, for example, those described in U.S. Pat. No. 9,499,788, International Patent Publication No. WO2016 / 198480, and UK Patent Application No. 2204926.6, each of which is incorporated herein by reference in its entirety. In some embodiments, suitable γδ T cells may be derived from tumor tissue (e.g., tumor-infiltrating γδ T cells). Alternatively, suitable γδ T cells that can be engineered to express a transgene may be derived from non-hematopoietic tissues according to the methods described below.

[0219] Isolation and expansion of γδ T cells from VA blood In some embodiments, the engineered γδ T-cells of the invention are derived from the blood (e.g., peripheral blood) of a subject. For example, the engineered γδ T-cells may be derived from blood-derived V52 cells or blood-derived V51 cells.

[0220] In some embodiments, peripheral blood mononuclear cells (PBMCs) can be collected from a subject according to any suitable method known in the art. PBMCs can be cultured in the presence of IL-2, aminobisphosphonates (e.g., zoledronic acid), synthetic phosphoantigens (e.g., bromohydrin pyrophosphate; BrHPP), 2M3B1PP, or 2-methyl-3-butenyl-1-pyrophosphate for 1-2 weeks to generate an enriched population of V52 cells. Alternatively, immobilized anti-TCR gamma delta (e.g., pan-TCR gamma delta) can induce preferential expansion of V52 cells from a population of PBMCs (e.g., for approximately 14 days) in the presence of IL-2. In some embodiments, preferential expansion of V52 cells from PBMCs can be achieved upon culture of immobilized anti-CD3 antibodies (e.g., OKT3) in the presence of IL-2 and IL-4. In some embodiments, the aforementioned culture is maintained for approximately 7 days before passaging in soluble anti-CD3, IL-2, and IL-4. Alternatively, artificial antigen presenting cells can be used to promote preferential expansion of γδ T cells, such as V52 cells. For example, PBMC-derived γδ T cells cultured in the presence of irradiated aAPCs, IL-2, and / or IL-21 can be expanded to generate a population of γδ T cells that includes a high percentage of V52 cells, a moderate percentage of V51 cells, and some double negative cells. In some embodiments of the aforementioned methods, the PBMCs can be pre-enriched or post-enriched (e.g., by positive selection with a TCRγδ specific agent or negative selection with a TCRαβ specific agent). Such methods and other suitable methods for the expansion of γδ T cells, such as V52 cells, are described in detail by Deniger et al., Frontiers in Immunology 5,636:1-10,2014, which is incorporated by reference in its entirety.

[0221] In some embodiments, V51 T cells may be engineered to express a transgene (e.g., a heterologous targeting construct). Any suitable method of obtaining a population of V51 T cells may be used. For example, Almeida et al. (Clinical Cancer Research, 22, 23; 5795-5805, 2016), incorporated herein by reference in its entirety, provides a suitable method of obtaining a population of V51 T cells that may be engineered to express a heterologous targeting construct as described herein. For example, in some embodiments, PBMCs are pre-enriched using magnetic bead sorting, which can recover more than 90% of γδ T cells. These cells can be cultured in gas-permeable bioreactor bags in the presence of one or more factors (e.g., TCR agonists, co-receptor agonists, and / or cytokines, e.g., IL-4, IL-15, and / or IFN-γ) for up to 21 days or more. Variations of this method, as well as other methods of obtaining V51 T cells, are suitable as part of the present invention. For example, blood-derived V51 T cells can be alternatively obtained using methods described, for example, in U.S. Pat. No. 9,499,788 and International Patent Publication No. WO2016 / 198480, each of which is incorporated herein by reference in its entirety.

[0222] VB Isolation and proliferation of non-hematopoietic tissue-resident γδ T cells from non-hematopoietic tissues Non-hematopoietic tissue-resident γδ T cells obtained as described below may exhibit good tumor penetration and retention and may therefore be suitable vehicles for the transgenes described herein. More detailed methods for the isolation and expansion of non-hematopoietic tissue-resident γδ T cells are described, for example, in PCT Publication Nos. WO2020 / 095058, WO2020 / 095059, WO2017 / 072367, and GB Patent No. 2006989.4, each of which is incorporated herein by reference in its entirety.

[0223] Non-hematopoietic tissue resident γδ T cells (e.g., skin-derived γδ T cells and / or non-V52 T cells, e.g., V51 T cells and / or DNT cells) can be isolated from any human or non-human animal non-hematopoietic tissue, which can be removed from the patient to obtain cells suitable for modification by the methods of the invention. In some embodiments, the non-hematopoietic tissue (from which γδ T cells are derived and expanded) is skin (e.g., human skin), which can be harvested by methods known in the art. In some embodiments, the skin is obtained by punch biopsy. Alternatively, the methods of isolating and expanding γδ T cells provided herein can be applied to the gastrointestinal tract (e.g., colon), breast, lung, prostate, liver, spleen, and pancreas. γδ T cells can also be present in human cancer tissue, e.g., breast or prostate tumors. In some embodiments, γδ T cells can be derived from human cancer tissue (e.g., solid tumor tissue). In other embodiments, γδ T cells may be derived from a non-hematopoietic tissue other than human cancer tissue (e.g., tissue that does not contain a substantial number of tumor cells). For example, γδ T cells may be derived from an area of ​​skin separate from the proximal or adjacent cancer tissue (e.g., healthy skin).

[0224] The predominant γδ T cells in blood are mainly Vδ2 T cells, whereas the predominant γδ T cells in non-hematopoietic tissues are mainly Vδ1 T cells, such that Vδ1 T cells comprise approximately 70-80% of the non-hematopoietic tissue-resident γδ T cell population. However, some Vδ2 T cells are also found in non-hematopoietic tissues, e.g., in the intestine, and they may comprise approximately 10-20% of γδ T cells. Some γδ T cells resident in non-hematopoietic tissues express neither Vδ1 nor Vδ2 TCRs, which we term double-negative (DN) γδ T cells. These DN γδ T cells predominantly express Vδ3, with a small number of T cells likely expressing Vδ5. Thus, the γδ T cells normally present in non-hematopoietic tissues and expanded by the method of the present invention are preferably non-Vδ2 T cells, e.g., Vδ1 T cells, which include a small number of DN γδ T cells.

[0225] In some embodiments, a key step is to carefully separate non-hematopoietic tissue-resident T cells (e.g., within a mixed lymphocyte population (which may include, e.g., αβ cells, natural killer (NK) cells, B cells, as well as γδ2 T cells and non-γδ2 T cells)) from non-hematopoietic cells of the tissue from which the T cells are derived (e.g., stromal cells, particularly fibroblasts), e.g., after days or weeks of culture. This allows preferential and rapid expansion of non-hematopoietic tissue-derived Vδ1 T cells and DNγδ T cells over the following days and weeks.

[0226] In general, non-hematopoietic tissue resident γδ T cells can naturally proliferate when physical contact with stromal cells (e.g., skin fibroblasts) is removed. Thus, the scaffold-based culture methods described above can be used to induce such detachment, resulting in de-repression of γδ T cells and inducing proliferation. Thus, in some embodiments, there is no substantial TCR pathway activation during the expansion step (e.g., no exogenous TCR pathway activators are included in the culture). Furthermore, the present invention provides a method of expanding non-hematopoietic tissue resident γδ T cells, which does not involve contact with feeder cells, tumor cells, and / or antigen-presenting cells.

