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Definitions

• y6 T cells represent a subset of T cells that express on their surface a distinct, defining y6 T-cell receptor (TCR). This TCR is made up of one gamma (y) and one delta (6) chain.
• Human y6 T cells can be broadly classified as one or two types: peripheral blood-resident y6 T cells and non-haematopoietic tissue-resident y6 T cells. Most blood-resident y6 T cells express a V62 TCR, whereas this is less common among tissueresident y6 T cells, which more frequently use V61 and/or other V6 chains.
• the invention features a method of producing a population of engineered y6 T cells by transducing a population of y6 T cells with a viral vector having a beta retroviral pseudotype and a Retroviridae family viral vector backbone.
• the betaretroviral pseudotype may be baboon endogenous virus (BaEV).
• the betaretroviral pseudotype may be RD114.
• the Retroviridae family viral vector backbone is a retroviral vector backbone (e.g., lentiviral backbone, gammaretroviral backbone, or alpharetroviral backbone).
• the engineered y6 T cells may be V61 T cells.
• the engineered y6 T cells may be V62 T cells.
• the engineered y6 T cells may be non-V61/V62 T cells.
• the viral vector includes 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 cytokine or a membrane-bound cytokine).
• the transgene encodes IL-15 (e.g., secreted IL-15 or membrane-bound IL-15).
• the viral vector includes a first transgene and a second transgene.
• the first transgene encodes a CAR
• 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).
• a cytokine e.g., IL-15
• secreted IL-15 e.g., secreted IL-15 or membrane-bound IL-15
• the CAR targets CD19, CD20, ROR1 , CD22, carcinoembryonic antigen, alphafetoprotein, 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, HERV-K, I L-IIRalpha, kappa chain, lambda chain, CSPG4, ERBB2, EGFRvlll, VEGFR2, HER2-HER3 in combination, HER1-HER2 in combination, NY-ESO-1 , synovial sarcoma X breakpoint 2 (SSX2), melanoma antigen (MAGE),
• the invention features a method of producing a population of engineered y6 T cells.
• the method includes providing a starting population of y6 T cells and culturing the starting population of y6 T cells for a first culture period in the absence of a viral vector to produce a population of primed y6 T cells.
• the method may further include culturing the population of primed y6 T cells for a second culture period in the presence of a viral vector having a betaretroviral 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 y6 T cells, thereby producing the population of engineered y6 T cells.
• a viral vector having a betaretroviral 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%,
• the viral vector is in an amount effective to transduce at least 20% of the primed y6 T cells.
• the first culture period is for 1 day or longer (e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or longer, e.g., 1-3 days, 3-5 days, 5-7 days, 7-10 days, or longer). In some embodiments, the first culture period is for 2 days or longer (e.g., 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or longer, e.g., 1-3 days, 3-5 days, 5-7 days, 7-10 days, or longer).
• the first culture period is for 5 days or longer (e.g., 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or longer, e.g., 5-7 days, 7-10 days, or longer). In some embodiments, the first culture period is for 7 days or longer (e.g., 7 days, 8 days, 9 days, 10 days, or longer, e.g., 7-10 days, or longer).
• the second culture period is for 2 days or longer (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 longer, e.g., 2-4 days, 4-7 days, 7-10 days, 10-14 days, or longer).
• the second culture period is for 7 days or longer (e.g., 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, or longer, e.g., 7-10 days, 10-14 days, or longer).
• the population of primed y6 T cells expresses ASCT-1 and/or ASCT-2. In some embodiments, the population of primed y6 T cells lacks functional expression of a VSV-G entry receptor (e.g., an LDL receptor).
• a VSV-G entry receptor e.g., an LDL receptor
• the viral vector is cultured with the primed y6 T cells at a multiplicity of infection (MOI) no greater than 10 (e.g., no greater than 5, e.g., from about 1 to about 5).
• MOI multiplicity of infection
• the invention features a method of producing a population of engineered y6 T cells by providing a starting population of y6 T cells; and culturing the starting population of y6 T cells in the presence of IL-15 and a viral vector having a betaretroviral pseudotype in an amount effective to transduce at least 3% (e.g., at least 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%m 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 y6 T cells, thereby producing the population of engineered y6 T cells.
• a viral vector having a betaretroviral pseudotype in an amount effective to transduce at least 3% (e.g., at least 4%, 5%, 6%, 7%
• the starting population of y6 T cells lack expression of ASCT-1 and/or ASCT-2.
• the population of engineered y6 T cells expresses ASCT-1 and/or ASCT-2.
• the starting population of y6 T cells may lack functional expression of a VSV-G entry receptor (e.g., an LDL receptor).
• the viral vector is cultured with the starting population of y6 T cells at an MOI no greater than 10 (e.g., no greater than 5, e.g., from about 1 to about 5).
• the viral vector has a beta retroviral pseudotype of BaEV or RD114.
• the viral vector includes a Retroviridae family viral vector backbone.
• the Retroviridae family viral vector backbone may be a retroviral vector backbone (e.g., lentiviral backbone, gammaretroviral backbone, or alpharetroviral backbone).
• the engineered y6 T cells may be V61 T cells.
• the engineered y6 T cells may be V62 T cells.
• the engineered y6 T cells may be non-V61/V62 T cells.
• the viral vector includes 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 cytokine or a membrane-bound cytokine).
• the transgene encodes IL-15 (e.g., secreted IL-15 or membrane-bound IL-15).
• the viral vector includes a first transgene and a second transgene.
• the first transgene encodes a CAR
• 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).
• a cytokine e.g., IL-15
• secreted IL-15 e.g., secreted IL-15 or membrane-bound IL-15
• the CAR targets CD19, CD20, ROR1 , CD22, carcinoembryonic antigen, alphafetoprotein, 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, HERV-K, I L-IIRalpha, kappa chain, lambda chain, CSPG4, ERBB2, EGFRvlll, VEGFR2, HER2-HER3 in combination, HER1-HER2 in combination, NY-ESO-1 , SSX2, MAGE, MART-1 , gp100, PSA, PSMA, PSCA, g9
• the invention features a method of producing a population of y6 T cells expressing a CAR by transducing a population of y6 T cells with a viral vector that includes a transgene encoding the CAR; a beta retroviral pseudotype; and a Retroviridae family viral vector backbone.
• the invention features a method of producing a population of y6 T cells expressing a CAR and an armor protein by transducing a population of y6 T cells with a viral vector that includes a first transgene encoding the CAR; a second transgene encoding the armor protein; a betaretroviral pseudotype; and a Retroviridae family viral vector backbone.
• 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).
• the betaretroviral pseudotype is BaEV. In other embodiments, the betaretroviral pseudotype is RD114.
• the viral vector includes a Retroviridae family viral vector backbone.
• the Retroviridae family viral vector backbone may be a retroviral vector backbone (e.g., lentiviral backbone, gammaretroviral backbone, or alpharetroviral backbone).
• the y6 T cells may be V61 T cells.
• the y6 T cells may be V62 T cells.
• the y6 T cells may be non-V61/V62 T cells.
• the invention features a method of producing a population of y6 T cells expressing a CAR by providing a starting population of y6 T cells and culturing the starting population of y6 T cells for a first culture period in the absence of a viral vector to produce a population of primed y6 T cells.
• the method may further include culturing the population of primed y6 T cells for a second culture period in the presence of a viral vector having a betaretroviral pseudotype and a transgene encoding the 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%m 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 y6 T cells, thereby producing the population of y6 T cells expressing the CAR.
• a viral vector having a betaretroviral pseudotype and a transgene encoding the CAR wherein the viral vector is in an amount effective to transduce at least 3% (e.g.
• the invention features a method of producing a population of y6 T cells expressing a CAR and an armor protein by providing a starting population of y6 T cells and culturing the starting population of y6 T cells for a first culture period in the absence of a viral vector to produce a population of primed y6 T cells.
• the method may further include culturing the population of primed y6 T cells for a second culture period in the presence of a viral vector having a betaretroviral pseudotype, a first transgene encoding the CAR, and a second transgene encoding the 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%m 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 y6 T cells, thereby producing the population of y6 T cells expressing the CAR and the armor protein.
• a viral vector having a betaretroviral pseudotype e.g., at least 4%, 5%,
• the transgene encodes IL-15 (e.g., secreted IL-15 or membranebound IL-15).
• the viral vector includes a first transgene and a second transgene.
• the first transgene encodes a CAR
• 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).
• the viral vector is in an amount effective to transduce at least 20% of the primed y6 T cells.
