Engineering gamma delta T cells and their compositions

Abstract

The present invention provides methods for engineering γδ T cells (e.g., vδ1 T cells and vδ2 T cells) by transduction with viral vectors (e.g., viral vectors having betaretrovirus pseudotypes and retroviridae viral vector backbones). Further provided are compositions of engineered γδ T cells and methods of using the same.

Description

[Background technology]

[0001] The growing interest in T cell immunotherapy for cancer has focused on the apparent potential of engineered T cells as therapeutic moieties. Gamma delta T cells (γδ T cells) represent a subset of T cells that express a distinct and definitive 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 as one or two types: peripheral blood resident γδ T cells and non-hematopoietic tissue resident γδ T cells. While most blood resident γδ T cells express the Vδ2 TCR, this is less common among tissue resident γδ T cells, which more frequently use Vδ1 and / or other Vδ chains.

[0002] As opposed to αβ T cells, there is a lack of methods for efficient transduction of γδ T cells to express a desired transgene. Thus, there is a need in the art for improved methods for transducing γδ T cells and generating populations of γδ T cells of sufficient quality and quantity for use as a therapy, e.g., as adoptive T cell therapy. Summary of the Invention

[0003] In one aspect, the invention features a method of producing an engineered population of γδ T cells by transducing a population of γδ T cells with a viral vector having a betaretrovirus pseudotype and a Retroviridae viral vector backbone. The betaretrovirus pseudotype can be Baboon Endogenous Virus (BaEV). The betaretrovirus pseudotype can be RD114.

[0004] In some embodiments, the Retroviridae viral vector backbone is a retroviral vector backbone (eg, a lentiviral backbone, a gammaretroviral backbone, or an alpharetroviral backbone).

[0005] The engineered γδ T cells may be Vδ1 T cells. The engineered γδ T cells may be Vδ2 T cells. The engineered γδ T cells may be non-Vδ1 / Vδ2 T cells.

[0006] In some embodiments, the viral vector comprises a transgene. The transgene may encode a cell surface receptor (e.g., a chimeric antigen receptor (CAR)) and / or a cytokine (e.g., a secreted or membrane-bound cytokine). In some embodiments, the transgene encodes IL-15 (e.g., secreted or membrane-bound IL-15). In some embodiments, the viral vector comprises a first transgene and a second transgene. In some embodiments, the first transgene encodes a CAR and the second transgene encodes an armor protein (e.g., a cytokine, e.g., IL-15, e.g., secreted or membrane-bound IL-15).

[0007] In some embodiments, the CAR is selected from the group consisting of CD19, CD20, ROR1, CD22, carcinoembryonic antigen, alpha fetoprotein, CA-125, 5T4, MUC-1, epithelial tumor antigen, prostate specific antigen, melanoma associated antigen, mutated p53, mutated ras, HER2 / Neu, folate binding protein, HIV-1 envelope glycoprotein gpl20, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD33, CD138, CD23, CD30, CD56, c-Met, mesothelin, GD3, H Targeting ERV-K, IL-llR alpha, kappa chain, lambda chain, CSPG4, ERBB2, EGFRvIII, VEGFR2, HER2-HER3 in combination, HER1-HER2 in combination, NY-ESO-1, synovial sarcoma X breakpoint 2 (SSX2), melanoma antigen (MAGE), melanoma antigen 1 recognized by T cells (MART-1), gp100, prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), g9d2, or combinations thereof.

[0008] In another aspect, the invention features a method of producing a population of engineered γδ T cells, the method includes providing a starting population of γδ T cells and culturing the starting population of γδ T cells for a first culture period in the absence of a viral vector to produce a population of primed γδ T cells. The method may further comprise culturing the population of primed γδ T cells for a second culture period in the presence of a viral vector having a betaretrovirus pseudotype in an amount 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, thereby producing a population of engineered γδ T cells.

[0009] In some embodiments, the viral vector is in an amount effective to transduce at least 20% of the primed γδ T cells.

[0010] In some embodiments, the first culture period is 1 day or more (e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days or more, e.g., 1 to 3 days, 3 to 5 days, 5 to 7 days, 7 to 10 days or more). In some embodiments, the first culture period is 2 days or more (e.g., 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days or more, e.g., 1 to 3 days, 3 to 5 days, 5 to 7 days, 7 to 10 days or more). In some embodiments, the first culture period is 5 days or more (e.g., 5 days, 6 days, 7 days, 8 days, 9 days, 10 days or more, e.g., 5 to 7 days, 7 to 10 days or more). In some embodiments, the first culture period is 7 days or more (e.g., 7 days, 8 days, 9 days, 10 days or more, e.g., 7 to 10 days or more).

[0011] In some embodiments, the second culture period is 2 days or more (e.g., 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., 2-4 days, 4-7 days, 7-10 days, 10-14 days or more). In some embodiments, the second culture period is 7 days or more (e.g., 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days or more, e.g., 7-10 days, 10-14 days or more).

[0012] In some embodiments, the primed population of γδ T cells expresses ASCT-1 and / or ASCT-2, In some embodiments, the primed population of γδ T cells lacks functional expression of a VSV-G entry receptor (e.g., an LDL receptor).

[0013] In some embodiments, the viral vectors are cultured with primed γδ T cells at a multiplicity of infection (MOI) of 10 or less (eg, 5 or less, eg, about 1 to about 5).

[0014] In another aspect, the invention features a method of producing a population of engineered γδ T cells by providing a starting population of γδ T cells and culturing the starting population of γδ T cells in the presence of IL-15 and a viral vector having a betaretrovirus pseudotype in an amount effective to transduce at least 3% (e.g., at least 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13% or 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 starting population of γδ T cells, thereby producing the starting population of engineered γδ T cells.

[0015] In some embodiments, the starting population of γδ T cells lacks expression of ASCT-1 and / or ASCT-2. In some embodiments, the engineered population of γδ T cells expresses ASCT-1 and / or ASCT-2. The starting population of γδ T cells may lack functional expression of a VSV-G entry receptor (e.g., an LDL receptor).

[0016] In some embodiments, the viral vector is cultured with the starting population of γδ T cells at an MOI of 10 or less (eg, 5 or less, eg, from about 1 to about 5).

[0017] In some embodiments, the viral vector has a BaEV or RD114 betaretrovirus pseudotype.

[0018] In some embodiments, the viral vector comprises a retroviridae viral vector backbone. The retroviridae viral vector backbone can be a retroviral vector backbone (e.g., a lentiviral backbone, a gamma retroviral backbone, or an alpha retroviral backbone).

[0019] The engineered γδ T cells may be Vδ1 T cells. The engineered γδ T cells may be Vδ2 T cells. The engineered γδ T cells may be non-Vδ1 / Vδ2 T cells.

[0020] In some embodiments, the viral vector comprises a transgene. The transgene may encode a cell surface receptor, e.g., a chimeric antigen receptor (CAR), and / or a cytokine (e.g., a secreted or membrane-bound cytokine). In some embodiments, the transgene encodes IL-15 (e.g., secreted or membrane-bound IL-15). In some embodiments, the viral vector comprises a first transgene and a second transgene. In some embodiments, the first transgene encodes a CAR and the second transgene encodes an armor protein (e.g., a cytokine, e.g., IL-15, e.g., secreted or membrane-bound IL-15).

[0021] In some embodiments, the CAR is a CAR specific for any of the following: CD19, CD20, ROR1, CD22, carcinoembryonic antigen, alpha fetoprotein, CA-125, 5T4, MUC-1, epithelial tumor antigen, prostate specific antigen, melanoma associated antigen, mutated p53, mutated ras, HER2 / Neu, folate binding protein, HIV-1 envelope glycoprotein gpl20, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD33, CD1 38, CD23, CD30, CD56, c-Met, mesothelin, GD3, HERV-K, IL-llR alpha, kappa chain, lambda chain, CSPG4, ERBB2, EGFRvIII, VEGFR2, HER2-HER3 in combination, HER1-HER2 in combination, NY-ESO-1, SSX2, MAGE, MART-1, gp100, PSA, PSMA, PSCA, g9d2, or combinations thereof.

[0022] In another aspect, the invention features a method of generating a population of γδ T cells expressing a CAR by transducing a population of γδ T cells with a viral vector comprising a transgene encoding a CAR, a betaretrovirus pseudotype, and a Retroviridae viral vector backbone.

[0023] In another aspect, the invention features a method of producing a population of γδ T cells expressing a CAR and an armor protein by transducing a population of γδ T cells with a viral vector comprising a first transgene encoding a CAR, a second transgene encoding an armor protein, a betaretrovirus pseudotype, and a Retroviridae viral vector backbone. In some embodiments, the armor protein is a cytokine (e.g., a membrane-bound cytokine or a secreted cytokine (e.g., membrane-bound IL-15 or secreted IL-15).