[0227] The expansion protocol comprises culturing non-hematopoietic tissue resident γδ T cells in the presence of an effective cocktail of biological factors that support efficient γδ T cell expansion. In one embodiment, a method for expanding γδ T cells comprises providing a population of γδ T cells obtained from a non-hematopoietic tissue (e.g., an isolated population of non-hematopoietic tissue-derived γδ T cells, e.g., a population isolated by a method described herein) and culturing the γδ T cells in the presence of IL-2 and IL-15, and optionally IL-1β, IL-4, and / or IL-21. These cytokines or analogs thereof can be cultured with the cells in an amount effective to produce an expanded population of γδ T cells for a period of time (e.g., at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 21 days, at least 28 days, or more, e.g., 5 to 40 days, 7 to 35 days, 14 to 28 days, or about 21 days).

[0228] A number of basal media suitable for use in priming and / or expanding γδ T cells are available, such as complete medium, OPTMIZER™, AIM-V, Iscoves medium, and RPMI-1640 (Life Technologies) and TXMACS™ (Miltenyi Biotec). The medium may be supplemented with other media factors such as serum, serum proteins, and selection agents such as antibiotics. For example, in some embodiments, the medium comprises RPMI-1640 containing 2 mM glutamine, 10% FBS, 10 mM HEPES (pH 7.2), 1% penicillin-streptomycin, sodium pyruvate (1 mM; Life Technologies), non-essential amino acids (e.g., 100 μM Gly, Ala, Asn, Asp, Glu, Pro, and Ser; 1×MEM non-essential amino acids Life Technologies), and 10 μl / L β-mercaptoethanol. Conveniently, cells are cultured in a suitable culture medium at 37° C. in a humidified atmosphere containing 5% CO 2 .

[0229] γδ T-cells may be cultured as described herein in any suitable system, including stirred tank fermenters, airlift fermenters, roller bottles, culture bags or dishes, and other bioreactors, such as hollow fiber bioreactors. The use of such systems is well known in the art. General methods and techniques for the culture of lymphocytes are well known in the art.

[0230] The methods described herein may include two or more selection steps, e.g., two or more depletion steps. Enrichment of a T cell population by negative selection may be achieved, for example, using a combination of antibodies directed to surface markers unique to negatively selected cells. One method is cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies directed to cell surface markers present on negatively selected cells.

[0231] VI. Transgenes The engineered γδ T-cells of the invention are engineered to express a desired transgene. The engineered γδ T-cells to express a transgene are suitable for use in cancer therapy (e.g., immunotherapy). The viral vectors described herein encode a transgene, which is then stably or transiently expressed in the transduced γδ T-cells. Transgenes that can be used in conjunction with the compositions and methods described herein include IL-15Rβ, IL-15Rα, IL-15, or any biologically active variants, mutant proteins, or truncated fragments thereof. Other transgenes include, for example, chimeric antigen receptors (CARs). The transgene may include an internal ribosome entry site (IRES) operably connected to the transgene.

[0232] In some embodiments, the γδ T-cells express two or more (e.g., two, three, four, or more) transgenes. Each of the transgenes may be expressed on a single polynucleotide and expressed in tandem. Each transgene may include a linker between them. The linker may encode a cleaving peptide or may include an IRES. The cleaving peptide may be, for example, a self-cleaving peptide, such as a 2A peptide. In some embodiments, the 2A peptide is an F2A peptide, an E2A peptide, a P2A peptide, or a T2A peptide. In some embodiments, the polynucleotide includes a separate IRES operably connected to each transgene on a single polynucleotide. In some embodiments, the polynucleotide includes a single IRES operably connected to all transgenes on a single polynucleotide.

[0233] In some examples, the transgene expressed by the engineered γδ T-cells of the invention comprises a selectable marker (e.g., a reporter gene) or a suicide gene. For example, a truncated epidermal growth factor receptor (EGFR), lacking the intracellular signaling domain, can be used as a transgene for in vivo depletion using an anti-EGFR monoclonal antibody, for example, in case of toxicity. Similarly, CD20 can be used as a transgene for in vivo depletion using an anti-CD20 monoclonal antibody. Another exemplary transgene is a suicide gene that facilitates drug-mediated control of the administered engineered γδ T-cells. In case of adverse events, the engineered cells can be depleted from the patient by using a suicide gene. In one example, the drug binding domain is fused to a caspase 9 apoptosis-promoting molecule. In some examples, the transgene is a cytosine deaminase. In some examples, the transgene is a thymidine kinase.

[0234] Additionally, or alternatively, a transgene for expression by an engineered γδ T-cell of the invention can be a membrane-bound protein, e.g., a membrane-bound receptor (e.g., αβTCR, a natural cytotoxicity receptor (e.g., NKp30, NKp44, or NKp46), a cytokine receptor (e.g., IL-12 receptor), and / or a chemokine receptor (e.g., CCR2 receptor), and / or a membrane-bound ligand or cytokine (e.g., membrane-bound IL-15, membrane-bound IL-7, membrane-bound CD40L, membrane-bound 4-1BB, membrane-bound 4-1BBL, membrane-bound CC In one embodiment, the transgene encodes a soluble ligand or cytokine (e.g., soluble IL-15, soluble IL-7, soluble IL-12, soluble CD40L, soluble 4-1BBL, and / or soluble CCL19). Membrane-bound ligands and cytokines include naturally occurring membrane-bound ligands and cytokines (e.g., trans-presented IL-15 and 4-1BBL) as well as synthetic membrane-bound constructs (e.g., ligands artificially fused to transmembrane proteins). Additionally or alternatively, the transgene expressed by the engineered γδ T-cell of the invention encodes a soluble ligand or cytokine (e.g., soluble IL-15, soluble IL-7, soluble IL-12, soluble CD40L, soluble 4-1BBL, and / or soluble CCL19).

[0235] In some examples, engineered γδ T-cells carrying a transgene encoding a CAR can be armored with an additional transgene that contributes to immunogenicity. Such armored CAR T-cells express an armor protein, such as any of the membrane-bound or soluble proteins described herein. For example, armor proteins include membrane-bound receptors (e.g., αβTCR, natural cytotoxicity receptors (e.g., NKp30, NKp44, or NKp46), cytokine receptors (e.g., IL-12 receptor), and / or chemokine receptors (e.g., CCR2 receptor), and / or membrane-bound ligands or cytokines (e.g., membrane-bound IL-15, membrane-bound IL-7, membrane-bound CD40L, membrane-bound 4-1BB, membrane-bound 4-1BBL, membrane-bound CCL19). Additionally or alternatively, armor proteins expressed by the engineered γδ CAR T cells of the invention comprise soluble ligands or cytokines (e.g., soluble IL-15, soluble IL-7, soluble IL-12, soluble CD40L, soluble 4-1BBL, and / or soluble CCL19).

[0236] In some embodiments, an engineered γδ T-cell of the invention is engineered to express one or more transgenes (e.g., one or more of any of the transgenes described herein) for arming the γδ T-cell (e.g., as an arming CAR T cell, as described in Yeku and Brentjens Biochem. Soc. Trans. 2016, 15:44, 2, 412-418, which is incorporated herein by reference in its entirety).

[0237] In some embodiments, the transgene is codon optimized.