• the first culture period is for 1 day or longer (e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or longer, e.g., 1-3 days, 3-5 days, 5-7 days, 7-10 days, or longer). In some embodiments, the first culture period is for 2 days or longer (e.g., 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or longer, e.g., 1-3 days, 3-5 days, 5-7 days, 7-10 days, or longer).
• the first culture period is for 5 days or longer (e.g., 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or longer, e.g., 5-7 days, 7-10 days, or longer). In some embodiments, the first culture period is for 7 days or longer (e.g., 7 days, 8 days, 9 days, 10 days, or longer, e.g., 7-10 days, or longer).
• the second culture period is for 2 days or longer (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 longer, e.g., 2-4 days, 4-7 days, 7-10 days, 10-14 days, or longer).
• the second culture period is for 7 days or longer (e.g., 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, or longer, e.g., 7-10 days, 10-14 days, or longer).
• the population of primed y6 T cells expresses ASCT-1 and/or ASCT-2.
• the population of primed y6 T cells lacks functional expression of a VSV-G entry receptor (e.g., an LDL receptor). In some embodiments, more than 95% of the population of primed y6 T cells lacks a sufficient level of a VSV-G entry receptor expression (e.g., LDL receptor) to mediate detectable VSV-G entry (e.g., as measured by BlaM-Vpr-based assay).
• a VSV-G entry receptor e.g., an LDL receptor
• more than 95% of the population of primed y6 T cells lacks a sufficient level of a VSV-G entry receptor expression (e.g., LDL receptor) to mediate detectable VSV-G entry (e.g., as measured by BlaM-Vpr-based assay).
• more than 96% of the population of primed y6 T cells lacks a sufficient level of a VSV- G entry receptor expression (e.g., LDL receptor) to mediate detectable VSV-G entry (e.g., as measured by BlaM-Vpr-based assay).
• more than 97% of the population of primed y6 T cells lacks a sufficient level of a VSV-G entry receptor expression (e.g., LDL receptor) to mediate detectable VSV-G entry (e.g., as measured by BlaM-Vpr-based assay).
• more than 98% of the population of primed y6 T cells lacks a sufficient level of a VSV- G entry receptor expression (e.g., LDL receptor) to mediate detectable VSV-G entry (e.g., as measured by BlaM-Vpr-based assay). In some embodiments, more than 99% of the population of primed y6 T cells lacks a sufficient level of a VSV-G entry receptor expression (e.g., LDL receptor) to mediate detectable VSV-G entry (e.g., as measured by BlaM-Vpr-based assay).
• a VSV- G entry receptor expression e.g., LDL receptor
• the viral vector is cultured with the primed y6 T cells at an MOI no greater than 10 (e.g., no greater than 5, e.g., from about 1 to about 5).
• the invention features a method of producing a population of y6 T cells expressing a CAR by providing a starting population of y6 T cells; and culturing the starting population of y6 T cells in the presence of IL-15 and a viral vector having a betaretroviral pseudotype and a transgene encoding the 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%m 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 y6 T cells, thereby producing the population of engineered y6 T cells expressing the CAR.
• the viral vector is in an amount effective to transduce at least 3% (
• the invention features a method of producing a population of y6 T cells expressing a CAR and an armor protein by providing a starting population of y6 T cells; and culturing the starting population of y6 T cells in the presence of IL-15 and a viral vector having a betaretroviral pseudotype, a first transgene encoding the 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%m 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 y6 T cells, thereby producing the population of engineered y6 T cells expressing
• the starting population of y6 T cells lack expression of ASCT-1 and/or ASCT-2.
• the population of engineered y6 T cells may express ASCT-1 and/or ASCT-2.
• the starting population of y6 T cells may lack functional expression of a VSV-G entry receptor (e.g., an LDL receptor).
• a VSV-G entry receptor e.g., an LDL receptor
• more than 95% of the population of primed y6 T cells lacks a sufficient level of a VSV-G entry receptor expression (e.g., LDL receptor) to mediate detectable VSV- G entry (e.g., as measured by BlaM-Vpr-based assay).
• more than 96% of the population of primed y6 T cells lacks a sufficient level of a VSV-G entry receptor expression (e.g., LDL receptor) to mediate detectable VSV-G entry (e.g., as measured by BlaM-Vpr-based assay).
• more than 97% of the population of primed y6 T cells lacks a sufficient level of a VSV- G entry receptor expression (e.g., LDL receptor) to mediate detectable VSV-G entry (e.g., as measured by BlaM-Vpr-based assay).
• more than 98% of the population of primed y6 T cells lacks a sufficient level of a VSV-G entry receptor expression (e.g., LDL receptor) to mediate detectable VSV-G entry (e.g., as measured by BlaM-Vpr-based assay). In some embodiments, more than 99% of the population of primed y6 T cells lacks a sufficient level of a VSV- G entry receptor expression (e.g., LDL receptor) to mediate detectable VSV-G entry (e.g., as measured by BlaM-Vpr-based assay).
• a VSV-G entry receptor expression e.g., LDL receptor
• the viral vector is cultured with the starting population of y6 T cells at an MOI no greater than 10 (e.g., no greater than 5, e.g., from about 1 to about 5).
• the betaretroviral pseudotype is BaEV or RD114.
• the viral vector includes a Retroviridae family viral vector backbone.
• the Retroviridae family viral vector backbone may be a retroviral vector backbone (e.g., lentiviral backbone, gammretroviral backbone, or alpharetroviral backbone).
• the engineered y6 T cells may be V61 T cells.
• the engineered y6 T cells may be V62 T cells.
• the engineered y6 T cells may be non-V61/V62 T cells.
• the CAR targets CD19, CD20, ROR1 , CD22, carcinoembryonic antigen, alphafetoprotein, 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, HERV-K, I L-IIRalpha, kappa chain, lambda chain, CSPG4, ERBB2, EGFRvlll, VEGFR2, HER2-HER3 in combination, HER1-HER2 in combination, NY-ESO-1 , SSX2, MAGE, MART-1 , gp100, PSA, PSMA, PSCA, g9
• the invention features a population of engineered y6 T cells produced by a method as described herein.
• 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
• at least 50% e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or substantially all
• the population of engineered y6 T cells expresses a CAR.
• 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
• 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 membranebound IL-15).
• a cytokine e.g., a secreted cytokine or a membrane-bound cytokine (e.g., IL-15, e.g., secreted IL-15 or membranebound 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 engineered y6 T cells expresses 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).
• 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 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
• 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 engineered y6 T cells expresses a CAR and 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).
• 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).
• the invention features a population of y6 T cells expressing a CAR produced by a method as described herein.
• the invention features a population of y6 T cells expressing a CAR and an armor protein produced by a method as described herein.
• 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).
• engineered y6 T cell refers to a y6 T cell that expresses a transgene (i.e., a gene that has been transduced into the engineered y6 T cell or a parental cell thereof).
• primed y6 T cell refers to a starting population (e.g., an endogenous population of y6 T cells) that has been affected by a culture condition.
• a primed y6 T cell has a different functional viral entry receptor profile relative to its unprimed counterpart before experiencing the culture condition.
• a population of primed y6 T cells is an expanded population of y6 T cells.
• an “expanded population of y6 cells” refers to a population of haematopoietic cells including y6 T cells that has been cultured in a condition and for a duration that has induced the expansion of y6 cells, i.e., increased y6 cell number.
• an “expanded population of V61 T cells,” as used herein, refers to a population of haematopoietic cells including V61 T cells that has been cultured in a condition and for a duration that has induced the expansion of V61 T cells, i.e., increased V61 cell number.
• an “expanded population of V62 T cells,” as used herein, refers to a population of haematopoietic cells including V62 T cells that has been cultured in a condition and for a duration that has induced the expansion of V62 T cells, i.e., increased V62 cell number.
• a “population” of y6 T cells refers to a group of three or more y6 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 1 °, at least 10 11 , at least 10 12 , or at least 10 13 ) y6 T cells (e.g., engineered y6 T cells).
• y6 T cells e.g., engineered y6 T cells
• a population of a particular cell type refers to the cells of that type and not to cells of a different type within a broader population. For example, if 10% of the cells of a starting population of 10 8 T cells are y6 T cells, the starting population of y6 T cells is 10 7 .
• an “armor protein” refers to a protein encoded by a transgene that, when expressed by a y6 T cell (e.g., a y6 T cell expressing a CAR), increases persistent or increased immunogenicity of the y6 T cell toward a target cell, e.g., through paracrine signaling (e.g., cytokine signaling) to improve, e.g., cell persistence, cell viability, activation and other desired characteristics.
