Methods for isolating and expanding cells

Abstract

To provide a method for isolating lymphocytes (in particular γδ T cells) from a non-hematopoietic tissue sample.SOLUTION: Provided is a method including the steps of: culturing a non-hematopoietic tissue sample which is an intact biopsy obtained from a non-hematopoietic tissue in the presence of Interleukin-2 (IL-2) and Interleukin-15 (IL-15); and collecting a population of lymphocytes cultured from the non-hematopoietic tissue sample. Provided are methods of subsequent expansion, as well as populations of isolated cells obtained by the method and uses thereof.SELECTED DRAWING: None

Description

Technical Field

[0001] (Field of the Invention) The present invention relates to a method for isolating and / or expanding non-hematopoietic tissue resident lymphocytes, particularly γδ T cells. Such γδ T cells include non-Vδ2 cells, such as Vδ1, Vδ3, and Vδ5 cells. Such non-hematopoietic tissues include skin and gastrointestinal tract. Such isolated and / or expanded non-hematopoietic tissue resident lymphocytes will be understood to be very useful in adoptive T cell therapy, chimeric receptor therapy, etc. The present invention also relates to cells produced by the methods described herein.

Background Art

[0002] (Background of the Invention) The increasing interest in T cell immunotherapy for cancer has focused on the apparent ability of subsets of CD8+ and CD4+ αβ T cells that recognize cancer cells and, particularly, mediate host-defensive functional potential when unregulated by clinically mediated antagonism of inhibitory pathways brought about by PD-1, CTLA-4, and other receptors. However, αβ T cells are MHC-restricted, which can result in graft-versus-host disease. Gamma delta T cells (γδ T cells) correspond to a subset of T cells that express a characteristic definitive γδ T cell receptor (TCR) on their surface. This TCR is composed of one gamma (γ) chain and one delta (δ) chain. Human γδ TCR chains are selected from three main δ chains, Vδ1, Vδ2, and Vδ3, and six γ chains. Specific γ and δ types are not exclusively, but

[0003] , often found in cells in one or more tissue types, human γδ T cells can be broadly classified based on their TCR chains. For example, most blood-resident γδ T cells express a Vδ2 TCR, such as Vγ9Vδ2, which is less common among tissue-resident γδ T cells. Instead, tissue-resident γδ T cells often use Vδ1 in the skin and Vγ4 in the gastrointestinal tract . .

[0004] Most methods for isolating lymphocytes rely on isolating these cell types from the blood. Non-hematopoietic tissue-resident lymphocytes such as αβ T cells, γδ T cells, and NK cells may have properties particularly suitable for specific applications, such as targeting non-hematopoietic tumors and other targets. However, isolating such tissue-resident lymphocytes in clinically significant amounts remains a challenge, especially when clinical doses ranging from 10 cells or more are required for many indications. Importantly, significant cell loss during production means that even more starting cells must be generated. 8

[0005] Non-hematopoietic tissue-resident lymphocytes, particularly αβ T cells, γδ T cells, and NK cells, are not easily obtained in large numbers, so they have not been well characterized or studied for therapeutic applications. Therefore, there is a need in this field for methods to isolate and expand non-hematopoietic tissue-resident lymphocytes, particularly γδ T cells, to sufficient amounts for research and potential adaptation as therapies, such as adoptive T cell therapy.

[0006] The literature of Clark et al. (2006) J. Invest. Dermatol. 126(5): 1059-70 describes a method for isolating skin resident T cells from normal skin and diseased skin. However, the method described therein, although due to the presence of animal products, in particular, the yield of the isolated cells is relatively low, that is, less than 10 cells per 1 cm of tissue 2 6 6 6 6 is not suitable for clinical use. In the method described in the literature of Clark et al., a disrupted sample is used, which results in an intentional destruction of the structural integrity of the tissue sample. WO2017072367 and WO 2018 / 202808 relate to a method for expanding non-hematopoietic tissue resident γδ T cells in vitro by culturing lymphocytes obtained from non-hematopoietic tissue in the presence of at least interleukin-2 (IL -2) and / or interleukin-15 (IL-15). WO2015189356 describes a composition for expanding lymphocytes obtained from a sample obtained by apheresis and containing at least two cytokines selected from IL-2, IL-15 , and IL-21. Therefore, there is still a need for a method for isolating tissue resident non-hematopoietic lymphocytes, for example, from the skin, which produces a larger amount of cells suitable for clinical use. SUMMARY OF THE INVENTION

[0007] (Summary of the Invention) According to a first aspect of the present invention, there is provided a method for isolating lymphocytes from a non-hematopoietic tissue sample, comprising: (i) an intact biopsy having a minimum cross-sectional area of at least 2 mm obtained from non-hematopoietic tissue, the non-hematopoietic culturing a tissue sample in the presence of interleukin-2 (IL-2) and interleukin-15 (IL-15); and (ii) recovering a population of lymphocytes cultured from the non-hematopoietic tissue sample : A method is provided that includes these steps.

[0008] According to a further aspect of the present invention, a method for isolating γδ T cells from a non-hematopoietic tissue sample, comprising (i) culturing a non-hematopoietic tissue sample, which is an intact biopsy having a minimum cross-sectional area of at least 2 mm obtained from non-hematopoietic tissue, in the presence of IL-2 and IL-15; and (ii) recovering a population of γδ T cells cultured from the non-hematopoietic tissue sample : A method is provided that includes these steps.

[0009] According to a further aspect of the present invention, a method for isolating lymphocytes from a non-hematopoietic tissue sample, comprising (i) culturing a non-hematopoietic tissue sample, which is an intact biopsy having a minimum cross-sectional area of at least 2 mm obtained from non-hematopoietic tissue, in the presence of IL-2 and IL-15; and 2 (ii) recovering a population of lymphocytes cultured from the non-hematopoietic tissue sample : A method is provided that includes these steps.

[0010] According to a further aspect of the present invention, a method for isolating γδ T cells from a non-hematopoietic tissue sample, comprising (i) culturing a non-hematopoietic tissue sample, which is an intact biopsy having a minimum cross-sectional area of at least 2 mm obtained from non-hematopoietic tissue, in the presence of IL-2 and IL-15; and 2 (ii) recovering a population of γδ T cells cultured from the non-hematopoietic tissue sample : A method is provided that includes these steps.