[0024] In some embodiments, the betaretrovirus pseudotype is BaEV. In other embodiments, the betaretrovirus pseudotype is RD114.

[0025] In some embodiments, the viral vector comprises a retroviridae viral vector backbone. The retroviridae viral vector backbone can be a retroviral vector backbone (e.g., a lentiviral backbone, a gamma retroviral backbone, or an alpha retroviral backbone).

[0026] The γδ T cell may be a Vδ1 T cell. The γδ T cell may be a Vδ2 T cell. The γδ T cell may be a non-Vδ1 / Vδ2 T cell.

[0027] In another aspect, the invention features a method of producing a population of γδ T cells that express a CAR by providing a starting population of γδ T cells and culturing the starting population of γδ T cells for a first culture period in the absence of a viral vector to produce a population of primed γδ T cells. The method may further comprise culturing the population of primed γδ T cells for a second culture period in the presence of a viral vector carrying a betaretroviral pseudotype and a transgene encoding a CAR, wherein the viral vector is in an amount 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, thereby producing a population of γδ T cells that express the CAR.

[0028] In another aspect, the invention features a method of producing a population of γδ T cells that expresses a CAR and an Armor protein by providing a starting population of γδ T cells and culturing the starting population of γδ T cells for a first culture period in the absence of a viral vector to produce a population of primed γδ T cells. The method may further comprise culturing the population of primed γδ T cells for a second culture period in the presence of a viral vector having a betaretroviral pseudotype, a first transgene encoding a CAR, and a second transgene encoding an armor protein, wherein the viral vector is in an amount 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, thereby producing a population of γδ T cells that express the CAR and the armor protein. In some embodiments, the transgene encodes IL-15 (e.g., secreted IL-15 or membrane-bound IL-15). In some embodiments, the viral vector comprises a first transgene and a second transgene. In some embodiments, the first transgene encodes a CAR and the second transgene encodes an armor protein (e.g., a cytokine, e.g., IL-15, e.g., secreted IL-15 or membrane-bound IL-15).

[0029] In some embodiments, the viral vector is in an amount effective to transduce at least 20% of the primed γδ T cells.

[0030] In some embodiments, the first culture period is 1 day or more (e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days or more, e.g., 1 to 3 days, 3 to 5 days, 5 to 7 days, 7 to 10 days or more). In some embodiments, the first culture period is 2 days or more (e.g., 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days or more, e.g., 1 to 3 days, 3 to 5 days, 5 to 7 days, 7 to 10 days or more). In some embodiments, the first culture period is 5 days or more (e.g., 5 days, 6 days, 7 days, 8 days, 9 days, 10 days or more, e.g., 5 to 7 days, 7 to 10 days or more). In some embodiments, the first culture period is 7 days or more (e.g., 7 days, 8 days, 9 days, 10 days or more, e.g., 7 to 10 days or more).

[0031] In some embodiments, the second culture period is 2 days or more (e.g., 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., 2-4 days, 4-7 days, 7-10 days, 10-14 days or more). In some embodiments, the second culture period is 7 days or more (e.g., 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days or more, e.g., 7-10 days, 10-14 days or more).

[0032] In some embodiments, the primed population of γδ T cells express ASCT-1 and / or ASCT-2. In some embodiments, the primed population of γδ T cells lacks functional expression of a VSV-G entry receptor (e.g., LDL receptor). In some embodiments, greater than 95% of the primed population of γδ T cells lacks a sufficient level of VSV-G entry receptor expression (e.g., LDL receptor) to mediate detectable VSV-G entry (e.g., as measured by a BlaM-Vpr-based assay). In some embodiments, greater than 96% of the primed population of γδ T cells lacks a sufficient level of VSV-G entry receptor expression (e.g., LDL receptor) to mediate detectable VSV-G entry (e.g., as measured by a BlaM-Vpr-based assay). In some embodiments, greater than 97% of the primed γδ T cell population lacks a sufficient level of VSV-G entry receptor expression (e.g., LDL receptor) to mediate detectable VSV-G entry (e.g., as measured by a BlaM-Vpr-based assay). In some embodiments, greater than 98% of the primed γδ T cell population lacks a sufficient level of VSV-G entry receptor expression (e.g., LDL receptor) to mediate detectable VSV-G entry (e.g., as measured by a BlaM-Vpr-based assay). In some embodiments, greater than 99% of the primed γδ T cell population lacks a sufficient level of VSV-G entry receptor expression (e.g., LDL receptor) to mediate detectable VSV-G entry (e.g., as measured by a BlaM-Vpr-based assay).

[0033] In some embodiments, the viral vectors are cultured with primed γδ T cells at an MOI of 10 or less (eg, 5 or less, eg, about 1 to about 5).

[0034] In another aspect, the invention features a method of producing a population of γδ T cells expressing a CAR by providing a starting population of γδ T cells and culturing the starting population of γδ T cells in the presence of IL-15 and a vector having a betaretroviral pseudotype and a transgene encoding a CAR, wherein the viral vector is in an amount 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 starting population of γδ T cells, thereby producing a starting population of engineered γδ T cells that expresses a CAR.

[0035] In another aspect, the invention provides a method for producing an engineered γδ T cell population comprising: providing a starting population of γδ T cells; and culturing the starting population of γδ T cells in the presence of IL-15 and a viral vector having a beta retrovirus pseudotype, a first transgene encoding a CAR, and a second transgene encoding an Armor protein, wherein the viral vector is in an amount 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 starting population of γδ T cells, thereby producing an engineered γδ T cell population that expresses the CAR and Armor protein. and generating a population of T cells that express a CAR and an armor protein. In some embodiments, the armor protein is a cytokine, e.g., IL-15, e.g., secreted IL-15 or membrane-bound IL-15.

[0036] In some embodiments, the starting population of γδ T cells lacks expression of ASCT-1 and / or ASCT-2. The engineered population of γδ T cells may express ASCT-1 and / or ASCT-2. The starting population of γδ T cells may lack functional expression of a VSV-G entry receptor (e.g., LDL receptor). In some embodiments, greater than 95% of the primed γδ T cell population lacks a sufficient level of VSV-G entry receptor expression (e.g., LDL receptor) to mediate detectable VSV-G entry (e.g., as measured by a BlaM-Vpr-based assay). In some embodiments, greater than 96% of the primed γδ T cell population lacks a sufficient level of VSV-G entry receptor expression (e.g., LDL receptor) to mediate detectable VSV-G entry (e.g., as measured by a BlaM-Vpr-based assay). In some embodiments, greater than 97% of the primed γδ T cell population lacks a sufficient level of VSV-G entry receptor expression (e.g., LDL receptor) to mediate detectable VSV-G entry (e.g., as measured by a BlaM-Vpr-based assay). In some embodiments, greater than 98% of the primed γδ T cell population lacks a sufficient level of VSV-G entry receptor expression (e.g., LDL receptor) to mediate detectable VSV-G entry (e.g., as measured by a BlaM-Vpr-based assay). In some embodiments, greater than 99% of the primed γδ T cell population lacks a sufficient level of VSV-G entry receptor expression (e.g., LDL receptor) to mediate detectable VSV-G entry (e.g., as measured by a BlaM-Vpr-based assay).

[0037] In some embodiments, the viral vector is cultured with the starting population of γδ T cells at an MOI of 10 or less (eg, 5 or less, eg, from about 1 to about 5).

[0038] In some embodiments, the betaretrovirus pseudotype is BaEV or RD114.

[0039] In some embodiments, the viral vector comprises a retroviridae viral vector backbone. The retroviridae viral vector backbone can be a retroviral vector backbone (e.g., a lentiviral backbone, a gamma retroviral backbone, or an alpha retroviral backbone).

[0040] The engineered γδ T cells may be Vδ1 T cells. The engineered γδ T cells may be Vδ2 T cells. The engineered γδ T cells may be non-Vδ1 / Vδ2 T cells.

[0041] In some embodiments, the CAR is a CAR specific for any of the following: CD19, CD20, ROR1, CD22, carcinoembryonic antigen, alpha fetoprotein, CA-125, 5T4, MUC-1, epithelial tumor antigen, prostate specific antigen, melanoma associated antigen, mutated p53, mutated ras, HER2 / Neu, folate binding protein, HIV-1 envelope glycoprotein gpl20, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD33, CD1 38, CD23, CD30, CD56, c-Met, mesothelin, GD3, HERV-K, IL-llR alpha, kappa chain, lambda chain, CSPG4, ERBB2, EGFRvIII, VEGFR2, HER2-HER3 in combination, HER1-HER2 in combination, NY-ESO-1, SSX2, MAGE, MART-1, gp100, PSA, PSMA, PSCA, g9d2, or combinations thereof.

[0042] In another aspect, the invention features a population of engineered γδ T cells produced by the methods described herein.