• An armor protein can be a membrane-bound protein or a soluble protein.
• armor proteins include membrane-bound proteins, such as a membrane-bound receptor (e.g., ap 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, membranebound 4-1 BB, membrane-bound 4-1 BBL, membrane bound CCL19).
• a membrane-bound receptor e.g., ap TCR, a natural cytotoxicity receptor (e.g., NKp30, NKp44, or NKp46)
• a cytokine receptor e.g., IL-12 receptor
• chemokine receptor e.g., CCR2 receptor
• 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 4-1 BBL, and/or soluble CCL19).
• soluble proteins such as soluble ligands or cytokines (e.g., soluble IL-15, soluble IL-7, soluble IL-12, soluble CD40L, soluble 4-1 BBL, and/or soluble CCL19).
• an armor protein is not antigen specific.
• IL-15 refers to native or recombinant IL-15 or a variant thereof that acts as an agonist for one or more IL-15 receptor (IL-15R) subunits (e.g., mutants, muteins, analogues, subunits, receptor complexes, fragments, isoforms, and peptidomimetics thereof).
• IL-15 like IL-2, is a known T-cell growth factor that can support proliferation of an IL-2-dependent cell line, CTLL-2.
• IL-15 was first reported by Grabstein et al. (Science 264.5161 : 965-969, 1994) as a 114-amino acid mature protein.
• IL-15 means native or recombinant IL-15 and muteins, analogs, subunits thereof, or complexes thereof (e.g., receptor complexes, e.g., sushi peptides, as described in PCT Pub. No. WO 2007/046006), and each of which can stimulate proliferation of CTLL-2 cells.
• CTLL-2 proliferation assays supernatants of cells transfected with recombinantly expressed precursor and in-frame fusions of mature forms of IL-15 can induce CTLL-2 cell proliferation.
• Human IL-15 can be obtained according to the procedures described by Grabstein et al. (Science 264.5161 : 965-969, 1994) or by conventional procedures such as polymerase chain reaction (PCR). A deposit of human IL-15 cDNA was made with the ATCC® on Feb. 19, 1993, and assigned accession number 69245.
• the amino acid sequence of human IL-15 (Gene ID 3600) is found in Genbank under accession locator NP000576.1 Gl: 10835153 (isoform 1) and NP_751915.1 Gl: 26787986 (isoform 2).
• the murine (Mus musculus) IL-15 amino acid sequence (Gene ID 16168) is found in Genbank under accession locator NP_001241676.1 Gl: 363000984.
• IL-15 can also refer to IL-15 derived from a variety of mammalian species, including, for example, human, simian, bovine, porcine, equine, and murine.
• An IL-15 "mutein” or “variant”, as referred to herein, is a polypeptide substantially homologous to a sequence of a native mammalian IL- 15 but that has an amino acid sequence different from a native mammalian IL-15 polypeptide because of an amino acid deletion, insertion, or substitution. Variants may comprise conservatively substituted sequences, meaning that a given amino acid residue is replaced by a residue having similar physiochemical characteristics.
• Naturally occurring IL-15 variants are also encompassed by the invention.
• examples of such variants are proteins that result from alternate mRNA splicing events or from proteolytic cleavage of the IL-15 protein, wherein the IL-15 binding property is retained. Alternate splicing of mRNA may yield a truncated but biologically active IL-15 protein.
• Variations attributable to proteolysis include, for example, differences in the N- or C-termini upon expression in different types of host cells, due to proteolytic removal of one or more terminal amino acids from the IL-15 protein (generally from 1-10 amino acids).
• the terminus of the protein can be modified to alter its physical properties, for example, with a chemical group such as polyethylene glycol (Yang et al. Cancer 76:687-694, 1995).
• the terminus or interior of the protein can be modified with additional amino acids (Clark-Lewis et al. PNAS 90:3574- 3577, 1993).
• non-haematopoietic cells include stromal cells and epithelial cells.
• Stromal cells are non-haematopoietic connective tissue cells of any organ and support the function of the parenchymal cells of that organ. Examples of stromal cells include fibroblasts, pericytes, mesenchymal cells, keratinocytes, endothelial cells, and non-hematological tumor cells.
• Epithelial cells are non-haematopoietic cells that line the cavities and surfaces of blood vessels and organs throughout the body. They are normally squamous, columnar, or cuboidal in shape and can be arranged as a single layer of cells, or as layers of two or more cells.
• non-haematopoietic tissue-resident y6 T cells refer to y6 T cells that were present in a non-haematopoietic tissue at the time the tissue is explanted.
• Non-haematopoietic tissue-resident y6 T cells may be obtained from any suitable human or non-human animal non-haematopoietic tissue.
• Non-haematopoietic tissue is a tissue other than blood or bone marrow.
• the y6 T cells are not obtained from particular types of samples of biological fluids, such as blood or synovial fluid.
• suitable human or non-human animal non-haematopoietic tissues include skin or a portion thereof (e.g., dermis or epidermis), the gastrointestinal tract (e.g., gastrointestinal epithelium, colon, small intestine, stomach, appendix, cecum, or rectum), mammary gland tissue, lung (preferably wherein the tissue is not obtained by bronchoalveolar lavage), prostate, liver, and pancreas.
• non-haematopoietic tissue-resident y6 T cells can be derived from a lymphoid tissue, such as thymus, spleen, or tonsil.
• the y6 T cells may also be resident in human cancer tissues, e.g., breast and prostate. In some embodiments, the y6 T cells are not obtained from human cancer tissue.
• Non-haematopoietic tissue samples may be obtained by standard techniques e.g., by explant (e.g., biopsy).
• Non-haematopoietic tissue-resident y6 T cells include e.g., V61 T cells, double negative (DN) T cells, V62 T cells, V63 T cells, and V65 T cells.
• the phrase “in an amount effective to” refers to an amount that induces a detectable result (e.g., a number of cells having a statistically significant increased number relative to its starting population, e.g., at a p ⁇ 0.05).
• an “expanded population of y6 cells” refers to a population of haematopoietic cells including y6 T cells that has been cultured in a condition and for a duration that has induced the expansion of y6 cells, i.e., increased y6 cell number.
• an “expanded population of V61 T cells,” as used herein, refers to a population of haematopoietic cells including V61 T cells that has been cultured in a condition and for a duration that has induced the expansion of V61 T cells, i.e., increased V61 cell number.
• an “expanded population of V62 T cells,” as used herein, refers to a population of haematopoietic cells including V62 T cells that has been cultured in a condition and for a duration that has induced the expansion of V62 T cells, i.e., increased V62 cell number
• marker herein to refers to a DNA, RNA, protein, carbohydrate, glycolipid, or cellbased molecular marker, the expression or presence of which in a patient's sample can be detected by standard methods (or methods disclosed herein).
• a cell or population of cells that “expresses” a marker of interest is one in which mRNA encoding the protein, or the protein itself, including fragments thereof, is determined to be present in the cell or the population.
• Expression of a marker can be detected by various means.
• expression of a marker refers to a surface density of the marker on a cell.
• Mean fluorescence intensity (MFI) for example, as used as a readout of flow cytometry, is representative of the density of a marker on a population of cells.
• MFI mean fluorescence intensity
• a person of skill in the art will understand that MFI values are dependent on staining parameters (e.g., concentration, duration, and temperature) and fluorochrome composition. However, MFI can be quantitative when considered in the context of appropriate controls.
• a population of cells can be said to express a marker if the MFI of an antibody to that marker is significantly higher than the MFI of an appropriate isotype control antibody on the same population of cells, stained under equivalent conditions.
• a population of cells can be said to express a marker on a cell-by-cell basis using a positive and negative gate according to conventional flow cytometry analytical methods (e.g., by setting the gate according to isotype or “fluorescence-minus-one” (FMO) controls).
• FMO fluorescence-minus-one
• 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 the target population of cells, as measured by a beta-lactamase-Vpr (BlaM-VpR)-based assay. See, e.g., Cavrois et al., Nat Biotechnol. 11 :1151-1154, 2002.
• a population of cells that “lacks functional expression of a VSV-G entry receptor” more than 95% of the cell population lacks the sufficient level of VSV-G entry receptor expression to mediate detectable VSV-G entry, as measured by a BlaM-VpR-based assay.
• a population when a population’s expression is stated as a percentage of positive cells and that percentage is compared to a corresponding percentage of positive cells of a reference population, the percentage difference is a percentage of the parent population of each respective population.
• population A is said to have a 9% greater frequency of marker-positive cells than population B (i.e., 10%-1 %, not 10%- %).