[0238] In some embodiments, at least 3% (e.g., at least 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or substantially all) of the population of engineered γδ T-cells (e.g., V51 or V52 cells) express a transgene, e.g., IL-15Rβ, CAR, or a membrane-bound or soluble protein. In some embodiments, at least 10% (e.g., at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or substantially all) of the population of engineered γδ T-cells (e.g., V51 or V52 cells) express a transgene, e.g., IL-15Rβ, CAR, or a membrane-bound or soluble protein. In some embodiments, at least 50% (e.g., at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or substantially all) of the population of engineered γδ T-cells (e.g., Vδ1 or Vδ2 cells) express a transgene, e.g., IL-15Rβ, CAR, or other membrane-bound or soluble protein. In some embodiments, 3%-95% (e.g., 5%-95%, 10%-95%, 20%-95%, 25%-95%, or 50%-95%) of the population of engineered γδ T-cells (e.g., Vδ1 or Vδ2 cells) express a transgene, e.g., IL-15Rβ, CAR, or other membrane-bound or soluble protein. In some embodiments, between 3% and 90% (e.g., between 5% and 90%, 10% and 90%, 20% and 90%, 25% and 90%, or 50% and 90%) of the population of engineered γδ T-cells (e.g., Vδ1 or Vδ2 cells) express a transgene, e.g., IL-15Rβ, CAR, or other membrane-bound or soluble protein.

[0239] VII. Treatment method Some embodiments of the present disclosure relate to a method of treating a patient in need thereof, comprising administering to the subject an engineered γδ T-cell as disclosed herein. In some embodiments, the subject is afflicted with cancer. Generally, the proliferation and / or lifespan of cancer cells exceeds and is not coordinated with the proliferation and / or lifespan of the surrounding normal cells and tissues. Cancer can be benign, pre-malignant, or malignant. Cancers arise in a variety of cells and tissues, including the oral cavity (e.g., mouth, tongue, pharynx, etc.), digestive system (e.g., esophagus, stomach, small intestine, colon, rectum, liver, bile duct, gallbladder, pancreas, etc.), respiratory system (e.g., larynx, lungs, bronchi, etc.), bones, joints, skin (e.g., basal cell, squamous cell, meningioma, etc.), breast, reproductive system, (e.g., uterus, ovaries, prostate, testes, etc.), urinary system (e.g., bladder, kidneys, ureters, etc.), eyes, nervous system (e.g., brain, etc.), endocrine system (e.g., thyroid, etc.), and hematopoietic system (e.g., lymphoma, myeloma, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, etc.).

[0240] In some embodiments, the disease or condition includes cancer, e.g., the subject is afflicted with cancer. In some embodiments, the cancer is bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, lung cancer (e.g., non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC)), uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, gastric cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, acute myeloid leukemia (AML), e.g., In some embodiments, the cancer is locally advanced. In some embodiments, the cancer is metastatic. In some embodiments, the cancer is refractory. In some embodiments, the cancer is recurrent. In some embodiments, the cancer is recurrent. In some embodiments, the cancer is refractory or recurrent after one or more prior anti-cancer therapies. In some embodiments, the one or more prior anti-cancer therapies include standard of care.

[0241] In some embodiments, the compositions disclosed herein are administered in combination with an additional anti-cancer therapy. In some embodiments, the additional anti-cancer therapy includes chemotherapy, immunotherapy, radiation therapy, surgery, or any combination thereof. In some embodiments, the additional anti-cancer therapy includes chemotherapy. In some embodiments, the additional anti-cancer therapy includes an immune checkpoint inhibitor. In some embodiments, the additional anti-cancer therapy includes a PD-1 antagonist, a PD-L1 antagonist, a CTLA-4 antagonist, a LAG-3 antagonist, a GITR antagonist, or any combination thereof. In some embodiments, the anti-cancer therapy includes an antibody or antigen-binding portion thereof that specifically binds and inhibits PD-1 (e.g., pembrolizumab or nivolumab). In some embodiments, the anti-cancer therapy includes an antibody or antigen-binding portion thereof that specifically binds and inhibits PD-L1 (e.g., atezolizumab, avelumab, or durvalumab).

[0242] In some embodiments, the method further comprises pretreating the subject prior to administering the population of immune cells. In some embodiments, the subject is administered chemotherapy prior to administering the population of immune cells. In some embodiments, the subject is administered immunodepleting chemotherapy prior to administering the population of immune cells. In some embodiments, the immunodepleting chemotherapy comprises cyclophosphamide, fludarabine, or both.

[0243] In some aspects, the method comprises administering to the subject (i) a modified innate lymphoid cell disclosed herein, and (ii) a cytokine. In some aspects, the cytokine comprises IL-2, an analog thereof, a variant thereof, or a fragment thereof.

[0244] In some embodiments, the cells of the present disclosure are administered at least about 1×10 6 Cells, at least about 2 x 10 6 Cells, at least about 3 x 10 6 Cells, at least about 4 x 10 6 Cells, at least about 5 x 10 6 cells, 1 x 107 Cells, at least about 2 x 10 7 Cells, at least about 3 x 10 7 Cells, at least about 4 x 10 7 Cells, at least about 5 x 10 7 cells, 1 x 10 8 Cells, at least about 2 x 10 8 Cells, at least about 3 x 10 8 Cells, at least about 4 x 10 8 Cells, at least about 5 x 10 8 cells, 1 x 10 9 Cells, at least about 2 x 10 9 Cells, at least about 3 x 10 9 Cells, at least about 4 x 10 9 cells, or at least about 5×10 9 A dose of cells is administered to the subject.

[0245] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of those in the art and are fully explained in the literature. For example, Sambrook et al., ed. (1989) Molecular Cloning A Laboratory Manual (2nd ed.; Cold Spring Harbor Laboratory Press); Sambrook et al., ed. (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); DNGlover ed., (1985) DNA Cloning, Volumes I and II; Gait, ed. Synthesis; Mullis et al. U.S. Patent No. 4,683,195; Hames and Higgins, eds. (1984) Nucleic Acid Hybridization; Hames and Higgins, eds. (1984) Transcription And Translation; Freshney (1987) Culture Of Animal Cells (Alan R. Liss, Inc.); Immobilized Cells And Enzymes (IRL Press)(1986);Perbal(1984)A Practical Guide To Molecular Cloning;the treatise,Methods In Enzymology(Academic Press,Inc.,NY);Miller and Calos eds.(1987)Gene Transfer Vectors For Mammalian Cells,(Cold Spring Harbor Laboratory);Wu et al.,eds.,Methods In Enzymology,Vols.154 and 155;Mayer and Walker,eds.(1987) Immunochemical Methods In Cell And Molecular Biology (Academic Press, London); Weir and Blackwell, eds., (1986) Handbook Of Experimental Immunology, Volumes I-IV; Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1986)); Crooke, Antisense drug Technology: Principles, Strategies and Applications,2. nd Ed. CRC Press (2007); and Ausubel et al. (1989) Current Protocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.).

[0246] All references cited above, and all references cited herein, are hereby incorporated by reference in their entirety.

[0247] The following examples are offered by way of illustration and not by way of limitation. EXAMPLES

[0248] Materials and Methods Isolation and expansion of Vδ1 T cells Vδ1 γδ T cell enriched product (GDX012) was generated using a modified protocol based on Almeida et al. (2016) Clin. Cancer Res. 22:5795-804. Briefly, αβ T cell depleted peripheral blood mononuclear cells were expanded in G-Rex (Wilson Wolf) culture vessels using serum-free culture medium (CTS OpTmizer, Thermo Fisher) supplemented with 2.5% autologous plasma and Glutamax (ThermoFisher). Isolated cells were expanded in the presence of recombinant IL-4 [rIL4] (100 ng / mL), recombinant interferon-γ [rIFNγ] (70 ng / mL), recombinant IL-21 [rIL21] (7 ng / mL), recombinant IL-1β [rIL1β] (15 ng / mL), and soluble OKT-3 monoclonal antibody (42 ng / mL). Cells were incubated overnight in a humidified incubator at 37° C. and 5% CO. Proliferating cells were periodically fed with fresh medium containing recombinant IL-15 [rIL15] ("high": day 5: 21 ng / mL and day 7: 100 ng / mL, or "low": day 5: 2.1 ng / mL and day 7: 10 ng / mL).

