Amplification of gamma-delta T cells, compositions and uses thereof

Abstract

The present invention provides methods for expanding γδ T cells from non-hematopoietic tissues as a cell source, compositions of the expanded γδ T cells, and methods for using the γδ T cells (e.g., as part of adoptive T cell therapy). [Solution] Provided is a method for expanding γδ T cells by the following steps: (i) providing a population of γδ T cells obtained from non-hematopoietic tissue; (ii) culturing the cells in the presence of IL-2, IL-15, and a factor selected from the group consisting of IL-4, IL-21, IL-6, IL-7, IL-8, IL-9, IL-12, IL-18, IL-33, IGF-1, IL-1β, human platelet lysate (HPL), and stromal cell-derived factor-1 (SDF-1) for at least 5 days to generate an expanded population of γδ T cells.

Description

Background Art

[0001] The increasing interest in T cell immunotherapy for cancer has focused particularly on the demonstrable ability of subsets of CD8+ and CD4+ αβ T cells to recognize cancer cells and mediate host-protective functional potential when derepressed by clinically mediated antagonism of inhibitory pathways exerted by PD1, CTLA4 and other receptors. Nevertheless, many questions remain. For example, there appear to be a number of major clinical situations where the effectiveness of such treatments seems poor. In many cases, serious adverse events occur, the ability to predict effectiveness or adverse events is extremely limited, and there is little explanation about the interactions (“immunogenicity”) that enable the host to sense tumor cells and that must precede the activation of conventional antigen-specific CD8+ and CD4+ αβ T cell responses.

[0002] Gamma delta T cells (γδ T cells) are a subset of T cells that express a distinctively characteristic gamma delta T cell receptor (TCR) on their surface. This TCR consists of one gamma (γ) chain and one delta (δ) chain. Human γδ T cells are broadly classified into one or two - peripheral blood resident γδ T cells and non-hematopoietic tissue resident γδ T cells. Most blood resident γδ T cells express the Vδ2 TCR, which is not common in tissue resident γδ TCR cells, where Vδ1 and / or other Vδ chains are used. It has not been easy to obtain a large amount of non-hematopoietic tissue resident γδ T cells, and there is no standard protocol for isolation and amplification, so characterization and research for therapeutic applications have not been fully advanced. Therefore, there are problems to be solved regarding methods for isolating and amplifying a sufficient amount of non-hematopoietic tissue resident γδ T cells for studying them and potentially applying them to therapies such as adoptive T cell therapy.

Summary of the Invention

[0003] The present invention provides a method for amplifying γδ T cells (e.g., skin-derived γδ T cells and / or non-Vδ2 T cells, e.g., Vδ1 T cells and / or DN T cells) from non-hematopoietic tissue sources (e.g., non-hematopoietic tissue-derived γδ T cells, e.g., non-hematopoietic tissue-derived Vδ1 T cells). The amplification method includes culturing γδ T cells (e.g., γδ T cells isolated from stromal cells of non-hematopoietic tissue) under conditions substantially free of TCR stimulation and / or in the presence of IL-4, IL-15, IL-21, and / or IL-2. Further provided are the amplified γδ T cells (e.g., non-hematopoietic tissue-derived γδ T cells, e.g., non-hematopoietic tissue-derived Vδ1 T cells), and methods of using the γδ T cells (e.g., as part of adoptive T cell therapy, e.g., for cancer treatment).

[0004] In one aspect, the present invention features a method for amplifying γδ T cells by the following steps: (i) providing a population of γδ T cells obtained from non-hematopoietic tissue; (ii) culturing for at least 5 days in the presence of IL-2 and a factor selected from the group consisting of IL-15, IL-4, IL-21, IL-6, IL-7, IL-8, IL-9, IL-12, IL-18, IL-33, IGF-1, IL-1β, human platelet lysate (HPL), and stromal cell-derived factor-1 (SDF-1) to generate an amplified population of γδ T cells. For example, the γδ T cells can be cultured in the presence of IL-2, IL-15, and IL-4; IL-2, IL-15, and IL-21; or IL-2, IL-15, IL-4, and IL-21.

[0005] In another aspect, the present invention provides a method for amplifying γδ T cells by the following steps: (i) providing a population of γδ T cells obtained from non-hematopoietic tissue; (ii) culturing the γδ T cells for at least 5 days in the presence of effective amounts of IL-2, IL-4, IL-15, and IL-21 to generate an amplified population of γδ T cells.

[0006] In some embodiments of both of the foregoing aspects, the γδ T cells are simultaneously exposed to IL-2, IL-4, IL-15, and IL-21 for at least 5 days. In some embodiments, step (ii) includes culturing the γδ T cells under conditions without an exogenous TCR pathway agonist. In some embodiments, the method further includes, after step (i), separating γδ T cells from non-hematopoietic cells to generate a population of isolated γδ T cells, and step (ii) includes: (a) culturing the γδ T cells substantially without contact with stromal cells; (b) culturing the γδ T cells substantially without contact with tumor cells; and / or (c) culturing the γδ T cells substantially without contact with support cells.

[0007] In other aspects, a method of amplifying γδ T cells includes the following steps: (i) providing a non-hematopoietic tissue, a tissue containing non-hematopoietic cells, and γδ T cells; (ii) separating γδ T cells from the non-hematopoietic cells to obtain a population of isolated γδ T cells; and (iii) culturing for at least 5 days in the presence of IL-2 and a factor selected from the group consisting of IL-15, IL-4, IL-21, IL-6, IL-7, IL-8, IL-9, IL-12, IL-18, IL-33, IGF-1, IL-1β, HPL, and SDF-1 to generate a population of amplified γδ T cells. In some embodiments, step (ii) includes culturing the γδ T cells in the presence of IL-2, IL-15, IL-4, and / or IL-21 (e.g., IL-2, IL-15, and IL-4; IL-2, IL-15, and IL-21; or IL-2, IL-15, IL-4, and IL-21). In some embodiments, the γδ T cells are simultaneously exposed to IL-2, IL-15, IL-4, and / or IL-21. In some embodiments, step (iii) includes culturing the γδ T cells under conditions substantially without contact between the γδ T cells and stromal cells. In some embodiments, step (iii) includes culturing the γδ T cells under conditions without an exogenous TCR pathway agonist.

[0008] In other aspects, the invention features a method of expanding γδ T cells that includes the following steps: (i) providing a population of γδ T cells obtained from non-hematopoietic tissue; (ii) culturing the γδ T cells in the presence of effective amounts of IL-2, IL-4, IL-15, and IL-21 for at least 5 days to generate an expanded population of γδ T cells. The γδ T cells may be simultaneously exposed to IL-2, IL-4, IL-15, and IL-21, for example, for at least 5 days, or may be exposed to one or more of the factors before being exposed to other factors. In some embodiments, step (ii) includes culturing the γδ T cells in the presence of effective amounts of human recombinant IL-2, human recombinant IL-4, human recombinant IL-15, and human recombinant IL-21 for at least 5 days to generate an expanded population of γδ T cells. In some embodiments, step (ii) includes culturing the γδ TCR cells under conditions without an exogenous TCR pathway agonist (e.g., anti-CD3), e.g., under conditions of substantial TCR pathway inactivation. In some embodiments, step (ii) includes culturing the γδ T cells in the presence of IL-21 at a concentration from 1 ng / mL to 1,000 ng / mL (e.g., about 10 ng / mL, or about 100 ng / mL). Step (ii) may include culturing the γδ T cells in the presence of one or more factors selected from the group consisting of IL-6, IL-7, IL-8, IL-9, IL-12, IL-18, IL-33, IGF-1, IL-1β, human platelet lysate (HPL), and stromal cell-derived factor-1 (SDF-1).

[0009] In another aspect, a method of amplifying γδ T cells is provided by the following steps: (i) providing a population of γδ T cells obtained from non-hematopoietic tissue; (ii) culturing the γδ T cells in the presence of effective amounts of IL-2 and IL-15 for at least 5 days to generate a population of amplified γδ T cells. In some embodiments of this aspect, the γδ T cells are exposed to IL-2 and IL-15 simultaneously. Step (ii) may include culturing the γδ T cells under conditions without an exogenous TCR pathway agonist or substantially without TCR pathway activation. In some embodiments, step (ii) includes culturing the γδ T cells in the presence of one or more factors selected from the group consisting of IL-4, IL-21, IL-6, IL-7, IL-8, IL-9, IL-12, IL-18, IL-33, IGF-1, IL-1β, HPL, and SDF-1. In some embodiments, the γδ T cells are cultured in the presence of IL-4 and / or IL-21. In some embodiments, step (ii) includes culturing the γδ T cells in the presence of IL-2, IL-4, IL-15, and IL-21. The concentration of IL-21 can be from 1 ng / mL to 1,000 ng / mL (e.g., 10 ng / mL or 100 ng / mL).

[0010] In yet another aspect, the present invention features a method for amplifying γδ T cells by the following steps: (i) providing a population of γδ T cells obtained from non-hematopoietic tissue; (ii) culturing the γδ T cells for at least 5 days under conditions substantially without activation of the TCR pathway to generate an amplified population of γδ T cells. In some embodiments, step (ii) includes culturing the γδ T cells in the presence of IL-2 and IL-15. In some examples, the γδ T cells are simultaneously exposed to IL-4 and IL-15. In some embodiments, step (ii) includes culturing the γδ T cells in the presence of one or more factors selected from the group consisting of IL-4, IL-21, IL-6, IL-7, IL-8, IL-9, IL-12, IL-18, IL-33, IGF-1, IL-1β, HPL, and SDF-1. For example, the γδ T cells may be cultured in the presence of IL-4, IL-21, or both. In some embodiments, the γδ T cells are cultured in the presence of IL-2, IL-4, IL-15, and IL-21. The concentration of IL-21 can be from 1 ng / mL to 1,000 ng / mL (e.g., 10 ng / mL or 100 ng / mL).

[0011] In some embodiments of any of the foregoing aspects, the method of the present invention further includes, after step (i), separating the γδ T cells from non-hematopoietic cells to generate a population of separated γδ T cells, and step (ii) includes culturing the γδ T cells under conditions substantially without contact with stromal cells, substantially without contact with tumor cells, and / or substantially without contact with feeder cells (e.g., irradiated feeder cells, B cells, or antigen-presenting cells).

[0012] In another aspect, provided herein is a method for amplifying γδ T cells, characterized by the following steps: (i) providing a non-hematopoietic tissue comprising non-hematopoietic cells and γδ T cells; (ii) separating the γδ T cells from the non-hematopoietic cells to obtain a population of isolated γδ T cells; (iii) culturing the γδ T cells for at least 5 days under conditions substantially free of contact between stromal cells and γδ T cells to obtain a population of amplified γδ T cells. In some embodiments, step (iii) comprises culturing the γδ T cells under conditions without an exogenous TCR pathway agonist and / or substantially without activation of the TCR pathway. In some embodiments, step (iii) comprises culturing the γδ T cells in the presence of IL-2 and IL-15. For example, the γδ T cells may be simultaneously exposed to IL-2 and IL-15. In some embodiments, step (iii) comprises culturing the γδ T cells in the presence of one or more factors selected from the group consisting of IL-4, IL-21, IL-6, IL-7, IL-8, IL-9, IL-12, IL-18, IL-33, IGF-1, IL-1β, HPL, and SDF-1. For example, in some instances, step (iii) comprises culturing the γδ T cells in the presence of IL-4, IL-21, or both. In some embodiments, step (iii) comprises culturing the γδ T cells in the presence of IL-2, IL-4, IL-15, and IL-21. The concentration of IL-21 can be from 1 ng / mL to 1,000 ng / mL (e.g., 10 ng / mL or 100 ng / mL).

[0013] In another aspect, the present invention provides a method for amplifying γδ T cells by the following steps: (i) providing a non-hematopoietic tissue, wherein the tissue is a tissue containing non-hematopoietic cells and γδ T cells; (ii) separating γδ T cells from non-hematopoietic cells to obtain a population of separated γδ T cells; (iii) culturing the γδ T cells in the presence of IL-2 and IL-15 for at least 5 days to obtain a population of amplified γδ T cells. In some embodiments, step (iii) includes culturing γδ T cells under conditions substantially free of contact between stromal cells and γδ T cells. In some embodiments, step (iii) includes culturing γδ T cells under conditions without an exogenous TCR pathway agonist or substantially without activation of the TCR pathway. In some embodiments, step (iii) includes culturing γδ T cells in the presence of IL-2 and IL-15. The γδ T cells may be simultaneously exposed to IL-2 and IL-15. In some examples, step (iii) includes culturing γδ T cells in the presence of one or more factors selected from the group consisting of IL-4, IL-21, IL-6, IL-7, IL-8, IL-9, IL-12, IL-18, IL-33, IGF-1, IL-1β, HPL, and SDF-1. For example, the γδ T cells may be cultured in the presence of IL-2 and / or IL-21. In some embodiments, step (iii) includes culturing γδ T cells in the presence of IL-2, IL-4, IL-15, and IL-21. The concentration of IL-21 can be from 1 ng / mL to 1,000 ng / mL (e.g., 10 ng / mL or 100 ng / mL).

[0014] In yet another aspect, provided herein is a method for expanding γδ T cells, characterized by the following steps: (i) providing a non-hematopoietic tissue, wherein the tissue is a tissue containing non-hematopoietic cells and γδ T cells; (ii) separating γδ T cells from non-hematopoietic cells to obtain a population of isolated γδ T cells; (iii) culturing the γδ T cells for at least 5 days under conditions substantially without activation of the TCR pathway to obtain a population of expanded γδ T cells. In some examples, step (iii) includes culturing γδ T cells under conditions without an exogenous TCR pathway agonist and / or substantially without contact between stromal cells and γδ T cells. In some embodiments, step (iii) includes culturing γδ T cells in the presence of IL-2 and IL-15. For example, γδ T cells may be simultaneously exposed to IL-2 and IL-15. In some embodiments, step (iii) includes culturing γδ T cells in the presence of one or more factors selected from the group consisting of IL-4, IL-21, IL-6, IL-7, IL-8, IL-9, IL-12, IL-18, IL-33, IGF-1, IL-1β, HPL, and SDF-1. For example, γδ T cells may be cultured in the presence of IL-2 and / or IL-21. In some embodiments, step (iii) includes culturing γδ T cells in the presence of IL-2, IL-4, IL-15, and IL-21. The concentration of IL-21 can be from 1 ng / mL to 1,000 ng / mL (e.g., 10 ng / mL or 100 ng / mL).

[0015] In some embodiments of any of the foregoing methods, the step of separating γδ T cells from non-hematopoietic cells comprises culturing γδ T cells on a synthetic scaffold constructed to release cells from non-hematopoietic tissue. In some examples, the separation of γδ T cells from non-hematopoietic cells comprises culturing γδ T cells and non-hematopoietic cells in the presence of IL-2, IL-15, or both. In some embodiments, the population of isolated lymphocytes comprises a population of isolated γδ T cells, and the population of isolated γδ T cells comprises a population of isolated Vδ1 T cells and / or double-negative (DN cells). In some embodiments, at a stage prior to the amplification step, 1-10% of the population of isolated lymphocytes is γδ T cells. In some embodiments, at a stage prior to the amplification step, 1-10% of the population of isolated lymphocytes is Vδ1 T cells. Prior to the amplification step, at least 80% of the population of isolated γδ T cells may be Vδ1 T cells, and / or less than 10% of the isolated γδ T cells may be Vδ2 T cells. In some embodiments, αβ T cells and / or NK cells are removed from the population of isolated γδ T cells (e.g., prior to the amplification step).

[0016] In some embodiments, prior to the amplification step, the population of isolated γδ T cells comprises at least 10% CCR3 + cells, at least 10% CCR4 + cells, at least 10% CCR7 + cells, at least 10% CCR8 + cells, or at least 10% CD103 + cells. In some embodiments, prior to the amplification step, the population of isolated γδ T cells comprises a greater number of CCR3 + cells, CCR4 + cells, CCR7 + cells, and / or CCR8 +It contains cells. In some embodiments, prior to the amplification step, the population of isolated Vδ1 T cells has a greater number of NKG2D + cells, CD56 + cells, CD69 + cells and / or TIM3 + cells as compared to a reference population (e.g., a population of reference blood resident Vδ2 T cells).

[0017] In some embodiments of any of the foregoing aspects, in a culture within 14 days during the amplification step, the population of amplified γδ T cells contains at least 20-fold the number of γδ T cells as compared to the population of isolated γδ T cells prior to the amplification step. In addition to, or alternatively to, in a culture within 21 days during the amplification step, the population of amplified γδ T cells may contain at least 50-fold the number of γδ T cells as compared to the population of isolated γδ T cells prior to amplification. The population of amplified γδ T cells includes the population of amplified Vδ1 T cells. In some embodiments, in a culture within 14 days during the amplification step, the population of amplified Vδ1 T cells contains at least 20-fold the number of Vδ1 T cells as compared to the population of isolated Vδ1 T cells prior to amplification. In addition to, or alternatively to, in a culture within 21 days during the amplification step, the population of amplified Vδ1 T cells contains at least 50-fold the number of Vδ1 T cells as compared to the population of isolated Vδ1 T cells prior to amplification.

[0018] In some embodiments of any of the foregoing aspects, the population of amplified γδ T cells expresses CD27. For example, the population of amplified γδ T cells may exhibit a median CD27 surface expression level higher than that of the population of isolated γδ T cells. In some examples, the population of amplified γδ T cells exhibits a median CD27 surface expression level at least 2-fold that of the population of isolated γδ T cells. In addition to, or alternatively to, the population of amplified γδ T cells may have a greater number of CD27 + cells. For example, the population of amplified γδ T cells has at least 5% more CD27 than the population of isolated γδ T cells+ may have cells. In some embodiments, the population of amplified Vδ1 T cells expresses CD27. In some embodiments, the population of amplified Vδ1 T cells exhibits a median CD27 surface expression level higher than that of the population of isolated Vδ1 T cells. For example, the population of amplified Vδ1 T cells may exhibit a median CD27 surface expression level at least 2-fold higher compared to the population of isolated Vδ1 T cells. In addition to, or instead of, this, the population of amplified Vδ1 T cells may have a higher frequency of CD27 + cells. For example, the population of amplified Vδ1 T cells may have a CD27 frequency at least 5% higher compared to the population of isolated Vδ1 T cells + cells.

[0019] In some embodiments of any of the foregoing aspects, the population of amplified γδ T cells exhibits a median TIGIT surface expression level lower than that of the population of isolated γδ T cells. For example, the population of amplified γδ T cells may exhibit a median TIGIT surface expression level at least 50% lower compared to the population of isolated γδ T cells. In addition to, or instead of, this, the population of amplified γδ T cells may have a lower frequency of TIGIT + cells. For example, the population of amplified γδ T cells may have a TIGIT frequency at least 20% lower compared to the population of isolated γδ T cells + cells. In some embodiments, the population of amplified Vδ1 T cells exhibits a median TIGIT surface expression level lower than that of the population of isolated Vδ1 T cells. For example, the population of amplified Vδ1 T cells may exhibit a median TIGIT surface expression level at least 50% lower compared to the population of isolated Vδ1 T cells. In addition to, or instead of, this, the population of amplified Vδ1 T cells may have a lower frequency of TIGIT+ cells. For example, the population of amplified Vδ1 T cells may have a TIGIT+ cell frequency at least 20% lower compared to the population of isolated Vδ1 T cells.

[0020] In some embodiments of any of the foregoing aspects, the surface expression level of one or more markers selected from the group consisting of CD124, CD215, CD360, CTLA4, CD1b, BTLA, CD39, CD45RA, Fas ligand, CD25, ICAM-1, CD31, KLRG1, CD30, and CD2 in the population of amplified γδ T cells shows a high value compared to a reference population (e.g., compared to a population of isolated γδ T cells, e.g., compared to a population of isolated γδ T cells before amplification). In addition to, or instead of, this, the number of cells expressing one or more markers selected from the group consisting of CD124, CD215, CD360, CTLA4, CD1b, BTLA, CD39, CD45RA, Fas ligand, CD25, ICAM-1, CD31, KLRG1, CD30, and CD2 in the population of amplified γδ T cells can be at a high frequency compared to a reference population (e.g., compared to a population of isolated γδ T cells, e.g., compared to a population of isolated γδ T cells before amplification). In some embodiments, the surface expression level of one or more markers selected from the group consisting of NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1 and CD64 in the population of amplified γδ T cells shows a low value compared to a reference population (e.g., compared to a population of isolated γδ T cells, e.g., compared to a population of isolated γδ T cells before amplification). In addition to, or instead of, this, the number of cells expressing one or more markers selected from the group consisting of NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1 and CD64 in the population of amplified γδ T cells can be at a low frequency compared to a reference population (e.g., compared to a population of isolated γδ T cells, e.g., compared to a population of isolated γδ T cells before amplification).

[0021] In some embodiments, the surface expression level of one or more markers selected from the group consisting of CD124, CD215, CD360, CTLA4, CD1b, BTLA, CD39, CD45RA, Fas ligand, CD25, ICAM-1, CD31, KLRG1, CD30, and CD2 in a population of amplified Vδ1 T cells is a high value compared to a reference population (e.g., compared to a population of isolated γδ T cells, e.g., compared to a population of isolated γδ T cells before amplification). In some embodiments, the number of cells expressing one or more markers selected from the group consisting of CD124, CD215, CD360, CTLA4, CD1b, BTLA, CD39, CD45RA, Fas ligand, CD25, ICAM-1, CD31, KLRG1, CD30, and CD2 in a population of amplified Vδ1 T cells is a high frequency compared to a reference population (e.g., compared to a population of isolated γδ T cells, e.g., compared to a population of isolated γδ T cells before amplification). In some embodiments, the surface expression level of one or more markers selected from the group consisting of NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1, and CD64 in a population of amplified γδ T cells is a low value compared to a reference population (e.g., compared to a population of isolated γδ T cells, e.g., compared to a population of isolated γδ T cells before amplification). In other embodiments, the number of cells expressing one or more markers selected from the group consisting of NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1, and CD64 in a population of amplified γδ T cells is a low frequency compared to a reference population (e.g., compared to a population of isolated γδ T cells, e.g., compared to a population of isolated γδ T cells before amplification).

[0022] In some embodiments of any of the foregoing aspects, step (iii) includes culturing γδ T cells that are substantially free of contact with stromal cells, substantially free of contact with support cells, and / or substantially free of contact with tumor cells. In some embodiments, the non-hematopoietic tissue is not a tumor cell.