• a frequency is multiplied through by the number of cells in the parent population, the difference in absolute number of cells is calculated. In the example given above, if there are 100 cells in population A, and 10 cells in population B, then population A has 100-fold the number of cells relative to population B, i.e., (10% x 100) + (1% x 10).
• An expression level of a 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 a 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® technique, in situ hybridization (e.g., FISH), or combinations thereof.
• SAGE serial analysis of gene expression
• MASSARRAY® technique e.g., FISH
• a “reference population” of cells refers to a population of cells corresponding to the cells of interest, against which a phenotype of the cells of interest are measured. For example, a level of expression of a marker on a separated population of non-haematopoietic tissue-derived y6 cells may be compared to the level of expression of the same marker on a haematopoietic tissue- derived y6 T cell (e.g., a blood-resident y6 cell, e.g., a blood-resident y6 cell derived from the same donor or a different donor) or a non-haematopoietic tissue-derived y6 T cell expanded under different conditions (e.g., in the presence of substantial TCR activation, in the presence of an exogenous TCR activation agent (e.g., anti-CD3), or in substantial contact with stromal cells (e.g., fibroblasts)).
• a population may also be compared to itself at an earlier state.
• a reference population can be a separated cell population prior to its expansion.
• the expanded population is compared to its own composition prior to the expansion step, i.e., its past composition, in this case, is the reference population.
• chimeric antigen receptor or alternatively a “CAR” refers to a recombinant polypeptide construct including an extracellular antigen binding domain, a transmembrane domain, and, optionally, an intracellular domain that propagates an activation signal that activates the cell and/or a costimulatory signal.
• the CAR includes an optional leader sequence at the N-terminus of the CAR fusion protein.
• FIGS. 1A and 1 B are graphs showing broad tropism VSV-G pseudotyped lentiviral vectors cannot transduce V81 y ⁇ T cells.
• Representative dot plots show y ⁇ T cells transduced with VSV-G (FIG. 1A) or BaEV (FIG. 1 B) pseudotyped GFP encoding lentiviral vectors using various multiplicity of infections at day 7 of the expansion culture. Transduction efficiency was determined by FACS analysis 72 hours post-transduction. UTD, untransduced controls; MOI, multiplicity of infection; NVP, nevirapine (RT inhibitor).
• FIGS. 2A and 2B are graphs showing ransduction of V81 y ⁇ T-cells with VSV-G pseudotyped CAR encoding lentiviral vectors result in pseudotransduction.
• UTD untransduced controls
• MOI multiplicity of infection
• CAR chimeric antigen receptor
• NVP nevirapine.
• FIGS. 3A and 3B are graphs showing that cytokine priming is a major determinant of V81 y8 T cells transduction by BaEV pseudotyped lentiviral vectors.
• FIG. 3B shows representative dot plots of cells transduced at day 14 of the expansion culture.
• UTD untransduced controls
• MOI multiplicity of infection
• GFP green fluorescent protein
• NVP nevirapine.
• FIGS. 4A and 4B are graphs showing transduction efficiency of V81 y8 T cells correlates with the multiplicity of infection (MOI).
• FIG. 4A shows percentage of CAR+ve V81 cells 3 days after transduction with CAR encoding BaEV pseudotyped lentiviral vectors with different MOIs. Cells were transduced on day 10 of the expansion.
• UTD untransduced controls; MOI, multiplicity of infection; CAR, chimeric antigen receptor; NVP, nevirapine.
• FIGS. 5A and 5B are graphs showing BaEV pseudotyped lentiviral vectors transduce both V81 and non-V81 (V82, V83) y8 T-cells.
• Dot plots show CAR (FIG. 5A) and GFP (FIG. 5B) expressing V81 and non-V81 (V82, V83) y8 T-cells.
• FIG. 6 is a set of graphs showing transduction of V81 y8 T cells with BaEV pseudotyped lentiviral vectors can be further enhanced by repeated transductions.
• FIG. 7 is a graph showing transduction in the presence of vectofusin is as efficient as in the presence of retronectin.
• V81 cells were transduced in the presence of retronectin (left) or vectofusin (right) with various MOIs and various frequencies (one or two hit). Cells were transduced on day 10 of the expansion and FACS analysis was conducted three days post-transduction.
• FIG. 8 is a set of graphs showing V81 cells can be transduced with RD114 pseudotyped viral vectors.
• the present invention provides methods of engineering y6 T cells (e.g., v61 T cells and v62 T cells) by transduction with a viral vector (e.g., a viral vector with a betaretroviral pseudotype and a Retroviridae family viral vector backbone). Further provided are compositions of engineered y6 T cells and methods of using the same.
• a viral vector e.g., a viral vector with a betaretroviral pseudotype and a Retroviridae family viral vector backbone.
• the present invention is based, in part, on the unexpected discovery that y6 T cells can be transduced with a betaretroviral pseudotyped viral vector to a high level.
• y6 T cells are non-permissive for retroviral transductions, e.g., using a VSV-G pseudotyped viral vector.
• VSV-G vectors easily transduce ap T cells as well as NK cells, which are the closest cell types to y6 T cells.
• a betaretroviral pseudotyped viral vector would be able to transduce y6 T cells.
• the present invention also based on the discovery of optimal culture conditions and durations of y6 T cells in the presence of a viral vector in order to transduce a population y6 T cells with the vector.
• the methods of transduction described herein allow efficient transduction of y6 T cells in order to produce an engineered population of y6 T cells expressing a desired transgene.
• the invention provides a method for producing a population of engineered y6 T cells by transducing a population of y6 T cells (e.g., V61 T cells, V62 T cells, and/or non-V61/V62 T cells) with a viral vector that includes a betaretroviral pseudotype and a Retroviridae family (e.g., retroviral) vector backbone.
• the retroviral vector backbone may be, e.g., a lentiviral backbone, a gammaretroviral backbone, or an alpharetroviral backbone.
• the betaretroviral psuedotype may be, e.g., BaEV or RD114. In some embodiments the betaretroviral psuedotype is BaEV. In some embodiments the betaretroviral psuedotype is RD114.
• the invention provides a method of producing a population of engineered y6 T cells by providing a starting population of y6 T cells, priming the y6 T cells in the absence of a viral vector, and culturing the population of primed y6 T cells in the presence of a 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 y6 T cells.
• at least 3% e.g., at least 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%,
• the population of primed y6 T cells is cultured in the presence of a viral vector in an amount effective to transduce at least 5% of the primed y6 T cells. In some embodiments, the population of primed y6 T cells is cultured in the presence of a viral vector in an amount effective to transduce at least 20% of the primed y6 T cells.
• the primed y6 T cells may be obtained by culturing the starting population of y6 T cells in the absence of a viral vector.
• the starting population of y6 T cells may be cultured for a first culture period of at least 1 hour (e.g., at least 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, or longer, e.g., from about 1 hourto about 14 days, from about 6 hours to about 14 days, from about 1 day to about 14 days, from about 2 days to about 14 days, from about 5 days to about 14 days, from about 7 days to about 14 days, from about 5 days to about 10 days, from about 5 days to about 7 days, or from about 7 days to about 10 days).
• the primed y6 T cells When the primed y6 T cells are obtained, e.g., following culturing of the cells in the absence of a viral vector, the primed y6 T cells may be further cultured for a second culture period of at least 1 day (e.g., at least 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 longer, e.g., from about 1 day to about 14 days, from about 2 days to about 14 days, from about 5 days to about 14 days, from about 7 days to about 14 days, from about 5 days to about 10 days, from about 5 days to about 7 days, or from about 7 days to about 10 days).
• a second culture period of at least 1 day e.g., at least 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 longer, e.g.
• the second culture period may be from about 1 day to about 14 days (e.g., from about 3 days to about 14 days, from about 3 days to about 12 days, from about 4 days to about 1 days, from about 5 days to about 10 days, or from about 5 days to about 7 days).
• the viral vector is cultured with the primed y6 T cells at a multiplicity of infection (MOI) of no greater than about 10, e.g., no greater than about 9, 8, 7, 6, 5, 4, 3, 2, 1 , 0.5, or 0.25. In some embodiments, viral vector is cultured with the primed y6 T cells at a multiplicity of infection (MOI) of no greater than about 5. In some embodiments, viral vector is cultured with the primed y6 T cells at a multiplicity of infection (MOI) of no greater than about 4. In some embodiments, viral vector is cultured with the primed y6 T cells at a multiplicity of infection (MOI) of no greater than about 3.