[0043] In some embodiments, at least 10% (e.g., at least 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 express a CAR. In some embodiments, at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or substantially all) of the population of engineered γδ T cells express a CAR. In some embodiments, at least 10% (e.g., at least 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 express an armor protein, e.g., a cytokine (e.g., a secreted cytokine or a membrane-bound cytokine (e.g., IL-15, e.g., secreted IL-15 or membrane-bound IL-15). In some embodiments, the engineered γδ T cells express an armor protein, e.g., a cytokine (e.g., a secreted cytokine or a membrane-bound cytokine (e.g., IL-15, e.g., secreted IL-15 or membrane-bound IL-15). At least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or substantially all) of the population of T cells express an armor protein, e.g., a cytokine (e.g., a secreted cytokine or a membrane-bound cytokine (e.g., IL-15, e.g., secreted IL-15 or membrane-bound IL-15). In some embodiments, an engineered γδ T cell expresses an armor protein, e.g., a cytokine that is secreted or membrane-bound. At least 10% (e.g., at least 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 T cells express a CAR and armor protein, e.g., a cytokine (e.g., a secreted cytokine or a membrane-bound cytokine (e.g., IL-15, e.g., secreted IL-15 or membrane-bound IL-15).In some embodiments, at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or substantially all) of the population of engineered γδ T cells express a CAR and armor protein, e.g., a cytokine (e.g., a secreted cytokine or a membrane-bound cytokine (e.g., IL-15, e.g., secreted IL-15 or membrane-bound IL-15).

[0044] In another aspect, the invention features a population of CAR-expressing γδ T cells produced by the methods described herein.

[0045] In another aspect, the invention features a population of γδ T cells expressing a CAR and an armor protein produced by the methods described herein. In some embodiments, the armor protein is a cytokine (e.g., a secreted cytokine or a membrane-bound cytokine (e.g., IL-15, e.g., secreted IL-15 or membrane-bound IL-15).

[0046] 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.

[0047] The term "about" as used herein refers to the normal error range of the respective value, which is readily known to those skilled in the art. Reference herein to a value or parameter of "about" includes (and describes) an embodiment that is directed to the value or parameter itself. In some cases, "about" encompasses a variation of +20%, in some cases +10%, in some cases +5%, in some cases +1%, or in some cases +0.1% from the specified value, as such variations are appropriate for carrying out the disclosed method.

[0048] As used herein, the term "engineered γδ T cell" refers to a γδ T cell that expresses a transgene (i.e., a gene transduced into the engineered γδ T cell or a parent cell thereof).

[0049] 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 cases, primed γδ T cells have a different functional viral entry receptor profile relative to their unprimed counterparts prior to experiencing the culture conditions. In some embodiments, a population of primed γδ T cells is an expanded population of γδ T cells.

[0050] 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 duration that induced γδ cell expansion, i.e., an increase in γδ cell numbers. Similarly, an "expanded population of V51 T cells" as used herein refers to a population of hematopoietic cells comprising V51 T cells cultured under conditions and for a duration that induced V51 T cell expansion, i.e., an increase in V51 cell numbers. Similarly, an "expanded population of V52 T cells" as used herein refers to a population of hematopoietic cells comprising V52 T cells cultured under conditions and for a duration that induced V52 T cell expansion, i.e., an increase in V52 cell numbers.

[0051] As used herein, a "population" of γδ T cells refers to three or more γδ T cells (e.g., at least 10, at least 10 2 , at least 10 3 , at least 10 4 , at least 10 5 , at least 10 6 , at least 10 7 , at least 10 8 , at least 10 9 , at least 10 10 , at least 10 11 , at least 10 12, or at least 10 13 A population of 10 γδ T cells (e.g., engineered γδ T cells) refers to a population of 10 γδ T cells (e.g., engineered γδ T cells). 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, 8 If 10% of the cells in a starting population of T cells are γδ T cells, then the starting population of γδ T cells is 10 7 There are 10 pieces.

[0052] As used herein, "armor protein" refers to a transgene-encoded protein that, when expressed by a γδ T cell (e.g., a γδ T cell expressing a CAR), increases the persistent or increased immunogenicity of the γδ T cell toward a target cell, e.g., through paracrine signaling (e.g., cytokine signaling), improving, e.g., cell persistence, cell survival, activation, and other desirable properties. Armor proteins can be membrane-bound or soluble proteins. For example, armor proteins include membrane-bound proteins such as membrane-bound receptors (e.g., αβ TCR, 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 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.

[0053] As used herein, "IL-15" refers to natural or recombinant IL-15 or variants thereof that act as agonists of one or more IL-15 receptor (IL-15R) subunits (e.g., mutants, muteins, analogs, subunits, receptor complexes, fragments, isoforms, and peptidomimetics thereof). IL-15, like IL-2, is a known T cell growth factor that can support the proliferation of the IL-2-dependent cell line, CTLL-2. IL-15 was first reported as a 114 amino acid mature protein by Grabstein et al. (Science 264.5161:965-969, 1994). As used herein, the term "IL-15" refers to natural or recombinant IL-15, and its muteins, analogs, subunits, or complexes thereof (e.g., receptor complexes, e.g., sushi peptides described in PCT Publication No. WO2007 / 046006), each of which can stimulate the proliferation of CTLL-2 cells. In a CTLL-2 proliferation assay, supernatants from cells transfected with recombinantly expressed precursor and in-frame fusions of the mature form of IL-15 are able to induce CTLL-2 cell proliferation.

[0054] Human IL-15 can be obtained according to the procedure described by Grabstein et al. (Science 264.5161:965-969, 1994) or by conventional procedures such as polymerase chain reaction (PCR). A human IL-15 cDNA was deposited with the ATCC on February 19, 1993 and assigned accession number 69245.

[0055] 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 under the accession locator NP_001241676.1 GI:363000984.

[0056] IL-15 may also refer to IL-15 from various mammalian species, including, for example, human, monkey, cow, pig, horse, and mouse. An IL-15 "mutein" or "variant" as referred to herein is a polypeptide that has an amino acid sequence that is substantially homologous to the sequence of a native mammalian IL-15, but differs from the native mammalian IL-15 polypeptide by amino acid deletion, insertion, or substitution. A variant may include a conservatively substituted 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, such as Ile, Val, Leu, or Ala, for each other, or the substitution of one polar residue for another, such as between Lys and Arg, between Glu and Asp, or between Gln and Asn. Other such conservative substitutions, such as the substitution of entire regions with similar hydrophobic properties, are well known. Naturally occurring 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, where IL-15 binding properties are retained. Alternate splicing of the mRNA can result in truncated but biologically active IL-15 proteins. Variations 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 from 1 to 10 amino acids) from the IL-15 protein. In some embodiments, the termini of the protein can be modified to alter its physical properties, for example, with chemical groups such as polyethylene glycol (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).

[0057] As used herein, "non-hematopoietic cells" includes stromal cells and epithelial cells. Stromal cells are non-hematopoietic connective tissue cells of any organ that support the function of the organ's parenchymal cells. Examples of stromal cells include fibroblasts, pericytes, mesenchymal cells, keratinocytes, endothelial cells, and non-hematopoietic tumor cells. Epithelial cells are non-hematopoietic cells that line the cavities and surfaces of blood vessels and organs throughout the body. They are usually scaly, columnar, or cuboidal in shape and can be arranged as a monolayer of cells or as layers of two or more cells.

[0058] As used herein, "non-hematopoietic tissue resident γδ T cells," "non-hematopoietic tissue derived," and "non-hematopoietic tissue native γδ T cells" refer to γδ T cells that were present in the non-hematopoietic tissue when the tissue was explanted. Non-hematopoietic tissue resident γδ T cells may be obtained from any suitable human or non-human animal non-hematopoietic tissue. A non-hematopoietic tissue is a tissue other than blood or bone marrow. In some embodiments, γδ T cells are not obtained from a sample of a particular type of biological fluid, such as blood or synovial fluid. Examples of such suitable human or non-human animal non-hematopoietic tissues include skin or a portion thereof (e.g., dermis or epidermis), gastrointestinal tract (e.g., gastrointestinal epithelium, colon, small intestine, stomach, appendix, cecum, or rectum), mammary tissue, lung (preferably, the tissue is not obtained by bronchoalveolar lavage), prostate, liver, and pancreas. In some embodiments, non-hematopoietic tissue resident γδ T cells may be derived from lymphoid tissue, such as the thymus, spleen, or tonsils. γδ T cells may also be resident in human cancer tissues, e.g., breast and prostate. In some embodiments, the γδ T cells are not obtained from human cancer tissue. Non-hematopoietic tissue samples may be obtained by standard techniques, e.g., explant (e.g., biopsy). Non-hematopoietic tissue resident γδ T cells include, for example, V51 T cells, double negative (DN) T cells, V52 T cells, V53 T cells, and V55 T cells.