[0023] In some embodiments of any of the foregoing aspects, the non-hematopoietic tissue is skin (e.g., human skin, e.g., skin obtained by punch biopsy). In other embodiments, the non-hematopoietic tissue is gastrointestinal tissue.

[0024] In any of the foregoing aspects and embodiments, the method of amplifying γδ T cells can be performed in vitro.

[0025] In any of the foregoing aspects and embodiments, the step of providing non-hematopoietic tissue can be a step of providing non-hematopoietic cells obtained from a subject (e.g., a human or non-human animal subject).

[0026] In another aspect, the present invention features amplified γδ T cells obtained by the method of any one of the foregoing aspects.

[0027] In another aspect, the present invention provides a pharmaceutical composition comprising the expanded γδ T cells of the foregoing aspect. In some embodiments, the pharmaceutical composition further comprises an additional therapeutic agent selected from the group consisting of an immunotherapeutic agent, a cytotoxic agent, a growth inhibitor, a radiotherapy agent, an angiogenesis inhibitor, and a combination of two or more agents. In some embodiments, the additional therapeutic agent is an immunotherapeutic agent (e.g., IL-2, e.g., low-dose IL-2, e.g., 0.3×10 6 ~3.0×10 6 IU of IL-2, e.g., 1.0×10 6 IU of IL-2 per day).

[0028] In another aspect, the present invention features the pharmaceutical composition of the foregoing aspect for use in a method of treating a subject by adoptive T cell therapy.

[0029] In other aspects, the present invention features expanded γδ T cells of any of the foregoing aspects for use in a method of treating a subject by adoptive T cell therapy.

[0030] In yet other aspects, the present invention provides the use of expanded γδ T cells of any of the foregoing aspects or a pharmaceutical composition thereof in the manufacture of a medicament for treating a subject's cancer (e.g., solid cancer), infection (e.g., cytomegalovirus (CMV) infection), or immune disorder.

[0031] In other aspects, the present invention provides expanded γδ T cells of any of the foregoing aspects or a pharmaceutical composition thereof for use in a method of treating a subject's cancer (e.g., solid cancer), infection (e.g., cytomegalovirus (CMV) infection), or immune disorder.

[0032] In other aspects, the present invention provides a method of treating a subject by adoptive T cell therapy, comprising administering to the subject in need of treatment an amplified γδ T cell obtained by the method of any of the foregoing embodiments in a therapeutically effective amount. In some embodiments, the therapeutically effective amount of the amplified γδ T cells is less than 10×10 12 cells per administration, or less than 10×10 12 cells over the course of treatment. In some embodiments, the method further comprises administering to the subject in need of treatment one or more additional therapeutic agents. The additional therapeutic agent can be selected from the group consisting of an immunotherapeutic agent, a cytotoxic agent, a growth inhibitor, a radiation therapy agent, an angiogenesis inhibitor, and combinations of two or more of these agents. The additional therapeutic agent can be administered simultaneously with, prior to, or after administration of the expanded γδ T cells. In some embodiments, the additional therapeutic agent is an immunotherapeutic agent. In one embodiment, the immunotherapeutic agent is IL-2 (e.g., low-dose IL-2, e.g., 0.3×10 6 to 3.0×10 6 IU of IL-2 per day, e.g., 1.0×106 (IL-2 of IU). These embodiments are applicable to any of the foregoing and following aspects related to the use of the amplified γδ T cells obtained by the method described herein (or a pharmaceutical composition containing these γδ T cells) in the method for treating a subject by adoptive T cell therapy.

[0033] In other aspects, the present invention features a method for treating a subject by adoptive T cell therapy, comprising administering to the subject in need of treatment a therapeutically effective amount of the pharmaceutical composition according to any of the foregoing aspects.

[0034] In some embodiments of any of the foregoing aspects, the subject is a human (e.g., a human cancer patient (e.g., a human cancer patient receiving treatment for solid cancer), or a human cancer patient receiving treatment for an infectious disease (e.g., a viral infectious disease such as CMV)).

[0035] In other aspects, the present invention features a method for amplifying γδ T cells, comprising: (i) providing a population of γδ T cells obtained from non-hematopoietic tissue; (ii) culturing the γδ T cells in the presence of an effective amount of (a) IL-2 or IL-9; (b) IL-15; and (c) IL-21 for at least 5 days to generate a population of amplified γδ T cells. In some embodiments, the γδ T cells are further cultured in the presence of IL-4 in step (ii).

[0036] In yet another aspect, the present invention features a method of amplifying γδ T cells by the following steps: (i) providing a population of γδ T cells obtained from non-hematopoietic tissue; (ii) culturing for at least 5 days in the presence of IL-2, a factor selected from the group consisting of IL-15, IL-21, stromal cell-derived factor (SDF, e.g., SDF-1), IL-1β, IL-12, IL-18, and IL-33 to generate an amplified population of γδ T cells. In some embodiments, step (ii) includes culturing the γδ T cells under conditions without an exogenous TCR pathway agonist. In some embodiments, step (ii) includes culturing the γδ T cells in a serum-free medium. In some embodiments, after step (i), the γδ T cells are separated from non-hematopoietic cells to generate a population of separated γδ T cells. Additionally, step (ii) includes culturing the γδ T cells under conditions substantially free of contact with stromal cells, substantially free of contact with tumor cells, and / or substantially free of contact with feeder cells.

[0037] In another aspect, the present invention features a method of amplifying γδ T cells through the following steps: (i) providing non-hematopoietic tissue, wherein the tissue is a tissue containing non-hematopoietic cells and γδ T cells; (iii) culturing for at least 5 days in the presence of IL-2, a factor selected from the group consisting of IL-15, IL-21, SDF, IL-1β, IL-12, IL-18, and IL-33 to generate an amplified population of γδ T cells. The γδ T cells may be cultured in the presence of IL-2, IL-15, and IL-21. In addition to or in place of this, the γδ T cells may be cultured in a serum-free medium.

[0038] In yet other aspects, the invention features a population of isolated γδ T cells having the phenotype of any of the aforementioned amplified populations of γδ T cells. For example, in some embodiments, at least 50% of the isolated γδ T cells express CD27 and substantially do not express TIGIT. In some embodiments, at least 50% of the isolated γδ T cells express Vδ1.

[0039] In other aspects, the invention includes a pharmaceutical composition of the isolated γδ T cells of the aforementioned aspects.

[0040] In other aspects, use of the pharmaceutical compositions described herein is provided.

[0041] In other aspects, the invention features a method of treating a subject by adoptive T cell therapy, comprising administering to a subject in need of treatment a therapeutically effective amount of the aforementioned amplified γδ T cells, the aforementioned isolated population, or the aforementioned pharmaceutical composition.

[0042] Within the scope of each aspect of the invention, any of the embodiments described herein can be combined with any other described embodiments.

Brief Description of the Drawings

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Embodiments for Carrying Out the Invention

[0044] I. Introduction Disclosed herein is a method for expanding γδ T cells (e.g., skin-derived γδ T cells and / or non-Vδ2 T cells, e.g., Vδ1 T cells, and / or double-negative T cells) using non-hematopoietic tissue as a cell source (e.g., non-hematopoietic tissue-derived γδ T cells, e.g., non-hematopoietic tissue-derived Vδ1 T cells). The expansion method includes culturing γδ T cells (e.g., γδ T cells isolated from stromal cells of non-hematopoietic tissue) under conditions substantially without TCR stimulation and / or in the presence of interleukin-4 (IL-4), interleukin-15 (IL-15), interleukin-21 (IL-21), and / or interleukin-2 (IL-2). Further provided are compositions of expanded γδ T cells (e.g., skin-derived γδ T cells and / or non-Vδ2 T cells, e.g., Vδ1 T cells, and / or DN T cells), and methods of using the expanded γδ T cells (e.g., adoptive T cell therapy, e.g., for treating cancer).

[0045] II. Definitions Aspects and embodiments of the invention described herein include "comprising", "consisting", and "consisting essentially of" aspects and embodiments. As used herein, the singular forms "a", "an", and "the" include the plural forms as well, unless specifically indicated otherwise.

[0046] As used herein, the term "about" indicates the normal error range at each value, readily known to those skilled in the art. When referring to a value or parameter "about", embodiments (and disclosures) that target the value or parameter itself are included. In some examples, "about" includes a variation of +20% from the specified value, in some examples +10%, in some examples +5%, in some examples +1%, or in some examples +0.1% from the specified value, such variations being suitable for carrying out the disclosed methods.

[0047] As used herein, the terms "substantial" and "substantially" indicate a qualitative condition indicating the entire or almost entire scope or degree of a feature or property of interest. Those of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely achieve and / or proceed towards completion, or achieve or avoid absolute results. Thus, as used herein, the term "substantially" is used to capture the potential inherent lack of completeness in many biological and chemical phenomena. When describing physical scenarios such as receptor / ligand interactions or cell-cell contacts, the scenario is substantial if the functional result can be detected by conventional means available to the practitioner of the method. For example, "substantial TCR activation" refers to a level at which TCR activation is detectable (e.g., a statistically significant degree of TCR activation) among a population of cells. In some embodiments, the TCR is substantially activated by exposure to a TCR pathway agonist (e.g., an antibody, such as anti-CD3, or a lectin) at up to 0.1%, up to 0.5%, up to 1%, up to 5%, up to 10%, up to 20%, up to 30%, or up to 40% of EC 50 on each cell population. Similarly, "substantial cell contact" (e.g., substantial support cell contact, substantial stromal cell contact, or substantial tumor cell contact) indicates the degree of cell-cell contact that can result in a detectable change (e.g., decreased amplification) in the amplified cells. In some examples, substantial cell contact occurs when a contaminating cell type (e.g., a support cell, stromal cell, or tumor cell) is present at a concentration of up to 0.1%, up to 0.5%, up to 1%, up to 5%, up to 10%, or up to 20% with respect to the population of amplified cells in culture. A "substantial number" of cells or a "substantial amount" of a drug similarly indicates the number or amount necessary to produce a substantial effect, as defined above.

[0048] As used herein, "non-hematopoietic cells" include stromal cells and epithelial cells. Stromal cells are non-hematopoietic connective tissue cells of any organ that support the functions 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-hematopoietic cells that cover the blood vessels of the whole body and the cavities and surfaces of organs. They usually have a flat, cylindrical, or cuboid shape and can be arranged as a single layer of cell layer or two or more cell layers.

[0049] As used herein, "non-hematopoietic tissue resident γδ T cells", "derived from non-hematopoietic tissue", and "native non-hematopoietic tissue γδ T cells" refer to γδ T cells present in non-hematopoietic tissue at the time the tissue was removed. Non-hematopoietic tissue resident γδ T cells can be obtained from any suitable non-hematopoietic tissue of a human or non-human animal. Non-hematopoietic tissue is tissue different from blood or bone marrow. In some embodiments, the γδ T cells are not obtained from a sample of a particular type of biological fluid such as blood or synovial fluid. Suitable examples of non-hematopoietic tissue of a human or non-human animal include skin or a part thereof (e.g., dermis or epidermis), gastrointestinal tract (e.g., gastrointestinal epithelium, colon, small intestine, stomach, appendix, cecum, or rectum), mammary tissue, lung (preferably not tissue obtained by bronchoalveolar lavage), prostate, liver, and pancreas. In some embodiments, non-hematopoietic tissue resident γδ T cells can be derived from lymphoid tissue such as the thymus, spleen, or tonsils. γδ T cells may reside, for example, in human cancer tissues such as breast and prostate. In some embodiments, γδ T cells are not obtained from human cancer tissues. Samples of non-hematopoietic tissue can be obtained by standard techniques such as explant (e.g., biopsy). Non-hematopoietic tissue resident γδ T cells include non-Vδ2 T cells such as, for example, Vδ1 T cells, double-negative (DN) T cells, Vδ3 T cells, and Vδ5 T cells.

[0050] As used herein, "IL-2" refers to native or recombinant IL-2 or variants thereof that act as agonists to one or more IL-2 receptor (IL-2R) subunits (e.g., mutants, muteins, analogs, subunits, receptor complexes, fragments, isoforms, and peptidomimetics thereof). These agents can support the growth of the IL-2-dependent cell line CTLL-2 (33; American Type Culture Collection (ATCC®) TIB 214). Mature human IL-2 results as a 133 amino acid sequence (lacking the signal peptide consisting of an additional 20 N-terminal amino acids) as described in Fujita, et al. Cell 1986. 46.3:401-407. IL-2 mutant proteins are polypeptides in which specific substitutions have been made to the interleukin-2 protein while retaining the ability to bind to IL-2Rβ, as described in U.S. Patent Application Publication No. 2014 / 0046026. IL-2 mutant proteins can be characterized by insertions, deletions, substitutions, and modifications of amino acids at one or more positions or other residues of the native IL-2 polypeptide chain. In accordance with the present disclosure, any of the above insertions, deletions, substitutions, and modifications result in IL-2 mutant proteins that retain IL-2Rβ binding activity. Examples of mutant proteins include those containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions.

[0051] Nucleic acids encoding human IL-2 can be obtained by conventional procedures such as polymerase chain reaction (PCR). The amino acid sequence of human IL-2 (Gene ID 3558) can be referenced at Genbank accession locator NP_000577.2 GI:28178861. The mouse (Mus musculus) IL-2 amino acid sequence (Gene ID 16183) can be referenced at Genbank accession locator NP_032392.1 GI:7110653.

[0052] IL-2 can represent IL-2 derived from a variety of mammalian species, including, for example, humans, monkeys, cows, pigs, horses, and mice. Variants may include conservative substitutions of the sequence, which means that a given amino acid residue is replaced by a residue with similar physicochemical properties. Examples of conservative substitutions include substitutions of one aliphatic residue for another, such as among Ile, Val, Leu, or Ala, or substitutions of one polar residue for another, such as between Lys and Arg; between Glu and Asp; between Gln and Asn. Other examples of such conservative substitutions include substitutions of entire regions with similar hydrophobic properties, which are well known. Naturally occurring IL-2 variants are also included in the present invention. Examples of such variants include proteins resulting from alternative mRNA splicing events or proteolytic cleavage of the IL-2 protein, provided that the binding properties of IL-2 are retained. Alternative splicing of mRNA can result in an IL-2 protein that has biological activity even when cleaved. Changes due to proteolysis include, for example, differences in the N- or C-terminus upon expression in different types of host cells due to proteolytic removal of one or more terminal amino acids (usually 1-10 amino acids) from the IL-2 protein. In some embodiments, the ends or interior of the protein can be modified using chemical groups such as polyethylene glycol to change its physical properties (Yang, et al. Cancer 1995. 76:687-694). In some embodiments, the ends or interior of the protein may be modified with additional amino acids (Clark-Lewis, et al. PNAS 1993. 90:3574-3577).

[0053] As used herein, "IL-15" refers to native or recombinant IL-15 or a variant thereof that acts as an agonist to one or more IL-15 receptor (IL-15R) subunits (e.g., its mutants, muteins, analogs, subunits, receptor complexes, fragments, isoforms, and peptidomimetics). IL-15, like IL-2, is a known T cell growth factor that can support the growth of the IL-2-dependent cell line CTLL-2. IL-15 was first reported by Grabstein et al. as a 114-amino acid mature protein (Grabstein, et al. Science 1994. 264.5161: 965-969). The term "IL-15" as used herein means native or recombinant IL-15 and its mutant proteins, analogs, subunits, or complexes (e.g., receptor complexes, such as sushi peptides, as described in WO 2007 / 046006), each of which can stimulate the growth of CTLL-2 cells. In an assay of CTLL-2 cell growth, cell supernatants into which in-frame fusions of recombinantly expressed precursor and mature IL-15 have been introduced can induce the growth of CTLL-2 cells.

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

[0055] The amino acid sequences of human IL-15 (Gene ID 3600) can be referenced by the Genbank 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) can be referenced by the Genbank accession locator NP_001241676.1 GI: 363000984.

[0056] IL-15 can also represent IL-15 derived from diverse mammalian species, including, for example, humans, monkeys, cows, pigs, horses, and mice. As used herein, an IL-15 “mutein” or “variant” is a polypeptide that is substantially homologous to the sequence of native mammalian IL-15 but has an amino acid sequence that differs from native mammalian IL-15 due to amino acid deletions, insertions, and substitutions. Variants may include conservative substitutions of the sequence, which means that a given amino acid residue is replaced by a residue having similar physicochemical properties. Examples of conservative substitutions include substitution of one aliphatic residue for another, such as between Ile, Val, Leu, or Ala, or substitution of one polar residue for another, such as between Lys and Arg; Glu and Asp; Gln and Asn. Other examples of such conservative substitutions include substitution of an entire region having similar hydrophobic properties. Naturally occurring IL-15 variants are also included in the present invention. Examples of such variants include proteins resulting from alternative mRNA splicing events or proteolytic cleavage of the IL-15 protein, which retain the binding properties of IL-15. Alternative splicing of mRNA can result in an IL-15 protein that has biological activity even when cleaved. Changes due to proteolysis include, for example, differences in the N or C terminus upon expression in different types of host cells due to proteolytic removal of one or more terminal amino acids (usually 1-10 amino acids) from the IL-15 protein. In some embodiments, the ends of the protein can be modified using chemical groups such as polyethylene glycol to change its physical properties (Yang, et al. Cancer 1995. 76:687-694). In some embodiments, the ends or interior of the protein may be modified with additional amino acids (Clark-Lewis, et al. PNAS 1993. 90:3574-3577).

[0057] As used herein, "IL-4" is native or recombinant IL-4 or a variant thereof that acts as an agonist to one or more IL-4 receptor (IL-4R) subunits (e.g., its mutants, muteins, analogs, subunits, receptor complexes, fragments, isoforms, and peptidomimetics). Such agents support the differentiation of naive helper T cells (Th0 cells) into Th2 cells. Mature human IL-4 results as a 129 amino acid sequence (short by the portion of the signal peptide consisting of an additional 24 N-terminal amino acids). IL-4 mutant proteins are polypeptides in which specific substitutions to the interleukin-4 protein have been made while retaining the ability to bind to IL-4Rα, as described in U.S. Patent No. 6,313,272. IL-4 mutant proteins can be characterized by insertions, deletions, substitutions, and modifications of amino acids at one or more locations or other residues of the native IL-4 polypeptide chain. In accordance with the present disclosure, any of the above insertions, deletions, substitutions, and modifications produce IL-4 mutant proteins that retain IL-2Rα binding activity. Examples of mutant proteins include those containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions.

[0058] Nucleic acids encoding human IL-4 can be obtained by conventional procedures such as polymerase chain reaction (PCR). The amino acid sequence of human IL-4 (Gene ID 3565) can be referenced at Genbank accession locator NG_023252. The mouse (Mus musculus) IL-4 amino acid sequence (Gene ID 16189) can be referenced at Genbank accession locator NC_000077.6.

[0059] IL-4 can also represent IL-4 derived from a variety of mammalian species, including, for example, humans, monkeys, cows, pigs, horses, and mice. Variants may include conservative substitutions of the sequence, which means that a given amino acid residue is replaced by a residue with similar physicochemical properties. Examples of conservative substitutions include substitutions of one aliphatic residue for another aliphatic residue, such as among Ile, Val, Leu, or Ala, or substitutions of one polar residue for another polar residue, such as between Lys and Arg; between Glu and Asp; between Gln and Asn. Other examples of such conservative substitutions include substitutions of entire regions with similar hydrophobic properties, which are well known. Native IL-4 variants are also included in the present invention. Examples of such variants include proteins resulting from alternative mRNA splicing events or proteolytic cleavage of the IL-4 protein, which retain the binding properties of IL-4. Alternative splicing of mRNA can result in an IL-4 protein that has biological activity even when cleaved. Changes due to proteolysis include, for example, differences in the N or C terminus upon expression in different types of host cells due to proteolytic removal of one or more terminal amino acids (usually 1-10 amino acids) from the IL-4 protein. In some embodiments, the ends of the protein can be modified using chemical groups, such as polyethylene glycol, to change its physical properties (Yang, et al. Cancer 1995. 76:687-694). In some embodiments, the ends or interior of the protein may be modified with additional amino acids (Clark-Lewis, et al. PNAS 1993. 90:3574-3577).

[0060] As used herein, "IL-21" refers to native or recombinant IL-21 or a variant thereof that acts as an agonist to one or more IL-21 receptor (IL-21R) subunits (e.g., its mutants, muteins, analogs, subunits, receptor complexes, fragments, isoforms, and peptidomimetics). Such agents can support the proliferation of natural killer (NK) and cytotoxic (CD8 + ) T cells. Mature human IL-21 results as a 133 amino acid sequence (lacking the signal peptide consisting of an additional 22 N-terminal amino acids). IL-21 mutant proteins are polypeptides in which specific substitutions have been made to the interleukin-21 protein while retaining the ability to bind to IL-21Rα, as described in U.S. Patent No. 9,388,241. IL-21 mutant proteins can be characterized by insertions, deletions, substitutions, and modifications of amino acids at one or more positions or other residues of the native IL-21 polypeptide chain. In accordance with the present disclosure, any of the above insertions, deletions, substitutions, and modifications produce an IL-21 mutant protein that retains IL-21Rα binding activity. Examples of mutant proteins include those containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions.

[0061] Nucleic acids encoding human IL-21 can be obtained by conventional procedures such as polymerase chain reaction (PCR). The amino acid sequence of human IL-21 (Gene ID 59067) can be referenced at Genbank accession locator NC_000004.12. The mouse (Mus musculus) IL-21 amino acid sequence (Gene ID 60505) can be referenced at Genbank accession locator NC_000069.6.