[0011]

[0011] ​​​​​According to a further aspect of the present invention, there is provided a method for isolating lymphocytes from a non-hematopoietic tissue sample, comprising: (i) culturing a non-hematopoietic tissue sample, which is an intact biopsy having a volume of at least 2 mm obtained from a non-hematopoietic tissue, in the presence of IL-2 and IL-15; and 3 a non-hematopoietic tissue (ii) recovering a population of lymphocytes cultured from the non-hematopoietic tissue sample. A method is provided which comprises:

[0012] According to a further aspect of the present invention, there is provided a method for isolating γδ T cells from a non-hematopoietic tissue sample, comprising: (i) culturing a non-hematopoietic tissue sample, which is an intact biopsy having a volume of at least 2 mm obtained from a non-hematopoietic tissue, in the presence of IL-2 and IL-15; and 3 a non-hematopoietic tissue (ii) recovering a population of γδ T cells cultured from the non-hematopoietic tissue sample. A method is provided which comprises:

[0013] According to a further aspect of the present invention, there is provided a method for isolating lymphocytes from a non-hematopoietic tissue sample, comprising: (i) placing the non-hematopoietic tissue sample in a container containing a gas-permeable material; (ii) culturing the non-hematopoietic tissue sample in the presence of IL-2 and IL-15; and (iii) recovering a population of lymphocytes cultured from the non-hematopoietic tissue sample. A method is provided which comprises:

[0014] According to a further aspect of the present invention, there is provided a method for isolating γδ T cells from a non-hematopoietic tissue sample, comprising: (i) placing the non-hematopoietic tissue sample in a container containing a gas-permeable material; (ii) culturing the non-hematopoietic tissue sample in the presence of IL-2 and IL-15; and (iii) recovering a population of γδ T cells cultured from the non-hematopoietic tissue sample. A method is provided that includes:

[0015] According to a further aspect of the present invention, a method for isolating and expanding lymphocytes from a non-hematopoietic tissue sample comprises: (i) isolating a population of lymphocytes from the non-hematopoietic tissue sample according to the method defined herein ; and (ii) further culturing the population of lymphocytes for at least 5 days to produce an expanded population of lymphocytes . A method is provided that includes:

[0016] According to a further aspect of the present invention, a method for isolating and expanding γδ T cells from a non-hematopoietic tissue sample comprises: (i) isolating a population of γδ T cells from the non-hematopoietic tissue sample according to the method defined herein ; and (ii) further culturing the population of γδ T cells for at least 5 days to produce an expanded population of γδ T cells . A method is provided that includes: BRIEF DESCRIPTION OF THE DRAWINGS

[0017] (Brief description of the drawings)

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Mode for Carrying Out the Invention

[0018] (Detailed Description of the Invention) According to a first aspect of the present invention, there is provided a method for isolating lymphocytes from a non-hematopoietic tissue sample, comprising: (i) culturing a non-hematopoietic tissue sample, which is an intact biopsy having a minimum cross-sectional area of at least 2 mm obtained from a non-hematopoietic tissue, in the presence of interleukin-2 (IL-2) and interleukin-15 (IL-15); and (ii) recovering a population of lymphocytes cultured from the non-hematopoietic tissue sample.

[0019] According to a further aspect of the present invention, there is provided a method for isolating γδ T cells from a non-hematopoietic tissue sample, comprising: (i) culturing a non-hematopoietic tissue sample, which is an intact biopsy having a minimum cross-sectional area of at least 2 mm obtained from a non-hematopoietic tissue, in the presence of IL-2 and IL-15; and (ii) recovering a population of γδ T cells cultured from the non-hematopoietic tissue sample.

[0020] As used herein, the term "isolation" or "isolating" of cells, particularly lymphocytes and / or γδ T cells, refers to the removal of cells from a tissue or pool of cells, A method or process of being isolated, purified, concentrated, or taken out in another form is meant. Such references will be understood to include the terms "isolated", "removed", "purified", "concentrated", and similar terms. The isolation of lymphocytes and / or γδ T cells includes the isolation or separation of cells from an intact non-hematopoietic tissue sample or from stromal cells of non-hematopoietic tissue (e.g., fibroblasts or epithelial cells). Such isolation may, instead of or in addition to, include the isolation or separation of γδ T cells from other hematopoietic cells (e.g., αβ T cells or other lymphocytes). Isolation begins when a tissue explant or biopsy is placed into an isolation culture and ends when the cells are recovered from the culture by, for example, centrifugation or other means for transferring the isolated cell population to an expansion culture, or used for other purposes, or when the original tissue explant or biopsy is removed from the culture. The isolation process may be for at least about 3 days to about 45 days. In one embodiment, the isolation process is for at least about 10 days to at least 28 days. In a further embodiment, the isolation process is for at least 14 days to at least 21 days. Therefore, the isolation process may be for at least 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 days, 25, 26, 27, 28, 29, 30, 31, 32, about 35 days, about 40 days, or about 45 days. During this isolation process, the proliferation of the isolated cells may not be significant, but it is understood that the proliferation of the cells does not necessarily not exist. In one embodiment, the isolation process is for at least about 10 days to at least 28 days. In a further embodiment, the isolation process is for at least 14 days to at least 21 days. Therefore, the isolation process may be for at least 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 days, 25, 26, 27, 28, 29, 30, 31, 32, about 35 days, about 40 days, or about 45 days. During this isolation process, the proliferation of the isolated cells may not be significant, but it is understood that the proliferation of the cells does not necessarily not exist. It is understood that although the proliferation of the isolated cells may not be significant during this isolation process, it does not necessarily mean that there is no proliferation of the cells. It can be done. In fact, for some those skilled in the art, it is also recognized that isolated cells can begin to divide and generate multiple cells of the same kind within an isolation container containing tissue and / or a scaffold. It can also be done. It is recognized that it can be done.

[0021] Therefore, references herein to "isolated lymphocytes", "isolated lymphocyte populations", "populations of isolated lymphocytes", "separated lymphocytes", "separated lymphocyte populations", "populations of separated lymphocytes", "isolated γδ T cells", "isolated γδ T cell populations", "populations of isolated γδ T cells", "separated γδ T cells", "separated γδ T cell populations", or "populations of separated γδ T cells" are understood to refer to hematopoietic cells or populations of hematopoietic cells containing γδ cells that have been isolated, separated, removed, purified, or concentrated from a non-hematopoietic tissue sample of origin such that the cells are substantially not in contact with non-hematopoietic cells or cells contained within intact non-hematopoietic tissue. Similarly, references herein to "populations of isolated or separated Vδ1 T cells" are understood to refer to hematopoietic cells containing Vδ1 T cells that have been isolated, separated, removed, purified, or concentrated from a non-hematopoietic tissue sample of origin such that the cells are substantially not in contact with non-hematopoietic cells or cells contained within intact non-hematopoietic tissue. Therefore, isolation or separation refers to the isolation, separation, removal, purification, or concentration of hematopoietic cells (e.g., γδ T cells or other lymphocytes) from non-hematopoietic cells (e.g., stromal cells, fibroblasts, and / or epithelial cells). The methods of isolating lymphocytes and / or γδ T cells as defined herein involve tissue disruption (e.g., such as from a non-hematopoietic tissue sample of origin such that the cells are substantially not in contact with non-hematopoietic cells or cells contained within intact non-hematopoietic tissue, isolated, separated, removed, purified, or concentrated γδ cells containing hematopoietic cells or populations of hematopoietic cells will be understood. Similarly, references herein to "populations of isolated or separated Vδ1 T cells" are understood to refer to hematopoietic cells containing Vδ1 T cells that have been isolated, separated, removed, purified, or concentrated from a non-hematopoietic tissue sample of origin such that the cells are substantially not in contact with non-hematopoietic cells or cells contained within intact non-hematopoietic tissue. Therefore, isolation or separation refers to the isolation, separation, removal, purification, or concentration of hematopoietic cells (e.g., γδ T cells or other lymphocytes) from non-hematopoietic cells (e.g., stromal cells, fibroblasts, and / or epithelial cells). such as from a non-hematopoietic tissue sample of origin such that the cells are substantially not in contact with non-hematopoietic cells or cells contained within intact non-hematopoietic tissue, isolated, separated, removed, purified, or concentrated Vδ1 T cells containing hematopoietic cells. Therefore, isolation or separation refers to the isolation, separation, removal, purification, or concentration of hematopoietic cells (e.g., γδ T cells or other lymphocytes) from non-hematopoietic cells (e.g., stromal cells, fibroblasts, and / or epithelial cells). For example, stromal cells, fibroblasts, and / or epithelial cells) from hematopoietic cells (e.g., γδ T cells or other lymphocytes).