[0059] As used herein, the phrase "in an effective amount" refers to an amount that induces a detectable result (e.g., a statistically significant increased number of cells relative to the starting population, e.g., at p<0.05).

[0060] 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 duration that induced γδ cell expansion, i.e., an increase in γδ cell numbers. Similarly, an "expanded population of V51 T cells" as used herein refers to a population of hematopoietic cells comprising V51 T cells cultured under conditions and for a duration that induced V51 T cell expansion, i.e., an increase in V51 cell numbers. Similarly, an "expanded population of V52 T cells" as used herein refers to a population of hematopoietic cells comprising V52 T cells cultured under conditions and for a duration that induced V52 T cell expansion, i.e., an increase in V52 cell numbers.

[0061] The term "marker" 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).

[0062] 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 the marker can be detected by a variety of 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), when used as a flow cytometry readout, represents the density of the marker on a cell population. Those skilled in the art will understand 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 cell population 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 on the same cell population stained under comparable conditions. Additionally or alternatively, a cell population can be said to express the 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). By this metric, a population can be said to "express" a marker if the number of cells detected as positive for the marker is significantly higher than background (eg, by gating on an isotype control).

[0063] As used herein, "functional expression of VSV-G entry receptor" refers to a level of VSV-G entry receptor expression sufficient to mediate detectable VSV-G entry in at least 5% of a target cell population, as measured by beta-lactamase-Vpr (BlaM-VpR)-based assay.See, for example, Cavrois et al., Nat Biotechnol.11:1151-1154,2002. Conversely, in a cell population that "lacks functional expression of VSV-G entry receptor," more than 95% of the cell population lacks a level of VSV-G entry receptor expression sufficient to mediate detectable VSV-G entry, as measured by BlaM-VpR-based assay.

[0064] As used herein, when the expression of a population is stated as the percentage of positive cells and the percentage is compared with the corresponding percentage of positive cells of a reference population, the percentage difference is the percentage of the parent population of each respective population. For example, if a marker is expressed in 10% of the cells of population A and the same marker is expressed in 1% of the cells of population B, population A is said to have a 9% higher frequency of marker-positive cells than population B (i.e., 10%-1%, not 10%÷1%). When the frequency is multiplied by the number of cells in the parent population, the difference in absolute number of cells is calculated. In the example given above, if population A has 100 cells and population B has 10 cells, population A has 100 times the number of cells compared to population B, i.e., (10%×100)÷(1%×10).

[0065] 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, serial analysis of gene expression (SAGE), MASSARRAY® technology, in situ hybridization (e.g., FISH), or a combination thereof.

[0066] As used herein, a "reference population" of cells refers to a cell population 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 γδ cells from a non-hematopoietic tissue can be compared to the expression level of the same marker on γδ T cells from a hematopoietic tissue (e.g., blood-resident γδ cells, e.g., blood-resident γδ cells from the same donor or a different donor), or on γδ T cells from a non-hematopoietic tissue 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 can also be compared to itself at a previous state. For example, the reference population can be an isolated cell population prior to its expansion. In this case, the expanded population is compared to its own composition prior to the expansion step, i.e., its past composition is the reference population in this case.

[0067] 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 that activates the cell. In some embodiments, the CAR includes an optional leader sequence at the N-terminus of the CAR fusion protein. [Brief description of the drawings]

[0068] [Figure 1] Figure 1 shows the inability of broadly tropic VSV-G pseudotyped lentiviral vectors to transduce Vδ1 γδ T cells. Representative dot plots show γδ T cells transduced with lentiviral vectors encoding GFP pseudotyped with VSV-G (A) or BaEV (B) using various multiplicities of infection on day 7 of expansion culture. Transduction efficiency was determined by FACS analysis 72 h after transduction. UTD, non-transduced control; MOI, multiplicity of infection; NVP, nevirapine (RT inhibitor). [Figure 2A]Graph showing that transduction of V51 γδ T cells with lentiviral vector encoding a VSV-G pseudotyped CAR results in mock transduction. Representative dot plots of CAR-positive V51 γδ T cells 4 days (top) or 8 days (bottom) after transduction with lentiviral vector encoding a VSV-G pseudotyped CAR at MOI=1 in the presence or absence of nevirapine. UTD, non-transduced control; MOI, multiplicity of infection; CAR, chimeric antigen receptor; NVP, nevirapine. [Figure 2B] Figure 1 shows that transduction of Vδ1 γδ T cells with lentiviral vectors encoding a VSV-G pseudotyped CAR results in pseudotransduction of Vδ1 γδ T cells 4 days (black bars) or 8 days (dotted bars) after transduction with lentiviral vectors encoding a VSV-G pseudotyped CAR at various MOIs (MOI = 5 to 0.1) in the presence or absence of nevirapine. UTD, non-transduced control; MOI, multiplicity of infection; CAR, chimeric antigen receptor; NVP, nevirapine. [Figure 3A] Figure 13: Cytokine priming is a major determinant of V51 γδ T cell transduction by BaEV pseudotyped lentiviral vectors. Bar graph showing percentage of GFP-positive V51 cells transduced at MOI=1 with BaEV pseudotyped lentiviral vectors encoding GFP 3 days after transduction. Cells were transduced at the beginning of culture (day 0) or on days 7, 10, 14, and 15 of the expansion phase. UTD, non-transduced control; MOI, multiplicity of infection; GFP, green fluorescent protein; NVP, nevirapine. [Figure 3B] Figure 1: Cytokine priming is a major determinant of Vδ1 γδ T cell transduction by BaEV pseudotyped lentiviral vectors. Representative dot plots of transduced cells on day 14 of expansion cultures are shown. UTD, non-transduced control; MOI, multiplicity of infection; GFP, green fluorescent protein; NVP, nevirapine. [Figure 4A]Graph showing that transduction efficiency of V51 γδ T cells correlates with multiplicity of infection (MOI). Percentage of CAR-positive V51 cells 3 days after transduction with BaEV pseudotyped lentiviral vector encoding a CAR with different MOIs is shown. Cells were transduced on day 10 of expansion. UTD, non-transduced control; MOI, multiplicity of infection; CAR, chimeric antigen receptor; NVP, nevirapine. [Figure 4B] Graph showing that transduction efficiency of V51 γδ T cells correlates with multiplicity of infection (MOI). Representative dot plots showing CAR-positive cells transduced at MOI=5 are shown. UTD, non-transduced control; MOI, multiplicity of infection; CAR, chimeric antigen receptor; NVP, nevirapine. [Figure 5A] Graph showing that BaEV pseudotyped lentiviral vectors transduce both V51 and non-V51 (V52, V53) γδ T cells. Dot plots show CAR (A) and GFP (B) expressing V51 and non-V51 (V52, V53) γδ T cells. Cells were transduced with BaEV pseudotyped vectors (MOI=5) and transduction efficiency was determined 3 days post-transduction by gating on pan γδ T cells followed by gating on V51 cells. [Figure 5B] Graph showing that BaEV pseudotyped lentiviral vectors transduce both V51 and non-V51 (V52, V53) γδ T cells. Dot plots show CAR (A) and GFP (B) expressing V51 and non-V51 (V52, V53) γδ T cells. Cells were transduced with BaEV pseudotyped vectors (MOI=5) and transduction efficiency was determined 3 days post-transduction by gating on pan γδ T cells followed by gating on V51 cells. [Figure 6]1 is a set of graphs showing that transduction of V51 γδ T cells with BaEV pseudotyped lentiviral vectors can be further enhanced by repeated transduction. V51 cells were transduced at MOI=1 with BaEV pseudotyped lentiviral vectors encoding CAR on either day 10 (1 hit) or on two consecutive days (2 hits: day 10 and day 11). The percentage of CAR positive cells was determined 72 hours after transduction. [Figure 7] Graph showing that transduction in the presence of vectofucin is as efficient as in the presence of retronectin. V51 cells were transduced in the presence of retronectin (left) or vectofucin (right) at different MOIs and different frequencies (1 or 2 hits). Cells were transduced on day 10 of expansion and FACS analysis was performed 3 days after transduction. [Figure 8] 1 is a set of graphs showing that V51 cells can be transduced with RD114 pseudotyped viral vectors. V51 cells were transduced with BaEV pseudotyped lentiviral vectors encoding CAR or RD114 pseudotyped gamma retroviral vectors at MOI=1. Dot plots show V51 cells expressing CAR 3 days after transduction. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0069] The present invention provides methods for engineering γδ T cells (e.g., vδ1 T cells and vδ2 T cells) by transduction with viral vectors (e.g., viral vectors having betaretrovirus pseudotypes and retroviridae viral vector backbones). Further provided are compositions of engineered γδ T cells and methods of using the same.