[0249] Retroviral transduction Proliferating γδ T cells were transduced with retroviral vectors in Retronectin-coated (20 mg / mL) cell expansion bags (PermaLife bags, OriGen Biomedical) at a defined multiplicity of infection (MOI). MOI refers to the number of infectious particles added per cell during transduction (measured by flow cytometry). Viral vectors were diluted in CTS OpTmizer medium. Transduction efficiency was measured periodically using flow cytometry 3 days after transduction.

[0250] Retroviral Vector Production and Titration Gammaretroviral vectors were produced by transient transfection of human embryonic kidney (HEK) cells with murine leukemia virus genomic plasmids. Vectors were harvested 96 hours post-transfection, filtered through 0.45 μm pore-size polyethersulfone (PES) filters, and concentrated using hollow fiber filtration.

[0251] Vector titers were measured by transducing serial dilutions of concentrated vector material into SupT1 (acute lymphoblastic leukemia) cell lines in the presence of Retronectin (20 μg / mL). Transduction efficiency was measured 3 days after transduction using a BD FACS Lyric flow cytometer. Infectious titers (TU / mL) were calculated using the following formula: TU / mL = ((number of cells transduced) x vector dilution x (transduction efficiency (%) / 100)) / volume of vector (mL).

[0252] Flow cytometry Immunophenotyping was performed using a BD FACS Lyric flow cytometer. Cells were analyzed for expression of surface markers using the antibodies in Table 5 below.

[0253] [Table 4]

[0254] Cytotoxicity assessment Expanded cells were co-cultured with floating tumor targets expressing firefly luciferase (e.g., NALM-6) at various effector:target ratios. Target cells in the absence of effectors served as controls (Ctrl). Maximum lysis was determined using target cells treated with staurosporine. After 20 hours, quantification of target viability was assessed using the ONE-Glo™ Luciferase Assay System kit (Promega) or the SYTOX AADvanced Dead Cell Stain kit (ThermoFisher). Luminescence was measured on a Biotek Synergy H4 plate reader (luciferase system) and dead cell staining with SYTOX AADvanced was measured by flow cytometry on a MACSQuant (Miltenyi).

[0255] IL-15 ELISA IL-15 production was measured using a commercially available human IL-15 Quantikine ELISA kit (Bio-Techne) according to the manufacturer's instructions. IL-15 production was measured after 72 hours of incubation.

[0256] Repeated antigen stimulation assay CD19 model

[0257] Repeated antigen stimulation (RAS) assays were performed to assess the proliferation of CAR-modified Vδ1 T cells and CAR-T cell enrichment. Freshly thawed preparations were resuspended in RPMI10 medium (RPMI, 10% FBS) in the presence or absence of IL-15 (10 ng / mL or 70 pg / mL). Target tumor cell lines were counted using an automated cell counter (NC250) and the concentration was adjusted to 1E+06 cells per mL. Two times the cytokine concentration was added to the target cells and 100 μL (1E+05 cells) was transferred to each well of a 96-well round-bottom plate. The concentration of effector cells was adjusted to 2E+06 viable cells per mL and 100 μL (2E+05 cells) was plated into wells containing target cells. Replicate wells were plated per condition depending on effector cell number. Co-cultures were maintained at 37°C, 5% CO2 for 14 days. On days 4, 7, and 10, duplicate wells were pooled, counted, and phenotyped using a BD FACSLyric flow cytometer. The remaining cells were then centrifuged, resuspended in fresh medium at a density of 2E+06 viable cells / mL, and plated onto another round of target cells prepared according to day 0.

[0258] A549-Mesothelin model

[0259] The formulations were thawed and co-cultured in 24-well plates (300,000 effectors to 150,000 targets) with A549-MLSN-fluc-gfp cells at an E:T ratio of 2:1 in the presence or absence of 1 ng / mL IL-15 in an Incucyte to follow GFP fluorescence over time as a surrogate for tumor cell lysis. On days 2 and 9, the cultures were fed with fresh medium with or without 1 ng / mL IL-15, and after centrifugation, 50% of the well volume was replaced by careful aspiration. On days 4 and 11, the cultures were fed with fresh medium containing targets (150,000 per well) with or without 1 ng / mL IL-15, and after centrifugation, 50% of the well volume was replaced by careful aspiration. On day 7, all wells were harvested, counted, and re-seeded at 2:1 E:T as on day 0. Tumor confluence was measured by following GFP fluorescence over time.

[0260] Viability assay Cryopreserved formulations were thawed and viable cell concentration was adjusted to 2E+06 cells / mL in RPMI10. Replicate wells were seeded with 2E+05 cells per well. On days 0, 3, 7, 10, and 14, replicate wells were stained by adding SytoxAADvanced dye directly to the medium for 10 minutes at room temperature in the dark. Forty microliter samples were analyzed on a MACSQuant flow cytometer without further processing for cell quantification and viability assessment. To determine the viability of CAR+V51+ T cells, replicate plates were stained for CAR, V51, and pan-γδ T cell markers and analyzed by flow cytometry.

[0261] In vivo efficacy CD19 model

[0262] Eight to ten week old NSG and transgenic IL-15 expressing NOG-IL-15 mice were implanted with 5x105 NALM6-fluc-GFP cells via intravenous (IV) injection. On day 7, mice were injected with vehicle (n=5 NSG mice, Cryostor CS5, group 1) or one of three formulations, muCAR19.b, muCAR19.ab, or muCAR19.a.15.b, across three experimental conditions: 1 μg IL-15 administered daily via IP injection (N=9, groups 2-4), NSG mice not receiving IL-15 (N=4-9, groups 5-7), and NOG-hIL15 mice not receiving IL-15 (N=6, groups 8-10). Figure 13 contains a table of groupings. Mice were imaged twice weekly using an IVIS camera after intraperitoneal (IP) injection of 150 mg / kg d-luciferin. On day 10 (3 days after formulation administration), N=4 mice from groups 2, 3, 4, and 7 were sacrificed and bone marrow aspirates from both femurs, spleens, and peripheral blood (collected in lithium heparin tubes) were harvested. Samples were processed into single cell suspensions and stained for flow cytometry analysis. Samples were analyzed using a BD FACSLyric flow cytometer.

[0263] A549-Mesothelin model

[0264] Six to eight week old NSG mice were implanted with 1x106 A549-MSLN-fluc-GFP cells via intravenous (IV) injection. 19 days after implantation, mice were injected with vehicle (n=7, Cryostor CS5, group 1), a total of 10 million P4γδ CAR T cells per mouse (n=7, group 2), 10 million P4α.15.βγδ CAR T cells per mouse (n=7, group 3), or 10 million P4.βγδ CAR T cells per mouse (n=7, group 4). Mice were imaged twice weekly using an IVIS camera following intraperitoneal (IP) injection of 150 mg / kg d-luciferin. Mice in group 4 were administered 1 µg IL-15 daily via IP injection. On day 19 after administration of the formulation, all mice were sacrificed and bone marrow aspirates from both femurs, spleens, lungs, and peripheral blood (collected in lithium heparin tubes) were harvested.