[0022] The methods of isolating lymphocytes and / or γδ T cells as defined herein involve tissue disruption (e.g., , including comminution) and subsequent separation of lymphocytes and / or γδ T cells from other cell types can be performed. Preferably, the method for isolating lymphocytes and / or γδ T cells as defined herein includes "creeping out" of lymphocytes and / or γδ T cells and other cell types from intact non-hematopoietic tissue samples or explants or biopsy tissue matrices, where tissue resident lymphocytes physically separate from the tissue matrix without the need for disruption of the tissue matrix . By maintaining the integrity of the tissue matrix, surprisingly, tissue resident lymphocytes can be easily removed at the end of isolation later and are retained in the explant or biopsy with little or no escape of inhibitory cell types such as fibroblasts from the tissue matrix . Thus, in some embodiments, the use of intact non-hematopoietic tissue samples or tissue matrices results in the release of a small number of fibroblasts into the tissue culture . Such a "creeping out" method that utilizes intact non-hematopoietic tissue or tissue matrix has the advantage of reducing the need for excessive processing of non-hematopoietic tissue samples or tissue matrices, maintains the structural integrity of the non-hematopoietic tissue or tissue matrix, and can provide the unexpected advantage of resulting in a higher isolated cell yield.

[0023] Therefore, the method for isolating non-hematopoietic tissue-derived lymphocytes as defined herein includes a method for isolating non-hematopoietic tissue-derived lymphocytes from an intact biopsy or explant of non-hematopoietic tissue. Such intact biopsies or explants are those in which the structural integrity of the biopsy or explant has not been intentionally disrupted within the excision perimeter that removes the biopsy or explant from the tissue sample . Such intact biopsies or explants are those that have not been intentionally disrupted within the excision perimeter that removes the biopsy or explant from the tissue sample. Such intact biopsies The biopsy or explant has a three-dimensional structure that is mostly maintained, except for minor disruptions caused by the manipulation. Therefore, this intact biopsy or explant has not been mechanically disrupted, for example, by mincing or cutting, nor has it been chemically or enzymatically disrupted. However, disrupted tissue may be used in the isolation method of the present invention. In one embodiment, the isolated lymphocytes are αβ T cells. In an alternative embodiment, the isolated lymphocytes are γδ T cells. In another embodiment, the isolated lymphocytes are NK cells. It can be understood that multiple types of lymphocytes can be isolated from the same isolation step. The isolation method of lymphocytes and / or γδ T cells utilizing the "creeping out" method defined herein can include culturing cells and / or non-hematopoietic tissues in the presence of cytokines and / or chemokines sufficient to induce the isolation or separation of γδ T cells and / or other lymphocytes defined herein. Thus, in one embodiment of the present invention, the isolation of lymphocytes and / or γδ T cells from non-hematopoietic tissues includes culturing non-hematopoietic tissues in the presence of IL-2 and IL-15. As used herein, "IL-2" refers to natural or recombinant IL-2 or a variant thereof that acts as an agonist (e.g., its mutants, muteins, analogs, subunits, receptor complexes, fragments, isoforms, and peptidomimetics) of one or more IL-2 receptor (IL-2R) subunits. Such agents are CTLL-2, an IL-2-dependent cell line (33; a

[0024]

[0025] ​​​​​​​​​​​​​​​can support the growth of the American Type Culture Collection (ATC C (registered trademark)) TIB 214). Mature human IL-2 is described in the article by Fujita et al., Cell 1986. 46.3:401-407, and results in a 133-amino acid sequence (subtracting the signal peptide consisting of an additional 20 N-terminal amino acids). An IL-2 mutein is a polypeptide that has specific substitutions made to the interleukin-2 protein while retaining the ability to bind to IL-2Rβ , for example, the polypeptide described in US 2014 / 0046026. An IL-2 mutein can be characterized by amino acid insertions, deletions, substitutions, and modifications at one or more sites in other residues of the native IL-2 polypeptide chain . According to the present disclosure, any such insertions, deletions, substitutions, and modifications result in an IL-2 mutein that retains IL-2Rβ binding activity. Exemplary muteins can include substitutions of 1, 2, 3, 4, 5, 6, 7, 8 , 9, 10, or more amino acids.

[0026] 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) is found in Genbank under the accession locator NP_00 0577.2 GI: 28178861. The murine (Mus musculus) IL-2 amino acid sequence (Gene ID 16183) is found in Genbank under the accession locator NP_032392. 1 GI: 7110653.

[0027] ​​​​​ IL-2 can also refer to IL-2 derived from various mammalian species, including, for example, humans, monkeys, cows, pigs, horses, and mice. Variants can include conservatively substituted sequences, meaning that a given amino acid residue has been replaced by a residue having similar physicochemical characteristics. Examples of conservative substitutions include the substitution of one aliphatic residue for another, for example, the substitution of Ile, Val, Leu, or Ala for each other, or the substitution of one polar residue for another, for example, the substitution between Lys and Arg; the substitution between Glu and Asp; or the substitution between Gln and Asn. Other such conservative substitutions, for example, the substitution of entire regions having similar hydrophobic characteristics, are well known. Natural IL-2 variants are also encompassed by the present invention. Examples of such variants are proteins resulting from alternative mRNA splicing events or proteolytic cleavage of the IL-2 protein, where the IL-2 binding characteristics are retained. Alternative splicing of mRNA can yield a cleaved but biologically active IL-2 protein. Mutations resulting from proteolysis include, for example, proteolytic removal of one or more terminal amino acids (usually 1 to 10 amino acids) from the IL-2 protein, resulting in various types of N- or C-terminal differences in expression in different host cells. In some embodiments, the terminal or internal portion of the protein can be modified with a chemical group, such as polyethylene glycol, to change its physical properties (Yang et al., Cancer 1995. 76: 687-694). In some embodiments, the terminal or internal portion of the protein can be modified with additional amino acids (Cla ims ​rk-Lewis et al., PNAS 1993. 90:3574-3577).