• MOI multiplicity of infection
• viral vector is cultured with the primed y6 T cells at a multiplicity of infection (MOI) of no greater than about 2. In some embodiments, viral vector is cultured with the primed y6 T cells at a multiplicity of infection (MOI) of no greater than about 1 . In some embodiments, viral vector is cultured with the primed y6 T cells at a multiplicity of infection (MOI) of no greater than about 0.5. In some embodiments, viral vector is cultured with the primed y6 T cells at a multiplicity of infection (MOI) of no greater than about 0.25. In some embodiments, viral vector is cultured with the primed y6 T cells at a multiplicity of infection (MOI) of from 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).
• MOI multiplicity of infection
• transduction of y6 T cells includes the use of a transduction enhancer to enhance transduction efficiency.
• Suitable transduction enhancers include, e.g., vectorfusin, spermid, and/or retronectin.
• the methods may include contacting the y6 T cells with the transduction enhancer during culturing. In some embodiments, the method further includes contact the cells with nevirapine.
• transduction of y6 T cells includes supplementing the culture medium with a IL-15, which can increase y6 T cell expression of ASCT-2, the viral entry receptor for a betaretroviral pseudotyped viral vector.
• y6 T cells may be spun e.g., by centrifugation, while being cultured with a viral vector (e.g., in combination with one or more additional agents described herein). This “spinoculation” process may occur with a centripetal force of, e.g., from about 200 x g to about 2,000 x g.
• the centripetal force may be, e.g., from about 300 x g to about 1 ,200 x g (e.g., about 300 x g, 400 x g, 500 x g, 600 x g, 700 x g, 800 x g, 900 x g, 1 ,000 x g, 1 ,100 x g, or 1 ,200 x g, or more).
• the y6 T cells are spun for from 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).
• the y6 T cells are spun at room temperature, such as at a temperature of about 25° C.
• Exemplary transduction protocols involving a spinoculation step are described, e.g., 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.
• compositions and methods described herein include the use of betaretroviral pseudotyped viral vectors for efficient transduction of y6 T cells.
• Viral genomes provide a rich source of vectors that can be used for the efficient delivery of exogenous genes into a mammalian cell.
• Viral genomes are particularly useful vectors for gene delivery as the polynucleotides contained within such genomes are typically incorporated into the nuclear genome of a mammalian cell by generalized or specialized transduction. These processes occur as part of the natural viral replication cycle, and do not require added proteins or reagents in order to induce gene integration.
• Examples of viral vectors that can be betaretroviral pseudotyped include retrovirus (e.g., Retroviridae family viral vector).
• retroviruses are: avian leukosis-sarcoma, avian C-type viruses, mammalian C-type, B- type viruses, D-type viruses, oncoretroviruses, HTLV-BLV group, lentivirus, alpharetrovirus, beta retrovirus, gammaretrovirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, Virology, Third Edition (Lippincott-Raven, Philadelphia, (1996))).
• murine leukemia viruses MMVs
• murine sarcoma viruses 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 ape leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus, and lentiviruses.
• vectors that can be pseudotyped with betraretrovirus for the present methods are described, for example, in McVey et al., (U.S. Pat. No. 5,801 ,030), the teachings of which are incorporated herein by reference.
• the viral vector used in the methods and compositions described herein is a retroviral vector.
• retroviral vector One type of retroviral vector that may be used in the methods and compositions described herein is a lentiviral vector.
• Lentiviral vectors LVs
• LVs Lentiviral vectors
• An overview 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.
• lentivirus-based gene transfer techniques relies on the in vitro production of recombinant lentiviral particles carrying a highly deleted viral genome in which the transgene of interest is accommodated.
• the recombinant lentivirus are recovered through the in trans coexpression in a permissive cell line of (1) the packaging constructs, i.e., a vector expressing the Gag-Pol precursors together with Rev (alternatively expressed in trans); (2) a vector expressing an envelope protein, generally of an heterologous nature; and (3) the transfer vector, consisting in the viral cDNA deprived of all open reading frames, but maintaining the sequences required for replication, encapsidation, and expression, in which the sequences to be expressed are inserted.
• a LV used in the methods and compositions described herein may include one or more of a 5'-Long terminal repeat (LTR), HIV signal sequence, HIV Psi signal 5'-splice site (SD), delta-GAG element, Rev Responsive Element (RRE), 3'-splice site (SA), elongation factor (EF) 1 -alpha promoter and 3'-self inactivating LTR (SIN-LTR).
• the lentiviral vector optionally includes a central polypurine tract (cPPT) and a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), as described in US 6,136,597, the disclosure of which is incorporated herein by reference as it pertains to WPRE.
• the lentiviral vector may further include a pHR' backbone, which may include for example as provided below.
• Lentigen LV described in Lu et al., Journal of Gene Medicine 6:963, 2004, may be used to express the DNA molecules and/or transduce cells.
• a LV used in the methods and compositions described herein may a 5'-Long terminal repeat (LTR), HIV signal sequence, HIV Psi signal 5'-splice site (SD), delta-GAG element, Rev Responsive Element (RRE), 3'-splice site (SA), elongation factor (EF) 1 -alpha promoter and 3'-self inactivating L TR (SIN-LTR). It will be readily apparent to one skilled in the art that optionally one or more of these regions is substituted with another region performing a similar function.
• Enhancer elements can be used to increase expression of modified DNA molecules or increase the lentiviral integration efficiency.
• the LV used in the methods and compositions described herein may include a nef sequence.
• the LV used in the methods and compositions described herein may include a cPPT sequence which enhances vector integration.
• the cPPT acts as a second origin of the (+)-strand DNA synthesis and introduces a partial strand overlap in the middle of its native HIV genome.
• the introduction of the cPPT sequence in the transfer vector backbone strongly increased the nuclear transport and the total amount of genome integrated into the DNA of target cells.
• the LV used in the methods and compositions described herein may include a Woodchuck Posttranscriptional Regulatory Element (WPRE).
• WPRE Woodchuck Posttranscriptional Regulatory Element
• the WPRE acts at the transcriptional level, by promoting nuclear export of transcripts and/or by increasing the efficiency of polyadenylation of the nascent transcript, thus increasing the total amount of mRNA in the cells.
• the addition of the WPRE to LV results in a substantial improvement in the level of transgene expression from several different promoters, both in vitro and in vivo.
• the LV used in the methods and compositions described herein may include both a cPPT sequence and WPRE sequence.
• the vector may also include an IRES sequence that permits the expression of multiple polypeptides from a single promoter.
• the vector used in the methods and compositions described herein may include multiple promoters that permit expression more than one polypeptide.
• the vector used in the methods and compositions described herein may include a protein cleavage site that allows expression of more than one polypeptide. Examples of protein cleavage sites that allow 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.
• retroviral vectors e.g., retroviral backbones
• retroviral vectors include gammaretroviral vectors.
• Exemplary gamma retroviral vectors are, or are derived from, chick syncytial virus, feline leukemia virus, finkel-biskis- jinkins murine sarcoma virus, gardner-arnstein feline sarcoma virus, gibbon ape leukemia virus, guinea pig type-c oncovirus, hardy-zuckerman feline sarcoma virus, harvey murine sarcoma virus, kirsten murine sarcoma virus, koala retrovirus, moloney murine sarcoma virus, murine leukemia virus, porcine type-c oncovirus, reticuloendotheliosis virus, snyder-theilen feline sarcoma virus, horrr duck spleen necros
• the viral vector backbone is derived from a lentivirus (LV). In certain embodiments, the viral vector backbone is derived from a third-generation self-inactivation (SIN) lentiviral vector (LV) (e.g., HIV, SIV, or EIAV). In certain embodiments, the viral vector backbone is derived from a LV (e.g.,) that is not self-inactivating.
• LV lentivirus
• LV third-generation self-inactivation lentiviral vector
• the viral vector backbone is derived from a LV (e.g.,) that is not self-inactivating.
• retroviral vectors e.g., retroviral backbones
• alpharetroviral vectors are, or are derived from, avian carcinoma mill hill virus 2, avian leukosis virus, avian myeloblastosis virus, avian myelocytomatosis virus 29, avian sarcoma virus ct10, fujinami sarcoma virus, rous sarcoma virus, ur2 sarcoma virus, and y73 sarcoma virus.
• the viral vectors used in conjunction with the compositions and methods described herein includes a betaretroviral psueodotyped envelope gene.
• the beta retroviral envelope gene may be from a canonical type B or type D betaretrovirus.
• the betaretoviral psueodtype may be derived from any suitable betaretrovirus.