[0265] Example 1: Schematic of "conventional" and novel IL-15 armoring constructs Currently used armoring strategies utilize either secreted (sIL-15) or membrane-tethered IL-15 (mbIL-15) constructs. These strategies have been shown to enhance the viability and proliferation of αβT cells (Hurton LV. et al, PNAS, 113, E7788-E7797, (2016)), NK cells (Liu et al. Leukemia 32, 520-531 (2018)), and iNK-T (Xu et al, Clin Cancer Res., 25, 7126-7138 (2019)) cells.

[0266] As part of the present invention, a set of novel constructs was designed and evaluated in V51 γδ T cells (Figure 1A). Multicistronic gammaretroviral vectors were generated in which sequences encoding chimeric antigen receptors (CARs) were fused in frame with individual IL-15 receptor chain components encoding wild type or IL-15 fusion (tethered) chain variants. Figures 1B and 1C show schematic diagrams of the gammaretroviral vectors used to co-express the CAR and the armouring moiety(s).

[0267] Example 2: "Conventional" IL-15 armouring strategies are suboptimal for the expansion of blood-borne V51 T cells To determine whether the currently used (conventional) IL-15 armoring strategy could drive the expansion of engineered Vδ1 T cells, isolated Vδ1 T cells were transduced with non-armoring secreted IL-15 (sIL-15) or membrane-bound IL-15 (mbIL-15) CAR constructs. After transduction, cells were expanded in the presence of IL-15 ("high" IL-15 feed) as described in the methods section. During the expansion phase, CAR expression was periodically monitored by flow cytometry and total cell numbers were enumerated by automated cell counting. The absolute number of viable CAR+Vδ1+ cells (total viable cell count multiplied by the percentage of CAR+Vδ1+ T) was plotted against the expansion day to determine the effect of armoring on the fold expansion of Vδ1 T cells (Figure 2A).

[0268] Figure 2A shows that in the presence of IL-15, non-armored CAR-modified V51 T cells are continuously enriched over time over the culture period. In contrast, conventional IL-15-armored V51 T cells show limited proliferation during the expansion procedure, suggesting that the armored cells are desensitized to exogenous IL-15. Analysis of the preparations revealed that the armored cells indeed express mbIL-15 on their surface (muCAR19.mbIL-15) and secrete IL-15 (muCAR19.sIL-15). Figure 2B shows the surface expression of IL-15 and IL-15Rα chain on mbIL-15-armored CAR+V51+ T cells. Figure 2C shows the functionality of sIL-15-armored CAR+V51+ T cells as measured by overnight IL-15 ELISA.

[0269] Example 3: "Conventional" IL-15 armouring strategies provide restrictive survival and proliferation signals for blood-borne V51 T cells Next, we tested the conventional Armoring strategy to improve the cytotoxicity, viability, and proliferation of Armored CAR-modified Vδ1 T cells. To this end, overnight cytotoxicity assays, viability assays, and repeated antigen stimulation assays (using NALM-6 target cells) were performed using freshly thawed preparations.

[0270] In overnight cytotoxicity assays, conventionally armored CAR+Vδ1+ T cells exhibited comparable lytic activity to non-armored CAR-modified Vδ1 T cells (Figure 3A). Figure 3B shows that in the absence of exogenous IL-15, non-armored and sIL-15 armored cells did not survive. Conversely, armoring cells with mbIL-15 slightly improved cell viability as assessed by flow cytometry (viable lymphocyte population).

[0271] Surprisingly, in repeated antigen stimulation assays, conventional IL-15 armored CAR+Vδ1+ T cells were not enriched in the presence of IL-15 (Figure 3C). For reference, the same phenomenon was observed in the absence of IL-15 in RAS assays (data not shown). This was in stark contrast to non-armored cells, where CAR-modified Vδ1 T cells were continually enriched upon repeated challenge with NALM-6. Overall, these results suggest that cells armored with conventional IL-15 armored constructs retain the ability to kill but lose the ability to proliferate and enrich upon target cell engagement. These data also show that conventional strategies (sIL-15) do not improve or only slightly improve (mbIL-15)Vδ1 T cell viability.

[0272] Example 4: Reducing IL-15 concentration during the expansion step does not rescue the expansion of mbIL-15-armored CAR-modified V51 T cells To further explore the effect of the mbIL-15 armoring strategy, non-armored and mbIL-15 armored CAR-transduced Vδ1 T cells were expanded in the presence of high and low concentrations of IL-15 and the fold expansion of the cells was monitored (Figure 4). Consistent with the results previously described, both high and low IL-15 concentrations supported the expansion of non-armored Vδ1+ T cells. In contrast, mbIL-15 armored CAR+Vδ1+ T cells could not be enriched even at a 10-fold lower IL-15 dose. Overall, these results suggest that mbIL-15 armored cells lose the ability to respond to exogenous IL-15 and that the expansion of armored cells is not rescued by lowering the dose of IL-15 during the expansion process.

[0273] Example 5: IL-15Rβ chain transfer restores proliferation of mbIL-15 armored CAR-modified Vδ1 T cells For ease of context, IL-2Rβ is referred to as IL-15Rβ throughout this specification.

[0274] Because mbIL-15 armored cells showed negligible proliferation both in the presence of exogenous IL-15 (Figs. 2A and 4) and during repeated antigen challenge (Fig. 3C), we hypothesized that overexpression of the common β chain might restore proliferation of the transduced cells.

[0275] We first investigated whether expanded blood-derived Vδ1 T cells expressed detectable levels of either IL-15Rβ chain on their surface. High-throughput flow cytometry analysis (LEGENDScreen) revealed that unmodified Vδ1 T cells expressed neither IL-15Rα nor IL-15Rβ chains (Figure 5). In contrast, Vδ1 T cells showed high-level and uniform expression of the common γ chain.

[0276] Having shown that V51 T cells express negligible amounts of IL-15Rβ chain on their surface, we further confirmed the effect of co-expressing IL-15Rβ chain with IL-15 tethered chain or wild-type IL-15Rα chain (Figure 1B and C). To this end, proliferating V51 T cells were transduced with CAR constructs armoured with membrane-tethered IL-15 (mbIL-15=α.IL-15), membrane-tethered IL-15, and IL-15Rβ (α.IL-15.β) and IL-15Rα and β (IL-15.α.β) chains. The transduced cells were then expanded in the presence of exogenous IL-15 and the fold expansion was continuously monitored during the expansion process. Figure 6A shows the fold expansion of armoured CAR+V51+T cells over time. These data revealed that transduction of cells with IL-15Rβ chain (both IL-15-tethered and wild-type) rescued proliferation of armored CAR+Vδ1+ T cells with IL-15Rα chain. Strikingly, in the presence of exogenous IL-15, IL-15Rα chain and β chain co-expressing cells showed significantly higher fold proliferation than tethered IL-15 and β chain expressing cells. Importantly, tethered IL-15 and β chain co-expressing cells showed similar fold proliferation as non-armored cells (Figure 6A, triangles vs. circles). This phenomenon was consistently observed across two different doses of IL-15 (Figure 6B), clearly demonstrating that co-expression of IL-15R β chain rescues proliferation of mbIL-15 modified cells in response to exogenous IL15.