[0070] The present invention is based, in part, on the unexpected discovery that γδ T cells can be transduced to high levels with betaretrovirus pseudotyped viral vectors. Contrary to other lymphocyte types, γδ T cells are not permissive to retroviral transduction, for example using VSV-G pseudotyped viral vectors. VSV-G vectors readily transduce αβ T cells as well as NK cells, the closest cell type to γδ T cells. Thus, it was not expected that betaretrovirus pseudotyped viral vectors can transduce γδ T cells. Furthermore, the present invention is also based on the discovery of optimal culture conditions and duration of γδ T cells in the presence of a viral vector to transduce a population of γδ T cells with the vector. The transduction methods described herein allow for efficient transduction of γδ T cells to produce an engineered population of γδ T cells expressing a desired transgene.

[0071] Transduction methods In one aspect, the invention provides a method of producing 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 viral vector comprising a betaretrovirus pseudotype and a retroviridae (e.g., retrovirus) vector backbone. The retroviral vector backbone can be, for example, a lentivirus backbone, a gammaretrovirus backbone, or an alpharetrovirus backbone. The betaretrovirus pseudotype can be, for example, BaEV or RD114. In some embodiments, the betaretrovirus pseudotype is BaEV. In some embodiments, the betaretrovirus pseudotype is RD114.

[0072] In another aspect, the invention provides a method of producing 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 the viral vector in an amount 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 the viral vector effective to transduce at least 5% of the primed γδ T cells, hi some embodiments, the population of primed γδ T cells is cultured in the presence of an amount of the viral vector effective to transduce at least 20% of the primed γδ T cells.

[0073] Primed γδ T cells can be obtained by culturing a starting population of γδ T cells in the absence of a viral vector. For example, the starting population of γδ T cells can 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). For example, when primed γδ T cells are obtained after culturing the cells in the absence of a viral vector, the primed γδ T cells can 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 can 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).

[0074] In some embodiments, the viral vector is cultured with primed γδ T cells at a multiplicity of infection (MOI) of about 10 or less, e.g., 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 primed γδ T cells at a multiplicity of infection (MOI) of about 5 or less. In some embodiments, the viral vector is cultured with primed γδ T cells at a multiplicity of infection (MOI) of about 4 or less. In some embodiments, the viral vector is cultured with primed γδ T cells at a multiplicity of infection (MOI) of about 3 or less. In some embodiments, the viral vector is cultured with primed γδ T cells at a multiplicity of infection (MOI) of about 2 or less. In some embodiments, the viral vector is cultured with primed γδ T cells at a multiplicity of infection (MOI) of about 1 or less. In some embodiments, the viral vector is cultured with 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).

[0075] 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, vectofucin, spermatocyte, 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 supplementing the medium with IL-15, which can increase γδ T cell expression of ASCT-2, a viral entry receptor for betaretroviral pseudotyped viral vectors.

[0076] Spinoculation In some embodiments of the present disclosure, γδ T cells may be spun, e.g., by centrifugation, while in culture with a viral vector (e.g., in combination with one or more additional agents described herein). This "spinoculation" process may occur at a centripetal force of, e.g., about 200xg to about 2,000xg. The centripetal force may be, e.g., about 300xg to about 1,200xg (e.g., about 300xg, 400xg, 500xg, 600xg, 700xg, 800xg, 900xg, 1,000xg, 1,100xg, or 1,200xg or more). In some embodiments, the γδ T cells are spun for about 10 minutes to about 3 hours (e.g., about 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 65 minutes, 70 minutes, 75 minutes, 80 minutes, 85 minutes, 90 minutes, 95 minutes, 100 minutes, 105 minutes, 110 minutes, 115 minutes, 120 minutes, 125 minutes, 130 minutes, 135 minutes, 140 minutes, 145 minutes, 150 minutes, 155 minutes, 160 minutes, 165 minutes, 170 minutes, 175 minutes, 180 minutes or more). In some embodiments, the γδ T cells are spun at room temperature, such as at a temperature of about 25° C.

[0077] Exemplary transduction protocols involving a spinoculation step are described, for example, in Millington et al., PLoS One 4:e6461, 2009; Guo et al., Journal of Virology 85:9824-9833, 2011; O'Doherty et al., Journal of Virology 74:10074-10080, 2000; and Federico et al., Lentiviral Vectors and Exosomes as Gene and Protein Delivery Tools, Methods in Molecular Biology 1448, Chapter 4, 2016, the disclosures of each of which are incorporated herein by reference.

[0078] Viral Vectors The compositions and methods described herein include the use of betaretrovirus pseudotyped viral vectors for efficient transduction of γδ T cells. Viral genomes provide a rich source of vectors that can be used for efficient delivery of exogenous genes into mammalian cells. Viral genomes are particularly useful vectors for gene delivery because polynucleotides contained within such genomes are typically 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 additional proteins or reagents to induce gene integration. Examples of viral vectors that can be betaretrovirus pseudotyped include retroviruses (e.g., Retroviridae viral vectors). Examples of retroviruses are avian leukemia sarcoma, avian C virus, mammalian C, B, and D viruses, oncoretroviruses, HTLV-BLV group, lentiviruses, alpharetroviruses, betaretroviruses, gammaretroviruses, and spumaviruses (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 mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus (BaEV), gibbon leukemia virus, Mason-Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus, and lentiviruses. Other examples of vectors that can be pseudotyped with betaretroviruses for the present methods are described, for example, in McVey et al. (U.S. Patent No. 5,801,030), the teachings of which are incorporated herein by reference.

[0079] Retroviral Vectors In some cases, 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), transduce a wide range of dividing and non-dividing cell types with high efficiency, resulting in stable, long-term expression of transgenes. A summary of optimization strategies for packaging and transducing LVs is provided in Delenda, The Journal of Gene Medicine 6:S125, 2004, the disclosure of which is incorporated herein by reference.

[0080] The use of lentivirus-based gene transfer techniques relies on the in vitro production of recombinant lentiviral particles with a highly deleted viral genome that harbors the transgene of interest. In particular, recombinant lentiviruses are recovered through in 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, typically of heterologous nature, and (3) a transfer vector consisting of a viral cDNA deprived of all open reading frames but maintaining sequences required for replication, encapsidation, and expression, into which the sequence to be expressed is inserted.

[0081] The LV used in the methods and compositions described herein may include one or more of a 5'-long terminal repeat (LTR), an HIV signal sequence, an HIV Psi signal 5'-splice site (SD), a delta-GAG element, a Rev response element (RRE), a 3'-splice site (SA), an elongation factor (EF) 1-alpha promoter, and a 3'-self-inactivating LTR (SIN-LTR). The lentiviral vector optionally includes a central polypurine tract (cPPT) and a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), as described in US 6,136,597, the disclosure of which is incorporated herein by reference. The lentiviral vector may further include a pHR' backbone, which may include, for example, those provided below.

[0082] Lentigen LV, described in Lu et al., Journal of Gene Medicine 6:963,2004, can be used to express DNA molecules and / or transduce cells. The LV used in the methods and compositions described herein can 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 response element (RRE), a 3'-splice site (SA), an elongation factor (EF) 1-alpha promoter, and a 3'-self-inactivating LTR (SIN-LTR). Optionally, one or more of these regions are replaced with another region performing a similar function, as will be readily apparent to one of skill in the art.

[0083] Enhancer elements may be used to increase expression of modified DNA molecules or to increase lentiviral integration efficiency. LVs used in the methods and compositions described herein may contain a nef sequence. LVs used in the methods and compositions described herein may contain a cPPT sequence that enhances integration of the vector. The cPPT acts as a second origin of (+)-strand DNA synthesis and introduces a partial strand duplication in the middle of the native HIV genome. Introduction of a cPPT sequence in the transfer vector backbone strongly increased nuclear transport and the total amount of genome integrated into the DNA of the target cell. LVs used in the methods and compositions described herein may contain a woodchuck posttranscriptional regulatory element (WPRE). The WPRE acts at the transcriptional level by promoting nuclear export of the transcript and / or by increasing the efficiency of polyadenylation of the nascent transcript, thus increasing the total amount of mRNA in the cell. The addition of the WPRE to the LV results in a significant improvement in transgene expression levels from several different promoters, both in vitro and in vivo. The LV used in the methods and compositions described herein can contain both a cPPT sequence and a WPRE sequence. The vector can also contain an IRES sequence, which allows expression of multiple polypeptides from a single promoter.

[0084] In addition to IRES sequences, other elements that allow for the expression of multiple polypeptides are useful. The vectors used in the methods and compositions described herein may contain multiple promoters that allow for the expression of more than one polypeptide. The vectors used in the methods and compositions described herein may contain protein cleavage sites that allow for the expression of more than one polypeptide. Examples of protein cleavage sites that allow for the expression of more than one polypeptide 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 more than one polypeptide. It will be readily apparent to one of skill in the art that other elements that allow for the expression of multiple polypeptides that are identified in the future will be useful and may be utilized in vectors suitable for use with the compositions and methods described herein.