[0062] IL-21 can also represent IL-21 derived from a variety of mammalian species, including, for example, humans, monkeys, cows, pigs, horses, and mice. Variants may include conservative substitutions of the sequence, which means that a given amino acid residue is replaced by a residue having similar physicochemical properties. Examples of conservative substitutions include substitutions of one aliphatic residue for another aliphatic residue, such as among Ile, Val, Leu, or Ala, or substitutions of one polar residue for another polar residue, such as between Lys and Arg; between Glu and Asp; between Gln and Asn. Other examples of such conservative substitutions include substitutions of entire regions having similar hydrophobic properties. Naturally occurring IL-21 variants are also included in the present invention. Examples of such variants include proteins resulting from alternative mRNA splicing events or proteolytic cleavage of the IL-21 protein, provided that the binding properties of IL-21 are retained. Alternative splicing of mRNA can result in an IL-21 protein that has biological activity even when cleaved. Changes resulting from proteolysis include, for example, differences in the N or C terminus upon expression in different types of host cells due to proteolytic removal of one or more terminal amino acids (usually 1-10 amino acids) from the IL-21 protein. In some embodiments, the ends of the protein can be modified using chemical groups, such as polyethylene glycol, for example, to change its physical properties (Yang, et al. Cancer 1995. 76:687-694). In some embodiments, the ends or interior of the protein may be modified with additional amino acids (Clark-Lewis, et al. PNAS 1993. 90:3574-3577).

[0063] As used herein, "IL-9" refers to native or recombinant IL-9 or a variant thereof that acts as an agonist to one or more IL-9 receptor (IL-9R) subunits (e.g., its mutants, muteins, analogs, subunits, receptor complexes, fragments, isoforms, and peptidomimetics). Mature human IL-9 results in a 144 amino acid sequence. An IL-9 mutant protein is a polypeptide in which specific substitutions to the interleukin-9 protein have been made while retaining the ability to bind to IL-9R. An IL-9 mutant protein can be characterized by insertions, deletions, substitutions, and modifications of amino acids at one or more positions of the native IL-9 polypeptide chain or other residues. In accordance with the present disclosure, any of the above insertions, deletions, substitutions, and modifications result in an IL-9 mutant protein that retains IL-9R binding activity. Examples of mutant proteins include those containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions.

[0064] Nucleic acids encoding human IL-9 can be obtained by conventional procedures such as polymerase chain reaction (PCR). The amino acid sequence of human IL-9 is provided by UniProtKB P15248.

[0065] IL-9 can also represent IL-9 derived from diverse mammalian species, including, for example, humans, monkeys, cows, pigs, horses, and mice. Variants may include conservative substitutions of the sequence, which means that a given amino acid residue is replaced by a residue having similar physicochemical properties. Examples of conservative substitutions include substitutions of one aliphatic residue for another aliphatic residue, such as between Ile, Val, Leu, or Ala, or substitutions of one polar residue for another polar residue, such as between Lys and Arg; between Glu and Asp; between Gln and Asn. Other examples of such conservative substitutions include substitutions of entire regions having similar hydrophobic properties, which are well known. Naturally occurring IL-9 variants are also included in the present invention. Examples of such variants include proteins resulting from alternative mRNA splicing events or proteolytic cleavage of the IL-9 protein, which retain the binding properties of IL-9. Alternative splicing of mRNA can result in an IL-9 protein that has biological activity even when cleaved. Changes due to proteolysis include, for example, differences in the N or C terminus upon expression in different types of host cells due to proteolytic removal of one or more terminal amino acids (usually 1-10 amino acids) from the IL-9 protein. In some embodiments, the termini of the protein can be modified using chemical groups, such as polyethylene glycol, to change its physical properties (Yang, et al. Cancer 1995. 76:687-694). In some embodiments, the termini or interior of the protein may be modified with additional amino acids (Clark-Lewis, et al. PNAS 1993. 90:3574-3577).

[0066] One or more of the above factors can be incorporated into the amplification protocol in an amount sufficient for the generation of amplified γδ T cells. As used herein, the phrase "in an amount effective to" refers to an amount that induces a detectable result (e.g., a statistically significantly increased number of cells compared to the starting population, e.g., p < 0.05). When multiple factors are present at once, the effective amount refers to the combined effect of all the factors (e.g., the combined effect of IL-2 and IL-15, or the combined effect of IL-2 or IL-9, IL-4, IL-15, and IL-21).

[0067] The term "T cell receptor (TCR) pathway agonist" or "agent that activates the TCR pathway" refers to a compound that induces, through TCR signaling, the proliferation of T cells such as αβ T cells and / or blood-resident γδ T cells, or other activation outcomes. T cell signaling modulators function through the sequential activation of Src-related protein tyrosine kinases (PTKs), Lck and Fyn, and the 70 kDA zeta chain (TCR)-associated protein kinase (ZAP70). These PTKs result in the phosphorylation of polypeptides, including the linker for activation of T cells (LAT), which leads to downstream stimulation via extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs), and nuclear factor of activated T cells (NFAT). Co-stimulation via, for example, CD28 and CD45 promotes phosphorylation and the TCR signaling pathway. Thus, substances that target a part of the TCR or co-stimulation pathway can activate T cell signaling. TCR pathway agonists include antibodies (e.g., monoclonal antibodies, e.g., anti-TCRVδ1, anti-TCRδTCS-1, anti-TCR PANγδ, and anti-CD3 antibodies), lectins (e.g., plant lectins, e.g., concanavalin A; lectins from Phaseolus vulgaris (PHA-P), Phytolacca Americana, Triticum vulgaris, Lens culinaris, Glycine max, Maackia amurensis, Pisum sativum, and Sambucus nigra), synthetic phosphorylated antigens (e.g., BrHPP (bromohydrin pyrophosphate), 2M3B1PP (2-methyl-3-butenyl-1-pyrophosphate), HMBPP ((E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate), or IPP (isopentenyl pyrophosphate)), and N-bisphosphonates (e.g., zoledronate).TCR pathway agonists include co-receptor agonists and include antibodies (e.g., monoclonal antibodies, e.g., anti-CD2, anti-CD6, anti-CD9, anti-CD28, anti-CD43, anti-CD94, anti-CD160, anti-SLAM, anti-NKGD2, anti-2B4, anti-HLA-A, anti-HLA-b, anti-HLA-C, and anti-ICAM-3 antibodies) and proteins (e.g., recombinant proteins, e.g., recombinant human proteins, e.g., CD7L, CD26, CD27L, CD30L, CD40L, OX40L, 4-1BBL, ICAM-1, fibronectin, hydrocortisone, and variants thereof, e.g., Fc fusion proteins, e.g., CD27L-Fc). TCR pathway agonists are soluble or membrane-bound and can be presented on cells such as artificial antigen-presenting cells (aAPCs), similar to the case of MHC or HLA complexes. aAPCs suitable for activation of T cell signaling are known in the art. Appropriate methods for activating T cells by exogenously adding TCR pathway agonists are well known in the art and are summarized in Figure 1 of Deniger et al. (Deniger, et al. Frontiers in Immunology, 2014. 5(636):1-10).

[0068] "Exogenous TCR pathway agonist" refers to a TCR pathway agonist that is not derived from non-hematopoietic tissue or its donor (i.e., is exogenously added). Thus, in some embodiments of the present invention, it will be understood that TCR pathway agonists (e.g., soluble fibronectin or cell-bound ICAM-1) may be present as residual substances from non-hematopoietic tissue in a culture. In some embodiments, the residual TCR pathway agonists are at negligible concentrations and do not substantially activate T cells.

[0069] As used herein, the terms "synthetic scaffold", "scaffold" and "grid" are used interchangeably and refer to a non-native three-dimensional structure suitable for supporting cell growth. An explant may be attached to a synthetic scaffold to facilitate the movement of lymphocytes from the explant to the scaffold. Synthetic scaffolds are constructed of natural and synthetic materials, such as polymers (e.g., natural or synthetic polymers such as polyvinylpyrrolidone, polymethylmethacrylate, methylcellulose, polystyrene, polypropylene, polyurethane), ceramics (e.g., tricalcium phosphate, calcium aluminate, calcium hydroxyapatite), or metals (tantalum, titanium, platinum, and metals of the same elemental group as combinations of platinum, niobium, hafnium, tungsten, and alloys thereof). Biological agents (e.g., collagen (e.g., collagen I or collagen II), fibronectin, laminin, integrin, angiogenic factors, anti-inflammatory factors, glycosaminoglycans, vitrogen, antibodies and fragments thereof, cytokines (e.g., IL-2 or IL-15, and combinations thereof) can be coated on the surface of the scaffold or encapsulated within the material of the scaffold to enhance cell adhesion, migration, survival, or proliferation according to methods known in the art. Using this and other methods, lymphocytes can be isolated from many other types of non-hematopoietic tissues, such as, for example, the gastrointestinal tract, prostate, and breast. Examples of synthetic scaffolds contemplated for use as part of the present invention include those used in the Clark protocol.

[0070] As used herein, the terms "separation", "separated", or "separating" refer to severing or inhibiting physical contact between different cell populations (e.g., separation of hematopoietic cells (e.g., lymphocytes) from non-hematopoietic cells). Separation can be effected, for example, by pipetting a mixed cell population to forcibly sever intermembrane linkages, or, as described by Carrasco et al., by culturing a cell population with a chemokine or cytokine, for example, by inducing "crawl out" of a cell population from a tissue matrix (Carrasco A. et al Journal of Immunological Methods 2013. 389(1-2):29-37). Separation can be maintained by a transwell culture system, or a similar culture method, that inhibits physical contact between different cell populations.

[0071] As used herein, a "population of isolated γδ cells" refers to a population of hematopoietic cells that includes γδ cells separated from the original non-hematopoietic tissue such that they are substantially not in contact with non-hematopoietic cells (e.g., in accordance with the separation protocols described herein). Similarly, a "population of isolated Vδ1 T cells" refers to a population of hematopoietic cells that includes Vδ1 T cells separated from the original non-hematopoietic tissue such that they are substantially not in contact with non-hematopoietic cells (e.g., in accordance with the separation protocols described herein). Thus, in these examples, separation refers to the separation of hematopoietic cells (e.g., lymphocytes) from non-hematopoietic cells (e.g., stromal cells and / or epithelial cells).

[0072] The term "antibody" is used in the broadest sense and covers, in particular, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity.

[0073] As used herein, the term "amplification step" refers to the stage of culturing after isolation, in which a certain number of γδ T cells increase by cell division. While cell division can also occur during the isolation stage while the γδ T cells are in contact with stromal cells, it will be understood that the amplification step does not begin until isolation is complete. Thus, when a population of isolated cells is characterized as being at a point "prior to the amplification step", this means a point in time after isolation culture and before amplification culture.

[0074] As used herein, the term "population of amplified γδ cells" refers to a population of hematopoietic cells that includes γδ T cells that have been continuously cultured under conditions that induce amplification of the γδ cells, i.e., an increase in the number of γδ cells. Similarly, the term "population of amplified Vδ1 T cells" as used herein refers to a population of hematopoietic cells that includes Vδ1 T cells that have been continuously cultured under conditions that induce amplification of the Vδ1 T cells, i.e., an increase in the number of Vδ1 cells.

[0075] As used herein, the term "support cell" refers to exogenous cells that are added to a culture to provide cell-to-cell surface contact with cells derived from non-hematopoietic tissue. The support cells may be primary cells (e.g., tissue-derived) or cells derived from cell lines. The support cells may be live cells or irradiated cells and include tumor cells, fibroblasts, B cells, and other antigen-presenting cells.

[0076] As used herein, the term "marker" 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).

[0077] A single cell or population of cells that "expresses" a marker of interest is determined to have the protein-encoding mRNA, or the protein itself, which contains a fragment thereof, present in said cell or population. Expression of the marker can be detected by a variety of methods. For example, in some embodiments, expression of the marker is indicated by the surface density of the marker on the cell. For example, the mean fluorescence intensity (MFI) used as a flow cytometry readout represents the density of the marker on the population of cells. One of ordinary skill in the art will understand that the value of the MFI depends on staining parameters (e.g., concentration, duration, and temperature) and the composition of the fluorescent dye. However, the MFI can be made quantitative when considered in the context of having appropriate controls. For example, if the MFI of an antibody against the marker is significantly higher than the MFI of an appropriate isotype control antibody on the same population of cells stained under equivalent conditions, the population of cells can be said to express that marker. In addition to, or instead of, this, a population of cells can detect the expression of 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 for isotype or "fluorescence minus one" (FMO) controls). By this measure, if the number of cells detected as positive for the marker is significantly greater than the background (e.g., by gating with an isotype control), the population can be said to "express" the marker.

[0078] As used herein, the expression of a population is described as the percentage of positive cells, and when that percentage is compared to the corresponding percentage of positive cells in a reference population, the difference in percentages indicates the percentage of the parent population of each population. For example, if a marker is expressed in 10% of the cells in population A and the same marker is expressed in 1% of the cells in population B, it can be said that population A has marker-positive cells at a 9% higher frequency (i.e., 10% - 1%, not 10% ÷ 1%). Multiplying the frequency by the number of cells in the parent population calculates the difference in the absolute number of cells. In the above example, if there are 100 cells in population A and 10 cells in population B, then population A has 100 times the number of cells compared to population B; i.e., (10% × 100) ÷ (1% × 10).

[0079] The expression level may be a nucleic acid expression level (e.g., DNA expression level or RNA expression level, e.g., mRNA expression level). Any suitable method for measuring nucleic acid expression may be used. In some embodiments, the nucleic acid expression level is measured 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 combinations thereof.

[0080] As used herein, a "reference population" of cells is a population of cells corresponding to the cells of interest, and is measured against the phenotype of the cells of interest. For example, the expression level of a marker on a separated population of γδ cells derived from non-hematopoietic tissue is compared to the expression level of γδ T cells derived from hematopoietic tissue (e.g., blood-resident γδ cells, e.g., blood-resident γδ cells from the same donor or other donors), or γδ T cells derived from non-hematopoietic tissue amplified under different conditions (e.g., conditions with substantial TCR activation, in the presence of an exogenous TCR activator (e.g., anti-CD3), or conditions with substantial contact with stromal cells (e.g., fibroblasts)). It can also be compared to an earlier state of the same population. For example, the reference population may be a separated population of cells before amplification. In this case, the amplified population is compared to the composition of the same population before the amplification step; that is, in this case, the past composition becomes the reference population.

[0081] "Cancer" refers to the abnormal proliferation of malignant cancer cells and includes leukemias such as acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL), lymphomas such as Hodgkin lymphoma, non-Hodgkin lymphoma and multiple myeloma, and solid cancers such as sarcoma, skin cancer, melanoma, bladder cancer, brain cancer, breast cancer, endometrial cancer, ovarian cancer, prostate cancer, lung cancer, colorectal cancer, cervical cancer, liver cancer, head and neck cancer, esophageal cancer, pancreatic cancer, kidney cancer, adrenal cancer, gastric cancer, testicular cancer, gallbladder cancer and bile duct cancer, thyroid cancer, thymic cancer, bone tumors and brain tumors.

[0082] Cancer cells in a cancer patient can be immunologically distinguished from the individual's normal somatic cells (e.g., a cancerous tumor may be immunogenic). For example, cancer cells can induce a systemic immune response against one or more antigens expressed by the cancer cells in the body of a cancer patient. Antigens that can induce an immune response may be tumor antigens or may be shared with normal cells. A patient with cancer may exhibit at least one recognizable sign, symptom, or laboratory finding sufficient to diagnose cancer according to clinical criteria known in the art. Examples of such clinical criteria can be found in medical books such as Harrison's Principles of Internal Medicine (Longo DL, Fauci AS, Kasper DL, Hauser SL, Jameson J, Loscalzo J. eds. 18e. New York, NY: McGraw-Hill; 2012). For example, the diagnosis of cancer in an individual may include the identification of characteristic cell types (e.g., cancer cells) in a sample of body fluid or tissue obtained from the individual.

[0083] As used herein, "solid cancer" is cancer of any body tissue other than blood, bone marrow, or the lymphatic system. Solid cancers can be further divided into cancers derived from epithelial cells and cancers derived from non-epithelial cells. Examples of solid tumors of epithelial cells include tumors of the gastrointestinal tract, colon, breast, prostate, lung, kidney, liver, pancreas, ovary, head and neck, oral cavity, stomach, duodenum, small intestine, large intestine, anus, gallbladder, labia, nasopharynx, skin, uterus, male genitalia, urinary tract, bladder, and skin. Examples of solid tumors of non-epithelial cells include sarcomas, brain tumors, and bone tumors.

[0084] Patients, subjects, or individuals suitable for the above treatment can be mammals such as rodents (e.g., guinea pigs, hamsters, rats, mice), murines (e.g., mice), canines (e.g., dogs), felines (e.g., cats), equines (e.g., horses), primates, simians (e.g., monkeys or apes), monkeys (e.g., marmosets or baboons), apes (e.g., gorillas, chimpanzees, orangutans or gibbons), or humans.

[0085] In some embodiments, the patient, subject or individual is a human. In other preferred embodiments, non-human mammals, particularly mammals conventionally used as models for demonstrating therapeutic effects in humans, can be used.

[0086] As used herein, "treatment" (and grammatical variations such as "treat" or "treating") refers to a clinical intervention in a human or animal (e.g., for veterinary applications) in which a desired therapeutic effect, such as suppression or delay of the progression of a medical condition, is achieved, including a decrease in the rate of progression, a halt in the rate of progression, improvement of the medical condition, cure or remission (partial or total) of the medical condition, prevention, delay, alleviation or halt of one or more symptoms and / or signs of the condition, or an extension of the lifespan of a subject or patient beyond that expected under untreated conditions.

[0087] Treatment as a prophylactic measure (e.g., prevention) is also included. For example, patients, subjects, or individuals at risk of or likely to develop cancer or recurrence of cancer can be treated as described herein. Such treatment can suppress or delay the onset or recurrence of cancer in the patient, subject, or individual.

[0088] In particular, treatment includes suppression of cancer growth, including complete remission of cancer and / or suppression of cancer metastasis. Cancer growth generally exhibits any one of several indicators that show a change to a more developed form within the cancer. Thus, indicators for measuring suppression of cancer growth include a decrease in the survival rate of cancer cells, a decrease in tumor size or morphology (e.g., determined using computed tomography (CT), ultrasound, or other imaging methods), a delay in tumor growth, destruction of tumor blood vessels, improvement in the performance of a delayed hypersensitivity skin test, enhancement of the activity of cytotoxic T lymphocytes, and a decrease in the level of tumor-specific antigens. Reducing immunosuppression in an individual's cancerous tumor can improve the individual's ability to resist the growth of cancer, particularly cancer that is already present in the subject, and / or decrease the individual's tendency for cancer growth.

[0089] In some embodiments, expanded γδ T cells (e.g., γδ T cells derived from non-hematopoietic tissue, such as Vδ1 T cells derived from non-hematopoietic tissue) are administered to delay the development of a disease or slow the progression of a disease or disorder.

[0090] As used herein, "administering" means giving a patient a single dose of therapy (e.g., adoptive T cell therapy including, for example, γδ T cells derived from non-hematopoietic tissue) or a composition (e.g., a pharmaceutical composition, e.g., a pharmaceutical composition including γδ T cells derived from non-hematopoietic cells). The compositions utilized in the methods described herein can be administered, for example, intramuscularly, intravenously, intradermally, transdermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrathoracically, intratracheally, subarachnoidally, intranasally, intravaginally, rectally, topically, intratumorally, peritoneally, subcutaneously, subconjunctivally, intravesically, mucosally, epicardially, intraumbilically, intraocularly, intraorbitally, intravitreally (e.g., by intravitreal injection), ophthalmically, orally, topically, transdermally, by inhalation, by injection, by transplantation, by infusion, by continuous infusion, by topical perfusion directly immersing target cells, by topical perfusion directly immersing target cells, by catheter, by lavage, by cream, or by lipid composition. Administration of the compositions utilized in the methods described herein can be systemic or local. The method of administration may be varied depending on a variety of factors (e.g., the therapeutic agent or composition to be administered, and the severity of the condition, disease or disorder to be treated).

[0091] "An amount effective for treatment (a therapeutically effective amount)" refers to the amount of a therapeutic agent for treating or suppressing a mammalian disease or disorder. In the case of cancer, an amount effective for treatment of a therapeutic agent (e.g., γδ T derived from non-hematopoietic tissue) is to reduce the number of cancer cells, shrink the size of the primary tumor, suppress the invasion of cancer cells into peripheral tissues (i.e., delay to some extent and preferably stop), suppress the metastasis of the tumor (i.e., delay to some extent and preferably stop), suppress the growth of the tumor for a certain period of time, and / or relieve to some extent one or more symptoms associated with the disorder. To the extent that the drug can prevent the growth of cancer cells and / or kill existing cancer cells, it may be cytostatic and / or cytotoxic. In the case of cancer treatment, the in vivo efficacy can be measured, for example, by evaluating survival time, time to disease progression (TTP), response rate (e.g., complete response (CR) and partial response (PR)), duration of response and / or quality of life.

[0092] As used herein, the term "simultaneously" refers to the administration of two or more therapeutic agents such that the administration times at least partially overlap. Thus, co - administration includes dosing schedules where the administration of one or more agents continues after the administration of one or more other agents has been discontinued. For example, in some embodiments, γδ T cells derived from non - hematopoietic tissue and IL - 2 can be administered simultaneously.

[0093] The term "pharmaceutical composition" refers to a formulation that assumes a form enabling the biological activity of one or more active ingredients contained therein and that does not contain additional ingredients having unacceptable toxicity for the patient to whom the formulation is administered.

[0094] III. Methods for Isolating and Amplifying γδ T Cells The present invention provides a method for isolating and amplifying γδ T cells (e.g., γδ T cells derived from skin and / or non - Vδ2 T cells, such as Vδ1 T cells and / or DN T cells) from non - hematopoietic tissue of a human or non - human animal that can be removed from or has been removed from a patient. In some embodiments, the non - hematopoietic tissue from which the γδ T cells are removed and amplified is skin (e.g., human skin) and is obtained by methods known in the art. In some embodiments, the skin is obtained by punch biopsy. Alternatively, the method for isolating and amplifying γδ T cells provided herein can be applied to the gastrointestinal tract (e.g., colon), mammary gland, lung, prostate, liver, spleen, pancreas. The γδ T cells may be resident in human cancer tissue, such as a tumor of the breast or prostate. In some embodiments, the γδ T cells may be derived from human cancer tissue (e.g., solid tumor tissue). In other embodiments, the γδ T cells may be non - hematopoietic tissue other than human cancer tissue (e.g., tissue having no substantial number of tumor cells). For example, the γδ T cells may be derived from a skin region (e.g., healthy skin) separated from the periphery of or adjacent to a cancer.