• Betaretroviruses include, for example, mouse mammary tumor virus (MMTV), enzootic nasal tumor virus types 1 and 2 (ENT-1 and ENT-2), siminan retrovirus types 1 , 2 (SRV-1 and SRV-2), and 3, jaagsiekte sheep retrovirus (JSRV), squirrel monkey retrovirus (SMRV), Trichosurus Vulpecula endogenous type D retrovirus (TvERV-D), Mus musculus type D retovirus (MusD), simian endogenous retrovirus (SERV), Mason-Pfizer monkey virus MPMV.
• the betaretroviral envelope gene is from a non-betaretroviral vector.
• viruses have potentially acquired the betaretroviral pseudotype through recombination and cross-species transmission. Suitable examples include BaEV, feline retrovirus RD114, sinopi virus (SNV), and reticuloendotheliosis virus (REV). Envelope genes that may be used in conjunction with the compositions and methods described herein include those from viruses described in Baillie et al., J. Virol. 78: 5784-5798, 2004, the disclosure of which is hereby incorporated by reference in its entirety. y6 T cells
• Gamma delta T cells represent a subset of T cells that express on their surface a distinct, defining y6 T-cell receptor (TCR).
• This TCR is made up of one gamma (y) and one delta (6) chain.
• Human y6 T cells can be broadly classified as one or two types — peripheral blood-resident y6 T cells and non-haematopoietic tissue-resident y6 T cells. Most blood-resident y6 T cells express a V62 TCR, whereas this is less common among tissue-resident y6 T cells, which more frequently use V61 and/or other V6 chains.
• the invention provides y6 T cells that are transduced with a viral vector encoding a desired transgene as described herein.
• suitable y6 T cells for use as a source for the presently described engineered y6 T cells include V61 cells, V62 cells, V63 cells, V65 cells, and V68 cells.
• the population of engineered y6 T cells is derived from a population of V61 cells or V62 cells. In some instances, the population of engineered y6 T cells is derived from a population of non- V61/V62 T cells. In some instances, population of engineered y6 T cells is derived from a mixed population of V61 cells and V62 cells.
• the y6 T cells described herein may lack a vesicular stomatis virus G glycoprotein (VSV-G) entry receptor (e.g., LDL).
• VSV-G vesicular stomatis virus G glycoprotein
• the y6 T cell e.g., endogenous y6 T cells or primed y6 T cells
• the y6 T cell may express ASCT-1 and/or ASCT-2.
• the expression of ASCT-1 and/or ASCT-2 may permit transduction with a betaretroviral pseudotyped vector (e.g., BaEV and RD114).
• the lack of expression of VSV-G may prevent transduction with a VSV-G psuedotyped vector.
• the invention provides a population of y6 T cells engineered to express one or more transgenes, which may encode a membrane-bound protein (e.g., a cell surface receptor, such as a chimeric antigen receptor (CAR), an ap 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, membranebound IL-7, membrane-bound CD40L, membrane-bound 4-1 BB, membrane-bound 4-1 BBL, membrane bound CCL19), a soluble protein (e.g., soluble ligands or cytokines, e.g., soluble IL-15, soluble IL-7, soluble IL-12, soluble CD40L, soluble 4
• the invention provides a population of y6 T cells engineered to express a CAR and one or more additional transgene-encoded proteins (e.g., an armor protein).
• the one or more transgenes are codon optimized.
• the y6 T cell is transduced with a viral vector encoding a transgene.
• the viral vector is a retroviral vector.
• the viral vector is a lentiviral vector.
• the cell may stably express the transgene.
• the cell may transiently express the transgene.
• the invention features a cell population (e.g., an isolated cell population) of engineered y6 T cells (e.g., at least 10, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 1 °, 10 11 , 10 12 , or 10 13 cells), wherein 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 are of engineered y6 T cells expressing the transgene (e.g., the CAR and/or one or more additional proteins).
• the transgene e.g., the CAR and/or one or more
• Engineered y6 T cells of the invention can be derived from any suitable autologous or allogeneic y6 T cell or population thereof.
• suitable y6 T cells for use as a source for the presently described engineered y6 T cells include V61 cells, V62 cells, V63 cells, V65 cells, and V68 cells.
• the population of engineered y6 T cell is derived from a population of V61 cells or V62 cells.
• suitable y6 T cells can be derived from blood (e.g., peripheral blood). Methods of isolating and expanding V61 cells from blood include those described, for example, in U.S. Patent No. 9,499,788 and International Patent Publication No. WO 2016/198480, each of which is incorporated herein by reference in its entirety.
• suitable y6 T cells can be derived from tumor tissue (e.g., tumor-infiltrating y6 T cells).
• suitable y6 T cells that can be engineered to express a transgene can be derived from non-haematopoietic tissue according to methods described below.
• the engineered y6 T cells of the present invention are derived from blood (e.g., peripheral blood) of a subject.
• engineered y6 T cells may be derived from blood-derived V62 cells or blood-derived V61 cells.
• peripheral blood mononuclear cells 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), 2M3B1 PP, or 2-methyl-3-butenyl-1 -pyrophosphate in the presence of IL-2 for one-to-two weeks to generate an enriched population of V62 cells.
• aminobisphosphonates e.g., zoledronic acid
• synthetic phosphoantigens e.g., bromohydrin pyrophosphate; BrHPP
• 2M3B1 PP e.g., 2-methyl-3-butenyl-1 -pyrophosphate
• immobilized anti- TCRyQ can induce preferential expansion of V62 cells from a population of PBMCs in the presence of IL-2, e.g., for approximately 14 days.
• preferential expansion of V62 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.
• the aforementioned culture is maintained for about seven days prior to subculture in soluble anti-CD3, IL-2, and IL-4.
• artificial antigen presenting cells can be used to promote preferential expansion of y6 T cells, such as V62 cells.
• PBMC-de rived y6 T cells cultured in the presence of irradiated aAPC, IL-2, and/or IL-21 can expand to generate a population of y6 T cells including a high proportion of V62 cells, moderate proportion of V61 cells, and some double negative cells.
• PBMCs can be pre-enriched or post-enriched (e.g., through positive selection with TCRyQ-specific agents or negative selection of TCRap-specific agents).
• TCRyQ-specific agents e.g., through positive selection with TCRyQ-specific agents or negative selection of TCRap-specific agents.
• V61 T cells can be engineered to express a transgene (e.g., a heterologous targeting construct).
• a transgene e.g., a heterologous targeting construct
• Any suitable method of obtaining a population of V61 T cells can be used.
• Almeida et al. (Clinical Cancer Research, 22, 23; 5795-5805, 2016), incorporated herein by reference in its entirety, provides suitable methods of obtaining a population of V61 T cells that can be engineered to express a heterologous targeting construct described herein.
• PBMCs are pre-enriched using magnetic bead sorting, which can yield greater than 90% y6 T cells.
• V61 T 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-y) in gas-permeable bioreactor bags for up to 21 days or more.
• factors e.g., TCR agonists, co-receptor agonists, and/or cytokines, e.g., IL-4, IL-15, and/or IFN-y
• blood derived V61 T cells can alternatively be obtained using methods described, for example, in U.S. Patent No. 9,499,788 and International Patent Publication No. WO 2016/198480, each of which is incorporated herein by reference in its entirety.
• Non-haematopoietic tissue-resident y6 T cells obtained as described below can be suitable vehicles for transgenes described herein, as they can exhibit good tumor penetration and retention capabilities. More detailed methods for isolation and expansion of non-haematopoietic tissue-resident y6 T cells can be found, for example, in PCT Pub. Nos. WO 2020/095058, WO 2020/095059, WO 2017/072367, and GB App. No. 2006989.4, each of which is incorporated herein by reference in its entirety.
• Non-haematopoietic tissue-resident y6 T cells can be isolated from any human or non-human animal non- haematopoietic tissue that can be removed from a patient to obtain cells suitable for engineering according to the methods of the present invention.
• the non-haematopoietic tissue from which the y6 T cells are derived and expanded is skin (e.g., human skin), which can be obtained by methods known in the art.
• the skin is obtained by punch biopsy.
• the methods of isolation and expansion of y6 T cells provided herein can be applied to the gastrointestinal tract (e.g., colon), mammary gland, lung, prostate, liver, spleen, and pancreas.
• the y6 T cells may also be resident in human cancer tissues, e.g., tumors of the breast or prostate.
• the y6 T cells may be from human cancer tissues (e.g., solid tumor tissues).