[0028] As used herein, "IL-15" refers to native or recombinant IL-15 or a variant thereof that acts as an agonist (e.g., a mutant, mutein, analog, subunit, receptor complex, fragment, isoform, and peptidomimetic) of one or more IL-15 receptor (IL-15R) subunits IL-15 is a known T cell growth factor that can support the growth of the IL-2-dependent cell line CTLL-2, similar to IL-2. IL-15 is a mature protein of 114 amino acids and was first reported by Grabstein et al. (Grabstein et al., Science 1994. 264.516 1: 965-969). The term "IL-15" as used herein encompasses native or recombinant IL-15 and its mutants, analogs, subunits, or complexes thereof IL-15 is a known T cell growth factor that can support the growth of the IL-2-dependent cell line CTLL-2, similar to IL-2. IL-15 is a mature protein of 114 amino acids and was first reported by Grabstein et al. (Grabstein et al., Science 1994. 264.516 1: 965-969). The term "IL-15" as used herein encompasses native or recombinant IL-15 and its mutants, analogs, subunits, or complexes thereof IL-15 is a known T cell growth factor that can support the growth of the IL-2-dependent cell line CTLL-2, similar to IL-2. IL-15 is a mature protein of 114 amino acids and was first reported by Grabstein et al. (Grabstein et al., Science 1994. 264.516 1: 965-969). The term "IL-15" as used herein encompasses native or recombinant IL-15 and its mutants, analogs, subunits, or complexes thereof IL-15 is a known T cell growth factor that can support the growth of the IL-2-dependent cell line CTLL-2, similar to IL-2. IL-15 is a mature protein of 114 amino acids and was first reported by Grabstein et al. (Grabstein et al., Science 1994. 264.516 1: 965-969). The term "IL-15" as used herein encompasses native or recombinant IL-15 and its mutants, analogs, subunits, or complexes thereof IL-15 is a known T cell growth factor that can support the growth of the IL-2-dependent cell line CTLL-2, similar to IL-2. IL-15 is a mature protein of 114 amino acids and was first reported by Grabstein et al. (Grabstein et al., Science 1994. 264.516 1: 965-969). The term "IL-15" as used herein encompasses native or recombinant IL-15 and its mutants, analogs, subunits, or complexes thereof IL-15 is a known T cell growth factor that can support the growth of the IL-2-dependent cell line CTLL-2, similar to IL-2. IL-15 is a mature protein of 114 amino acids and was first reported by Grabstein et al. (Grabstein et al., Science 1994. 264.516

[0029] Human IL-15 can be obtained according to the procedures 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 (registration ​ It was conducted under the trademark) and attributed with accession number 69245.

[0030] The amino acid sequence of human IL-15 (Gene ID 3600) is NP000576.1 GI: 10835 in Genbank 153 (isoform 1) and NP_751915.1 GI: 26787986 (isoform 2) are found by accession locators. The murine (Mus musculus) IL-15 amino acid sequence (Gene ID 16168) is found by the accession locator NP_001241676.1 GI: 363000984 in Genbank. IL-15 can also refer to IL-15 derived from various mammalian species, including, for example, humans, monkeys, cows, pigs, horses, and mice. The IL-15 "mutants" or "variants" referred to herein are substantially homologous to the sequence of native mammalian IL-15, but have an amino acid sequence different from that of native mammalian IL-15 due to amino acid deletions, insertions, or substitutions. Variants can include conservatively substituted sequences, meaning that a given amino acid residue is replaced by a residue having similar physicochemical characteristics. Examples of conservative substitutions include the substitution of one aliphatic residue for another, such as the substitution of Ile, Val, Leu, or Ala for each other, or the substitution of one polar residue for another, such as the substitution between Lys and Arg; the substitution between Glu and Asp; or the substitution between Gln and Asn. Other such conservative substitutions, such as the substitution of entire regions having similar hydrophobic characteristics, are well known. It is found by the accession locator.

[0031] IL-15 can also refer to IL-15 derived from various mammalian species, including, for example, humans, monkeys, cows, pigs, horses, and mice. The IL-15 "mutants" or "variants" referred to herein are substantially homologous to the sequence of native mammalian IL-15, but have an amino acid sequence different from that of native mammalian IL-15 due to amino acid deletions, insertions, or substitutions. Variants can include conservatively substituted sequences, meaning that a given amino acid residue is replaced by a residue having similar physicochemical characteristics. Examples of conservative substitutions include the substitution of one aliphatic residue for another, such as the substitution of Ile, Val, Leu, or Ala for each other, or the substitution of one polar residue for another, such as the substitution between Lys and Arg; the substitution between Glu and Asp; or the substitution between Gln and Asn. Other such conservative substitutions, such as the substitution of entire regions having similar hydrophobic characteristics, are well known. Examples of conservative substitutions include the substitution of one aliphatic residue for another, such as the substitution of Ile, Val, Leu, or Ala for each other, or the substitution of one polar residue for another, such as the substitution between Lys and Arg; the substitution between Glu and Asp; or the substitution between Gln and Asn. Other such conservative substitutions, such as the substitution of entire regions having similar hydrophobic characteristics, are well known. Other such conservative substitutions, such as the substitution of entire regions having similar hydrophobic characteristics, are well known. ​​​Native IL-15 variants are also encompassed by the present invention. Examples of such variants are proteins resulting from alternative mRNA splicing events or proteolytic cleavage of the IL-15 protein, where the IL-15 binding properties are retained. Alternative splicing of the mRNA can produce biologically active IL-15 proteins that are truncated. Mutations resulting from proteolysis include, for example, differences in the N- or C-terminus upon expression in various types of host cells due to proteolytic removal of one or more terminal amino acids (usually 1 to 10 amino acids) from the IL-15 protein. In some embodiments, the termini or interior of the protein can be modified with chemical groups such as polyethylene glycol to alter its physical properties (Yang et al., Cancer 1995. 76: 687-694). In some embodiments, the termini or interior of the protein can be modified with additional amino acids (Clark-Lewis et al., PNAS 1993. 90:3574-3577). It will be understood that in certain embodiments, the isolation of lymphocytes and / or γδ T cells by the methods defined herein may further comprise culturing non-hematopoietic tissue in the presence of at least one additional cytokine. It will be further understood that in certain embodiments, the methods defined herein may further comprise culturing non-hematopoietic tissue in the presence of at least one additional agent, such as a chemokine. The chemokine is further selected according to the isolated γδ T cells or other lymphocytes. Further, the chemokine varies widely depending on the non-hematopoietic tissue used for the isolation of γδ T cells or lymphocytes and is selected accordingly. although truncated, can give rise to biologically active IL-15 proteins. Mutations resulting from proteolysis include, for example, differences in the N- or C-terminus upon expression in various types of host cells due to proteolytic removal of one or more terminal amino acids (usually 1 to 10 amino acids) from the IL-15 protein. In some embodiments, the termini or interior of the protein can be modified with chemical groups such as polyethylene glycol to alter its physical properties (Yang et al., Cancer 1995. 76: 687-694). In some embodiments, the termini or interior of the protein can be modified with additional amino acids (Clark-Lewis et al., PNAS 1993. 90:3574-3577). In some embodiments, the termini or interior of the protein can be modified with chemical groups such as polyethylene glycol to alter its physical properties (Yang et al., Cancer 1995. 76: 687-694). In some embodiments, the termini or interior of the protein can be modified with additional amino acids (Clark-Lewis et al., PNAS 1993. 90:3574-3577). In some embodiments, the isolation of lymphocytes and / or γδ T cells by the methods defined herein may further comprise culturing non-hematopoietic tissue in the presence of at least one additional cytokine. It will be further understood that in certain embodiments, the methods defined herein may further comprise culturing non-hematopoietic tissue in the presence of at least one additional agent, such as a chemokine. The chemokine is further selected according to the isolated γδ T cells or other lymphocytes. Further, the chemokine varies widely depending on the non-hematopoietic tissue used for the isolation of γδ T cells or lymphocytes and is selected In some embodiments, the termini or interior of the protein can be modified with chemical groups such as polyethylene glycol to alter its physical properties (Yang et al., Cancer 1995. 76: 687-694). In some embodiments, the termini or interior of the protein can be modified with additional amino acids (Clark-Lewis et al., PNAS 1993. 90:3574-3577). accordingly. In some embodiments, the isolation of lymphocytes and / or γδ T cells by the methods defined herein may further comprise culturing non-hematopoietic tissue in the presence of at least one additional cytokine. It will be further understood that in certain embodiments, the methods defined herein may further comprise culturing non-hematopoietic tissue in the presence of at least one additional agent, such as a chemokine. The chemokine is further selected according to the isolated γδ T cells or other lymphocytes. Further, the chemokine varies widely depending on the non-hematopoietic tissue used for the isolation of γδ T cells or lymphocytes and is selected accordingly. In some embodiments, the isolation of lymphocytes and / or γδ T cells by the methods defined herein may further comprise culturing non-hematopoietic tissue in the presence of at least one additional cytokine. It will be further understood that in certain embodiments, the methods defined herein may further comprise culturing non-hematopoietic tissue in the presence of at least one additional agent, such as a chemokine. The chemokine is further selected according to the isolated γδ T cells or other lymphocytes. Further, the chemokine varies widely depending on the non-hematopoietic tissue used for the isolation of γδ T cells or lymphocytes and is selected