[0095] Most of the γδ T cells that make up the majority of γδ T cells in the blood are mainly Vδ2 T cells, while most of the γδ T cells in non-hematopoietic tissues are mainly Vδ1 T cells, and Vδ1 T cells contain 70-80% of the population of non-hematopoietic tissue-resident γδ T cells. However, some Vδ2 T cells are also found in non-hematopoietic tissues, such as the digestive tract, where they account for 10-20% of the γδ T cells (Figure 6). Some of the γδ T cells resident in non-hematopoietic tissues do not express either Vδ1 or Vδ2 TCR. Such cells are named double-negative (DN) in this specification. Most DN γδ T cells are Vδ3-expressing T cells, and a small number are likely to be Vδ5-expressing T cells. Therefore, the γδ T cells resident in non-hematopoietic tissues and amplified by the method of the present invention are preferably not Vδ2 T cells, but rather Vδ1 T cells containing, for example, a small number of DN γδ T cells.

[0096] Those skilled in the art will understand that certain non-hematopoietic tissues are highly angiogenic, and in fact, peripheral blood resident cells are likely to contaminate samples of non-hematopoietic tissues. To avoid or minimize such contamination, care can be taken to remove peripheral blood from the separation and amplification culture, following methods known in the art, such as thoroughly washing the tissue with an appropriate buffer to remove cells. For example, in some embodiments, the population of γδ T cells isolated from lung tissue is not obtained by bronchoalveolar lavage.

[0097] Isolation of non-hematopoietic tissue-resident γδ T cells from non-hematopoietic tissues In some embodiments, an important step is to separate, for example, after several days or weeks of culture, non-hematopoietic tissue-resident T cells (e.g., within a mixed lymphocyte population including αβ cells, natural killer (NK) cells, B cells, and γδ2 T cells, and non-γδ2 T cells, etc.) from non-hematopoietic cells (e.g., stromal cells, especially fibroblasts) of the tissue from which the T cells were obtained, in accordance with the purpose. This enables preferential and rapid amplification of Vδ1 T cells and DN γδ T cells from non-hematopoietic tissues for several days and weeks.

[0098] The present invention provides a method comprising the isolation of γδ T cells (e.g., non-Vδ2 T cells, e.g., Vδ1 T cells and / or DN T cells) from non-hematopoietic tissue (e.g., skin, e.g., skin obtained by punch biopsy). In certain embodiments, the isolation of γδ T cells from non-hematopoietic cells comprises culturing γδ T cells and non-hematopoietic cells on a synthetic scaffold configured to promote the release of cells from non-hematopoietic tissue. Any scaffold suitable for the isolation of lymphocytes from solid tissue can be used. The synthetic scaffold can be composed of natural and / or synthetic materials (e.g., natural or native polymers, e.g., polyvinylpyrrolidone, polymethylmethacrylate, methylcellulose, polystyrene, polypropylene, polyurethane), ceramics (e.g., tricalcium phosphate, calcium aluminate, calcium hydroxyapatite) or metals (tantalum, titanium, platinum, and metals of the same elemental group as combinations of platinum, niobium, hafnium, tungsten, and alloys thereof). Biological factors (e.g., collagen (e.g., collagen I or collagen II), fibronectin, laminin, integrin, angiogenic factors, anti-inflammatory factors, glycosaminoglycans, vitronectin, antibodies and fragments thereof, cytokines (e.g., IL-2 or IL-15) and combinations thereof), chemokines, and / or chemoattractants can be coated on the scaffold surface or encapsulated within the scaffold material to promote cell adhesion, migration, survival, or proliferation according to methods known in the art. In some embodiments, the synthetic scaffold is a Cellfoam scaffold as described in the Clerk protocol. Alternatively, other methods, such as enzyme-based degradation of extracellular matrix components (e.g., collagenase), can be used to isolate lymphocytes from many other non-hematopoietic tissue types.

[0099] The separation culture may be carried out for any period between 1 hour (for example, in the case of simple digestion) and 42 days (for example, in the case of culture on a scaffold). For example, when the separation step is carried out on a scaffold, the culture may be carried out for at least 5 days (for example, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 12 days, at least 14 days, at least 16 days, at least 18 days, at least 20 days, at least 21 days, at least 24 days, at least 28 days, at least 30 days, at least 35 days, or at least 40 days, for example, between 7 days and 14 days, between 14 days and 21 days, or between 21 days and 35 days, for example, about 14 days, or about 21 days).

[0100] During the isolation of γδ T cells (e.g., skin-derived γδ T cells and / or non-Vδ2 T cells, such as Vδ1 T cells and / or DN T cells), non-hematopoietic tissues and cells derived therefrom can be cultured in the presence of biological factors to promote their escape from the tissue or to promote the survival of one or more subpopulations of cells. In some embodiments, the isolation culture contains IL-2, e.g., IL-2 at a concentration of at least 10 IU / mL (e.g., 10 IU / mL to 1,000 IU / mL, 20 IU / mL to 800 IU / mL, 25 IU / mL to 750 IU / mL, 30 IU / mL to 700 IU / mL, 40 IU / mL to 600 IU / mL, 50 IU / mL to 500 IU / mL, 75 IU / mL to 250 IU / mL or 100 IU / mL to 200 IU / mL, e.g., 10 IU / mL to 20 IU / mL, 20 IU / mL to 30 IU / mL, 30 IU / mL to 40 IU / mL, 40 IU / mL to 50 IU / mL, 50 IU / mL to 75 IU / mL, 75 IU / mL to 100 IU / mL, 100 IU / mL to 150 IU / mL, 150 IU / mL to 200 IU / mL, 200 IU / mL to 500 IU / mL or 500 IU / mL to 1,000 IU / mL). In some embodiments, the isolation culture contains about 100 IU / mL of IL-2. In addition to, or instead of, this, the isolation culture contains IL-15, e.g., IL-15 at a concentration of at least 0.1 ng / mL (e.g., 0.1 ng / mL to 10,000 ng / mL, 1.0 ng / mL to 1,000 ng / mL, 5 ng / mL to 800 ng / mL, 10 ng / mL to 750 ng / mL, 20 ng / mL to 500 ng / mL, 50 ng / mL to 400 ng / mL or 100 ng / mL to 250 ng / mL, e.g., 0.1 ng / mL to 1.0 ng / mL, 1.0 ng / mL to 5.0 ng / mL, 5.0 ng / mL to 10 ng / mL, 10 ng / mL to 20 ng / mL, 20 ng / mL to 50 ng / mL, 50 ng / mL to 100 ng / mL, 100 ng / mL to 200 ng / mL, 200 ng / mL to 500 ng / mL or 500 ng / mL to 1,000 ng / mL). In some embodiments, the isolation culture contains about 20 ng / mL of IL-15.

[0101] In some embodiments, the isolation of γδ T cells from non-hematopoietic tissue involves culturing under conditions in which both IL-2 and IL-15 (each at any of the concentrations listed above) are present. In some cases, the concentration of IL-2 is about 100 IU / mL and the concentration of IL-15 is 20 ng / mL.

[0102] γδ T cells (e.g., skin-derived γδ T cells and / or non-Vδ2 T cells, such as Vδ1 T cells and / or DN T cells) can be cultured under conditions in which IL-6, IL-23, and IL-1β are absent or these cytokines are present at low concentrations (e.g., less than 20 ng / mL); because the addition of this combination of cytokines can act to reduce the proliferation of γδ T cells derived from non-hematopoietic cells (e.g., skin-derived γδ T cells and / or non-Vδ2 T cells, such as Vδ1 T cells and / or DN T cells).

[0103] In isolation from non-hematopoietic tissue (e.g., skin), generally, γδ T cells are part of a larger lymphocyte population that includes, for example, αβ T cells, B cells, and natural killer (NK) cells. In some embodiments, 1% to 10% of the isolated population of lymphocytes are γδ T cells prior to amplification (e.g., 1% to 10% of the isolated population of skin-derived lymphocytes are γδ T cells prior to amplification). In most cases, the population of γδ T cells (e.g., the population of skin-derived γδ T cells) includes a large population of Vδ1 T cells. In some embodiments, 1% to 10% of the isolated population of lymphocytes (e.g., skin-derived lymphocytes) are Vδ1 T cells prior to amplification (e.g., among the isolated population of γδ T cells prior to amplification, Vδ1 T cells can be present at more than 50%, more than 60%, more than 70%, more than 80%, or more than 90% of the population). In some examples, less than 10% of the isolated population of γδ T cells are Vδ2 T cells prior to amplification (e.g., less than 10% of the isolated population of skin-derived γδ T cells are Vδ2 T cells prior to amplification).

[0104] Non-Vδ1 T cells or non-DN T cells, such as Vδ2 T cells, αβ T cells, B cells, or NK cells, can be removed from an isolated population of γδ T cells (e.g., before, during, or after amplification).

[0105] Prior to amplification, isolated γδ T cells (e.g., γδ T cells isolated from the skin, e.g., Vδ1 T cells isolated from the skin) have a different phenotype from cells derived from the corresponding hematopoietic tissue (e.g., γδ T cells derived from blood. E.g., Vδ2 T cells derived from blood). For example, an isolated population of γδ T cells can express higher levels of CCR3, CCR4, CCR7, CCR8, or CD103 than a reference population, such as a population of TCR-activated γδ T cells that are resident in non-hematopoietic tissue, or a population of cells derived from the corresponding hematopoietic tissue (e.g., γδ T cells derived from blood, e.g., Vδ2 T cells derived from blood). In some embodiments, the isolated population of γδ T cells comprises at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more CCR3 + cells; at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more CCR4 + cells; at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more CCR7 + cells; 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more CCR8 + cells; and / or 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more CD103 + cells. The isolated population of γδ T cells can express 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, or all 6 of CCR3, CCR4, CCR7, CCR8, or CD103.

[0106] In some embodiments, the isolated population of γδ T cells expresses higher levels of NKGD2, CD56, CD69, and / or TIM3 than a reference population, such as a population of TCR-activated γδ T cells resident in non-hematopoietic tissues, or the corresponding cell population derived from hematopoietic tissues (e.g., blood-derived γδ T cells, such as blood-derived Vδ2 T cells). In some embodiments, the isolated population of γδ T cells is at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more NKGD2 + cells; at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more CD56 + cells, at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more CD69 + cells; at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more TIM3 + cells. The isolated population of γδ T cells can express 1 or more, 2 or more, 3 or more, 4 or more, or all 5 of NKGD2, CD56, CD69, and / or TIM3.

[0107] An isolated population of γδ T cells derived from non-hematopoietic tissue (e.g., γδ T cells derived from skin, e.g., Vδ1 T cells derived from skin) can be characterized by its function. Functional assays exemplified in Example 3, known in the art, can measure the functional differences between the cells of the present invention derived from non-hematopoietic tissue (e.g., an isolated population of γδ T cells, e.g., a population of Vδ1 T cells derived from skin, or an amplified population of γδ T cells, e.g., Vδ1 T cells derived from skin) and reference cells (e.g., a population of TCR-activated non-hematopoietic tissue resident γδ T cells. Or a population of corresponding hematopoietic tissue-derived cells, e.g., γδ T cells derived from blood, e.g., Vδ2 T cells derived from blood). In some embodiments, an isolated population of γδ T cells derived from non-hematopoietic tissue (e.g., a population of γδ T cells isolated substantially without contact activating the TCR pathway) secretes a higher level of IL-13 than a reference population (e.g., a population of TCR-activated non-hematopoietic tissue resident γδ T cells, e.g., a population of anti-CD3-activated γδ T cells derived from non-hematopoietic tissue). For example, a population of isolated γδ T cells derived from non-hematopoietic tissue (e.g., γδ T cells and / or non-Vδ2 T cells derived from skin, e.g., Vδ1 T cells and / or DN T cells) can secrete IL-13 at a concentration 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1,000-fold or higher than that of a reference population of cells (e.g., a population of TCR-activated non-hematopoietic tissue resident γδ T cells, e.g., a population of anti-CD3-activated non-hematopoietic tissue resident γδ T cells). Similarly, the number or frequency of non-hematopoietic tissue-derived γδ T cells secreting IL-13 contained in an isolated population of cells can be higher than that of a reference population of cells (e.g., a population of TCR-activated non-hematopoietic tissue resident γδ T cells, e.g., a population of anti-CD3-activated non-hematopoietic tissue resident γδ T cells).For example, the frequency of IL-13 secreting cells in a population of isolated γδ T cells (e.g., the frequency of IL-13 secreting cells in a population of isolated Vδ1 T cells) can be higher than that in a reference population of cells (e.g., a population of TCR-activated non-hematopoietic tissue resident γδ T cells, e.g., a population of anti-CD3 activated non-hematopoietic tissue resident γδ T cells). In some embodiments, the frequency of IL-13 secreting cells in a population of isolated γδ T cells (e.g., the frequency of IL-13 secreting cells in a population of isolated DN T cells or Vδ1 T cells of the present invention) is at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, up to 100% greater than that in a reference population of cells (e.g., a population of TCR-activated non-hematopoietic tissue resident γδ T cells, e.g., a population of anti-CD3 activated non-hematopoietic tissue resident γδ T cells).

[0108] Expansion of non-hematopoietic tissue resident γδ T cells The present invention features methods for amplifying non-hematopoietic tissue-resident γδ T cells (e.g., skin-derived γδ T cells and / or non-Vδ2 T cells, such as Vδ1 T cells and / or DN T cells). These methods can be carried out in vitro. In some embodiments, the non-hematopoietic tissue-resident γδ T cells are amplified from a population of γδ T cells isolated from non-hematopoietic tissue isolated by the above methods. Generally, non-hematopoietic tissue-resident γδ T cells can be automatically amplified if physical contact with stromal cells (e.g., skin fibroblasts) is removed. Thus, the above scaffold-based culture method can be used to induce such separation, relieve the suppression of γδ T cells, and cause amplification. Thus, in some embodiments, in the amplification step, there is substantially no activation of the TCR pathway (e.g., no exogenous TCR pathway activator is included in the culture medium). Further, the present invention provides a method for amplifying non-hematopoietic-derived resident γδ TCR cells, which method does not include contact with support cells, tumor cells, and / or antigen-presenting cells.

[0109] The inventors of the present invention have developed an amplification protocol that includes culturing non-hematopoietic tissue-resident γδ T cells in the presence of a cocktail of biological factors effective to support efficient amplification of the γδ T cells. In certain embodiments, the present invention provides a population of γδ T cells obtained from non-hematopoietic tissue (e.g., a population of γδ T cells isolated from non-hematopoietic tissue, e.g., a population of γδ T cells isolated from non-hematopoietic tissue by the methods described herein), and provides a method of amplifying the γδ T cells by culturing the γδ T cells in the presence of IL-2, IL-4, IL-15, and / or IL-21. These cytokines or their analogs can be cultured with the cells in an amount effective to produce a population of amplified γδ T cells for a period of time (e.g., at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 21 days, at least 28 days or longer, e.g., 5 days to 40 days, 7 days to 35 days, 14 to 28 days, or about 21 days).

[0110] In some embodiments, an amount of IL-2 effective to generate a population of amplified γδ T cells is from 1 IU / mL to 2,000 IU / mL (e.g., from 5 IU / mL to 1,000 IU / mL, from 10 IU / mL to 500 IU / mL, from 20 IU / mL to 400 IU / mL, from 50 IU / mL to 250 IU / mL or about 100 IU / mL, e.g., from 5 IU / mL to 10 IU / mL, from 10 IU / mL to 20 IU / mL, from 20 IU / mL to 30 IU / mL, from 30 IU / mL to 40 IU / mL, from 40 IU / mL to 50 IU / mL, from 50 IU / mL to 60 IU / mL, from 60 IU / mL to 70 IU / mL, from 70 IU / mL to 80 IU / mL, from 80 IU / mL to 90 IU / mL, from 90 IU / mL to 100 IU / mL, from 100 IU / mL to 120 IU / mL, from 120 IU / mL to 140 IU / mL, from 140 IU / mL to 150 IU / mL, from 150 IU / mL to 175 IU / mL, from 175 IU / mL to 200 IU / mL, from 200 IU / mL to 300 IU / mL, from 300 IU / mL to 400 IU / mL, from 400 IU / mL to 500 IU / mL, from 500 IU / mL to 1,000 IU / mL, from 1,000 IU / mL to 1,500 IU / mL, from 1,500 IU / mL to 2,000 IU / mL or higher concentrations). In some embodiments, an amount of IL-2 effective to generate a population of amplified γδ T cells is about 100 IU / mL.

[0111] In some embodiments, an effective amount of IL-4 for generating a population of amplified γδ T cells (e.g., skin-derived γδ T cells and / or non-Vδ2 T cells, such as Vδ1 T cells and / or DN T cells) is at least 0.1 ng / mL (e.g., 0.1 ng / mL to 10,000 ng / mL, 1.0 ng / mL to 1,000 ng / mL, 5 ng / mL to 800 ng / mL, 10 ng / mL to 750 ng / mL, 20 ng / mL to 500 ng / mL, 50 ng / mL to 400 ng / mL, 100 ng / mL to 250 ng / mL, 0.1 ng / mL to 1.0 ng / mL, 1.0 ng / mL to 5.0 ng / mL, 5.0 ng / mL to 10 ng / mL, 10 ng / mL to 20 ng / mL, 20 ng / mL to 50 ng / mL, 50 ng / mL to 100 ng / mL, 100 ng / mL to 200 ng / mL, 200 ng / mL to 500 ng / mL, 500 ng / mL to 1,000 ng / mL). In some embodiments, an effective amount of IL-4 for generating amplified γδ T cells is about 5 ng / mL.

[0112] In some embodiments, an effective amount of IL-15 for generating a population of amplified γδ T cells (e.g., skin-derived γδ T cells and / or non-Vδ2 T cells, such as Vδ1 T cells and / or DN T cells) is at least 0.1 ng / mL (e.g., 0.1 ng / mL to 10,000 ng / mL, 1.0 ng / mL to 1,000 ng / mL, 5 ng / mL to 800 ng / mL, 10 ng / mL to 750 ng / mL, 20 ng / mL to 500 ng / mL, 50 ng / mL to 400 ng / mL, 100 ng / mL to 250 ng / mL, e.g., 0.1 ng / mL to 1.0 ng / mL, 1.0 ng / mL to 5.0 ng / mL, 5.0 ng / mL to 10 ng / mL, 10 ng / mL to 20 ng / mL, 20 ng / mL to 50 ng / mL, 50 ng / mL to 100 ng / mL, 100 ng / mL to 200 ng / mL, 200 ng / mL to 500 ng / mL, or 500 ng / mL to 1,000 ng / mL). In some embodiments, an effective amount of IL-15 for generating amplified γδ T cells is about 10 ng / mL.

[0113] In some embodiments, an effective amount of IL-21 to generate a population of amplified γδ T cells (e.g., skin-derived γδ T cells and / or non-Vδ2 T cells, such as Vδ1 T cells and / or DN T cells) is at least 0.1 ng / mL (e.g., 0.1 ng / mL to 10,000 ng / mL, 1.0 ng / mL to 1,000 ng / mL, 5 ng / mL to 800 ng / mL, 10 ng / mL to 750 ng / mL, 20 ng / mL to 500 ng / mL, 50 ng / mL to 400 ng / mL, 100 ng / mL to 250 ng / mL, e.g., 0.1 ng / mL to 1.0 ng / mL, 1.0 ng / mL to 5.0 ng / mL, 5.0 ng / mL to 10 ng / mL, 10 ng / mL to 20 ng / mL, 20 ng / mL to 50 ng / mL, 50 ng / mL to 100 ng / mL, 100 ng / mL to 200 ng / mL, 200 ng / mL to 500 ng / mL, or 500 ng / mL to 1,000 ng / mL). In some embodiments, an effective amount of IL-21 for the generation of amplified γδ T cells is about 10 ng / mL.

[0114] Substitution or addition of other factors in the amplification culture of non-hematopoietic tissue-resident γδ T cells is also provided herein. For example, in some embodiments, one or more factors selected from the group consisting of IL-6, IL-7, IL-8, IL-9, IL-12, IL-18, IL-33, IGF-1, IL-1β, human platelet lysate (HPL), and stromal cell-derived factor-1 (SDF-1) are included in a form that is added to or substituted for any of IL-2, IL-4, IL-15, and IL-21. Appropriate concentrations of each factor are provided in Table 2 of Example 3.

[0115] It will be understood that the amount of each of the above cytokines required for the generation of a population of amplified γδ T cells depends on the concentration of one or more other cytokines. For example, if the concentration of IL-2 increases or decreases, the concentration of IL-15 will decrease or increase accordingly. As noted above, herein, an effective amount for generating an amplified population refers to the combined effect of the effects of all factors in cell amplification.

[0116] In some embodiments, γδ T cells are exposed to each factor simultaneously (e.g., γδ T cells are exposed to IL-2, IL-4, IL-15, and IL-21 simultaneously, e.g., for 5 days). In other examples, γδ T cells are exposed to a specific factor prior to culture with other factors. For example, during the expansion culture, additional factors may be gradually provided over the expansion course, or γδ T cells may be transferred from a culture medium containing one factor or a group of factors to another culture medium.

[0117] In some embodiments, γδ T cells are expanded by culturing for a period of several hours (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 18, or 21 hours) to about 35 days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 days). In certain embodiments, a population of γδ T cells is expanded in 14 to 21 days. Thus, for the steps of separation and amplification, including the separation culture period (e.g., 1 to 40 days, e.g., 14 to 21 days), in some embodiments, between 28 days and 56 days, or about 41 days, may be required.

[0118] The amplification method provides a population of amplified γδ T cells that is greater in number than a reference population. In some embodiments, the population of amplified γδ T cells is greater in number than the population of isolated γδ T cells prior to the amplification step (e.g., at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1,000-fold, at least 5,000-fold, at least 10,000-fold, or more, compared to the population of isolated γδ T cells prior to the amplification step).