• the y6 T cells may be from non-haematopoietic tissue other than human cancer tissue (e.g., a tissue without a substantial number of tumor cells).
• the y6 T cells may be from a region of skin (e.g., healthy skin) separate from a nearby or adjacent cancer tissue.
• the y6 T cells that are dominant in the blood are primarily V62 T cells, while the y6 T cells that are dominant in the non-haematopoietic tissues are primarily V61 T cells, such that V61 T cells include about 70-80% of the non-haematopoietic tissue-resident y6 T cell population.
• V62 T cells are also found in non-haematopoietic tissues, e.g., in the gut, where they can include about 10-20% of y6 T cells.
• Some y6 T cells that are resident in non-haematopoietic tissues express neither V61 nor V62 TCR and we have named them double negative (DN) y6 T cells.
• DN y6 T cells are likely to be mostly V63-expressing with a minority of V65-expressing T cells. Therefore, the y6 T cells that are ordinarily resident in non-haematopoietic tissues and that are expanded by the method of the invention are preferably non-V62 T cells, e.g., V61 T cells, with the inclusion of a smaller amount of DN y6 T cells.
• a critical step is the deliberate separation, e.g., after some days or weeks of culture, of non-haematopoietic tissue-resident T cells (e.g., within a mixed lymphocyte population, which may for example include ap cells, natural killer (NK) cells, B cells, and y62 and non- y62 T cells) away from the non-haematopoietic cells (e.g., stromal cells, particularly fibroblasts) of the tissue from which the T cells were obtained.
• a mixed lymphocyte population which may for example include ap cells, natural killer (NK) cells, B cells, and y62 and non- y62 T cells
• stromal cells particularly fibroblasts
• non-haematopoietic tissue-resident y6 T cells are capable of spontaneously expanding upon removal of physical contact with stromal cells (e.g., skin fibroblasts).
• stromal cells e.g., skin fibroblasts
• the scaffold-based culture methods described above can be used to induce such separation, resulting in de-repression of the y6 T cells to trigger expansion.
• no substantial TCR pathway activation is present during the expansion step (e.g., no exogenous TCR pathway activators are included in the culture).
• the invention provides methods of expanding non-haematopoietic tissue-resident y6 T cells, wherein the methods do not involve contact with feeder cells, tumor cells, and/or antigen-presenting cells.
• Expansion protocols involve culturing non-haematopoietic tissue-resident y6 T cells in the presence of effective cocktails of biological factors to support efficient y6 T cell expansion.
• the method of expanding y6 T cells includes providing a population of y6 T cells obtained from a non-haematopoietic tissue (e.g., a separated population of non-haematopoietic tissue-derived y6 T cells, e.g., a population separated according to the methods described herein) and culturing the y6 T cells in the presence of IL-2 and, IL-15, and optionally IL-1 p, IL-4, and/or IL-21 .
• a non-haematopoietic tissue e.g., a separated population of non-haematopoietic tissue-derived y6 T cells, e.g., a population separated according to the methods described herein
• IL-2 and, IL-15 optionally IL-1
• cytokines or analogues thereof can be cultured with the cells for a duration (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 longer, e.g., from 5 days to 40 days, from 7 days to 35 days, from 14 days 28 days, or about 21 days) in an amount effective to produce an expanded population of y6 T cells.
• a duration 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 longer, e.g., from 5 days to 40 days, from 7 days to 35 days, from 14 days 28 days, or about 21 days
• basal culture media suitable for use in the priming and/or expansion of yG T cells are available, such as complete media, OPTIMIZERTM, AIM-V, Iscoves medium and RPMI-1640 (Life Technologies) and TEXMACSTM (Miltenyi Biotec).
• the medium may be supplemented with other media factors, such as serum, serum proteins and selective agents, such as antibiotics.
• a media includes RPMI-1640 containing 2 mM glutamine, 10% FBS, 10 mM HEPES, pH 7.2, 1% penicillin-streptomycin, sodium pyruvate (1 mM; Life Technologies), non- essential amino acids (e.g., 100 pM Gly, Ala, Asn, Asp, Glu, Pro and Ser; 1X MEM non-essential amino acids Life Technologies), and 10 pl/L p-mercaptoethanol.
• cells are cultured at 37°C in a humidified atmosphere containing 5% CO2 in a suitable culture medium.
• the y6 T cells may be cultured as described herein in any suitable system, including stirred tank fermenters, airlift fermenters, roller bottles, culture bags or dishes, and other bioreactors, such as hollow fiber bioreactors.
• suitable system including stirred tank fermenters, airlift fermenters, roller bottles, culture bags or dishes, and other bioreactors, such as hollow fiber bioreactors.
• stirred tank fermenters airlift fermenters, roller bottles, culture bags or dishes, and other bioreactors, such as hollow fiber bioreactors.
• Other bioreactors such as hollow fiber bioreactors.
• General methods and techniques for culture of lymphocytes are well-known in the art.
• the methods described herein can include more than one selection step, e.g., more than one depletion step.
• Enrichment of a T cell population by negative selection can be accomplished, e.g., with a combination of antibodies directed to surface markers unique to the negatively selected cells.
• One method is cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected.
• the engineered y6 T cells of the present invention are engineered to express a desired transgene.
• y6 T cells engineered to express a transgene are suitable for use in cancer treatment (e.g., immunotherapy).
• the viral vectors described herein encode the transgene, which is then stably or transiently expressed in the transduced y6 T cells.
• Transgenes that can be used in conjunction with the compositions and methods described herein include chimeric antigen receptors (CARs).
• the CAR targets CD19, CD20, ROR1 , CD22, carcinoembryonic antigen, alphafetoprotein, 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, HERV-K, I L-IIRalpha, kappa chain, lambda chain, CSPG4, ERBB2, EGFRvlll, VEGFR2, HER2-HER3 in combination, HER1-HER2 in combination, NY-ESO-1 , synovial sarcoma X breakpoint 2 (SSX2), melanoma antigen (MAGE),
• a transgene to be expressed by the engineered y6 T cells of the present invention includes a selectable marker (e.g., a reporter gene) or a suicide gene.
• a selectable marker e.g., a reporter gene
• a suicide gene e.g., truncated epidermal growth factor receptor (EGFR), lacking the intracellular signaling domain, can be used as a transgene for in vivo depletion in the event of, e.g., toxicity, using anti-EGFR monoclonal antibodies.
• EGFR epidermal growth factor receptor
• CD20 can be used as a transgene for in vivo depletion using anti-CD20 monoclonal antibodies.
• Another exemplary transgene is a suicide gene to facilitate drug-mediated control of administered engineered y6 T cells.
• modified cells can be depleted from the patient in case of an adverse event.
• a drug-binding domain is fused to the caspase9 pro-apoptotic molecule.
• the transgene is cytosine deaminase. In some instances, the transgene is thymidine kinase.
• transgenes for expression by the engineered y6 T cells of the present invention encode membrane-bound proteins, such as a membrane-bound receptor (e.g., ap 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, membranebound 4-1 BB, membrane-bound 4-1 BBL, membrane bound CCL19).
• a membrane-bound receptor e.g., ap TCR, a natural cytotoxicity receptor (e.g., NKp30, NKp44, or NKp46), a cytokine receptor (e.g., IL-12 receptor), and/or a chemokine receptor
• Membrane-bound ligands and cytokines include naturally membrane-bound ligands and cytokines (e.g., trans-presented IL-15 and 4-1 BBL) and synthetic membrane-bound configurations (e.g., ligands that have been artificially fused to a trans-membrane protein). Additionally, or alternatively, transgenes to be expressed by the engineered y6 T cells of the present invention encode soluble proteins, such as soluble ligands or cytokines (e.g., soluble IL-15, soluble IL-7, soluble IL-12, soluble CD40L, soluble 4-1 BBL, and/or soluble CCL19).
• soluble proteins such as soluble ligands or cytokines (e.g., soluble IL-15, soluble IL-7, soluble IL-12, soluble CD40L, soluble 4-1 BBL, and/or soluble CCL19).
• engineered y6 T cells having a transgene that encodes a CAR can be armored with an additional transgene that contributes to immunogenicity.
• Such armored CAR T cells express an armor protein, such as any of the membrane-bound or soluble proteins described herein.
• armor proteins include membrane-bound proteins, such as a membrane-bound receptor (e.g., ap 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 4-1 BB, membrane-bound 4-1 BBL, membrane bound CCL19).