[0032] In certain embodiments, the isolation of lymphocytes and / or γδ T cells by the methods defined herein may further comprise culturing non-hematopoietic tissue in the presence of at least one additional cytokine. It will be further understood that in certain embodiments, the methods defined herein may further comprise culturing non-hematopoietic tissue in the presence of at least one additional agent, such as a chemokine. The chemokine is further selected according to the isolated γδ T cells or other lymphocytes. Further, the chemokine varies widely depending on the non-hematopoietic tissue used for the isolation of γδ T cells or lymphocytes and is selected accordingly. In certain embodiments, the methods defined herein may further comprise culturing non-hematopoietic tissue in the presence of at least one additional agent, such as a chemokine. The chemokine is further selected according to the isolated γδ T cells or other lymphocytes. Further, the chemokine varies widely depending on the non-hematopoietic tissue used for the isolation of γδ T cells or lymphocytes and is selected accordingly. In certain embodiments, the methods defined herein may further comprise culturing non-hematopoietic tissue in the presence of at least one additional agent, such as a chemokine. The chemokine is further selected according to the isolated γδ T cells or other lymphocytes. Further, the chemokine varies widely depending on the non-hematopoietic tissue used for the isolation of γδ T cells or lymphocytes and is selected accordingly. accordingly. will be selected.

[0033] In certain embodiments, the methods defined herein typically include at least 10 IU / m L, for example, at least 100 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 / m L to 250 IU / mL, or 100 IU / mL to 200 IU / mL, for example, 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 certain embodiments, the methods defined herein typically , include IL-2 at a concentration of less than 1,000 IU / mL, for example, less than 500 IU / mL. In some embodiments the method includes IL-2 at a concentration of about 100 IU / mL.

[0034] In further embodiments, the methods defined herein typically include at least 0. 1 ng / mL, for example, at least 10 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, for example, 0.1 ng / mL to 1.0 ng / mL, 1.0 ng / mL to 5.0 ng / mL to 10 ng / mL, 10 ng / mL to 20 ng / mL, 20 ng / mL to 100 ng / mL, 20 ng / mL to 50 ng / mL, 40 ng / mL to 70 n g / mL, 50 ng / mL to 100 ng / mL, 50 ng / mL to 60 ng / mL, 100 ng / mL to 200 ng / mL, 200 ng / mL to 500 ng / m L, or 500 ng / mL to 1,000 ng / mL). In a further embodiment, the method defined in the present specif ication typically comprises IL-15 at a concentration of less than 500 ng / mL, for example, less than 100 ng / mL . In some embodiments, the method comprises IL-15 at a concentration of about 50 ng / mL.

[0035] In some embodiments, the isolation of lymphocytes and / or γδ T cells from non-hematopoietic tissue samples comprises culturing in the presence of both IL-2 and IL-15 at any of the above concentrations. Optionally, the concentration of IL-2 is about 100 IU / mL and the concentration of IL-15 is 55 ng / mL.

[0036] References herein to "non-hematopoietic tissue" or "non-hematopoietic tissue sample" include skin (e.g., human skin) and gastrointestinal tract (e.g., human gastrointestinal tract). Non-hematopoietic tissue is tissue other than blood, bone marrow, or thymus tissue. In one embodiment, the non-hematopoietic tissue sample is skin (e.g., human skin). In a further embodiment, the non-hematopoietic tissue sample is gastrointestinal tract or digestive tract (e.g., human gastrointestinal tract or human digestive tract). In some embodiments, lymphocytes and / or γδ T cells are not obtained from a particular type of sample of biological fluid, such as blood or synovial fluid. In some embodiments, the non-hematopoietic tissue sample from which lymphocytes and / or γδ T cells are isolated according to the method defined herein is skin (e.g., human skin), which is well known in the art. It can be obtained by known methods. Alternatively, the method for isolating lymphocytes and / or γδ T cells provided herein can be applied to the digestive tract (e.g., colon or gastrointestinal tract), breast, lung, prostate, liver kidney, spleen, pancreas, uterus, vagina, and other cutaneous membranes, mucous membranes, or serous membranes. Lymphocytes and / or γδ T cells can be resident in human cancer tissue samples, such as tumors of the breast or prostate. In some embodiments, the lymphocytes and / or γδ T cells can be from a human cancer tissue sample (e.g., solid tumor tissue). In other embodiments, the lymphocytes and / or γδ T cells can be from a non-hematopoietic tissue sample other than human cancer tissue (e.g., tissue not containing a significant number of tumor cells). For example, the lymphocytes and / or γδ T cells can be from a region of skin (e.g., healthy skin) adjacent to or remote from an adjacent cancer tissue. Thus, in some embodiments, γδ T cells are not obtained from human cancer tissue. In a further embodiment, lymphocytes are not obtained from human cancer tissue.

[0037] In one embodiment, the non-hematopoietic tissue sample of the method defined herein is obtained from a human. In an alternative embodiment, the non-hematopoietic tissue sample of the method defined herein is obtained from a non-human animal subject.

[0038] Methods for obtaining such tissues are known in the art. Examples of such methods include scalpel explants or punch biopsies, and the methods can vary in size. In some embodiments, the non-hematopoietic tissue sample is obtained by punch biopsy.