[0119] Accordingly, the present invention provides means for generating large populations of γδ T cells derived from non-hematopoietic tissues (e.g., γδ T cells derived from skin and / or non-Vδ2 T cells, such as Vδ1 T cells and / or DN T cells) with high efficiency (e.g., by exclusion of stromal cell contact and / or TCR stimulation, or by culturing in the presence of an effective amount of a factor). In some embodiments, the amplification step described herein amplifies γδ T cells with a short cell doubling time, which is obtained by the following formula: [Equation 1] Doubling time = period × log(2) / (log(final concentration) - log(initial concentration)) Based on the information in this specification, for example, the information in Example 3 described below, those skilled in the art will recognize that the present invention provides a method for amplifying non-hematopoietic tissue-derived γδ T cells (for example, skin-derived γδ T cells and / or non-Vδ2 T cells, such as Vδ1 T cells and / or DN T cells) with a doubling time of less than 5 days (for example, less than 4.5 days, less than 4.0 days, less than 3.9 days, less than 3.8 days, less than 3.7 days, less than 3.6 days, less than 3.5 days, less than 3.4 days, less than 3.3 days, less than 3.2 days, less than 3.1 days, less than 3.0 days, less than 2.9 days, less than 2.8 days, less than 2.7 days, less than 2.6 days, less than 2.5 days, less than 2.4 days, less than 2.3 days, less than 2.2 days, less than 2.1 days, less than 2.0 days, less than 46 hours, less than 42 hours, less than 38 hours, less than 35 hours, less than 32 hours).

[0120] In some embodiments, within 7 days of culturing, the amplified population of γδ T cells (e.g., an amplified population of Vδ1 T cells and / or DN T cells) contains at least 10-fold the number of γδ T cells compared to the population of isolated γδ T cells before amplification (e.g., at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1,000-fold, at least 2,000-fold, at least 3,000-fold, at least 4,000-fold, at least 5,000-fold, at least 6,000-fold, at least 7,000-fold, or at least 8,000-fold the number compared to the population of isolated γδ T cells before amplification). In some embodiments, within 14 days of culturing, the amplified population of γδ T cells (e.g., an amplified population of Vδ1 T cells and / or DN T cells) contains at least 20-fold the number of γδ T cells compared to the population of isolated γδ T cells before amplification (e.g., at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1,000-fold, at least 2,000-fold, at least 3,000-fold, at least 4,000-fold, at least 5,000-fold, at least 6,000-fold, at least 7,000-fold, at least 8,000-fold, at least 9,000-fold, or at least 10,000-fold the number).In some embodiments, within 21 days of culture, the amplified population of γδ T cells (e.g., an amplified population of Vδ1 T cells and / or DN T cells) contains at least 50-fold the number of γδ T cells compared to the isolated population of γδ T cells before amplification (e.g., at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1,000-fold, at least 2,000-fold, at least 3,000-fold, at least 4,000-fold, at least 5,000-fold, at least 6,000-fold, at least 7,000-fold, at least 8,000-fold, at least 9,000-fold, or at least 10,000-fold the number compared to the isolated population of γδ T cells before amplification). In some embodiments, within 28 days of culture, the amplified population of γδ T cells (e.g., an amplified population of Vδ1 T cells and / or DN T cells) contains at least 100-fold the number of γδ T cells compared to the isolated population of γδ T cells before amplification (e.g., at least 110-fold, at least 120-fold, at least 130-fold, at least 140-fold, at least 150-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1,000-fold, at least 2,000-fold, at least 3,000-fold, at least 4,000-fold, at least 5,000-fold, at least 6,000-fold, at least 7,000-fold, at least 8,000-fold, at least 9,000-fold, at least 10,000-fold, at least 12,000-fold, or at least 15,000-fold the number compared to the isolated γδ T cells before amplification).

[0121] γδ T cells derived from non-hematopoietic tissues (e.g., γδ T cells derived from skin and / or non-Vδ2 T cells, such as Vδ1 T cells and / or DN T cells) amplified by the methods described herein may have a phenotype suitable for anti-tumor ability. In some embodiments, a population of amplified γδ T cells (e.g., Vδ1 T cells derived from skin) exhibits a higher average CD27 expression level than a reference population (e.g., a population of isolated γδ T cells prior to the amplification step). In some embodiments, the average CD27 expression level of a population of amplified γδ T cells is at least 2-fold (e.g., at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1,000-fold, at least 5,000-fold, at least 10,000-fold, at least 20,000-fold, or more) compared to a population of isolated γδ T cells.

[0122] A distinct portion of the population of amplified γδ T cells (γδ T cells derived from skin and / or non-Vδ2 T cells, such as Vδ1 T cells and / or DN T cells) has an upregulated CD27 level, while other portions may have low CD27 or no CD27. - In this case, the frequency of CD27+ cells in the amplified population may be higher compared to the population of isolated γδ T cells. For example, a population of amplified γδ T cells has a higher frequency of CD27+ cells compared to the population of isolated γδ T cells prior to amplification. + +Can have cells at a frequency that is at least 5% higher (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or up to 100% higher) than a population of isolated γδ T cells before amplification, CD27 + Cells have a high frequency. In some embodiments, the number of CD27 + Cells in the amplified population compared to a population of isolated γδ T cells may be increased. For example, the population of amplified γδ T cells may have at least twice the number of CD27 + Cells (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or up to 100% higher) than a population of isolated γδ T cells before amplification, CD27 + Cells have a high frequency).

[0123] The expansion methods provided herein, in some embodiments, result in a population of non-hematopoietic tissue-derived 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) that have lower TIGIT expression compared to a reference population (e.g., a population of isolated γδ T cells prior to the expansion step). In some embodiments, the expanded population of γδ T cells exhibits lower mean TIGIT expression than a reference population (e.g., a population of isolated γδ T cells prior to the expansion step). In some embodiments, the expanded population of γδ T cells exhibits at least 10% lower mean TIGIT expression compared to the isolated population of γδ T cells (e.g., at least 20% lower, at least 30% lower, at least 40% lower, at least 50% lower, at least 60% lower, at least 70% lower, at least 80% lower, at least 90% lower, or up to 100% lower compared to the isolated population of γδ T cells).

[0124] A distinct portion of the expanded γδ T cell (e.g., skin-derived γδ T cells and / or non-Vδ2 T cells, e.g., Vδ1 T cells and / or DN T cells) population expresses TIGIT, e.g., high levels of TIGIT, whereas other portions express low levels of TIGIT or no TIGIT. - In this case, it is possible that TIGIT expression in the expanded population is higher than in the isolated population of γδ T cells. + For example, the expanded population of γδ T cells may have a lower frequency of TIGIT cells compared to the isolated population of γδ T cells prior to expansion. + The frequency of TIGIT cells may be at least 5% lower (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or up to 100% lower than the isolated population of γδ T cells prior to expansion). +(where the frequency of cells is low). In some embodiments, TIGIT of the amplified population, compared to the population of isolated γδ T cells before amplification + The number of cells can be low. For example, TIGIT in the population of isolated γδ T cells before amplification + Compared to the number of cells, TIGIT of the amplified population of γδ T cells + The number of cells is at least 10% less (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or up to 100% less TIGIT + The number of cells) compared to the number of TIGIT cells in the population of isolated γδ T cells before amplification. + (where the number of cells is low).

[0125] In some embodiments, a population of amplified γδ T cells (e.g., skin-derived γδ T cells or non-Vδ2 T cells, such as Vδ1 T cells and / or DN T cells) has a high frequency of CD27 + Cells and a low frequency of TIGIT + Cells. In some embodiments, a population of amplified γδ T cells has a high frequency of CD27 compared to a reference population (e.g., compared to the population of isolated γδ T cells before amplification) + TIGIT - Cells. For example, a population of amplified γδ T cells can have at least 5% higher frequency of CD27 + TIGIT - Cells compared to the population of isolated γδ T cells before amplification (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or up to 100% higher frequency of CD27 + TIGIT -cells). In some embodiments, compared to isolated γδ T cells, the number of CD27 + TIGIT - cells may be increased. For example, compared to a population of isolated γδ T cells before amplification, the amplified population of γδ T cells may have at least twice the number of CD27 + TIGIT - cells (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or up to 100% higher frequency of CD27 + TIGIT - cells) compared to the population of isolated γδ T cells before amplification).

[0126] In some embodiments, the mean expression level of TIGIT in a population of amplified γδ T cells (e.g., skin-derived γδ T cells or non-Vδ2 T cells, such as Vδ1 T cells and / or DNT cells) is lower compared to a reference population. In some embodiments, the amplified population of CD27 + γδ T cells has a lower mean expression level of TIGIT compared to a reference population (e.g., the population of isolated CD27 + γδ T cells before amplification). In some embodiments, the amplified population of CD27 + γδ T cells has a lower mean expression level of TIGIT compared to the population of isolated CD27 + γδ T cells. In some embodiments, the amplified population of CD27 + γδ T cells has a TIGIT expression level that is at least 10% lower (e.g., at least 20% lower, at least 30% lower, at least 40% lower, at least 50% lower, at least 60% lower, at least 70% lower, at least 80% lower, at least 90% lower, or up to 100% lower) compared to the population of isolated CD27 + γδ T cells).

[0127] In addition to, or instead of, TIGIT in a population of amplified γδ T cells (e.g., skin-derived γδ T cells or non-Vδ2 T cells, such as Vδ1 T cells and / or DNT cells) - The median CD27 expression level in the population of γδ T cells is high compared to the reference population. For example, amplified TIGIT - The population of γδ T cells is the isolated TIGIT before amplification - compared to the population of γδ T cells, CD27 + The frequency of cells is at least 5% higher (e.g., the isolated TIGIT - compared to the population of γδ T cells, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or up to 100% higher CD27 + frequency of cells). In some embodiments, the isolated TIGIT - compared to the population of γδ T cells, CD27 in the amplified population + The number of cells may be increased. For example, the isolated TIGIT before amplification - compared to the population of γδ T cells, the amplified TIGIT - The population of γδ T cells may have at least twice the number of CD27 + cells (e.g., compared to the population of isolated TIGIT - compared to the population of γδ T cells, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or up to 100% higher CD27 + frequency of cells).

[0128] Increased or decreased expression of other markers, including CD124, CD215, CD360, CTLA4, CD1b, BTLA, CD39, CD45RA, Fas ligand, CD25, ICAM-1, CD31, KLRG1, CD30, CD2, NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1, and CD64, can be used additionally or alternatively to characterize a population of amplified γδ T cells derived from one or more non-hematopoietic tissues (e.g., skin-derived γδ T cells or non-Vδ2 T cells, such as Vδ1 T cells and / or DN T cells). In some examples, a population of amplified γδ T cells (e.g., skin-derived γδ T cells or non-Vδ2 T cells, such as Vδ1 T cells and / or DNT cells) has a higher average expression level of one or more markers selected from the group consisting of CD124, CD215, CD360, CTLA4, CD1b, BTLA, CD39, CD45RA, Fas ligand, CD25, ICAM-1, CD31, KLRG1, CD30, and CD2, compared to a population of isolated γδ T cells before amplification. In addition to or instead of this, a population of amplified γδ T cells (e.g., skin-derived γδ T cells or non-Vδ2 T cells, such as Vδ1 T cells and / or DNT cells) has a higher frequency of cells expressing one or more markers selected from the group consisting of CD124, CD215, CD360, CTLA4, CD1b, BTLA, CD39, CD45RA, Fas ligand, CD25, ICAM-1, CD31, KLRG1, CD30, and CD2, compared to a population of isolated γδ T cells. In some embodiments, a population of amplified γδ T cells (e.g., skin-derived γδ T cells or non-Vδ2 T cells, such as Vδ1 T cells and / or DNT cells) has a lower average expression level of one or more markers selected from the group consisting of NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1, and CD64, compared to a population of isolated γδ T cells.Similarly, compared to a population of isolated γδ T cells, the amplified population has a lower number of cells expressing one or more markers selected from the group consisting of NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1, and CD64.

[0129] Thus, the non-hematopoietic tissue-resident γδ T cells generated by the methods of the invention can have one or more of the following properties: (i) exhibit a phenotype of high CD69, high TIM3, and low / no CD28; (ii) up-regulate one or more of CCR3, CD39, CD11b, and CD9; (iii) generate IFN-γ in response to NKG2D ligands in the absence of TCR agonists; (iv) generate IL-13 in the absence of TCR agonists; (v) generate one or more of IFN-γ, TNF-α, and GM-CSF in response to TCR activation; (vi) do not generate, or substantially do not generate, IL-17 in response to TCR activation; (vii) grow in a medium containing IL-2 in the absence of additional growth factors; (viii) exhibit a cytotoxic TCR cell response in the absence of TCR agonists; and / or (ix) exhibit selective cytotoxicity against tumor cells compared to normal cells.

[0130] In some examples, the non-hematopoietic tissue-resident γδ T cells generated by the methods of the invention generate IL-13 in the absence of TCR agonists and / or generate IFN-γ in response to NKG2D in the absence of TCR agonists.

[0131] A number of basal media suitable for use in the proliferation of γδ T cells are available, particularly complete media such as AIM-V, Iscoves medium, RPMI-1640 (Life Technologies), etc. Other medium components may be added to the medium, such as serum, serum proteins, and selective agents such as antibiotics. For example, in some embodiments, RPMI-1640 medium containing 2 mM glutamine, 10% FBS, 10 mM HEPES, pH 7.2, 1% penicillin-streptomycin, sodium pyruvate (1 mM; Life Technologies), non-essential amino acids (e.g., 100 μM of Gly, Ala, Asn, Asp, Glu, Pro, and Ser, 1×MEM non-essential amino acids, Life Technologies), and 10 μL / L β-mercaptoethanol. For convenience, cells are cultured at 37°C in a humidified atmosphere containing 5% CO2 in an appropriate medium.

[0132] γδ T cells can be cultured as described herein in any suitable system including stirred tank fermenters, air-lift fermenters, roller bottles, culture bags or dishes, and other bioreactors, particularly 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.

[0133] The methods described herein can include multiple selection steps, such as multiple depletion steps. Enrichment of a T cell population by negative selection can be achieved, for example, by combining antibodies against surface markers specific to the cells being negatively selected. In one method, a cocktail of monoclonal antibodies against cell surface markers present on the cells being negatively selected is used to perform cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry.

[0134] IV. Pharmaceutical Compositions and Therapeutic Methods The γδ T cells obtained by the method of the present invention can be used as a medicament, for example, for adoptive T cell therapy. This includes transplanting the γδ T cells obtained by the method of the present invention into a patient. The treatment may be autologous transplantation, that is, transplantation in which the γδ T cells are returned to the same patient from whom they were taken, or allogeneic transplantation, that is, transplantation of γδ T cells from one human to a different patient. In an example including allogeneic transplantation, the γδ T cells may be substantially free of αβ T cells. For example, αβ T cells can be depleted from a population of γδ T cells using any suitable means well known in the art (e.g., by negative selection, e.g., using magnetic beads), for example, after amplification. The treatment method may include providing a sample of non-hematopoietic cells obtained from a donor, culturing γδ T cells from the sample to generate an amplified population, and administering the amplified population of γδ T cells to an individual recipient.

[0135] The patient or subject to be treated is preferably a human cancer patient (e.g., a human cancer patient undergoing treatment for a solid tumor) or a viral infection patient (e.g., a CMV-infected or HIV-infected patient). In some examples, the patient has been and / or is undergoing treatment for solid cancer.

[0136] Since tissue-resident Vδ1 T and DN γδ T cells usually reside in non-hematopoietic tissues, they are more likely to home to and be retained in tumor masses compared to systemic blood residency, and adoptive transfer of these cells is more effective in targeting solid tumors and immune diseases associated with other non-hematopoietic tissues that can occur.

[0137] Since γδ T cells are not restricted by MHC, they do not recognize the host receiving transplantation as foreign, which means that graft-versus-host disease is less likely to occur. This means that they can be used as "off the shelf" and transplanted for any recipient, for example, for adoptive T cell therapy of allogeneic transplantation.

[0138] The non-hematopoietic tissue-resident γδ T cells obtained by the method of the present invention express NKG2D and respond to NKG2D ligands (such as MICA) strongly associated with malignant tumors. They are also effective in killing tumor cells because they exhibit cytotoxicity even under non-activated conditions. For example, the obtained non-hematopoietic tissue-resident γδ T cells described herein express one or more, preferably all, of IFN-γ, TNF-α, GM-CSF, CCL4, IL-13, granulysin, granzymes A and B, and perforin under non-activated conditions. IL-17A may not be expressed.

[0139] Accordingly, the findings reported herein provide compelling evidence regarding the utility and suitability of non-hematopoietic tissue-resident γδ T cells obtained by the method of the present invention for clinical application as an "off-the-shelf" immunotherapeutic agent. These cells have innate killing ability, are not restricted by MHC, and exhibit better homing and / or retention properties within tumors than other T cells.

[0140] In some embodiments, a method of treating an individual having a tumor in non-hematopoietic tissue may include the steps of: providing a sample of the non-hematopoietic tissue from a donor individual; culturing γδ T cells from the sample to generate an amplified population; and administering the amplified population of γδ T cells to the individual having the tumor.

[0141] The pharmaceutical composition may comprise the non-hematopoietic tissue-resident amplified γδ T cells described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include neutral buffered saline, buffers such as phosphate buffered saline, carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA and glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The cryopreservation solution that can be used in the pharmaceutical composition of the present invention may contain, for example, DMSO. The composition can be formulated, for example, for intravenous administration.

[0142] In certain embodiments, the pharmaceutical composition is, for example, free of detectable levels of contaminants such as endotoxin or mycoplasma and is substantially free of contaminants.

[0143] In some instances, an amount of the amplified T cells obtained by any of the above methods that is therapeutically effective can be administered to a subject in a therapeutically effective amount (e.g., for the treatment of cancer, e.g., for the treatment of solid tumors). In some cases, a therapeutically effective amount of the amplified γδ T cells (e.g., skin-derived γδ T cells or non-Vδ2 T cells, e.g., Vδ1 T cells and / or DN T cells) is less than 10×10 12 cells per dose (e.g., less than 9×10 12 cells per dose, less than 8×10 12 cells per dose, less than 7×10 12 cells per dose, less than 6×10 12 cells per dose, less than 5×10 12 cells per dose, less than 4×10 12 cells per dose, less than 3×10 12 cells per dose, less than 2×10 12 cells per dose, less than 1×10 12 cells per dose, less than 9×10 11 cells per dose, less than 8×10 11 cells per dose, less than 7×10 11less than cells, 6×10 per administration 11 less than cells, 5×10 per administration 11 less than cells, 4×10 per administration 11 less than cells, 3×10 per administration 11 less than cells, 2×10 per administration 11 less than cells, 1×10 per administration 11 less than cells, 9×10 per administration 10 less than cells, 7.5×10 per administration 10 less than cells, 5×10 per administration 10 less than cells, 2.5×10 per administration 10 less than cells, 1×10 per administration 10 less than cells, 7.5×10 per administration 9 less than cells, 5×10 per administration 9 less than cells, 2.5×10 per administration 9 less than cells, 1×10 per administration 9 less than cells, 7.5×10 per administration 8 less than cells, 5×10 per administration 8 less than cells, 2.5×10 per administration 8 less than cells, 1×10 per administration 8 less than cells, 7.5×10 per administration 7 less than cells, 5×10 per administration 7 less than cells, 2.5 x 10 per administration 7 less than cells, 1×10 per administration 7 less than cells, 7.5×10 per administration 6 less than cells, 5×10 per administration 6 less than cells, 2.5×10 per administration 6 less than cells, 1×10 per administration 6 less than cells, 7.5×10 per administration 5 less than cells, 5×10 per administration 5 less than cells, 2.5×10 per administration 5 less than cells or 1×10 per administration 5 less than cells).

[0144] In some embodiments, an effective amount for the treatment of expanded γδ T cells (e.g., skin-derived γδ T cells or non-Vδ2 T cells, such as Vδ1 T cells and / or DN T cells) is less than 10×10 12 cells over the course of treatment (e.g., less than 9×10 12 cells, less than 8×10 12 cells, less than 7×10 12 cells per dose, less than 6×10 12 cells, less than 5×10 12 cells per dose, less than 4×10 12 cells, less than 3×10 12 cells, less than 2×10 12 cells, less than 1×10 12 cells, less than 9×10 11 cells, less than 8×10 11 cells, less than 7×10 11 cells, less than 6×10 11 cells, less than 5×10 11 cells, less than 4×10 11 cells, less than 3×10 11 cells, less than 2×10 11 cells, less than 1×10 11 cells, less than 9×10 10 cells, less than 7.5×10 10 cells, less than 5×10 10 cells, less than 2.5×10 10 cells, less than 1×10 10 cells, less than 7.5×10 9 cells, less than 5×10 9 cells, less than 2.5×10 9 cells, less than 1×10 9 cells, less than 7.5×10 8 cells, less than 5×10 8 cells, less than 2.5×10 8 cells, less than 1×10 8 cells, less than 7.5×10 7 cells, less than 5×10 7 cells, less than 2.5 x 10 7 cells, less than 1×10 7 cells, less than 7.5×10 6 cells, less than 5×10 6 cells, less than 2.5×10 6Less than cells, 1×10 6 Less than cells, 7.5×10 5 Less than cells, 5×10 5 Less than cells, 2.5×10 5 Less than cells or 1×10 5 Less than cells).

[0145] In some embodiments, one dosage of the amplified non-hematopoietic tissue-resident γδ T cells described herein is about 1×10 6 , 1.1×10 6 , 2×10 6 , 3.6×10 6 , 5×10 6 , 1×10 7 , 1.8×10 7 , 2×10 7 , 5×10 7 , 1×10 8 , 2×10 8 or 5×10 8 cells / kg. In some embodiments, one dosage of the amplified non-hematopoietic tissue-resident γδ T cells (e.g., skin-derived γδ T cells or non-Vδ2 T cells, e.g., Vδ1 T cells and / or DN T cells) is at least about 1×10 6 , 1.1×10 6 , 2×10 6 , 3.6×10 6 , 5×10 6 , 1×10 7 , 1.8×10 7 , 2×10 7 , 5×10 7 , 1×10 8 , 2×10 8 or 5×10 8 cells / kg. In some embodiments, one dosage of the amplified non-hematopoietic tissue-resident γδ T cells (e.g., skin-derived γδ T cells or non-Vδ2 T cells, e.g., Vδ1 T cells and / or DN T cells) is at most about 1×10 6 , 1.1×10 6 , 2×10 6 , 3.6×10 6 , 5×10 6 , 1×10 7 , 1.8×10 7, 2×10 7 , 5×10 7 , 1×10 8 , 2×10 8 or 5×10 8 cells / kg. In some embodiments, a single dose of the expanded non-hematopoietic tissue-resident γδ T cells (e.g., skin-derived γδ T cells or non-Vδ2 T cells, e.g., Vδ1 T cells and / or DN T cells) is about 1.1×10 6 ~1.8×10 7 cells / kg. In some embodiments, a single dose of the expanded non-hematopoietic tissue-resident γδ T cells (e.g., skin-derived γδ T cells or non-Vδ2 T cells, e.g., Vδ1 T cells and / or DN T cells) is about 1×10 7 , 2×10 7 , 5×10 7 , 1×10 8 , 2×10 8 , 5×10 8 , 1×10 9 , 2×10 9 or 5×10 9 cells. In some embodiments, a single dose of the expanded non-hematopoietic tissue-resident γδ T cells (e.g., skin-derived γδ T cells or non-Vδ2 T cells, e.g., Vδ1 T cells and / or DN T cells) is at least about 1×10 7 , 2×10 7 , 5×10 7 , 1×10 8 , 2×10 8 , 5×10 8 , 1×10 9 , 2×10 9 or 5×10 9 cells. In some embodiments, a single dose of the expanded non-hematopoietic tissue-resident γδ T cells (e.g., skin-derived γδ T cells or non-Vδ2 T cells, e.g., Vδ1 T cells and / or DN T cells) is at most about 1×10 7 , 2×10 7 , 5×10 7 , 1×10 8 , 2×10 8 , 5×10 8 , 1×10 9 , 2×109 or 5×10 9 contains cells.