• a membrane-bound receptor e.g., ap TCR, a natural cytotoxicity receptor (e.g., NKp30, NKp44, or NKp46)
• a cytokine receptor e.g., IL-12 receptor
• chemokine receptor e.g., CCR2 receptor
• armor proteins to be expressed by the engineered y6 CAR T cells of the present invention include soluble proteins, such as soluble ligands or cytokines (e.g., soluble IL-15, soluble IL- 7, soluble IL-12, soluble CD40L, soluble 4-1 BBL, and/or soluble CCL19).
• soluble proteins such as soluble ligands or cytokines (e.g., soluble IL-15, soluble IL- 7, soluble IL-12, soluble CD40L, soluble 4-1 BBL, and/or soluble CCL19).
• the engineered y6 T cells of the present invention are engineered to express one or more transgenes (e.g., one or more of any of the transgenes described herein) for armoring the y6 T cells (e.g., as an armored 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).
• one or more transgenes e.g., one or more of any of the transgenes described herein
• the y6 T cells e.g., as an armored 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.
• the transgene is codon-optimized.
• 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
• the engineered population of y6 T cells e.g., V61 or V62 cells
• express the transgene e.g., the CAR or other membrane-bound or soluble protein.
• 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
• the engineered population of y6 T cells e.g., V61 or V62 cells
• express the transgene e.g., the CAR or other membrane-bound or soluble protein.
• At least 50% e.g., at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or substantially all
• the engineered population of y6 T cells express the transgene, e.g., the CAR or other membrane-bound or soluble protein.
• 3%-95% e.g., 5%-95%, 10%-95%, 20%-95%, 25%-95%, or 50%-95%) of the engineered population of y6 T cells (e.g., V61 or V62 cells) express the transgene, e.g., the CAR or other membrane-bound or soluble protein.
• 3%-90% e.g., 5%-90%, 10%- 90%, 20%-90%, 25%-90%, or 50%-90%) of the engineered population of y6 T cells (e.g., V61 or V62 cells) express the transgene, e.g., the CAR or other membrane-bound or soluble protein.
• Lentiviral vectors were produced by transient transfection of HEK293 cells with a third- generation self-inactivating vector platform consisting of genome (GFP or anti-CD19 chimeric antigen receptor), gag/pol, reverse transcriptase (rev) and envelope (VSV-G, BaEV) encoding plasmids.
• GFP genome
• rev reverse transcriptase
• VSV-G envelope
• BaEV envelope
• Gamma retroviral vectors were produced by transient transfection of FLYRD18 cells with murine leukaemia virus genome plasmid (GFP or anti-CD19 chimeric antigen receptor). Vectors were harvested 48 hours post-transfection, filtered through 0.45 urn pore size polyethersulfone (PES) filters and concentrated using low-speed centrifugation (6,000 g at 4 °C).
• GFP murine leukaemia virus genome plasmid
• PES polyethersulfone
• TU/mL ((number of transduced cells) x vector dilution x (% transduction efficiency/100))/volume of vector (mL).
• Immunophenotyping was performed using a BD FACS Lyric flow cytometer.
• Cells were analysed for the expression of surface markers using PerCP- Vio700 anti-TCR a/ (Miltenyi), APC anti-TCR y/8 (Miltenyi) and VioBlue anti-TCR V81 (Miltenyi) antibodies.
• Viable cells were detected using eFluor 780 fixable viability dye.
• CAR19 expression was detected using FITC labelled human CD19 protein (AcroBiosystems). yd T-cell isolation and expansion
• V81 y8 T-cell enriched product (GDX012) was produced using a modified protocol based on Almeida et al. Clin. Cancer Res. 22: 5795-804, 2016. Briefly, ap-depleted peripheral blood mononuclear cells were expanded using serum-free culture medium (CTS OpTmizer, Thermo Fisher) supplemented with 2.5% autologous plasma and Glutamax (ThermoFisher).
• CTS OpTmizer serum-free culture medium
• Thermo Fisher serum-free culture medium supplemented with 2.5% autologous plasma and Glutamax (ThermoFisher).
• the isolated cells were grown in the presence of recombinant IL-4 [rlL4] (100 ng/mL), recombinant interferon-y [rlFNy] (70 ng/mL), recombinant IL-21 [rlL21] (7 ng/mL), recombinant IL-1 p [rlL1 p] (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 incubattor. Expanding cells were regularly fed with fresh medium containing recombinant IL-15 [rlL15] (70 ng/mL), IFNy (30 ng/mL), and OKT3 (1 mg/mL).
• y8 T-cells were transduced with retroviral vectors at defined multiplicity of infection (MOI).
• MOI refers to the number of infectious particles (measured by flow cytometry) that were added per cell during transduction.
• y8 T cells (1 E+06/mL) were transduced in RetroNectin coated (20
• Viral vector was diluted in CTS OpTmizer medium supplemented with cytokines, OKT-3 and 2.5% autologous plasma (as above).
• y ⁇ T cells and vector stocks were spinoculated at 1 ,000 x g for 2 hours at 37 °C. Transduction efficiency was determined using flow cytometry after three days posttransduction at regular intervals.
• the culture media was supplemented with nevirapine (NVP), a non-nucleoside reverse transcriptase inhibitor, at 10
• NTP nevirapine
• GFP encoding lentiviral vectors were pseudotyped with vesicular stomatitis virus G (VSV-G) or baboon endogenous virus (BaEV) envelope, respectively.
• VSV-G vesicular stomatitis virus G
• BaEV baboon endogenous virus
• Expanded y ⁇ T-cells consisting of V81 , V82 and non-V81/V82 cells
• MOI multiplicity of infections
• VSV-G psdeudotyped lentiviral vectors fail to transduce y8 T cells even at high MOIs (MOI 50 and above, FIG. 1A).
• MOI 50 and above, FIG. 1A MOI 50 and above, FIG. 1A
• transduction with BaEV enveloped lentiviral vector resulted in high transduction efficiencies even at low multiplicity of infections (FIG. 1 B).
• Pre-treatment of y8 T cells with the reverse transcriptase inhibitor NVP abolished GFP expression, indicating that GFP expression was a result of successful transduction and GFP expression in V81 cells.
• V81 y8 T cells were transduced with chimeric antigen receptor encoding lentiviral vectors in the presence or absence of nevirapine (NVP).
• NTP nevirapine
• Nevirapine is a reverse transcriptase inhibitor that blocks viral transduction by inhibiting the reverse transcription of viral RNA to cDNA.
• incubation of the cells exposed to lentiviral vectors in the presence of nevirapine should diminish transgene expression.
• CAR expression was completely abolished when transduction with BaEV pseudotyped vector was performed in the presence of nevirapine, demonstrating that CAR expression did not result from pseudotransduction (FIGS. 4B and 6).
• VSV-G pseudotyped vectors are not able to transduce V81 cells, and transgene (CAR) expression is a result of pseudotransduction.
• CAR transgene
• Pseudotransduction was further confirmed by monitoring CAR expression over extended periods of time following transductions (4 and 8-days post-transduction). Monitoring the vector treated cells by FACS analysis revealed that the CAR expression was gradually lost over the time (FIG. 2A). This phenomenon was also demonstrated across various multiplicity of infections in the presence or absence NVP (FIG. 2B). Overall, the results suggest that VSV-G pseudotyped lentiviral vectors cannot enter to y8 T cells.
• Cytokine priming is a major determinant of V81 y8 T cells transduction by BaEV pseudotyped lentiviral vectors
• V81 cells were transduced at different time points during the cell expansion process.
• Transduced cells were analysed by flow cytometry for GFP expression three days post-transduction. Transduction efficiencies were gradually increased during the cell expansion phase and reached the highest level of transduction at day 15 (FIG. 3A).
• Treatment of the cells with NVP demonstrated that the GFP expression was a consequence of successful vector integration (FIG. 3B).
• FIG. 3B Treatment of the cells with NVP demonstrated that the GFP expression was a consequence of successful vector integration
• CAR chimeric antigen receptor
• Example 5 BaEV pseudotyped lentiviral vectors transduce both V81 and non-V81 (V82, V83) y8 T cells
• Example 7 Transduction in the presence of vectofusin is as efficient as in the presence of retronectin
• transduction enhancer has any influence on V81 transduction efficiencies.
• two widely used transduction enhancer retronectin and vectofusin were evaluated.
• V81 cells were transduced with various MOIs in the presence of retronectin or vectofusin and transduction efficiencies were determined three days post-transduction.
• FACS analysis revealed that vectofusin was as efficient to increase retroviral gene transfer as retronectin (FIG. 7).
• Example 8 V81 cells can be transduced with RD114 pseudotyped viral vectors

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