[0277] Example 6: IL-15R α- and β-chain co-expressing CAR-modified V51 T cells are cytotoxic and proliferate during repeated antigen challenge Having established that co-expression of IL-15R α and β chains could restore exogenous IL-15-induced proliferation of CAR-modified Vδ1+ T cells, we next investigated the functionality of the modified cells. Figure 7A shows representative short-term (overnight) cytotoxicity results from two donors. Armoring of Vδ1 T cells with additional IL-15R chain components did not cause any detrimental effects on the cytotoxic activity of these cells. Importantly, β chain co-expressing cells showed comparable cytotoxicity to mbIL-15 armored cells.

[0278] In a next step, we confirmed whether CAR-transduced IL-15R α- and β-chain co-expressing Vδ1 T cells could be enriched upon repeated antigen stimulation with CD19+ NALM-6 acute lymphoblastic leukemia cells. To this end, freshly thawed armoured preparations were co-cultured with NALM-6 target cells in the presence (10 ng / mL) or absence of exogenous IL-15 as described in the methods section. Effector cells were restimulated with fresh target cells every 3-4 days. The fold expansion and enrichment of CAR+ Vδ1+ effectors, as well as the clearance of target cells, were monitored over the course of the assay.

[0279] As shown above (Figure 3C), conventional mbIL-15 armored CAR+V51+ T cells failed to proliferate in any of the conditions examined (Figures 7B and 7C, left panels, open boxes). This was reflected in a rapid decline in the number of CAR+V51+ T cells. Compared to mbIL-15 armored cells, the number of IL-15Rα and β chain co-expressing cells declined even more rapidly in the absence of exogenous IL-15 (Figure 7B, left panel, closed boxes). Conversely, in the absence of IL-15, tethered IL-15 (α.IL-15) and β chain co-expressing cells were enriched over the course of the assay (Figure 7B, left and center panels, triangles). These results indicate that in the absence of exogenous IL-15, tethered IL-15 acts as a proliferation- and survival-inducing signal for IL-15Rα and β co-expressing cells. Enumeration of the number of remaining target cells over the course of the assay revealed that only tethered IL-15 and IL-15Rβ chain co-expressing cells were able to demonstrate long-term tumor control (Figure 7B, right panel, triangles).

[0280] Surprisingly, in the presence of supraphysiological levels of IL-15 (10 ng / mL), IL-15Rα and β co-expressing cells showed enhanced proliferation compared to tethered IL-15 and β chain co-expressing cells (Figure 7C, left panel). These data suggest that the availability of exogenous IL-15 may fine-tune the activity of tethered IL-15 and β chain co-expressing cells. Overall, IL-15Rβ chain co-expression did not adversely affect the cytotoxicity of the cells, and most importantly, it was shown that for tethered IL-15, tumor control could be achieved for a long period of time in the absence of exogenous IL-15.

[0281] Example 7: V51 T cells co-expressing IL-15Rα and β chains exhibit enhanced proliferation in the presence of exogenous IL-15 compared to cells expressing only the β chain Having shown that the presence of the IL-15Rβ chain was required to restore proliferation of transduced Vδ1 T cells, the next objective was to determine whether arming cells with the β chain alone could drive cell proliferation. To examine the function of the β chain, Vδ1 T cells were transduced with CAR constructs armed with the IL15Rβ chain alone or in combination with the IL-15Rα chain or IL-15-tethered IL-15Rα chain. Figure 8A shows the percentage of the CAR+Vδ1+ T cell population 2 days after transduction (N=2 representative donors). As expected, CAR gene transfer efficiency was negatively correlated with the size of the viral vector construct. At a fixed multiplicity of infection, the smaller (3150 bp) IL-15Rβ arming construct consistently yielded higher CAR transduction efficiency than the larger (IL15Rα.β or IL-15Rα.15.β) sized (3993 bp and 4413 bp, respectively) constructs. Figure 8B shows the expression of individual chain components and IL-15 expression within a CAR-transduced V51 T cell population. Importantly, these representative flow plots show that CAR-modified V51 T cells express equimolar levels of CAR and armouring components on their surface.

[0282] After transduction with the aforementioned IL-15Rβ chain co-expressing constructs, the number of CAR+Vδ1+ T cells was monitored throughout the cell expansion procedure. Consistent with the previous experiments (Figure 6A), IL-15Rα chain and β chain co-expressing cells showed the highest level of proliferation, followed by IL-15Rβ chain (alone) armoured cells (Figure 9A). To test whether proliferation of β chain expressing cells depends on the availability of exogenous IL-15, the expansion procedure was repeated in the presence of high and low concentrations of IL-15. Figure 9B shows the fold expansion of CAR+Vδ1+ T cells armoured with three different β chain co-expressing constructs. These data indicate that IL-15Rα chain and β chain co-expressing cells were more sensitized to exogenous IL-15 than β chain alone or tethered IL-15 and β chain co-expressing cells.

[0283] Example 8: Armouring cells are selectively enriched during culture and show enhanced expression of DNAM-1 and NKp30 After expansion, the transduced drug substance was harvested, formulated and cryopreserved. Transduction efficiency was determined upon thawing (Figure 10A). Flow cytometry analysis did not reveal significant differences in CAR transduction efficiency between the different arming conditions. In general, expanding cells using low dose IL-15 slightly increased the percentage of transduced cells.

[0284] Next, surface expression of CAR and individual Armouring molecules was assessed, Figure 10B shows that freshly thawed cells retained equimolar surface expression of CAR and IL-15R Armouring molecules.

[0285] Comparing the CAR transduction efficiency 3 days after transduction and at the end of the expansion process revealed that armored cells were selectively enriched during the culture period (Figure 10C). Notably, cells co-expressing α and β chains showed the highest level of enrichment on days 7-14 of the culture period. This effect was even more pronounced in the low concentration IL-15 feed culture condition. In contrast, non-armored CAR modified cells were not enriched during the expansion procedure. Proliferating cells showed steady-state CAR expression during the culture process. This further supports the finding that IL-15Rα and β chain armored cells are selectively enriched during the culture period.

[0286] We next proceeded to determine the cellular composition of the cells: FACS analysis revealed no significant qualitative or quantitative differences regarding pan-γδ, Vδ1 γδ T cell, and non-γδ T cell populations (data not shown).

[0287] Phenotypic characterization of Armoring CAR-transduced and non-transduced V51 T cell populations revealed marginal differences in the expression of NKG2D, NKp30, DNAM-1, PD-1, and TIGIT (Figure 10D).

[0288] Compared to the non-transduced V51 T cell population, the percentage of cells expressing the natural cytotoxic receptor NKp30 doubled in the CAR-expressing population of all Armouring cells except for membrane-tethered IL-15 and β-chain co-expressing cells. Similarly, an increased frequency of activated DNAM-1 receptor expressing cells was observed in the CAR-expressing Armouring cell population. In contrast, the opposite trend was observed in the case of NKG2D-expressing V51 T cells, i.e., the percentage of V51 T cells expressing NKG2D was slightly decreased in the CAR-expressing cell fraction compared to the non-transduced fraction. These data suggest that co-expression of β-chain (with respect to wild type and IL-15α-chain) may prime cells towards a more cytotoxic phenotype.

[0289] Example 9: V51 T cells engineered with novel IL-15 armoring constructs show equivalent or improved potency The cytotoxic activity of the novel β-chain Armoured CAR-modified Vδ1 T cells was assessed in an overnight killing assay in the presence and absence of exogenous IL-15. For the CD19-targeted CAR, Armoured cells showed no significant difference in potency when targeting a CD19-expressing tumor target (NALM-6) in the absence of IL-15 (Figure 11A), but in the presence of IL-15 there was a trend towards improved cytotoxic activity in IL-15Rα and β co-expressing cells (Figure 11B).