[0085] Other retroviral vectors (e.g., retroviral backbones) that can be used in conjunction with the compositions and methods described herein include gamma retroviral vectors. Exemplary gamma retroviral vectors are or are derived from chicken 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-Zeullen feline sarcoma virus, Troeger duck spleen necrosis virus, viper retrovirus, and woolly monkey sarcoma virus.

[0086] 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., ).

[0087] Other retroviral vectors (e.g., retroviral backbones) that can be used in conjunction with the compositions and methods described herein include alpharetroviral vectors. Exemplary alpharetroviral vectors are or are derived from avian cancer Mill Hill Virus 2, avian leukosis virus, avian myeloblastosis virus, avian myelomasis virus 29, avian sarcoma virus ct10, Fujinami sarcoma virus, Rous sarcoma virus, ur2 sarcoma virus, and y73 sarcoma virus.

[0088] Betaretrovirus pseudotypes The viral vectors used in conjunction with the compositions and methods described herein include a betaretrovirus pseudotyped envelope gene. The betaretrovirus envelope gene may be derived from a canonical type B or type D betaretrovirus. The betaretovirus pseudotype may be derived from any suitable betaretrovirus. Betaretroviruses include, for example, mouse mammary tumor virus (MMTV), endemic nasal tumor virus type 1 and type 2 (ENT-1 and ENT-2), simian retrovirus type 1, type 2 (SRV-1 and SRV-2), and type 3, Jaagsiekte sheep retrovirus (JSRV), squirrel monkey retrovirus (SMRV), brushtail possum endogenous type D retrovirus (TvERV-D), Mus musculus type D retrovirus (MusD), simian endogenous retrovirus (SERV), Mason-Pfizer monkey virus MPMV. In some embodiments, the betaretrovirus envelope gene is derived from a non-betaretrovirus vector. These viruses potentially acquire betaretrovirus pseudotypes through recombination and cross-species transmission. Suitable examples include BaEV, feline retrovirus RD114, Sin Nombre virus (SNV), and reticuloendotheliosis virus (REV). Envelope genes that may be used in conjunction with the compositions and methods described herein include those described in Baillie et al., J. Virol. 78:5784-5798, 2004, the disclosure of which is incorporated herein by reference in its entirety.

[0089] γδ T cells Gamma delta T cells (γδ T cells) represent a subset of T cells that express a distinct and definitive 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 as one or two types: peripheral blood resident γδ T cells and non-hematopoietic tissue resident γδ T cells. While most blood resident γδ T cells express Vδ2 TCR, this is less common among tissue resident γδ T cells, which more frequently use Vδ1 and / or other Vδ chains. The present invention provides γδ T cells transduced with a viral vector encoding a desired transgene as described herein.

[0090] In some embodiments, suitable γδ T cells for use as a source of engineered γδ T cells described herein include V51 cells, V52 cells, V53 cells, V55 cells, and V58 cells. In some embodiments, the engineered population of γδ T cells is derived from a population of V51 cells or V52 cells. In some cases, the engineered population of γδ T cells is derived from a population of non-V51 / V52 T cells. In some cases, the engineered population of γδ T cells is derived from a mixed population of V51 and V52 cells.

[0091] 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.

[0092] In one aspect, the invention provides a population of γδ T cells engineered to express one or more transgenes, which may encode a membrane-bound protein (e.g., a membrane-bound protein such as a chimeric antigen receptor (CAR)), an αβ TCR, a natural cytotoxicity receptor (e.g., NKp30, NKp44, or NKp46), a cytokine receptor (e.g., IL-12 receptor), 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 CCL19), a soluble protein (e.g., 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), a selectable marker (e.g., a reporter gene), or a suicide gene. In some cases, the invention provides a population of γδ T cells engineered to express a CAR and a protein encoded by one or more additional transgenes (e.g., an armor protein). In some embodiments, the one or more transgenes are codon optimized.

[0093] In some embodiments, the γδ T cells are transduced with a viral vector encoding a transgene. 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 cells may stably express the transgene. In some embodiments, the cells may transiently express the transgene.

[0094] In one aspect, the present invention provides an improved method for the treatment of γδ T cells (e.g., at least 10, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 1012 , or 10 13 In one embodiment, a cell population (e.g., an isolated cell population) of 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 cell population is a population of engineered γδ T cells that express a transgene (e.g., a CAR and / or one or more additional proteins).

[0095] How to harvest and expand γδ T cells 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 of engineered γδ T cells described herein include V51, V52, V53, V55, and V58 cells. In some embodiments, the population of engineered γδ T cells is derived from a population of V51 or V52 cells.

[0096] For example, provided herein are methods of 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 of isolating and expanding V51 cells from blood include, for example, those described in U.S. Pat. No. 9,499,788 and International Patent Publication No. WO 2016 / 198480, 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 may be engineered to express a transgene may be derived from non-hematopoietic tissue according to the methods described below.

[0097] Isolation and expansion of γδ T cells from 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 can be derived from blood-derived V52 cells or blood-derived V51 cells.

[0098] In some embodiments, peripheral blood mononuclear cells (PBMCs) can be obtained from a subject according to any suitable method known in the art. PBMCs can be cultured in the presence of aminobisphosphonates (e.g., zoledronic acid), synthetic phosphoantigens (e.g., bromohydrin pyrophosphate; BrHPP), 2M3B1PP, or 2-methyl-3-butenyl-1-pyrophosphate in the presence of IL-2 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 in the presence of IL-2, for example, for about 14 days. 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 cultures are maintained for about 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, γδ T cells from PBMCs 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., through positive selection with a TCR γδ specific agent or negative selection with a TCR αβ specific agent). Such methods and other suitable methods for expansion of γδ T cells, such as V52 cells, are described in Deniger et al., Frontiers in Immunology 5,636:1-10, 2014, which is incorporated herein by reference in its entirety.

[0099] In some embodiments, V51 T cells can be engineered to express a transgene (e.g., a heterologous targeting construct). Any suitable method of obtaining a population of V51 T cells can 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 can 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 give rise to more than 90% γδ T cells. These cells can be cultured 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 in a gas-permeable bioreactor bag. Variations of this method, and other methods of obtaining V51 T cells, are suitable as part of the invention. For example, blood-derived V51 T cells can alternatively be obtained using, for example, the methods described 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.

[0100] Isolation and expansion of non-hematopoietic tissue-resident γδ T cells from non-hematopoietic tissues Non-hematopoietic tissue resident γδ T cells obtained as described below can exhibit good tumor infiltration and retention capabilities and therefore may 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 UK Patent Application No. 2006989.4, each of which is incorporated herein by reference in its entirety.

[0101] 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 DN T cells) can be isolated from any human or non-human animal non-hematopoietic tissue that can be removed from a patient to obtain cells suitable for manipulation 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 obtained 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 resident in human cancer tissues, e.g., breast or prostate tumors. In some embodiments, γδ T cells can be derived from human cancer tissues (e.g., solid tumor tissues). In other embodiments, γδ T cells can be derived from non-hematopoietic tissues other than human cancer tissues (e.g., tissues that do not contain significant numbers of tumor cells). For example, γδ T cells may be derived from an area of ​​skin (eg, healthy skin) that is separate from nearby or adjacent cancer tissue.

[0102] While the γδ T cells that predominate in blood are primarily Vδ2 T cells, the γδ T cells that predominate in non-hematopoietic tissues are primarily 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., intestine, and they may comprise approximately 10-20% of the γδ T cells. Some γδ T cells that reside in non-hematopoietic tissues express neither Vδ1 nor Vδ2 TCRs, and they are termed double-negative (DN) γδ T cells. These DN γδ T cells are likely to be mostly Vδ3 expressing and contain a small number of Vδ5 expressing T cells. Thus, the γδ T cells that are normally resident in non-hematopoietic tissues and expanded by the methods of the present invention are preferably non-Vδ2 T cells, e.g., Vδ1 T cells, with a smaller number of DN γδ T cells.

[0103] In some embodiments, a key step is the deliberate separation of 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, and γδ2 and non-γδ2 T cells) from non-hematopoietic cells (e.g., stromal cells, in particular fibroblasts) of the tissue from which the T cells were obtained, e.g., after days or weeks of culture. This allows preferential and rapid expansion of non-hematopoietic tissue-derived Vδ1 T cells and DN γδ T cells over the following days and weeks.

[0104] In general, non-hematopoietic tissue resident γδ T cells are capable of spontaneously expanding upon removal of physical contact with stromal cells (e.g., skin fibroblasts). 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 invention provides methods of expanding non-hematopoietic tissue resident γδ T cells, which do not involve contact with supporting cells, tumor cells, and / or antigen-presenting cells.