[0039] ​​In some embodiments of the present invention, the non-hematopoietic tissue sample is a non-invasive biopsy. References herein to "non-invasive" biopsies or "explants" are biopsies or explants that are not substantially disrupted or disrupted at all, such that the structural integrity of the biopsy or explant includes tissue and tissue samples that are not intentionally disrupted within the perimeter of the excision that removes the biopsy or explant from the tissue sample. Such non-invasive biopsies or explants have a three-dimensional structure that is mostly maintained except for minor disruptions caused by manipulation. Therefore, this non-invasive biopsy or explant is not mechanically disrupted, for example, by crushing or mincing, nor is it chemically or enzymatically disrupted, for example. A non-invasive biopsy or non-invasive tissue sample can include the entire tissue, a complete tissue, a part of the tissue, or all elements of the tissue. For example, in one embodiment, the non-invasive biopsy includes all layers of the skin. In a further embodiment, the biopsy includes the epithelial and dermal layers of the skin. In such embodiments where the biopsy is non-invasive, it will be understood that the separation and distinction of such layers are maintained. Thus, references herein to "non-invasive" further include biopsies of all layers of the non-hematopoietic tissue sample. Therefore, in a particular embodiment of the present invention, the non-hematopoietic tissue sample is not minced. In a further embodiment, the non-invasive biopsy is a punch biopsy. In yet a further embodiment, the non-invasive biopsy is obtained by punch biopsy. The embodiments presented herein where the non-hematopoietic tissue sample is a non-invasive biopsy provide the surprising advantage of obtaining a large number of isolated or separated cells from a non-hematopoietic tissue sample that is not minced and / or is non-invasive. Further, for example, by crushing or mincing, nor is it chemically or enzymatically disrupted, for example. A non-invasive biopsy or non-invasive tissue sample can include the entire tissue, a complete tissue, a part of the tissue, or all elements of the tissue. For example, in one embodiment, the non-invasive biopsy includes all layers of the skin. In a further embodiment, the biopsy includes the epithelial and dermal layers of the skin. In such embodiments where the biopsy is non-invasive, it will be understood that the separation and distinction of such layers are maintained. Thus, references herein to "non-invasive" further include biopsies of all layers of the non-hematopoietic tissue sample. Therefore, in a particular embodiment of the present invention, the non-hematopoietic tissue sample is not minced. In a further embodiment, the non-invasive biopsy is a punch biopsy. In yet a further embodiment, the non-invasive biopsy is obtained by punch biopsy. The embodiments presented herein where the non-hematopoietic tissue sample is a non-invasive biopsy provide the surprising advantage of obtaining a large number of isolated or separated cells from a non-hematopoietic tissue sample that is not minced and / or is non-invasive. Further, in such embodiments where the biopsy is non-invasive, it will be understood that the separation and distinction of such layers are maintained. Thus, references herein to "non-invasive" further include biopsies of all layers of the non-hematopoietic tissue sample. Therefore, in a particular embodiment of the present invention, the non-hematopoietic tissue sample is not minced. In a further embodiment, the non-invasive biopsy is a punch biopsy. In yet a further embodiment, the non-invasive biopsy is obtained by punch biopsy. The embodiments presented herein where the non-hematopoietic tissue sample is a non-invasive biopsy provide the surprising advantage of obtaining a large number of isolated or separated cells from a non-hematopoietic tissue sample that is not minced and / or is non-invasive. Further,

[0040] Therefore, in a particular embodiment of the present invention, the non-hematopoietic tissue sample is not minced. In a further embodiment, the non-invasive biopsy is a punch biopsy. In yet a further embodiment, the non-invasive biopsy is obtained by punch biopsy. The embodiments presented herein where the non-hematopoietic tissue sample is a non-invasive biopsy provide the surprising advantage of obtaining a large number of isolated or separated cells from a non-hematopoietic tissue sample that is not minced and / or is non-invasive. Further, in such embodiments where the biopsy is non-invasive, it will be understood that the separation and distinction of such layers are maintained. Thus, references herein to "non-invasive" further include biopsies of all layers of the non-hematopoietic tissue sample. The embodiments presented herein where the non-hematopoietic tissue sample is a non-invasive biopsy provide the surprising advantage of obtaining a large number of isolated or separated cells from a non-hematopoietic tissue sample that is not minced and / or is non-invasive. Further, in such embodiments where the biopsy is non-invasive, it will be understood that the separation and distinction of such layers are maintained. Thus, references herein to "non-invasive" further include biopsies of all layers of the non-hematopoietic tissue sample. The embodiments presented herein where the non-hematopoietic tissue sample is a non-invasive biopsy provide the surprising advantage of obtaining a large number of isolated or separated cells from a non-hematopoietic tissue sample that is not minced and / or is non-invasive. Further, As shown herein, cells obtained from non-crushed and / or intact non-hematopoietic tissue samples can retain phenotypes useful for subsequent expansion and / or manipulation methods known in the art.