[0290] Conversely, exogenous IL-15, tethered IL-15 and IL-15Rβ co-expressing, anti-CD123 (Figure 11D) or anti-mesothelin targeted (Figure 11E) CAR V51 T cells showed improved cytotoxicity compared to non-armored cells. To further test whether armoring affects V51 T cell potency in the absence of CAR binding, the short-term cytotoxicity of non-armored and armored CD19-targeted CAR-modified V51 T cells was compared against CD19-negative (but V51 T cell sensitive) targets (MV4-11 or MOLM-13) (Figure 11C). As expected, the cytolytic activity of tethered IL-15 and IL-15Rβ co-expressing cells was significantly improved.

[0291] Example 10: CAR-modified V51 T cells co-expressing tethered IL-15 and IL-15Rβ chain survive in the absence of exogenous IL-15 The next question was to explore whether these novel armouring strategies could support the survival of armoured CAR-modified V51 T cells. Freshly thawed preparations (n=2 donors) were plated at 2E+06 cells per mL in 96-well plates in the absence of exogenous cytokine support. The total number of live cells and the percentage of CAR+V51+ T cells were monitored periodically for 2 weeks. Figures 12A and 12C show that only tethered IL-15 and IL-15Rβ co-expressing cells survived and proliferated. IL-15Rβ alone and IL15Rα and β co-expressing cells died rapidly in the absence of exogenous IL-15 support. This is reflected in the rapid decrease in the total number of CAR+V51+ T cells and the percentage of viable lymphocytes during the assay (Figures 12A and 12D). Figure 12C shows the percentage of CAR+V51+ T cells over time. The assay enriched for only tethered IL-15 and IL-15Rβ chain co-expressing cells. Flow cytometry analysis revealed that this was the result of a rapid loss of the non-transduced Vδ1 T cell population, which could not survive in the absence of exogenous IL-15 (Figure 12B). Interestingly, tethered IL-15 and IL-15Rβ chain co-expressing cells appeared to be able to support the survival of non-transduced cells from day 3 until the end of the assay (Figure 12B).

[0292] To show that the effect of Armoring is independent of the CAR molecule selected, the above experiment was repeated with mesothelin-targeted CAR-modified Armored Vδ1 T cells (Figure 15A). Indeed, tethered IL-15 and IL-15Rβ co-expressing mesothelin-targeted CAR cells survived for 14 days after cryopreservation, whereas non-armored cells were rapidly depleted. Importantly, the survival advantage of Armored cells remained the same regardless of the type of mesothelin-targeted binder (YP218 or P4) used in the CAR construct (Figure 15A). These results indicate that the functional attributes conferred by co-expression of tethered IL-15 and IL-15Rβ chains are independent of the CAR and / or binder used.

[0293] Overall, these results indicate that in the absence of exogenous IL-15, co-expression of IL-15Rα and β is necessary but not sufficient to support survival of CAR-modified Vδ1 T cells: IL-15 needs to be tethered to the IL-15Rα chain to achieve long-term cell survival.

[0294] Example 11: CAR-modified V51 T cells co-expressing tethered IL-15 and IL-15Rβ chain are enriched upon repeated antigen stimulation in the presence of physiological levels of IL-15 We next assessed whether transfer of the IL-15Rβ chain alone or in combination with the IL-15Rα chain was sufficient to drive selective enrichment of CAR-modified Vδ1 T cells during repeated tumor challenge. Furthermore, we also investigated whether physiological (pg / mL) IL-15 concentrations could support the proliferation of Armouring cells. To this end, repeated antigen stimulation assays were performed in the presence of two IL-15 concentrations: supraphysiological (10 ng / mL) and physiological (70 pg / mL).

[0295] Figures 13A-13C (left panels) show that Armouring constructs were able to support the proliferation and enrichment of CAR-modified Vδ1 T cells at supraphysiological concentrations of IL-15, with IL-15Rα.β and IL15-Rβ being the most effective. Consistent with this, Armouring CAR-modified cells were able to eliminate repeated challenges of NALM-6 cells over the duration of the assay (Figure 11C).

[0296] This was in stark contrast to the results using physiological concentrations (pg / mL) of IL-15 (Figures 13D-13F, right panels). In the presence of low concentrations of IL-15, only membrane-tethered IL-15 and IL-15R.β chain co-expressing cells could be continuously expanded and enriched (Figures 11D and 11E). Nevertheless, in the presence of physiological concentrations of IL-15, these cells showed an overall lower fold proliferation than that observed in the presence of supraphysiological concentrations of IL-15.

[0297] After three tumor challenges, both β-chain alone and α- and β-chain co-expressing Armored Vδ1 T cells rapidly declined in number, consistent with loss of tumor control and proliferation of NALM-6 target cells. Surprisingly, this was not the case for membrane-bound IL-15 and IL-15Rβ-chain co-expressing cells, which maintained the ability to clear repeated challenges of NALM-6 cells.

[0298] To show that the effect of armoring is independent of the CAR molecule selected, the above experiments were repeated with anti-mesothelin CAR-expressing non-armored and tethered IL-15 and IL-15Rβ chain armored Vδ1 T cells (Figure 15B-Figure 15E). Figure 15B shows that in the absence of exogenous IL-15, tethered IL-15 and IL-15Rβ chain armored mesothelin CAR T cells were able to control repeated challenges of mesothelin-expressing A549 cells. Importantly, the above observations were not affected by the choice of mesothelin-binding agent (YP218 or P4). Moreover, the improved tumor control shown by tethered IL-15 and IL-15Rβ chain armored CAR T cells did not correlate with the proliferation of these cells. Instead, the proliferation potential appeared to be driven by the CAR molecule itself and was specific to the binder used. Proliferation was observed with the YP218 binder but not with the P4 binder (Figure 15C). IL-15Rβ chain armored P4 CAR T cells were also able to provide superior tumor control over the course of repeated tumor challenge in the presence of low levels of IL-15 (1 ng / mL) (Figure 15D). The improved functionality correlated significantly with increased sensitivity to exogenous IL-15 and increased proliferation over the course of repeated tumor challenge (Figure 15E).

[0299] These results clearly demonstrate that at physiological IL-15 concentrations, cells expressing membrane-bound IL-15 and IL-15Rβ chain can proliferate, survive and exhibit productive tumor control.

[0300] Example 12: NALM-6 in vivo efficacy study using NSG and NOG-IL15 mice To confirm the in vitro findings, the in vivo efficacy of the formulations was evaluated in a NALM6-FLuc systemic tumor model, tracking tumor burden via non-invasive bioluminescence imaging (BLI) twice weekly (Figure 14A). Each formulation was evaluated under three conditions: NSG mice administered 1 μg IL-15 daily (Figure 14B), NSG mice without exogenous IL-15 (Figure 14C), or NOG IL-15 transgenic mice (Taconic, hIL-15NOG (model no. 13683), Figure 14D). In the presence of exogenous IL-15, all three constructs provided comparable tumor control, with all groups showing BLI comparable to background by day 6 post-dosing (study day 13) (Figure 14B). In transgenic NOG mice, IL-15 concentrations in peripheral blood were close to human physiological levels (~50 pg / mL), and all groups showed comparable tumor control in this case as well (Figure 14D). However, in the absence of exogenous IL-15, while all constructs were able to control tumor growth to some extent, only the muCAR19.α.15.β armored construct was able to reduce tumor burden below detectable levels by day 10 post-dosing (study day 17) (Figure 14C).