[0105] Expansion protocols involve culturing non-hematopoietic tissue resident γδ T cells in the presence of an effective cocktail of biological factors to support efficient γδ T cell expansion. In one embodiment, a method of expanding γδ T cells comprises providing a population of γδ T cells obtained from a non-hematopoietic tissue (e.g., an isolated population of γδ T cells from a non-hematopoietic tissue, e.g., a population isolated according to the methods 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 duration 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).

[0106] 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, as well as RPMI-1640 (Life Technologies) and TEXMACS™ (Miltenyi Biotec). The medium may be supplemented with other media factors, such as serum, serum proteins, and selective agents, such as antibiotics. For example, in some embodiments, the medium comprises RPMI-1640 containing 2 mM glutamine, 10% FBS, 10 mM HEPES at 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 medium at 37° C. in a humidified atmosphere containing 5% CO 2 .

[0107] γδ T cells can 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.

[0108] The methods described herein can include more than one selection step, for example, more than one depletion step. Enrichment of T cell population by negative selection can be achieved, for example, by using a combination of antibodies that target surface markers specific to the negatively selected cells. One method is cell sorting and / or selection via negative magnetic immunoadhesion or flow cytometry, using a cocktail of monoclonal antibodies that target cell surface markers present on the negatively selected cells.

[0109] Transgene The engineered γδ T cells of the present invention are engineered to express a desired transgene. The γδ T cells engineered 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 may be used in conjunction with the compositions and methods described herein include chimeric antigen receptors (CARs).

[0110] In some embodiments, the CAR is selected from the group consisting of CD19, CD20, ROR1, CD22, carcinoembryonic antigen, alpha fetoprotein, CA-125, 5T4, MUC-1, epithelial tumor antigen, prostate specific antigen, melanoma associated antigen, mutated p53, mutated ras, HER2 / Neu, folate binding protein, HIV-1 envelope glycoprotein gpl20, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD33, CD138, CD23, CD30, CD56, c-Met, mesothelin, GD3, H Targeting ERV-K, IL-llR alpha, kappa chain, lambda chain, CSPG4, ERBB2, EGFRvIII, VEGFR2, HER2-HER3 in combination, HER1-HER2 in combination, NY-ESO-1, synovial sarcoma X breakpoint 2 (SSX2), melanoma antigen (MAGE), melanoma antigen 1 recognized by T cells (MART-1), gp100, prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), g9d2, or combinations thereof.

[0111] In some cases, 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 an intracellular signaling domain can be used as a transgene for in vivo depletion using an anti-EGFR monoclonal antibody, for example, in the 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 to facilitate drug-mediated control of the administered engineered γδ T cells. Through the use of a suicide gene, modified cells can be depleted from the patient in the case of an adverse event. In one example, a drug binding domain is fused to a caspase 9 pro-apoptotic molecule. In some cases, the transgene is a cytosine deaminase. In some cases, the transgene is a thymidine kinase.

[0112] Additionally or alternatively, the transgene for expression by the engineered γδ T of the present disclosure encodes a membrane-bound protein, such as a membrane-bound receptor (e.g., αβ TCR, 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 CCL19). Membrane-bound ligands and cytokines include natural 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 engineered γδ T of the present disclosure encodes a membrane-bound protein, such as a membrane-bound receptor (e.g., αβ TCR, 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). The transgene expressed by the T cells encodes a soluble protein, such as 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).

[0113] In some cases, engineered γδ T cells carrying a transgene encoding a CAR can be protected with an additional transgene that contributes to immunogenicity. Such protective 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 proteins such as membrane-bound receptors (e.g., αβ TCR, 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 include 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).

[0114] In some embodiments, an engineered γδ T cell of the invention is engineered to express one or more transgenes (e.g., any one or more of the transgenes described herein) to protect the γδ T cell (e.g., as a protective 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).

[0115] In some embodiments, the transgene is codon optimized.

[0116] 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 an engineered population of γδ T cells (e.g., V51 or V52 cells) express a transgene, e.g., a CAR or other 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 an engineered population of γδ T cells (e.g., V51 or V52 cells) express a transgene, e.g., a CAR or other 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 an engineered population of γδ T cells (e.g., Vδ1 or Vδ2 cells) express a transgene, e.g., a CAR or other membrane-bound or soluble protein. In some embodiments, between 3% and 95% (e.g., between 5% and 95%, between 10% and 95%, between 20% and 95%, between 25% and 95%, or between 50% and 95%) of an engineered population of γδ T cells (e.g., Vδ1 or Vδ2 cells) express a transgene, e.g., a 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 an engineered population of γδ T cells (e.g., Vδ1 or Vδ2 cells) express a transgene, e.g., a CAR or other membrane-bound or soluble protein. EXAMPLES

[0117] Materials and Methods Retroviral vector production and titration Lentiviral vectors were produced by transient transfection of HEK293 cells with a third-generation self-inactivating vector platform consisting of plasmids encoding genome (GFP or anti-CD19 chimeric antigen receptor), gag / pol, reverse transcriptase (rev), and envelope (VSV-G, BaEV).

[0118] Gammaretroviral vectors were produced by transient transfection of FLYRD18 cells with murine leukemia virus genomic plasmids (GFP or anti-CD19 chimeric antigen receptor). Vectors were harvested 48 hours after transfection, filtered through 0.45 um pore size polyethersulfone (PES) filters, and concentrated using low speed centrifugation (6,000 g at 4°C).

[0119] Vector titers were determined by transduction of a human cervical cancer cell line (HeLa) with serial dilutions of concentrated vector material in the presence of polybrene (8ug / mL). Transduction efficiency was determined 3 days after transduction using a BD FACS Lyric flow cytometer. The infectious titer (TU / mL) was calculated using the following formula: TU / mL = ((number of transduced cells) x vector dilution x (% transduction efficiency / 100)) / volume of vector (mL).

[0120] Flow cytometry Immunophenotyping was performed using a BD FACS Lyric flow cytometer. Cells were analyzed for expression of surface markers using PerCP-Vio700 anti-TCR α / β (Miltenyi), APC anti-TCR γ / δ (Miltenyi), and VioBlue anti-TCR Vδ1 (Miltenyi) antibodies. Viable cells were detected using eFluor 780 fixable viability dye. CAR19 expression was detected using FITC-labeled human CD19 protein (AcroBiosystems).

[0121] Isolation and expansion of γδ T cells A Vδ1 γδ T cell enriched product (GDX012) was produced using a modified protocol based on Almeida et al. Clin. Cancer Res. 22:5795-804, 2016. Briefly, αβ-depleted peripheral blood mononuclear cells were expanded using serum-free medium (CTS OpTmizer, Thermo Fisher) supplemented with 2.5% autologous plasma and Glutamax (ThermoFisher). The isolated cells were expanded in the presence of recombinant IL-4 [rIL4] (100 ng / mL), recombinant interferon gamma [rIFNγ] (70 ng / mL), recombinant IL-21 [rIL21] (7 ng / mL), recombinant IL-1β [rIL1β] (15 ng / mL), and soluble OKT-3 anti-CD3 monoclonal antibody (70 ng / mL). Cells were incubated at 37°C and 5% CO2 in a humidified incubator. The expanding cells were periodically fed with fresh medium containing recombinant IL-15 [rIL15] (70 ng / mL), IFNγ (30 ng / mL), and OKT3 (1 mg / mL).

[0122] Retroviral transduction Proliferating γδ T cells were transduced with retroviral vectors at a defined multiplicity of infection (MOI). MOI refers to the number of infectious particles added per cell during transduction (measured by flow cytometry). γδ T cells (1E+06 / mL) were transduced in non-tissue culture treated 24-well plates coated with retronectin (20 μg / mL) or in 24-well plates in the presence of vectofucin (1 μg / mL). Viral vectors were diluted in CTS OpTmizer medium supplemented with cytokines, OKT-3, and 2.5% autologous plasma (as above). γδ T cells and vector stocks were spun at 1,000×g for 2 h at 37° C. Transduction efficiency was determined using flow cytometry 3 days after transduction at regular intervals. In certain experiments, media was supplemented with the non-nucleoside reverse transcriptase inhibitor nevirapine (NVP) at a final concentration of 10 μM to inhibit reverse transcriptase activity.

[0123] Example 1. Broadly tropic VSV-G pseudotyped lentiviral vectors fail to transduce γδ T cells Lentiviral vectors encoding GFP were pseudotyped with vesicular stomatitis virus G (VSV-G) or baboon endogenous virus (BaEV) envelopes, respectively. Expanded γδ T cells (consisting of Vδ1, Vδ2, and non-Vδ1 / Vδ2 cells) were transduced with concentrated viral vector stocks at a defined multiplicity of infection (MOI). Transduction efficiency was determined using flow cytometry 3 days after transduction.