[0041] In a further embodiment, the intact biopsy is skin (e.g., human skin), or the intact biopsy is gastrointestinal (e.g., human gastrointestinal). In one embodiment, the non-hematopoietic tissue sample has a minimum cross-sectional area of at least 2 mm. It will be understood that the "minimum cross-sectional area" refers to the minimum or shortest length measured through the centroid of the tissue sample. It will be further understood that the "maximum cross-sectional area" refers to the maximum or longest length measured through the centroid of the tissue sample. The term "centroid" as used herein is the average or mean position of all points of the tissue sample. According to a further embodiment, the non-hematopoietic tissue sample has a minimum cross-sectional area of at least 2 mm, at least 3 mm, at least 4 mm, at least 5 mm, at least 6 mm, at least 7 mm, or at least 8 mm. It will be understood that in a further embodiment, the non-hematopoietic tissue sample has a minimum cross-sectional area of 8 mm or less, 7 mm or less, 6 mm or less, 5 mm or less, 4 mm or less, or 3 mm or less. In one embodiment, the non-hematopoietic tissue sample has a minimum cross-sectional area of 2 mm to 8 mm (including both ends), for example, 2 mm to 4 mm. In a particular embodiment, the non-hematopoietic tissue sample has a minimum cross-sectional area of about 3 mm. In a particular embodiment, the non-hematopoietic tissue sample has a cross-sectional area of about 3 mm. According to a further embodiment, the non-hematopoietic tissue sample has a minimum cross-sectional area of at least 2 mm, at least 3 mm, at least 4 mm, at least 5 mm, at least 6 mm It will be understood that the term "centroid" as used herein is the average or mean position of all points of the tissue sample. According to a further embodiment, the non-hematopoietic tissue sample has a minimum cross-sectional area of at least 2 mm, at least 3 mm, at least 4 mm, at least 5 mm, at least 6 mm, at least 2 mm, at least 3 mm, at least 4 mm, at least 5 mm, at least 6 mm, at least 7 mm, or at least 8 mm. It will be understood that in a further embodiment, the non-hematopoietic tissue sample has a minimum cross-sectional area of 8 mm or less, 7 mm or less, 6 mm or less, 5 mm or less, 4 mm or less, 7 mm, or at least 8 mm. It will be understood that in a further embodiment, the non-hematopoietic tissue sample has a minimum cross-sectional area of 8 mm or less, 7 mm or less, 6 mm or less, 5 mm or less, 4 mm or less, or 3 mm or less. In one embodiment, the non-hematopoietic tissue sample has a minimum cross-sectional area of 2 mm to 8 mm (including both ends), for example, 2 mm to 4 mm. In a particular embodiment, the non-hematopoietic tissue sample has a minimum cross-sectional area of about 3 mm. In a particular embodiment, the non-hematopoietic tissue sample has a cross-sectional area of about 3 mm. According to a further embodiment, the non-hematopoietic tissue sample has a minimum cross-sectional area of at least 2 mm, at least 3 mm, at least 4 mm, at least 5 mm, at least 6 mm or 3 mm or less. In one embodiment, the non-hematopoietic tissue sample has a minimum cross-sectional area of 2 mm to 8 mm (including both ends), for example, 2 mm to 4 mm. In a particular embodiment, the non-hematopoietic tissue sample has a minimum cross-sectional area of about 3 mm. In a particular embodiment, the non-hematopoietic tissue sample has a cross-sectional area of about 3 mm. According to a further embodiment, the non-hematopoietic tissue sample has a minimum cross-sectional area of at least 2 mm, at least 3 mm, at least 4 mm, at least 5 mm, at least 6 mm (including both ends), for example, 2 mm to 4 mm. In a particular embodiment, the non-hematopoietic tissue sample has a minimum cross-sectional area of about 3 mm. In a particular embodiment, the non-hematopoietic tissue sample has a cross-sectional area of about 3 mm. According to a further embodiment, the non-hematopoietic tissue sample has a minimum cross-sectional area of at least 2 mm, at least 3 mm, at least 4 mm, at least 5 mm, at least 6 mm and / or intact non-hematopoietic tissue samples can retain phenotypes useful for subsequent expansion and / or manipulation methods known in the art. 2 mm, at least 3 mm, at least 4 mm, at least 5 mm, at least 6 mm It will be understood to have a maximum cross-section of at least 7 mm or at least 8 mm. In a further embodiment, the non-hematopoietic tissue sample has a maximum cross-section of 8 mm or less, 7 mm or less, 6 mm or less, 5 mm or less, 4 mm or less, 3 mm or less, or 2 mm or less. In one embodiment, the non-hematopoietic tissue sample has a maximum cross-section of 1 mm to 8 mm (including both ends), for example, 2 mm to 4 mm. In a specific embodiment, the non-hematopoietic tissue sample has a maximum cross-section of about 3 mm.

[0042] According to a further embodiment, the non-hematopoietic tissue sample has a minimum cross-sectional area of at least 2 mm 2 and it is understood that the "minimum cross-sectional area" refers to the area of the smallest cross-section measured around the centroid of the tissue sample. It will be further understood that the "maximum cross-sectional area" refers to the area of the largest cross-section measured around the centroid of the tissue sample. The term "centroid" used herein is the average or mean position of all points of the tissue sample. In a further embodiment, the non-hematopoietic tissue sample has a minimum cross-sectional area of at least 2 mm and at least 3 mm 2 and at least 2 also 4 mm and at least 5 mm 2 and at least 6 mm 2 and at least 7 mm 2 and at least 8 mm 2 and at least 9 mm 2 and at least also 10 mm 2 or at least 10 mm 2 and. In a further embodiment, the non-hematopoietic tissue sample is 50 mm 2 or less, 40 mm 2 or less, 30 mm 2 or less, 25 mm 2 or less, 20 mm 2 or less, 15 mm2 The following, 10 mm or less, or 8 mm 2 or less, has a minimum cross-sectional area. In one embodiment, the non-hematopoietic 2 tissue sample is 2 mm to 50 mm 2 , for example, 3 mm 2 to 12 mm 2 and has a minimum cross-sectional area. In a particular embodiment 2 , the non-hematopoietic tissue sample has a minimum cross-sectional area of about 7 mm . In a further embodiment 2 , the non-hematopoietic tissue sample is at least 2 mm , at least 3 mm 2 , at least 4 mm 2 , at least 2 5 mm , at least 6 mm 2 , at least 7 mm 2 , at least 8 mm 2 , at least 9 mm 2 , at least 2 or at least 10 mm and has a maximum cross-sectional area. In a further embodiment, the non-hematopoietic tissue 2 sample has a maximum cross-sectional area of 50 mm or less, 40 mm 2 or less, 30 mm 2 or less, 25 mm 2 or less, 20 mm 2 or less, 15 mm 2 or less, 10 mm 2 or less, 2 or 8 mm 2 or less and has a maximum cross-sectional area. In one embodiment, the non-hematopoietic tissue sample is 1 mm 2 to 50 mm 2 , for example, 3 mm 2 to 12 mm 2 and has a maximum cross-sectional area. In a particular embodiment , the non-hematopoietic tissue sample has a maximum cross-sectional area of about 7 mm 2 .

[0043] According to a further embodiment, the non-hematopoietic tissue sample has a volume of at least 2 mm 3 In a further embodiment, the non-hematopoietic tissue sample has a volume of at least 4 mm In a further embodiment, the non-hematopoietic tissue sample has a volume of at least 4 mm 3 at least 5 mm 3 at least also 8 mm 3 at least 10 mm 3 at least 15 mm 3 at least 20 mm 3 at least 25 mm 3 at least also 30 mm 3 at least 35 mm 3 at least 40 mm 3 at least 50 mm 3 or at least 60 mm 3 In a further embodiment, the non-hematopoietic tissue sample has a volume of 250 mm or less, 200 mm or less, for example, 180 mm or less, 160 mm or less, 140 mm or less, 120 mm or less, 100 mm or less, 80 mm or less, 60 mm or less, 50 mm or less, or 40 mm or less 3 In one embodiment, the non-hematopoietic tissue sample has a volume of 5 mm to 250 mm, for example, 15 mm to 65 mm 3 In a specific embodiment, the non-hematopoietic tissue sample has a volume of about 35 mm 3 In one embodiment, the non-hematopoietic tissue sample is a punch biopsy. The punch biopsy can be of any shape 3 In one embodiment, the non-hematopoietic tissue sample is a punch biopsy. The punch biopsy can be of any shape 3 In one embodiment, the non-hematopoietic tissue sample is a punch biopsy. The punch biopsy can be of any shape 3 In one embodiment, the non-hematopoietic tissue sample is a punch biopsy. The punch biopsy can be of any shape 3 In one embodiment, the non-hematopoietic tissue sample is a punch biopsy. The punch biopsy can be of any shape 3 In one embodiment, the non-hematopoietic tissue sample is a punch biopsy. The punch biopsy can be of any shape 3 In one embodiment, the non-hematopoietic tissue sample is a punch biopsy. The punch biopsy can be of any shape 3 In one embodiment, the non-hematopoietic tissue sample is a punch biopsy. The punch biopsy can be of any shape 3 In one embodiment, the non-hematopoietic tissue sample is a punch biopsy. The punch biopsy can be of any shape In one embodiment, the non-hematopoietic tissue sample has a volume of 5 mm 3 to 250 mm 3 For example, 15 mm 3 to 65 mm 3 In a specific embodiment, the non-hematopoietic tissue sample has a volume of about 35 mm In a specific embodiment, the non-hematopoietic tissue sample has a volume of about 35 mm 3 In a specific embodiment, the non-hematopoietic tissue sample has a volume of about 35 mm