[0146] In certain embodiments, the subject is administered from 10 4 to 10 6 amplified non-hematopoietic tissue-resident γδ T cells (e.g., skin-derived γδ T cells or non-Vδ2 T cells, e.g., Vδ1 T cells and / or DN T cells) per kg of the subject's body weight. In certain embodiments, the subject is administered a first dose of a population of non-hematopoietic tissue-resident γδ T cells (e.g., a first dose of from 10 4 to 10 6 γδ T cells per kg of the subject's body weight, e.g., a first dose of from 10 4 to 10 5 γδ T cells), followed by one or more (e.g., 2, 3, 4, or 5) administrations of amplified non-hematopoietic tissue-resident γδ T cells (e.g., one or more subsequent administrations of from 10 4 to 10 6 γδ T cells per kg of the subject's body weight, e.g., one or more subsequent administrations of from 10 4 to 10 5 γδ T cells per kg of the subject's body weight). In certain embodiments, one or more subsequent administrations are administered less than 15 days after the previous administration, e.g., less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days after the previous administration, e.g., less than 4, 3, or 2 days after the previous administration. In certain embodiments, the subject receives a total of from 10 6 γδ T cells per kg of the subject's body weight over the course of at least three administrations of a population of γδ T cells. For example, the subject receives a first dose of 1×10 5 γδ T cells, a second dose of 3×10 5 γδ T cells, and a third dose of 6×10 5 γδ T cells, and, e.g., each administration is administered less than 4, 3, or 2 days after the previous administration.

[0147] The non-hematopoietic tissue-resident γδ T cells obtained by the method of the present invention can also be used in CAR-T therapy. This involves the generation of a modified T cell receptor (TCR) for reprogramming T cells to have new specificities, such as the specificities of monoclonal antibodies. The modified TCR can make T cells specific for malignant cells and is thus useful for cancer immunotherapy. For example, T cells can recognize cancer cells expressing tumor antigens, such as tumor-associated antigens not expressed in normal somatic cells from the subject's tissue. Therefore, CAR-modified T cells can be used, for example, in adoptive T cell therapy for cancer patients.

[0148] The use of blood-resident γδ T cells for CAR has been reported. However, the non-hematopoietic tissue-resident γδ T cells obtained by the method of the present invention can be transduced with a chimeric antigen-specific TCR while retaining the unique function of recognizing transformed cells, making them a particularly good vehicle for the CAR-T approach. They are also likely to have superior tumor infiltration and retention capabilities compared to blood-resident γδ T cells or conventional systemic αβ T cells. Furthermore, since they do not have MHC-dependent antigen presentation, they can reduce the possibility of graft-versus-host and target tumors expressing low levels of MHC. Similarly, since they do not depend on conventional co-stimulation (e.g., co-stimulation by the involvement of CD28), they can also target tumors with low expression levels of co-stimulatory receptor ligands.

[0149] In some embodiments, one or more additional therapeutic agents can be administered to the subject. The additional therapeutic agents can be selected from the group consisting of immunotherapy agents, cytotoxic agents, growth inhibitors, radiotherapy agents, angiogenesis inhibitors, or combinations of two or more thereof. The additional therapeutic agents can be administered simultaneously with, before, or after the administration of the expanded γδ T cells. The additional therapeutic agent can be an immunotherapy agent that acts on targets in the subject's body (e.g., the subject's own immune system) and / or the transplanted γδ T cells.

[0150] Administration of the composition can be carried out by any convenient method. The compositions described herein can be administered to a patient by transarterial, subcutaneous, intradermal, intratumoral, intranodular, intramedullary, intramuscular, intravenous injection, or intraperitoneal, for example, intradermal or subcutaneous injection. Compositions of non-hematopoietic cell resident γδ T cells may be injected directly into tumors, lymph nodes, or sites of infection.

Examples

[0151] In most adults, Vδ2 cells, during the resting phase, constitute only a very diverse minority (0.01 - 5%) of blood T cells, but after stimulation by a wide range of substances including numerous bacteria and parasites, these cells rapidly amplify and can temporarily reach up to about 25% of CD3 + cells. The major basis for this response is the Vδ2 TCR-mediated recognition of low molecular weight "phospho moieties", including hydroxymethylbuta-2-enyl pyrophosphate (HMBPP), which are intermediates in the important microbial synthetic pathways for modifying proteins (e.g., by geranylation or farnesylation) using cholesterol and other lipids. In primates, this synthesis occurs via the mevalonate pathway, and one of its intermediates, isopentenyl pyrophosphate (IPP), is expressed at very high levels in virus-infected cells and transformed cells, which is also a target for Vδ2 TCR-mediated recognition.

[0152] In addition, most Vδ2 T cells express high levels of the NKG2D receptor, which can activate or co-stimulate (along with the T cell receptor (TCR)) the cell's cytolytic ability when engaged with NKG2D ligands (e.g., MICA, MICB, and ULBP). Those ligands are host proteins that are upregulated when cells are exposed to factors such as oxidative stress or osmotic stress or ultraviolet light. These factors promote hyper-active signaling of the epidermal growth factor receptor (EGFR) pathway, which is also generally dysregulated in human solid tumors.

[0153] The ability of Vδ2 T cells to detect transformed cells using TCR and / or NKG2D, together with their potent cytolytic ability and the distinct ability to present antigens to CD8 + T cells, has collectively led to the view that Vδ2 T cells can be clinically exploited to deliver cancer immunotherapy. This can be achieved by adoptive transfer of cells, and in this regard, the fact that γδ T cells are not restricted by MHC significantly and beneficially limits graft-versus-host disease (GvHD). To achieve this, by adding cytokines (such as interleukin-2 (IL-2)), together with exogenous TCR activators (such as the phospho moiety (e.g., BrHPP)), or together with clinically approved bisphosphonates (e.g., zoledronic acid) (which inhibit farnesyl pyrophosphate synthase in the mevalonate pathway, thereby inducing the accumulation of IPP, which is the TCR-activating moiety), blood-resident Vγ9Vδ2 γδ T cells can be amplified ex vivo. However, chronic activation of Vγ9Vδ2 γδ T cells via factors such as BrHPP can gradually lead to cell exhaustion and a decrease in cytotoxic ability.

[0154] Alternatively, a patient's own γδ T cells can be activated in situ using pharmacologically modified forms of HMBPP, or clinically approved aminobisphosphonates. Over 250 patients have been treated with these approaches, and these approaches appear to be safe, but complete remissions have occurred only rarely. One major concern regarding the limited clinical efficacy of the cells is their tendency to become irreversibly exhausted by chronic antigen exposure. A second major concern is that they do not appear to home efficiently to solid tumors and the tissues that harbor those tumors.

[0155] Chimeric antigen receptor T cell (CAR-T) therapy has been shown to be clinically promising for B cell malignancies. However, for the treatment of solid tumors, the performance of CAR-T cells is currently lower than expected, the efficiency of producing a complete tumor response is not high, and a high incidence of cytotoxicity outside the tumor has been shown. Regarding peripheral blood γδ T cells, the main obstacle to the success of the CAR-T approach for solid tumors is that systemic CAR-T cells are probably not efficient in migrating to the site of the malignant tumor and remaining there in a functionally effective state. In addition, based on conventional αβ T cells, CAR-T cells have to overcome immunosuppressive signals in the tumor microenvironment (e.g., those transmitted via the PD1 receptor).

[0156] Since γδ T cells can be transduced with a tumor-reactive chimeric antigen-specific TCR while retaining their innate ability to recognize transformed cells using receptors such as NKG2D, there may be advantages associated with using γδ T cells in the CAR-T approach. That is, γδ T cells can be made to have both tumor-compatible (TCR)-mediated action and innate (NKG2D)-mediated action simultaneously. However, the problem remains that human blood γδ T cells may be inefficient in homing to and being maintained in an active form within solid tissue tumors. This consideration usually leads to a more detailed examination of γδ T cells that normally reside in non-hematopoietic tissues.

[0157] Such T cells migrate to non-hematopoietic tissues as part of their development and are thus distinct from T cells that infiltrate tissues after systemic priming (e.g., tissue-resident TCRαβ memory T cells (so-called TRM cells)). Tissue-resident γδ T cells have been most extensively studied in mice, where they have been shown to be prevalent in, among other sites, the skin, gastrointestinal tract, and reproductive tissues. A number of such cells have been shown to retain an innate-like functional capacity to respond, thereby, to sensitization through activation of the NKG2D receptor. The inventors have recently obtained data demonstrating that human skin and intestine similarly harbor a large proportion of non-hematopoietic tissue-resident γδ T cells with innate-like activity. In the study of malignancies, inflammation, atopy, allergy, and other pathological conditions that form within non-hematopoietic tissues, the potential impact of these innate-like human T cells that reside within the tissues where lesions occur has not been fully explored.

[0158] Human γδ T cells resident in non-hematopoietic tissues have been little studied because their localization makes it difficult to obtain the cells and because there are no established means for culturing them. This subtype includes diverse cells with non-MHC-restricted cytolytic activity that do not respond at all to low molecular weight phospho moieties because they do not express a Vδ2-containing TCR. Little is known about the precise TCR specificities of such cells, but the available data suggest that the cells are reactive to self-antigens such as endothelial protein C receptor (EPCR) that are overexpressed by cytomegalovirus (CMV)-infected cells and a number of solid tumors. Non-hematopoietic tissue-associated γδ T cells also generally express NKG2D. Considering these properties, as well as the physiological residence of the cells within non-hematopoietic tissues such as the skin and gastrointestinal tract, adoptive transfer of such cells into cancer patients may be considerably more effective in targeting solid tumors and potentially other immunopathologies.

[0159] To utilize non-Vδ2 cells with respect to immunotherapy, either means for amplifying these cells in situ or means for harvesting them and amplifying them ex vivo prior to reinfusion are required. Since there are no known TCR activators that have been proven to be capable of amplifying a large number of non-Vδ2 cells in situ, the latter approach has been adopted. To overcome the difficulty of limited availability of non-hematopoietic tissues, some researchers have attempted to amplify a very small number of non-Vδ2 T cells derived from blood, which is a dominant subset of Vδ2-expressing cells, assuming that these cells are equivalent to tissue-resident non-Vδ2 T cells. The small number of non-Vδ2 γδ T cells found in the blood significantly amplify during active CMV infection, show superior reactivity against CMV compared to Vδ2 T cells, and seem to be able to protect human fetuses in cases of intrauterine CMV infection. In addition, CMV-reactive non-Vδ2 γδ T cells seem to protect transplant patients from CMV reactivation and reduce the risk of secondary malignancies through cross-reactivity with transformed cells during immunosuppression. Similarly, there is data suggesting that γδ T cells act beneficially in the control of HIV infection, and in this example, non-Vδ2 γδ T cells are amplified in the blood compared to Vδ2 T cells.

[0160] Blood resident non-Vδ2 cells have been amplified ex vivo either by adding exogenous factors that directly activate TCR signaling (e.g., by using substances such as anti-CD3 antibodies, pan-γδ TCR-specific antibodies or phytohemagglutinin (PHA)), or by co-culturing stimulated non-Vδ2 T cells with artificial antigen-presenting cells (aAPCs), in which case direct contact between the γδ T cells and the aAPCs is required for ex vivo non-Vδ2 T cell expansion). Alternatively, cells have been amplified by promoting NKG2D receptor signaling using immobilized recombinant MICA (an NKG2D ligand), similar to its use to maintain the expansion of ex vivo γδ T cell cultures derived from, for example, tumor-infiltrating lymphocytes (TILs) of epithelial cancers. Overall, current methods for ex vivo expansion of Vδ2-expressing blood γδ T cells or non-Vδ2 blood γδ T cells always require the addition of substances that promote activation of the TCR and / or NKG2D receptor, together with accessory cytokines such as IL-2. This combination of receptor activation signals and cytokines reflects the standard approach widely adopted by the community for culturing and expanding T cells. To date, methods for significantly expanding γδ T cells that reside in non-hematopoietic tissues have not been described. Such methods are described herein.

[0161] As part of the phenotypic and functional characterization of human non-hematopoietic tissue-resident γδ T cells (e.g., skin γδ T cells), the inventors have isolated distinct large populations of γδ T cells that normally reside in non-hematopoietic tissues and have unique properties compared to αβ T cells and blood-resident γδ T cells. The inventors have found that the cells exhibit strong TCR-independent innate-like responses to NKG2D ligands and to cytokines. While efforts at the expansion of primary αβ T cells have generally used co-culture with other supportive cells as a source of beneficial growth factors, the inventors have unexpectedly shown that γδ T cells resident in the skin and other non-hematopoietic tissues are potently and specifically suppressed by co-culturing these cells in contact with autologous skin fibroblasts and potentially other stromal components (such as keratinocytes and endothelial cells). Removal of such interactions enables the cells to be expanded in large numbers and rapidly, for potential clinical applications.

[0162] Furthermore, compared to current efforts to expand blood-derived and tumor-derived γδ T cells, the inventors have shown that such non-hematopoietic tissue-resident γδ T cells can be expanded without the intentional addition of any exogenous substances that activate their TCR or NKG2D signaling pathways.

[0163] Disclosed herein are novel means for efficiently and reproducibly isolating and expanding γδ T cells from human or non-human animal non-hematopoietic tissues such as the skin and intestine. Expansion is promoted by disrupting the contact of non-hematopoietic tissue-derived non-Vδ2 T cells with autologous fibroblasts and potentially other stromal components and is maintained by culture in IL-2, IL-15, IL-4, and / or IL-21.

[0164] The following examples are presented to provide a complete disclosure and description to those skilled in the art of how the methods and compounds claimed herein are made, made, and evaluated, and are intended to be merely illustrative of the invention and are not intended to limit the scope of what the inventors regard as their invention.

[0165] Example 1 Analytical Method Unless otherwise specified, the following methods were utilized to obtain the results of the following examples.

[0166] Flow Cytometry Flow cytometry was performed using the following antibody-fluorochrome conjugates: Ki-67-BV421, CD3-BV510, Vδ1-PeVio770, TIM-3-PE, CD9-PE, CCR3-BV421, and CD39-BV421. Samples were also stained for viability using eFluor770NIR. Commercially available antibodies were purchased from Biolegend or Miltenyi. The viability dye (near-infrared) was obtained from eBioscience. Ki-67 staining was performed on fixed and permeabilized cells using the Foxp3 staining buffer set (eBioscience). At the end of each experiment, the cell population was washed in PBS and split in half. Cells were stained for viability using eFluor770 NIR, washed, and subsequently stained with TrueStain (Biolegend) to avoid non-specific binding of the staining antibodies. Half of the samples were stained for the indicated surface markers, while the other half was stained for cell lineage markers only (CD3, Vδ1) and with the corresponding isotype controls for the surface markers used. Matching mouse isotype antibodies conjugated to the same fluorochrome were used at the same concentration. Isotype controls do not bind to known human antigens and thus show non-specific binding or false positives. Histograms are shown compared to their corresponding isotype controls or as FMO (Figures 1D, 2A, 3B, 4B, 6B, 7A, and 11-13). Data summary shows the percentage of cells positive for the indicated markers being compared, i.e., stained at a higher level than the isotype. Flow cytometry data analysis was performed using FlowJo (version 10.1).

[0167] RNA sequencing Vδ1 T cells derived from human skin and human blood Vδ1 T cells (after T cell receptor-induced amplification) were sorted (FACS), centrifuged, and the cell pellet was resuspended in RLT buffer. RNA was prepared using the RNA-Micro-plus kit (QIAGEN). RNA libraries were generated using the KAPA Stranded RNA-seq Kit with RiboErase (HMR) (KAPA BIOSYSTEMS). Paired-end sequencing on the HiSeq 2500 (illumina) used rapid run chemistry (read length: 100 bp). 101-base pair paired-end reads were aligned and quantified using RSEM (v1.2.11) together with Bowtie2. Reads were aligned against the human transcriptome, and the count values were log2-transformed and quantile-normalized.

[0168] Cytokine quantification Vδ1 T cells derived from human skin were stimulated for 24 hours with PMA and ionomycin or plate-bound anti-CD3 mAb (OKT3, 5 μg / mL). Subsequently, the supernatant was obtained and analyzed using the ProcartaPlex Human Cytokine & Chemokine Panel 1A (34 plex) (eBioscience). The assay was analyzed using Luminex FlexMap3D (Luminex). Data were analyzed in Microsoft Excel and represent the mean of three donors (duplicate experiments). Error bars indicate the standard deviation.

[0169] Co-culture with fibroblasts For each grid culture setting, the inventors prepared two Petri dishes (100 × 25 mm, Corning) that were scratched at several locations using a scalpel. The minced skin pieces were placed in the scratches. After drying in air for 5 - 10 minutes, the skin pieces were properly attached to the dishes and 10 mL of Skin - T medium was added. The medium was changed once a week, and after 3 weeks of growth, primary fibroblasts were recovered following treatment with ACCUTASE® (Life Technologies). The fibroblasts were seeded at 1 × 10 4 in a 48 - well plate, or in the case of a transwell experiment, at 2 × 10 4 in the bottom chamber of a 24 - well plate. After 2 - 3 days, the fibroblasts reached confluence, and in the case of a 48 - well plate, 2 × 10 5 mixed skin lymphocytes, or in the case of a 24 - well plate, bottom well and transwell, 3 × 10 5 lymphocytes were added to initiate the co - culture experiment.

[0170] Amplification of blood - derived γδ T cells Blood - derived γδ T cells within PBMCs can be amplified only when stimulated using a TCR ligand (e.g., IPP, HMBPP, bisphosphonates for Vδ2), or addition of an antibody to cross - link the TCR receptor (mAb) or TCR - associated kinase CD3. The same effect of TCR cross - linking can also be achieved using lectins such as PHA. In the absence of the addition of such TCR stimulants, γδ T cells in PBMCs survive for several days but cannot be amplified and remain at the initial composition of a T - cell subset with little diversity.

[0171] To isolate PBMC, blood from healthy volunteers was used, whole blood was layered on Ficoll, and subsequently centrifuged at 400 g for 20 minutes to separate red blood cells, plasma, and white lymohocyte / monocytes. The white blood cells were carefully collected through a stripett and washed 4 times in cold PBS. The cells were resuspended at a density of 1×10 6 / mL in RPMI-1640 medium (Life Technologies) containing 10% heat-inactivated fetal bovine serum (Life Technologies), L-glutamine (292 μg / mL; Life Technologies), penicillin (100 units / mL; Life Technologies), streptomycin (100 μg / mL; Life Technologies), N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES; 0.01 M; Life Technologies), sodium pyruvate (1 mM; Life Technologies), and minimum essential medium (MEM) non-essential amino acids (1×; Life Technologies), and IL-2 (100 IU / mL) was added. Ninety minutes prior to cell migration, the cells were transferred to 24-well plates coated with a pan-γδ TCR monoclonal antibody (20 μg / mL, clone B1, Biolegend). The cells were grown for 14 days, the medium was changed every 2 - 3 days, and fresh cytokines were added. When confluence was reached, the cells were split 1:1. Under these conditions, after 14 days, the original minor population of γδ T cells was highly activated normally through their TCRs (as indicated by the upregulation of CD69 and CD25) and consisted mainly of Vδ2 T cells, but also included Vδ1 T cells (up to 30% of all γδ T cells) and was significantly enriched. Vδ1 T cells can then be isolated using FACS for functional or phenotypic analysis (e.g., genetic analysis).

[0172] Example 2 Isolation of non-hematopoietic tissue-resident γδ T cells from skin and gastrointestinal tract A three-dimensional skin explant protocol was constructed using the Clark protocol. A Cellfoam matrix (Cytomatrix Pty Ltd, Victoria, Australia) or equivalent with dimensions of 9 mm × 9 mm × 1.5 mm was autoclaved and subsequently incubated in a 100 mg / mL rat tail type I collagen (BD Biosciences) solution (in PBS) at room temperature for 30 minutes, and then rinsed once in PBS. Samples of adult human skin were obtained within 3 - 6 hours from skin surgery. Subcutaneous fat was removed and the remaining skin tissue was minced into fragments approximately 1 mm × 1 mm in size. Approximately 5 skin fragments / explants were placed and pressed onto the surface of each matrix. Each matrix was placed in a separate well of a 24-well plate (Corning) containing 2 mL of "Skin-T" medium (Iscove's modified Dulbecco's medium (IMDM; Life Technologies) containing 10% heat-inactivated fetal bovine serum (Life Technologies), L-glutamine (292 μg / mL; Life Technologies), penicillin (100 units / mL; Life Technologies), streptomycin (100 μg / mL; Life Technologies), N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES; 0.01 M; Life Technologies), sodium pyruvate (1 mM; Life Technologies), minimum essential medium (MEM) non-essential amino acids (1×; Life Technologies) and 2-mercaptoethanol (3.5 μL / L; Life Technologies)). Amphotericin (2.5 μg / mL; Life Technologies) was added to the medium for the first 7 days of culture. The medium was changed three times a week by aspirating and removing the upper 1 mL of medium from each well and replacing it with fresh medium.Human recombinant IL-2 (PROLEUKIN®; Novartis Pharmaceutical UK Ltd) at 100 IU / mL and human recombinant IL-15 (Biolegend) at 20 ng / mL were added at the beginning of the culture and were added to the medium until lymphocytes were isolated 21 - 35 days later as shown in Table 1. Up to 96 wells (4 of 24-well plates) were set up for each donor during the culture.