[0124] Flow cytometry analysis revealed that VSV-G pseudotyped lentiviral vectors were unable to transduce γδ T cells even at high MOIs (MOI 50 or higher, Figure 1A). Conversely, transduction with BaEV envelope lentiviral vectors resulted in high transduction efficiency even at low multiplicities of infection (Figure 1B). Pretreatment of γδ T cells with the reverse transcriptase inhibitor NVP abolished GFP expression, indicating that GFP expression was the result of successful transduction and GFP expression in Vδ1 cells.

[0125] Example 2. Transduction of V51 γδ T cells with lentiviral vectors encoding a VSV-G pseudotyped CAR results in pseudotransduction To determine whether CAR expression was the result of vector integration or mock transduction, Vδ1 γδ T cells were transduced with a lentiviral vector encoding a chimeric antigen receptor in the presence or absence of nevirapine (NVP). Nevirapine is a reverse transcriptase inhibitor that blocks viral transduction by inhibiting reverse transcription of viral RNA into cDNA. Thus, incubation of cells exposed to lentiviral vectors in the presence of nevirapine should decrease transgene expression. CAR expression was completely abolished when transduction with BaEV pseudotyped vectors was performed in the presence of nevirapine, demonstrating that CAR expression does not result from mock transduction (Figure 4B and Figure 6). Conversely, treatment of Vδ1 cells with nevirapine did not withdraw CAR expression in cells transduced with VSV-G pseudotyped lentiviral vectors. This result demonstrates that VSV-G pseudotyped vectors are unable to transduce Vδ1 cells and that transgene (CAR) expression is the result of mock transduction. Pseudotransduction was further confirmed by monitoring CAR expression over time after transduction (days 4 and 8 post-transduction). Monitoring vector-treated cells by FACS analysis revealed a gradual loss of CAR expression over time (Figure 2A). This phenomenon was also demonstrated across various multiplicities of infection in the presence or absence of NVP (Figure 2B). Overall, the results suggest that VSV-G pseudotyped lentiviral vectors are unable to enter γδ T cells.

[0126] Example 3. Cytokine priming is a major determinant of V51 γδ T cell transduction by BaEV pseudotyped lentiviral vectors To investigate whether BaEV transduction efficiency depends on the length of cytokine priming during γδ T cell expansion, Vδ1 cells were transduced at different time points during the cell expansion process. Cells were transduced with MOI=1 at the beginning of culture (day 0) or on days 7, 10, 14, and 15 of the expansion phase. Transduced cells were analyzed by flow cytometry for GFP expression 3 days after transduction. Transduction efficiency gradually increased during the cell expansion phase, reaching the highest level of transduction on day 15 (Figure 3A). Treatment of cells with NVP demonstrated that GFP expression was a result of successful vector integration (Figure 3B). Overall, the results suggest that an early "cytokine priming" phase is necessary for successful Vδ1 transduction with BaEV pseudotyped lentiviral vectors.

[0127] Example 4. Transduction efficiency of V51 γδ T cells correlates with multiplicity of infection (MOI) To investigate whether BaEV transduction efficiency depends on viral vector dose (MOI), Vδ1 γδ T cells were transduced with lentiviral vectors encoding increasing amounts of BaEV envelope pseudotyped anti-CD19 chimeric antigen receptors (CARs). Three days after transduction, cells were analyzed by flow cytometry for CAR expression. Increasing the MOI significantly increased the percentage of transduced Vδ1 cells (Figure 4A). Representative dot plots of CAR transduction at MOI=5 in the presence or absence of NVP are shown in Figure 4B.

[0128] Example 5. BaEV pseudotyped lentiviral vectors transduce both V51 and non-V51 (V52, V53) γδ T cells To test whether BaEV pseudotyped vectors exclusively transduce V51 cells or can also transduce other γδ T cell subtypes, transduction efficiency was determined within pan-γδ and V51 cell populations. γδ T cells were expanded and transduced with BaEV envelope lentiviral vectors encoding GFP or CAR at MOI=1 on day 10 of expansion. FACS analysis using pan-γδ and V51 specific antibodies revealed that BaEV envelope vectors transduce both V51 and non-V51 (V52, V53, and others) γδ T cells (Figure 5).

[0129] Example 6. Transduction of V51 γδ T cells with BaEV pseudotyped lentiviral vectors can be further enhanced by repeated transduction Studies were performed to determine whether sequential transduction could further enhance CAR expression in expanded V51 γδ T cells. To this end, V51 cells were transduced either once (day 10) or twice (days 10 and 11) at MOI=1. After 3 days, cells were collected and analyzed by FACS. Flow cytometry analysis revealed that a single vector hit could efficiently transduce V51 cells, an effect that could be further enhanced by dual transduction on consecutive days (Figure 6).

[0130] Example 7. Transduction in the presence of vectofucin is as efficient as in the presence of retronectin To test whether the choice of transduction enhancer has any effect on V51 transduction efficiency, two widely used transduction enhancers (retronectin and vectofusin) were evaluated. On day 10 of cell expansion, V51 cells were transduced at various MOIs in the presence of retronectin or vectofusin, and transduction efficiency was determined 3 days after transduction. FACS analysis revealed that vectofusin was as efficient as retronectin to increase retroviral gene transfer (Figure 7).

[0131] Example 8. V51 cells can be transduced with RD114 pseudotyped viral vectors To test whether V51 γδ T cells could be transduced by other betaretrovirus viral envelope pseudotyped vectors, V51 γδ T cells were also transduced with RD114 envelope pseudotyped gammaretroviral vectors. Cells were grown as before and transduced at MOI=1 on day 10 of expansion. FACS analysis revealed that RD114 envelope gammaretroviral vectors, like BaEV pseudotyped lentiviral vectors, were able to transduce V51 γδ T cells with high efficiency (Figure 8).

[0132] 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.

[0133] While the invention has been described in relation to specific embodiments thereof, it will be understood that further modifications are possible, and this application is generally intended to cover any variations, uses, or adaptations of the invention, including such departures from the present disclosure that come within known practice or custom within the art to which the invention pertains, in accordance with the principles of the invention, and may be applied to the essential features hereinbefore described, and which follow in the claims.

[0134] Other embodiments are within the scope of the claims.

Claims

1. 1. A method for producing a population of engineered γδ T cells, comprising transducing a population of γδ T cells with a viral vector comprising a betaretrovirus pseudotype and a Retroviridae viral vector backbone; wherein said Retroviridae viral vector backbone is a retroviral vector backbone that is a lentiviral backbone, a gammaretroviral backbone, or an alpharetroviral backbone.

2. 2. The method of claim 1, wherein the betaretrovirus pseudotype is RD114.

3. 2. The method of claim 1, wherein the retroviral vector backbone is a lentiviral backbone.

4. 2. The method of claim 1, wherein the retroviral vector backbone is a gammaretroviral backbone.

5. 2. The method of claim 1, wherein the retroviral vector backbone is an alpharetroviral backbone.

6. 2. The method of claim 1, wherein the engineered γδ T cells are Vδ1 T cells or Vδ2 T cells.

7. 2. The method of claim 1, wherein the engineered γδ T cells are non-Vδ1 / Vδ2 T cells.

8. The method described in claim 1, wherein the viral vector further comprises an introduced gene encoding a cell surface receptor.

9. 9. The method of claim 8, wherein the cell surface receptor is a chimeric antigen receptor (CAR).

10. The method described in claim 1, wherein the viral vector further comprises an introduced gene encoding a cytokine.

11. 1. A method for producing a population of engineered γδ T cells, comprising: (i) providing a starting population of γδ T cells; (ii) culturing the starting population of γδ T cells for a first culture period in the absence of a viral vector to produce a population of primed γδ T cells; (iii) culturing the population of primed γδ T cells for a second culture period in the presence of a viral vector comprising a betaretrovirus pseudotype and a Retroviridae viral vector backbone in an amount effective to transduce at least 3% of the primed γδ T cells, thereby producing the population of engineered γδ T cells; wherein said Retroviridae viral vector backbone is a retroviral vector backbone that is a lentiviral backbone, a gammaretroviral backbone, or an alpharetroviral backbone.

12. i. The first culture period is one day or more. ii. the second culture period is 2 days or longer; or iii. Both of i. and ii. above; The method of claim 11.

13. 1. The population of primed γδ T cells: i. expresses ASCT-1 and / or ASCT-2, or ii. lacking functional expression of the VSV-G entry receptor; The method of claim 11.

14. 12. The method of claim 11, wherein the viral vector is in an amount effective to transduce at least 20% of the primed γδ T cells.

15. 12. The method of claim 11, wherein the viral vector is cultured with the primed γδ T cells at a multiplicity of infection (MOI) of 10 or less.

16. 2. A population of engineered γδ T cells produced by the method of claim 1.

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