[0301] Satellite mice were also sacrificed 3 days after dosing (study day 10) to assess the relative numbers of V51+ and CAR+ cells. In the presence of exogenous IL-15, all constructs were comparable in terms of both total cell numbers (Figure 14E) and percentage of CAR-expressing cells (Figure 14F) compared to pre-injection formulations. In the absence of exogenous IL-15, muCAR19.α.15.β-treated mice had comparable numbers of V51+, CAR+ cells, but a loss of CAR-non-transduced cells was observed as a loss in absolute numbers of CAR- cells and an increase in V51+ CAR (%), indicating a survival advantage within the armored compartment. This survival advantage in the absence of IL-15 was further enhanced by the end of the study 13 / 14 days after dosing (Figure 14J), with muCAR19.α.15.β-treated mice exhibiting a greater than 100-fold increase in CAR+Vδ1 T cell survival compared to muCAR19.α.β- and muCAR19.β-treated mice (Figure 14L). This increased survival was also associated with a complete lack of detectable residual NALM-6 tumor cells in the bone marrow of muCAR19.α.15.β-treated mice, while muCAR19.α.β- and muCAR19.β-treated mice had, on average, hundreds or thousands of residual NALM6 cells (respectively) in bone marrow harvested from the hind femur (Figure 14M). In the presence of exogenous IL-15, all three treatment groups showed an increase in both CAR+ and CAR-Vδ1 T cells beginning 3 days after dosing (Figure 14K vs. Figure 14E). The greatest percentage of CAR+ cells at the end of the study was observed in mice treated with muCAR19.β (Figure 14J), with the other groups showing no change (muCAR19.α.β) or a decrease (muCAR19.α.15.β) in CAR relative ratios. The trends observed in the control of NALM6 tumors based on whole-body imaging data were not altered by acquisition of additional time points (10 vs. 12 days after treatment): NSG mice treated with 1 μg IL-15 daily (Figure 14B vs. Figure 14G), NSG mice without exogenous IL-15 (Figure 14C vs. Figure 14H), or NOG IL-15 transgenic mice (Taconic, hIL-15 NOG (model number 13683), Figure 14D vs. Figure 14I).

[0302] Overall, these results show that all constructs are potent and capable of controlling disease in the NALM6 model in the presence of exogenous or transgenically expressed IL-15, but in its absence, muCAR19.α.15.β is uniquely capable of reducing residual tumors to below detectable levels.

[0303] Example 13: In vivo efficacy test of A549-MSLN using NSG mice To confirm the in vitro findings obtained with Armouring mesothelin-targeting CAR Vδ1 T cells, we evaluated the in vivo efficacy of the formulation in a disseminated A549-Meso-FLuc tumor model. Six to eight week old NSG mice were administered 1 × 10 6 A549-MSLN-fluc-GFP cells were implanted. 19 days after implantation, mice were injected with vehicle (n=7, Cryostor CS5, group 1), a total of 10 million P4 unarmored γδ CAR T cells per mouse (n=7, group 2), a total of 10 million P4 α.15.βγδ CAR T cells per mouse (n=7, group 3), or a total of 10 million P4 β γδ CAR T cells per mouse (n=7, group 4). Tumor burden was tracked via non-invasive bioluminescence imaging (BLI) twice weekly (Figure 16A). Figure 16B shows that in the absence of exogenous IL-15, α.15.β armoured Vδ1 P4 CAR T cells showed higher tumour control than non-armoured Vδ1 P4 CAR T cells, with mice treated with P4α.15.β having on average a 10-fold lower BLI signal than mice administered non-armoured cells at the end of the study (day 19 post-treatment). Consistent with the in vitro experiments, in the presence of IL-15, β armoured γδ CAR T cells show faster tumour clearance kinetics initially, but overall comparable tumour control to the α.15.β armoured (non-IL-15 supported) γδ CAR T treated group.

[0304] Other embodiments All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0305] While the invention has been described in relation to specific embodiments thereof, it will be understood that the invention is capable of further modifications, and this application is intended to cover any alterations, uses, or adaptations of the invention which are generally in accordance with the principles of the invention and which come within known or customary practice in the art to which the invention pertains, and which may be applied to the essential features described above, including such departures from the present disclosure as may be applicable, subject to the scope of the claims.

[0306] Other embodiments are within the scope of the claims.

Claims

1. γδ T cells expressing recombinant IL-15 receptor β subunit (IL-15Rβ).

2. The γδ T cells according to claim 1, further expressing recombinant IL-15 receptor α subunit (IL-15Rα) or a fragment thereof.

3. The γδ T cell according to claim 2, wherein the IL-15Rα or a fragment thereof is tethered to IL-15 or a variant thereof.

4. The γδ T cell according to claim 3, further comprising a linker between the IL-15Rβ and the IL-15Rα or a fragment thereof.

5. The linker contains a cleavable peptide, If desired, the cleavable peptide is a self-cleaving peptide. Furthermore, if desired, the self-cleaving peptide may be peptide 2A. Furthermore, if desired, the 2A peptide may be foot-and-mouth disease virus 18 2A (F2A) peptide, equine rhinitis A virus 2A (E2A) peptide, porcine rhinitis virus-1 2A (P2A) peptide, or thosea assigna virus 2A (T2A) peptide, and Furthermore, if desired, the 2A peptide may be a P2A peptide. The γδT cell according to claim 4.

6. It further expresses chimeric antigen receptors (CARs), The γδ T cell according to any one of claims 1 to 5, wherein the CAR optionally lacks an intracellular signaling domain.

7. The aforementioned γδT cells, (i) The endogenous gene encoding IL-15 is deleted. (ii) Expressing secreted IL-15 or a variant thereof, (iii) Expressing recombinant IL-15Rβ and recombinant IL-15 or its variants, (iv) Any combination of (i) to (iii) above, The γδ T cell according to any one of claims 1 to 5.

8. It further expresses recombinant IL-15Rα or a fragment thereof, If desired, the fragment of IL-15Rα may include a soluble fragment. Furthermore, if desired, the soluble fragment may include a sushi domain, and Furthermore, if desired, the sushi domain may be bound to the Fc domain. The γδT cell according to claim 7.

9. The γδ T cell according to claim 8, wherein the IL-15Rα or a fragment thereof is tethered to the IL-15 or a variant thereof.

10. A second linker is further included between the IL-15Rβ and the IL-15Rα or a fragment thereof. If desired, the second linker contains a cleavable peptide. Furthermore, if desired, the cleavable peptide may be a self-cleaving peptide. Furthermore, if desired, the self-cleaving peptide may be peptide 2A. Furthermore, optionally, the 2A peptide may be F2A peptide, E2A peptide, P2A peptide, or T2A peptide, and Furthermore, if desired, the 2A peptide may be a P2A peptide. The γδT cell according to claim 9.

11. It further expresses CAR, The γδ T cell according to claim 6, wherein, optionally, the CAR lacks an intracellular signaling domain.

12. The γδT cells according to claim 6, wherein the γδT cells lack an endogenous gene encoding IL-15.

13. The γδ T cell according to any one of claims 1 to 5, wherein the γδ T cell is a Vδ1+γδ T cell.

14. The γδ T cell according to any one of claims 1 to 5, wherein the γδ T cell is a blood-derived Vδ1+γδ T cell.

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