[0173] To isolate lymphocytes, the matrix was transferred to 50 mL conical tubes (Corning) containing 10 mL of Hank's balanced salt solution (HBSS; Life Technologies) with 0.01 mM HEPES (up to 12 matrices / tube). The matrix was washed with cell suspension using a 10 mL pipette and the cell suspension was placed into a new 50 mL conical tube (Corning) through a 70 μm filter (BD Biosciences). This washing of the matrix was repeated two more times. The medium from the culture wells was also aspirated and placed into a new 50 mL conical tube (Corning) through a 70 μm filter (BD Biosciences). The wells were washed two more times with 1 mL of 0.01 mM HEPES / HBSS and passed through a 70 μm filter (BD Biosciences). Subsequently, the cells were isolated by centrifugation (1600 rpm, 15 minutes). The pellet was resuspended in "Skin-T" medium. The final cell pellet was resuspended in "Skin-T" medium for subsequent flow cytometry analysis or functional studies. If cell counts were required, white blood cells were counted at this stage by either: (1) trypan blue staining (0.4%) (Life Technologies) and a hemocytometer, or (2) CASY® Model TT cell counter and analyzer (Roche). Exemplary study results are shown in Table 1 below.

[0174]

Table 1

[0175] Since untreated gastrointestinal samples are prone to contamination, the obtained biopsy samples were first washed twice with IMDM containing 10% FCS, penicillin (500 units / mL), streptomycin (500 μg / mL), gentamicin (100 μg / mL), amphotericin B (12.5 μg / mL), and metronidazole (5 μg / mL), then minced and placed on the scaffold. Gastrointestinal scaffold cultures were grown in "Gut-T" medium (IMDM, 10% FCS, penicillin 100 units / mL, streptomycin 100 μg / mL, gentamicin 20 μg / mL, metronidazole 1 μg / mL). Similar to the case of the skin, amphotericin B 2.5 μg / mL was also used during the first week of culture. IL-2 (100 IU / mL) and IL-15 (20 ng / mL) were also added to the medium and changed three times a week. Since the structure of the gastrointestinal tract is looser than that of the skin, lymphocytes were removed after one week.

[0176] Example 3 Characterization of Non-Hematopoietic Tissue Resident γδ T Cells Human γδ T cells are abundant in the skin, and most are Vδ2 - and are involved in the human lymphoid stress surveillance response Figures 1A - 1D show that human skin contains a prominent population of resident γδ T cells. Using the Clark protocol, the inventors expanded tissue resident lymphocytes over a 3-week period using human surplus skin samples supplemented with IL-2 and IL-15. The average yield was 240,000 lymphocytes per scaffold. Consistent with previous reports, the inventors were able to identify a distinct subset of skin resident lymphocytes, along with the majority of cells expressing the conventional αβ TCR, most of which are of the tissue resident "TRM" type. Overall, 59.9% (±8.6%) of CD45 + cells were CD4 + and 18.3% (±2.8%) were CD8 + αβ T cells, and an NK cell fraction of 8.7% (±3.6%) was included. In addition, the inventors identified a substantial population of γδ T cells (CD45 +An average of 8.513% ± 6.564% of the cells were found in our donors (Figures 1A and 3D). This lymphocyte phenotype was highly reproducible in ~100 donors after organotypic culture and equivalent to fresh digested skin samples, differing only by a slight increase in the γδ population, but was practically useful and provided a much larger and purer lymphocyte population compared to standard tissue digestion protocols. Consistent with the literature on tissue localization of human γδ T cells based on their TCR delta chains, the majority of human skin γδ T cells expressed Vδ1 TCR chains paired with various γ chains, as identified by flow cytometry. This represents a single specific TCR heterodimer of the Vδ2 chain linked to Vγ9 and was in contrast to the majority of peripheral blood γδ T cells, which were rarely present in human skin samples. However, it is important to note a subset that did not express either Vδ1 TCR or Vδ2 TCR (designated in this invention as "double negative" γδ T cells (DN γδ T cells)) (Figure 1C).

[0177] Skin resident γδ T cells grown in this manner exhibited an immature memory phenotype, did not express CD45RA, and expressed various levels of the co-stimulatory molecule CCR7. Compared to conventional systemic T cells, skin resident γδ T cells exhibited high expression of the surface marker CD69, and along with it, expression of the programmed cell death receptor 1 (PD-1); low to non-existent levels of the IL-2 receptor α (CD25); and absence of the co-stimulatory molecule CD28, which suggests previous or chronic activation (Figure 1D). Consistent with their tissue localization, Vδ1 cells and DN cells exhibited expression of skin and tissue homing markers such as CCR4, CCR8, and integrin αE (CD103) (Figure 7). This combination of tissue homing markers was thought to demonstrate being beneficial in an immunotherapy setting. Additionally, skin resident γδ T cells exhibited high levels of expression of the activating receptor NKG2D (Figure 2A), which implies the possibility of a role for these cells in the lymphoid stress surveillance response. NKG2D ligands such as MICA, MICB, and ULBP are each upregulated by cells in response to DNA damage, EGF receptor activation, and oxidative stress, and, thus, T cells expressing NKG2D may be able to identify and eliminate stressed or transformed cells, thereby enabling the maintenance of tissue homeostasis. Along these lines, the inventors found that skin resident γδ T cells amplified by the method of the present invention are activated upon exposure to recombinant ligands (MICA, ULBP2) for the NKG2D receptor, which demonstrated degranulation as measured by upregulation of the lysosome-associated membrane protein CD107a (Figure 2A). This innate-like feature was limited to Vδ1 + T cells and DN γδ T cells only, as other tissue resident T cells (Figure 2C) and systemic γδ T cells lacked this response (Figure 10B).

[0178] Overall, activated skin resident Vδ1 +T cells and DN γδ T cells, when activated by PMA / ionomycin or NKG2D ligands (e.g., recombinant MICA protein), executed a pro-inflammatory Th1-biased cytokine program (staining positive for IFN-γ, TNF-α, and GM-CSF) (Figures 2A and 2B), thereby revealing the innate-like response of the cells. Indeed, the response to MICA was almost completely abrogated by blocking the NKG2D receptor with an antibody (Figures 2B and 2C).

[0179] γδ T cells are known to secrete IL-17 in certain disease settings such as psoriasis and within some types of tumors. γδ T cells amplified by the methods of the present invention produce low levels of IL-17 or do not produce IL-17 even upon thorough activation (Figures 2B and 8). Conversely, tissue-resident CD4-expressing αβ T cells produced IL-17 upon TCR activation (Figure 2B). Overall, αβ T cells were Vδ1 + T cells and DN γδ T cells (which were limited to the Th1-biased program associated with host protection) showed a far more diverse cytokine repertoire in response to PMA / ionomycin.

[0180] Isolation from tissue causes activation and substantial expansion of human tissue γδ T cells To further study human tissue γδ T cells, mixed cutaneous lymphocytes were transferred into cell culture wells and IL-2 was added to maintain long-term viability. Interestingly, by separating from stromal and epithelial cells present during organotypic culture, Vδ1 T cells uniquely showed signs of activation and proliferation without any added stimuli. Over a 7-day period, Vδ1 + T cells and DN γδ T cells uniquely and substantially upregulated the nuclear factor Ki-67 and increased the surface expression of the IL-2 receptor α (CD25) (Figures 3B and 4B). Notably, over a 3-week period and in the presence of IL-2 only, tissue-derived Vδ1 +T cells and DN γδ T cells proliferated more than all other T cell subsets, thereby accounting for up to 65% of all skin lymphocytes, with numbers increasing on average up to 127.18-fold. In contrast, αβ T cells increased only 5.21-fold (p = 0.0124) when measured by absolute cell numbers (Figure 3A). The MFI of the cell cycle-related nuclear factor Ki-67 was + in T cells and DN γδ T cells increased from 2,664.5 (±1,876.1) to 8,457.7 (±4,574.2) over 14 days, while in αβ T cells, the MFI decreased from 592.8 (±390.5) to 284.7 (±140.1) over the same period (Figure 3C). This phenomenon of selective cutaneous resident γδ T cell proliferation could be further assisted using additional recombinant IL-15, which increased lymphocyte survival and total numbers.

[0181] Cutaneous γδ T cells are significantly inhibited by fibroblasts in a contact-dependent manner The above Vδ 1 + The prominent amplification of T cells and DN γδ T cells did not occur at all in an organotypic culture system in which a large amount of fibroblast proliferation was present. Vδ1 + To directly examine whether their co-culture with Vδ 1 T cells and DN γδ T cells inhibits T cell amplification, autologous fibroblasts were grown. After 3 weeks of scaffold culture, mixed skin lymphocytes were seeded into wells that were either empty or contained a confluent monolayer of pre-established fibroblasts, and in each case, exogenous IL-2 was added to the medium to maintain T cell growth. In addition, a transwell was used to prevent T lymphocytes from directly contacting the fibroblasts in the same well but to allow T cells to be affected by soluble factors secreted by the fibroblasts. During the 14-day co-culture, in wells without fibroblasts and in wells where T cells were prevented from directly contacting the fibroblasts, Vδ 1 +T cells and DN γδ T cells began to proliferate. As before, αβ T cell proliferation was low under all conditions. When T cells were brought into direct contact with fibroblasts, Vδ 1 + The two-week fertility rates of T cells and DN γδ T cells decreased significantly from 22.6 (±8.07)-fold to 3.3 (±0.17)-fold in wells without fibroblast contact (Figure 4A). This contact-mediated inhibition was further confirmed by the absence of upregulation of CD25, Ki-67, and the transcription factor T-bet in Vδ1 T cells over a seven-day period when compared to lymphocytes grown alone (Figure 4B). While it will be clear that some forms of tissue-mediated control of the immune system are fundamental to the maintenance of tissue homeostasis, this is because the potential for persistent inflammation would arise without this control. Vδ 1 by stromal fibroblasts + The suppressive regulation of T cells and DN γδ T cells would seem to be an example of such control.

[0182] Collectively, the phenotype of cutaneous resident Vδ 1 + T cells and DN γδ T cells and their prominent functional potential reflect pre-activated T cells that are normally suppressed by adjacent skin fibroblasts via contact-dependent mechanisms. By inactivating this mechanism by liberating T cells from contact with fibroblasts, Vδ 1 + Amplification of T cells and DN γδ T cells is selectively enabled, while other T cells in the skin are not affected.

[0183] Release of contact-mediated inhibition promotes a cytotoxic TH1-biased cytokine response by cutaneous Vδ1 T cells Hybrid skin-derived lymphocytes were amplified for 14 days, and fluorescence associated cell sorting was used to remove αβ T cells from γδ T cells, enabling a maximum purity of 90%. These highly enriched cells were placed into cell culture wells at a concentration of 150,000 cells / well in RPMI medium containing 10% FCS. After 24 hours, the supernatant was collected and evaluated for a wide range of effector cytokines using an array based on LUMINEX®. Completely unexpectedly, the γδ T cells during amplification (induced only by their isolation from fibroblasts) spontaneously produced large amounts of TH1-related cytokines such as IFN-γ (12,383.46 ± 16,618.90 pg / mL), GM-CSF (4,316.73 ± 4,534.96 pg / mL), as well as the pro-inflammatory chemokines CCL4 (14,877.34 ± 10,935.64 pg / mL) and CCL3 (1,303.07 ± 757.23 pg / mL) (Figure 5A).

[0184] Furthermore, the cells spontaneously produced large amounts of IL-13, which is associated with an atopic response, during amplification and in contrast to freshly isolated skin-derived TCR-activated γδ T cells. Other cytokines such as IL-17 were produced at much lower levels or not at all (Figure 8). The high effector capacity of the cells could be further increased after stimulation with recombinant MICA (NKG2D ligand), anti-CD3, or PMA / ionomycin. To evaluate the cytotoxic ability of the amplified γδ T cells against malignant target cells, the inventors used established transformed cell lines during a 24-hour co-culture experiment. Vδ 1 +T cells and DNγδ T cells showed very high cytotoxic activity against Hela cells (cervical cancer) and Caco2 cells (colon cancer) in a dose-dependent manner, far exceeding that of conventional tissue αβ T cells (Figure 5B). Furthermore, γδ cell-mediated cytotoxicity can be strongly inhibited by blocking the NKG2D receptor with a soluble monoclonal antibody, indicating that this receptor is at least partially involved in tumor surveillance by derepressing human skin-derived γδ T cells. Additionally, the cytotoxic ability of these cells was confirmed using other targets: HCT1954 cells (breast cancer), MDAMB231 cells (breast cancer), and HCT116 cells (colon) (Figure 9).

[0185] Non-hematopoietic tissue-resident γδ T cells generated by the method of the present invention can be further distinguished from other blood-derived γδ T cells in that they respond to the NKG2D ligand (MICA). Non-hematopoietic tissue-resident γδ T cells show a strong association with malignant tumors, for example, under conditions without ligand stimulation of the T cell receptor, as evidenced by increased production of TNFα, IFNγ, and CD107a (Figures 2 and 10). Also, they execute a cytotoxic response of T cells in the absence of activation of the T cell receptor by externally administered drugs or through ligand mediation, i.e., they are cytotoxic under conditions without stimulation (Figures 3 and 5). This indicates that non-hematopoietic tissue-resident γδ T cells generated by the method of the present invention are unique in their response and proliferative ability under conditions without the addition of exogenous drugs that activate T cell receptor signaling, compared to other γδ T cells, αβ T cells, or NK cells (Figure 3). Moreover, non-hematopoietic tissue-resident γδ T cells generated by the method of the present invention were positively stained with CD69 and PD-1, did not express CD28, and expressed only low levels of CD25 (see Figure 1D). This combination of markers is not expressed in blood-derived γδ T cells. Furthermore, they showed high expression of tissue-homing receptors such as CCR4 and CCR8 compared to blood-derived amplified Vδ2γδ T cells (Figure 7B).

[0186] Tissue-resident γδ T cells in the human gastrointestinal tract A population of non-hematopoietic tissue-resident γδ T cells derived from the human colon that express the Vδ1 T cell receptor was also identified (Figure 6). These cells could be amplified in three donors over a period of 4 - 5 weeks using the same method as used for skin cells. During amplification, colon-derived Vδ1 + T cells and DN γδ T cells showed a similar Ki-67 upregulation pattern after their isolation from fibroblast-rich organotypic cell cultures. Similarly, colon-resident Vδ1 + T cells and DN γδ T cells were strongly stimulated by the supply of ligands for the NKG2D receptor. Blood-derived γδ T cells are fully capable of performing antibody-dependent cell-mediated cytotoxicity via CD16 expression, which demonstrates enhanced cytotoxicity targeted against CD20-positive B cell lineage lymphomas when combined with rituximab. Similarly, chronic lymphocytic leukemia (CLL) and HER2-positive breast cancer cells are killed more effectively when targeted with monoclonal antibodies. To evaluate the ability of skin-derived Vδ1 T cells to target antibody-opsonized target cells, the expression levels of three IgG1-related Fc receptors, CD16, CD32, and CD64, were quantified. Skin-derived Vδ1 T cells express a low level of the Fc receptor CD16 but show a good expression level for the high-affinity IgG receptor CD64. Therefore, tissue-derived Vδ1 T cells may be fully capable of being used as adjuvants for monoclonal antibody therapies such as CD20 therapy or Her2 therapy, since they will be induced by antibodies towards malignant tumors and metastases, recognize opsonized tumor cells, and kill the targets via ADCC.

[0187] Example 4 Optimization of amplification conditions for non-hematopoietic tissue-resident γδ T cells Amplification of skin-derived γδ T cells After 3 to 4 weeks of scafold culture, the mixed lymphocytes were removed, washed with PBS, centrifuged, and resuspended in Roswell Park Memorial Institute 1640 medium (RPMI-1640; Life Technologies) containing 10% heat-inactivated fetal bovine serum (Life Technologies), L-glutamine (292 μg / mL; Life Technologies), penicillin (100 units / mL; Life Technologies), streptomycin (100 μg / mL; Life Technologies), N-2-hydroxyethylpiperazine N-2-ethanesulfonic acid (HEPES; 0.01 M; Life Technologies), sodium pyruvate (1 mM; Life Technologies), minimum essential medium (MEM) non-essential amino acid solution (1×; Life Technologies), and 50 μM 2-mercaptoethanol (Life Technologies) to a cell concentration of 1×10 6 cells / mL. The Vδ1 + cells of the initial population were 1.12% of the lymphocytes. 2×10 5 cells / well were seeded in 96-well flat-bottom plates (Corning), or 2×10 6 cells / well were seeded in 24-well plates and amplified by adding factors at the concentrations shown in Table 2.

[0188]

Table 2

[0189] Cells were monitored daily under a microscope and fresh medium and cytokines were provided three times per week. When complete confluence and cell aggregation occurred, the cells were split 1:1 into additional wells and plates as needed. After 21 days, the cells were removed using ACCUTASE® (eBioscience) and counted and analyzed using flow cytometry. Figures 17A and 17B show representative flow cytometry plots before and after amplification. Table 3 shows the amplification factor corresponding to Figure 17C, in addition to the relative expression levels of CD27 and TIGIT in the amplified cells of each treatment group. The final amplification factor of Vδ1 was calculated from the total numbers of Vδ1 before and after amplification (%CD3 + pan-γδ + Vδ1 + cells ÷ 100) × (total cell number).

[0190] [Table 3]

[0191] As shown in Figure 17C, the amplification of Vδ1 T cells increased by adding other factors compared to the case of IL-2 and IL-15 alone. Since the yield of Vδ1 T cells was high in response to IL-2, IL-15, IL-4, and IL-21, these factor combinations were further investigated as shown in Figures 17D to 17H.

[0192] Initial and final Vδ1 + The phenotype of each population of T cells was measured using the mean fluorescence intensity (MFI), including the expression of CD27 and TIGIT, and the samples of each group were averaged taking the median of the MFI. The expression levels of CD27 and TIGIT by Vδ1 + T cells under each amplification condition are shown in Table 4.

[0193] [Table 4]

[0194] As shown in Fig. 18A, compared with the case of only IL-2 and IL-15, an increase in the expression of CD27 measured by mean fluorescence intensity was observed upon addition of other factors. Notably, upon addition of IL-4 and IL-21, the CD27 MFI increased approximately eight-fold compared with that of IL-2 and IL-15 alone. Moreover, the highest CD27 expression was observed upon combination of the four cytokines IL-2, IL-15, IL-4, and IL-21, compared with their other combinations (Figs. 18B and 18D). Notably, when CD27 expression on T cells is low, it is often associated with a phenotype that has little potential for further long-term proliferation, is exhausted, and is terminally differentiated.

[0195] Amplified Vδ1 + Regarding the expression of TIGIT by T cells, different trends were observed (Fig. 19). In particular, the expression of TIGIT decreased in response to IL-4 and IL-21 in combination with IL-2 and IL-15. To further explore this trend, the expression level of TIGIT was plotted against the expression level of CD27 (Fig. 20). A negative correlation was observed between TIGIT and CD27. When the expression level of TIGIT is high, T cells are more susceptible to inhibition by the tumor microenvironment. In the tumor microenvironment, the expression of the T cell ligand, poliovirus receptor (PVR; CD155), is increased.

[0196] Example 5 Culturing of Four Cytokines Serves as an Alternative to Cell Culture Serum Conventionally, when manufacturing T cells derived from tissues or tumors, it has been common to add multiple blood-derived sera and plasma to the culture medium. However, the use of these serum or plasma components can lead to undesirable situations due to batch-to-batch differences in these components, their high costs, supply limitations considering the high demand across the advanced therapy medicinal products (ATMP) industry, and an increased risk of secondary contamination by exogenous substances from these components. Therefore, as described herein, after successfully identifying cytokines that support the proliferation and enrichment of the desired γδ T cells, it was tested whether the use of such cytokines could eliminate the need for multiple serum / plasma components commonly used for the proliferation of tissue-derived γδ T cells. To further test this, immune cells were released from skin samples, and it was tested whether plasma / serum was still necessary to support the proliferation and enrichment of γδ T cells + / − serum. Therefore, the released cells were removed and seeded into two media on "day 0". The first medium was one containing an animal derived component free (ADCF) medium (TexMACS, Miltenyi) supplemented with 10% human-derived serum and cytokines. The second medium was a similar mixture of ADCF medium / cytokines, including a standard defined supplement (CTS™, containing purified human serum albumin, recombinant insulin, and transferrin), but without serum. The results of this study are shown in Figure 21. Surprisingly, equivalent enrichment and equivalent amplification rates were observed regardless of the presence or absence of serum. This indicates that it is possible to amplify and enrich tissue-derived γδ T cells without animal-derived factors or human serum.

[0197] Next, this approach was evaluated with respect to the preferential amplification of γδ T cells from a population of mixed lymphoid cells taken from standard organotypic cultures. Such preferential amplification would be highly desirable if achievable, particularly if the protocol could result in a population of lymphocytes that, upon final amplification, contains more than 50% γδ T cells. Indeed, previous conventional enrichment protocols have required the use of depletion or enrichment techniques such as magnetic immunodepletion technology (e.g., from Miltenyi or Dynal) or flow cytometry sorting technology (e.g., from BD Biosciences) to physically separate a small number of γδ cells or physically deplete the majority of αβ cells. Instead, in this study, we evaluated whether the method of the present invention can enrich γδ T cells present in a mixed lymphocyte population without the need for such physical separation or depletion protocols. Surprisingly, such enrichment was achieved using these amplification methods by the selectivity of the protocol for amplifying γδ cells over other cell types present in the initial population. This resulted in a more enriched and highly purified population of γδ T cells representing 50% of all cells present in the culture. Furthermore, as shown in FIGS. 21 and 22A - 22D, this enrichment was achieved regardless of the presence or absence of serum, with γδ T cells being amplified more than 100 - fold and the purity of γδ T cells rising from less than 50% to more than 50% at all sites of this tissue sample.

[0198] Isolation and amplification method After 3 weeks of sca-hold culture, mixed lymphocytes were obtained from the tissue using the Clark protocol described above and equivalent methods described in Example 2. The characteristics of the obtained cells were equivalent to those described in Example 3. These obtained cells were washed with HBSS + HEPES, centrifuged, and resuspended in TexMACS medium (Miltenyi) containing (i) 10% human AB serum (Life Science Production) in addition to IL-2 (100 IU / L), IL-4 (Biolegend, 5 ng / mL), IL-15 (Biolegend, 20 ng / ml), and IL-21 (Biolegend, 5 ng / ml), or (ii) 5% CTS™ (Thermo Fisher Scientific) in addition to IL-2 (100 IU / L), IL4 (Biolegend, 5 ng / mL), IL-15 (Biolegend, 20 ng / ml), and IL-21 (Biolegend, 5 ng / ml). Penicillin / streptomycin antibiotics (100 units / mL and 100 μg / mL, respectively, Life Technologies) were also added to both the medium containing serum and the serum-free medium. The cells were then seeded at 2 million cells / well in a 24-well plate (Corning). When the cells became confluent, they were split between 1 / 2 and 1 / 4 into new wells containing the same medium for amplification / passaging. On day 21, the cells were removed using ACCUTASE® cell dissociation means (Thermo-Fisher) and analyzed by flow cytometry to determine the final cell characteristics as shown in FIGS. 21 and 22A-22D.

[0199] Example 6 Functional Relevance of TIGIT Expression As shown in FIG. 23, TIGIT is constitutively expressed on gut-resident Vδ1 cells. Data were generated using Vδ1 cells isolated from the gut by conventional cell digestion. The constitutive expression of TIGIT on tissue-resident γδ T cells is not only relevant to skin-derived Vδ1 cells, and the expression of TIGIT is not an artifact (synthetic product) of the Clark protocol (grid-based isolation procedure).

[0200] Furthermore, when the expression of IFNγ (Figure 24A) and TNFα (Figure 24B) was measured in cells cultured with only IL-2 and IL-15, the poliovirus receptor (PVR) particularly inhibited TCR signaling. When IFNγ (Figure 25A) or TNFα (Figure 25B) was measured in cells cultured in the presence of IL-2, IL-15, IL-4 and IL-21, the PVR inhibitory effect was lost in TIGIT-negative Vδ1 + / Vδ3 + In. This indicates that the TIGIT-mediated inhibition of Vδ1 + / Vδ3 + Activation of T cells is preferentially prevented by TIGIT negativity caused by the four mixed cytokines.

[0201] Example 7 Substitution of IL-2 with IL-9 in Amplification Culture Skin tissues from three donors (TS052, TS056 and SK073) were placed on a 9 mm grid and cultured for 3 weeks in a medium supplemented with IL-2 and IL-15. The isolated lymphocytes were cultured in a medium supplemented with IL-2, IL-4, IL-15 and IL-21 (Figures 26A and 26B, left bars) or IL-4, IL-9, IL-15 and IL-21 (Figures 26A and 26B, right bars). The final yields of γδ T cells / grid (Figure 26A) and Vδ1 cells / grid (Figure 26B) after 3 weeks of amplification were calculated.

[0202] As shown in Figures 27A to 27C, IL-9 was sufficient to substitute for the function of IL-2 in the amplification of skin γδ T cells, as measured by changes in the amplification factor (Figure 27A), the percentage of γδ TCR + T cells (%) (Figure 27B) and the percentage of Vδ1 + T cells (%) (Figure 27C). The skin tissues were from six donors (SK073, SK075, SK077, TS052, TS053 and TS056). The two cytokine cocktails contained IL-2 and IL-15, and the four cytokine cocktails contained IL-2, IL-15, IL-21 and IL-4.

[0203] As shown in FIGS. 28A and 28B, IL-9 is involved in the amplification of skin γδ T cells, Vδ1 + The mean fluorescence intensity (MFI) of CD27 expression on T cells (FIG. 28A) and the MFI corrected value of CD27 expression of Vδ1 + T cells (FIG. 28B), as measured, were sufficient to substitute for the function of IL-2. No difference in CD27 expression was seen compared to standard culture conditions. Skin tissues were from 4 donors (SK073, TS052, TS053, and TS056). Two cytokine cocktails contained IL-2 and IL-15, and four cytokine cocktails contained IL-2, IL-15, IL-21, and IL-4.

[0204] Amplification of skin-derived γδ T cells After 3 weeks of scafold culture, mixed lymphocytes were removed, washed with phosphate-buffered saline (PBS), centrifuged, and resuspended in RPMI-1640 with 10% heat-inactivated fetal bovine serum (Life Technologies), L-glutamine (292 μg / mL; Life Technologies), penicillin (100 units / mL; Life Technologies), streptomycin (100 μg / ml; Life Technologies), N-2-hydroxyethylpiperazine N-2-ethanesulfonic acid (HEPES; 0.01 M; Life Technologies), sodium pyruvate (1 mM; Life Technologies), minimum essential medium (MEM) non-essential amino acid solution (1×; Life Technologies), and 50 mM 2-mercaptoethanol (Life Technologies) to a cell concentration of 1×10 6 cells / mL. 2×10 6Cells / wells were seeded and cytokines were added and amplified to the following final concentrations: IL-2: 100 U / mL, IL-4: 5 ng / mL, IL-9: 10 ng / mL, IL-15: 20 ng / mL, and IL-21: 10 ng / mL.

[0205] Cells were monitored daily by microscopy and fresh medium and cytokines were provided by replacing 1 mL of the medium three times a week with 1 mL of fresh medium containing cytokines at twice the strength (i.e., IL-2: 200 U / mL, IL-4: 10 ng / mL, IL-9: 20 ng / mL, IL-15: 40 ng / mL, and IL-21: 20 ng / mL).

[0206] If complete confluence and cell aggregation occurred, the cells were split 1:1 into additional wells and plates as needed. After 21 days, the cells were removed using ACCUTASE® (eBioscience) and counted and analyzed using flow cytometry. The final numbers of γδ T cells and Vδ1 cells were calculated using the numbers before and after amplification, and the final cell yield per grid after amplification was shown.

[0207] Other embodiments All publications, patents, and patent applications mentioned herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

[0208] Although the invention has been described in connection with its particular embodiments, further modifications are possible, and this application generally covers any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and fall within the essential features set forth in the claims.

[0209] Other embodiments are within the scope of the claims. The present invention includes the following embodiments. (1) The following steps: (i) providing a population of γδ T cells obtained from non-hematopoietic tissue; (ii) culturing said γδ T cells in an effective amount of (a) IL-2 or IL-9; (b) IL-15; and (c) IL-21 for at least 5 days to generate a population of amplified γδ T cells; A method for amplifying γδ T cells, comprising: (2) The method according to (1), wherein step (ii) further comprises culturing the γδ T cells in the presence of IL-4. (3) The following steps: (i) providing a population of γδ T cells obtained from non-hematopoietic tissue; (ii) culturing said γδ T cells in the presence of IL-2, IL-15, at least one factor selected from the group consisting of IL-21, stromal cell-derived factor (SDF), IL-1β, IL-12, IL-18 and IL-33 for at least 5 days to generate a population of amplified γδ T cells; A method for amplifying γδ T cells, comprising: (4) The method according to any one of (1) to (3), wherein step (ii) comprises culturing the γδ T cells in the absence of an exogenous TCR pathway agonist. (5) The method according to any one of (1) to (4), wherein step (ii) comprises culturing the γδ T cells in a serum-free medium. (6) After step (i), further comprising the step of separating said γδ T cells from non-hematopoietic cells to generate a population of isolated γδ T cells, and step (ii) comprises: (a) culturing the γδ T cells substantially without contact with stromal cells; (b) culturing the γδ T cells substantially without contact with tumor cells; and / or (c) culturing the γδ T cells substantially without contact with feeder cells; (1)~(5) The method according to any one of (1) to (5). (7) The following steps: (i) providing a non-hematopoietic tissue comprising non-hematopoietic cells and γδ T cells; (ii) separating γδ T cells from the non-hematopoietic cells to obtain a population of isolated γδ T cells; (iii) culturing the γδ T cells in the presence of IL-2, IL-15, a factor selected from the group consisting of IL-21, SDF, IL-1β, IL-12, IL-18, and IL-33 for at least 5 days to generate an amplified population of γδ T cells; A method for amplifying γδ T cells, comprising: (8) The method according to (7), wherein the non-hematopoietic tissue is obtained from a human or non-human animal subject. (9) The method according to (7) or (8), wherein step (ii) comprises culturing γδ T cells in the presence of IL-2, IL-15, and IL-21. (10) The method according to any one of (7) to (9), wherein step (ii) comprises culturing γδ T cells in a serum-free medium. (11) The method according to any one of (7) to (10), wherein step (iii) comprises culturing γδ T cells under conditions substantially free of contact between stromal cells and the γδ T cells. (12) The method according to any one of (7) to (11), wherein step (iii) comprises culturing γδ T cells in the absence of an exogenous TCR pathway agonist. (13) The method according to any one of (6) to (12), wherein the step of separating γδ T cells from non-hematopoietic cells comprises culturing γδ T cells on a synthetic scaffold constructed to release cells from the non-hematopoietic tissue. (14) The method according to any one of (6) to (13), wherein the step of separating γδ T cells from non-hematopoietic cells comprises culturing γδ T cells and non-hematopoietic cells in the presence of IL-2 and / or IL-15. (15) The method according to any one of (6) to (14), wherein the isolated lymphocyte population comprises an isolated γδ T cell population, and the isolated γδ T cell population comprises an isolated Vδ1 T cell population. (16) The method according to (15), wherein 1 to 10% of the isolated lymphocyte population is γδ T cells before amplification. (17) The method according to (15) or (16), wherein 1 to 10% of the isolated lymphocyte population is Vδ1 T cells before amplification. (18) The method according to any one of (6) to (17), wherein at least 80% of the isolated γδ T cell population is Vδ1 T cells before amplification. (19) The method according to any one of (6) to (18), wherein less than 10% of the isolated γδ T cell population is Vδ2 T cells before amplification. (20) The method according to any one of (6) to (19), wherein αβ T cells and / or NK cells are removed from the isolated γδ T cell population. (21) Before amplification, the isolated γδ T cell population comprises at least 10% CCR3 + cells, at least 10% CCR4 + cells, at least 10% CCR7 + cells, at least 10% CCR8 + cells, or at least 10% CD103 + cells, and the method according to any one of (6) to (20). (22) Before amplification, the isolated γδ T cell population has a high frequency of CCR3 + cells, CCR4 + cells, CCR7 + cells and / or CCR8 + cells as compared to a reference population of blood-resident Vδ2 T cells, and the method according to any one of (6) to (21). (23) The isolated Vδ1 T cell population has a high frequency of NKG2D + cells, CD56 + cells, CD69 +Cells and / or TIM3 + The method according to any one of (14) to (22), comprising cells. (24) In a culture within 14 days, the population of amplified γδ T cells contains at least 20 times the number of γδ T cells compared to the population of isolated γδ T cells before amplification, according to any one of (1) to (23). (25) In a culture within 21 days, the population of amplified γδ T cells contains at least 50 times the number of γδ T cells compared to the population of isolated γδ T cells before amplification, according to any one of (1) to (24). (26) The method according to any one of (1) to (25), wherein the population of amplified γδ T cells contains a population of amplified Vδ1 T cells. (27) In a culture within 14 days, the population of amplified Vδ1 T cells contains at least 20 times the number of Vδ1 T cells compared to the population of isolated Vδ1 T cells before amplification, according to (26). (28) In a culture within 21 days, the population of amplified Vδ1 T cells contains at least 50 times the number of Vδ1 T cells compared to the population of isolated Vδ1 T cells before amplification, according to (26) or (27). (29) The method according to any one of (1) to (28), wherein the population of amplified γδ T cells expresses CD27. (30) The method according to (29), wherein the population of amplified γδ T cells shows a median CD27 expression level higher than that of the population of isolated γδ T cells. (31) The method according to (30), wherein the population of amplified γδ T cells shows a median CD27 expression level at least 2 times higher than that of the population of isolated γδ T cells. (32) The population of amplified γδ T cells has a high frequency of CD27 compared to the population of isolated γδ T cells. + The method according to (29), having cells. (33) The population of amplified γδ T cells has a high frequency of CD27 at least 5% higher compared to the population of isolated γδ T cells. + The method according to (32), having cells. (34) The method according to any one of (26) to (28), wherein the population of amplified Vδ1 T cells expresses CD27. (35) The method according to (34), wherein the population of amplified Vδ1 T cells exhibits a higher median CD27 expression level than the population of isolated Vδ1 T cells. (36) The method according to (35), wherein the population of amplified Vδ1 T cells exhibits a median CD27 expression level that is at least 2-fold higher than that of the population of isolated Vδ1 T cells. (37) The method according to any one of (34) to (36), wherein the population of amplified Vδ1 T cells has a higher frequency of CD27 + cells compared to the population of isolated Vδ1 T cells. (38) The method according to (37), wherein the population of amplified Vδ1 T cells has a frequency of CD27 that is at least 5% higher + compared to the population of isolated Vδ1 T cells. (39) The method according to any one of (1) to (38), wherein the population of amplified γδ T cells exhibits a lower mean TIGIT expression level than the population of isolated γδ T cells. (40) The method according to (39), wherein the population of amplified γδ T cells exhibits a mean TIGIT expression level that is at least 50% lower than that of the population of isolated γδ T cells. (41) The method according to (39) or (40), wherein the population of amplified γδ T cells has a lower frequency of TIGIT + cells compared to the population of isolated γδ T cells. (42) The method according to (41), wherein the population of amplified γδ T cells has a frequency of TIGIT+ cells that is at least 20% lower than that of the population of isolated γδ T cells. (43) The method according to any one of (26) to (28), or (34) to (38), wherein the population of amplified Vδ1 T cells exhibits a lower mean TIGIT expression level than the population of isolated Vδ1 T cells. (44) The method according to (43), wherein the population of amplified Vδ1 T cells exhibits a mean TIGIT expression level that is at least 50% lower than that of the population of isolated Vδ1 T cells. (45) The population of amplified Vδ1 T cells has a lower frequency of TIGIT than the population of isolated Vδ1 T cells. + The method according to any one of (26) to (28) or (34) to (38), which has cells. (46) The population of amplified Vδ1 T cells has at least 20% lower frequency of TIGIT than the population of isolated Vδ1 T cells. + The method according to (45), which has cells. (47) The average expression level of one or more markers selected from the group consisting of CD124, CD215, CD360, CTLA4, CD1b, BTLA, CD39, CD45RA, Fas ligand, CD25, ICAM-1, CD31, KLRG1, CD30, and CD2 in the population of amplified γδ T cells is higher compared to the population of isolated γδ T cells. The method according to any one of (1) to (46). (48) The cells expressing one or more markers selected from the group consisting of CD124, CD215, CD360, CTLA4, CD1b, BTLA, CD39, CD45RA, Fas ligand, CD25, ICAM-1, CD31, KLRG1, CD30, and CD2 in the population of amplified γδ T cells are at a higher frequency compared to the population of isolated γδ T cells. The method according to any one of (1) to (47). (49) The average expression level of one or more markers selected from the group consisting of NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1, and CD64 in the population of amplified γδ T cells is lower compared to the population of isolated γδ T cells. The method according to any one of (1) to (48). (50) The cells expressing one or more markers selected from the group consisting of NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1, and CD64 in the population of amplified γδ T cells are at a lower frequency compared to the population of isolated γδ T cells. The method according to any one of (1) to (49). (51) The method according to any one of (26)-(28), (34)-(38), (43) or (45), wherein the average expression level of one or more markers selected from the group consisting of CD124, CD215, CD360, CTLA4, CD1b, BTLA, CD39, CD45RA, Fas ligand, CD25, ICAM-1, CD31, KLRG1, CD30, and CD2 in the population of amplified Vδ1 T cells is higher compared to the population of isolated Vδ1 T cells. (52) The method according to any one of (26)-(28), (34)-(38), (43), (45) or (51), wherein the cells expressing one or more markers selected from the group consisting of CD124, CD215, CD360, CTLA4, CD1b, BTLA, CD39, CD45RA, Fas ligand, CD25, ICAM-1, CD31, KLRG1, CD30, and CD2 in the population of amplified γδ T cells are at a higher frequency compared to the population of isolated γδ T cells. (53) The method according to any one of (26)-(28), (34)-(38), (43), (45), (51) or (52), wherein the average expression level of one or more markers selected from the group consisting of NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1 and CD64 in the population of amplified γδ T cells is lower compared to the population of isolated γδ T cells. (54) The method according to any one of (26)-(28), (34)-(38), (43), (45) or (51)-(53), wherein the cells expressing one or more markers selected from the group consisting of NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1 and CD64 in the population of amplified γδ T cells are at a lower frequency compared to the population of isolated γδ T cells. (55) The method according to any one of (8)-(54), wherein step (iii) comprises culturing γδ T cells substantially without contact with stromal cells. (56) The method according to any one of (8) to (55), wherein step (iii) comprises culturing γδ T cells substantially without contact with Sertoli cells. (57) The method according to any one of (8) to (55), wherein step (iii) comprises culturing γδ T cells substantially without contact with tumor cells. (58) The method according to any one of (1) to (57), wherein the non-hematopoietic tissue is not a tumor tissue. (59) The method according to any one of (1) to (58), wherein the non-hematopoietic tissue is skin. (60) Amplified γδ T cells obtained by the method according to any one of (1) to (59). (61) A population of isolated γδ T cells, wherein at least 50% of the isolated population of γδ T cells express CD27 and substantially do not express TIGIT. (62) The population of isolated γδ T cells according to claim 61, wherein at least 50% of the isolated population of γδ T cells express Vδ1. (63) A pharmaceutical composition comprising the amplified γδ T cells according to (60), or the population of isolated γδ T cells according to (61) or (62). (64) The pharmaceutical composition according to (63), for use in a method of treating cancer or an infectious disease in a subject. (65) Use of the pharmaceutical composition according to (63) in the manufacture of a medicament for treating cancer or an infectious disease in a subject. (66) A method of treating a subject by adoptive T cell therapy, comprising administering to the subject in need of treatment a therapeutically effective amount of amplified γδ T cells obtained by the method according to any one of (1) to (59), the amplified γδ T cells according to claim 60, the isolated population according to (61) or (62), or the pharmaceutical composition according to (63). (67) The therapeutically effective amount of the amplified γδ T cells is less than 10×10 12 cells per administration, according to (66). (68) The method according to (66) or (67), comprising administering to the subject in need of treatment one or more additional therapeutic agents. (69) The method according to (68), wherein the one or more additional therapeutic agents are selected from the group consisting of immunotherapeutic agents, cytotoxic agents, growth inhibitors, radiotherapy agents, angiogenesis inhibitors, and combinations thereof. (70) The method according to (68) or (69), wherein the one or more additional therapeutic agents are administered simultaneously with the expanded γδ T cells. (71) The method according to (68) or (69), wherein the one or more additional therapeutic agents are administered after the administration of the expanded γδ T cells. (72) The method according to any one of (68) to (71), wherein the additional therapeutic agent is an immunotherapeutic agent. (73) A method of treating a subject by adoptive T cell therapy, comprising the step of administering to the subject in need of treatment a therapeutically effective amount of the pharmaceutical composition according to (63). (74) The method according to any one of (66) to (73), wherein the subject is a human. (75) The method according to (74), wherein the human is a human cancer patient. (76) The method according to (75), wherein the human cancer patient is undergoing treatment for solid cancer. (77) The method according to (76), wherein the human is undergoing treatment for viral infection.

Claims

**Claim 1** The following steps: (i) Separating γδ T cells from stromal cells and epithelial cells present in a skin sample taken from a subject to generate a population of γδ T cells; and (ii) Culturing the population of γδ T cells in the presence of IL-2, IL-4, IL-15 and IL-21 for at least 5 days to generate an amplified population of γδ T cells; comprising wherein the proportion of stromal cells in the cells in the culture of step (ii) is less than 10%, A method for amplifying γδ T cells. **Claim 2** The method according to claim 1, wherein the proportion of stromal cells in the cells in the culture of step (ii) is less than 1%. **Claim 3** Step (ii) is (a) in the presence of at least one factor selected from the group consisting of stromal cell-derived factor (SDF), IL-1, IL-12, IL-18 and IL-33; (b) in a serum-free medium; or (c) under both conditions of (a) and (b); The method according to claim 1 or 2, further comprising culturing the population of γδ T cells. **Claim 4** Step (i) is (a) culturing the skin sample on a synthetic scaffold constructed to release cells from the skin sample; (b) culturing the skin sample in the presence of IL-2 and / or IL-15; or (c) both (a) and (b); The method according to any one of claims 1 to 3. **Claim 5** The method according to any one of claims 1 to 4, wherein the population of γδ T cells comprises Vδ1 T cells. **Claim 6** (a) at least 80% of the population of γδ T cells before step (ii) are Vδ1 T cells; (b) the proportion of Vδ2 T cells in the population of γδ T cells before step (ii) is less than 10%; or (c) having both characteristics of (a) and (b); The method according to any one of claims 1 to 5. **Claim 7** The method according to any one of claims 1 to 6, further comprising removing αβ T cells and / or NK cells from the population of γδ T cells. **Claim 8** The population of γδ T cells before step (ii) is (a) High frequencies of CCR3 + cells, CCR4 + cells, CCR7 + cells, and / or CCR8 + cells; (b) A high frequency of NKG2D + cells, CD56 + cells, CD69 + cells, and / or TIM3 + cells; or, (c) both (a) and (b); The method according to any one of claims 1 to 7. **Claim 9** The method according to any one of claims 1 to 8, wherein within 14 days from the start of the culture in step (ii), the amplified population of γδ T cells contains at least 20 times the number of γδ T cells compared to the population of γδ T cells before step (ii).

10. The method according to any one of claims 1 to 9, wherein within 21 days from the start of the culture in step (ii), the amplified population of γδ T cells contains at least 50 times the number of γδ T cells compared to the population of γδ T cells before step (ii).

11. The method according to any one of claims 1 to 10, wherein within 14 days from the start of the culture in step (ii), the amplified population of γδ T cells contains at least 20 times the number of Vδ1 T cells compared to the Vδ1 T cells of the population of γδ T cells before step (ii).

12. The method according to any one of claims 1 to 11, wherein within 21 days from the start of the culture in step (ii), the amplified population of γδ T cells contains at least 50 times the number of Vδ1 T cells compared to the Vδ1 T cells of the population of γδ T cells before step (ii).

13. The method according to any one of claims 1 to 12, wherein the skin sample comprises a sample obtained by punch biopsy.

14. The method according to any one of claims 1 to 13, wherein the skin sample is minced before step (i).

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