[0044] In one embodiment, the non-hematopoietic tissue sample is a punch biopsy. The punch biopsy can be of any shape It may be in the shape of, but preferably is, a circular cross-section, and preferably has a diameter of at least 1 mm. In a further embodiment, the non-hematopoietic tissue sample is a punch biopsy having a diameter of at least 2 mm , for example, at least 3 mm in diameter, at least 4 mm in diameter, at least 5 mm in diameter, at least 6 mm in diameter, at least 7 mm in diameter, or at least 8 mm in diameter. In a further embodiment , the non-hematopoietic tissue sample includes a punch biopsy having a diameter of 8 mm or less, for example, 7 mm or less in diameter, 6 mm or less in diameter, 5 mm or less in diameter, or 3 mm or less in diameter. In one embodiment, the non-hematopoietic tissue sample includes a punch biopsy having a diameter of 1 mm to 8 mm, for example, 2 mm to 4 mm in diameter. In a particular embodiment , the non-hematopoietic tissue sample includes a punch biopsy having a diameter of 3 mm.

[0045] In one embodiment, the non-hematopoietic tissue sample includes a biopsy by size, area, volume, and / or diameter as defined above (e.g., a punch biopsy, particularly a punch biopsy with a circular cross-section), and the maximum depth is determined by the site from which the biopsy is obtained (although the depth may be reduced). In one embodiment, the biopsy is a skin biopsy and includes the epithelial and dermal layers. In a further embodiment, the biopsy substantially does not include subcutaneous fat. Thus, in one embodiment , the biopsy includes the epithelial and dermal layers and substantially does not include a layer of subcutaneous fat. In a further embodiment , the biopsy does not include subcutaneous fat. Alternatively, the subcutaneous fat is not removed and is therefore present (or at least partially present) during the biopsy. Thus, in a further embodiment , the biopsy consists of the epithelial and dermal layers. In one embodiment, the biopsy includes the entire layer of the non-hematopoietic tissue sample .

[0046] Accordingly, according to one aspect of the present invention, there is provided a method for isolating lymphocytes from a non-hematopoietic tissue sample that (i) culturing a non-hematopoietic tissue sample, which is an intact biopsy having a minimum cross-sectional area of at least 2 mm 2 in the presence of IL-2 and IL-15; and (ii) recovering the lymphocytes from the non-hematopoietic tissue : A method is provided that includes the steps of

[0047] According to a further aspect of the present invention, there is provided a method for isolating γδ T cells from a non-hematopoietic tissue sample, which (i) culturing a non-hematopoietic tissue sample, which is an intact biopsy having a minimum cross-sectional area of at least 2 mm 2 in the presence of IL-2 and IL-15; and (ii) recovering the γδ T cells from the non-hematopoietic tissue : A method is provided that includes the steps of

[0048] According to one aspect of the present invention, there is provided a method for isolating lymphocytes from a non-hematopoietic tissue sample, (i) culturing a non-hematopoietic tissue sample, which is an intact biopsy having a volume of at least 2 mm 3 in the presence of IL-2 and IL-15; and (ii) recovering the lymphocytes from the non-hematopoietic tissue : A method is provided that includes the steps of

[0049] According to yet a further aspect of the present invention, there is provided a method for isolating γδ T cells from a non-hematopoietic tissue sample, which (i) culturing a non-hematopoietic tissue sample, which is an intact biopsy having a volume of at least 2 mm 3 in the presence of IL-2 and IL-15; and (ii) recovering the γδ T cells from the non-hematopoietic tissue sample : A method is provided that includes the steps of ​

[0050] The method of the present invention involves culturing a non-hematopoietic tissue sample as defined herein. References herein to "culturing" include adding to a medium containing growth factors and / or essential nutrients required and / or preferred by cells and / or non-hematopoietic tissue samples, wherein the cells are isolated, separated, removed, purified, or concentrated from the non-hematopoietic tissue sample. Such culturing conditions can be adapted with respect to cells or cell populations that will be isolated from the non-hematopoietic tissue sample according to the present invention, or it will be understood that they can be adapted with respect to cells or cell populations that will be isolated and expanded from the non-hematopoietic tissue sample. In certain embodiments, culturing of the non-hematopoietic tissue sample is for a period sufficient for isolation of γδ T cells from the non-hematopoietic tissue sample. In alternative embodiments, culturing of the non-hematopoietic tissue sample is for a period sufficient for isolation of lymphocytes other than γδ T cells (e.g., αβ T cells and / or NK (natural killer) cells) from the non-hematopoietic tissue sample. In certain embodiments, the culturing period by the method defined herein is at least 14 days. In certain embodiments, the culturing period by the method defined herein is less than 45 days, e.g., less than 30 days, e.g., less than 25 days. In further embodiments, the culturing period by the method defined herein is from 14 days to 35 days, e.g., from 14 days to 21 days. In still further embodiments, the culturing period by the method defined herein is about 21 days. In certain embodiments, culturing of the non-hematopoietic tissue sample is for a period sufficient for isolation of γδ T cells from the non-hematopoietic tissue sample. In alternative embodiments, culturing of the non-hematopoietic tissue sample is for a period sufficient for isolation of lymphocytes other than γδ T cells (e.g., αβ T cells and / or NK (natural killer) cells) from the non-hematopoietic tissue sample. In certain embodiments, the culturing period by the method defined herein is at least 14 days. In certain embodiments, the culturing period by the method defined herein is less than 45 days, e.g., less than 30 days, e.g., less than 25 days. In further embodiments, the culturing period by the method defined herein is from 14 days to 35 days, e.g., from 14 days to 21 days. In still further embodiments, the culturing period by the method defined herein is about 21 days.

[0051] In certain embodiments, culturing of the non-hematopoietic tissue sample is for a period sufficient for isolation of γδ T cells from the non-hematopoietic tissue sample. In alternative embodiments, culturing of the non-hematopoietic tissue sample is for a period sufficient for isolation of lymphocytes other than γδ T cells (e.g., αβ T cells and / or NK (natural killer) cells) from the non-hematopoietic tissue sample. In certain embodiments, the culturing period by the method defined herein is at least 14 days. In certain embodiments, the culturing period by the method defined herein is less than 45 days, e.g., less than 30 days, e.g., less than 25 days. In further embodiments, the culturing period by the method defined herein is from 14 days to 35 days, e.g., from 14 days to 21 days. In still further embodiments, the culturing period by the method defined herein is about 21 days. In certain embodiments, culturing of the non-hematopoietic tissue sample is for a period sufficient for isolation of γδ T cells from the non-hematopoietic tissue sample. In alternative embodiments, culturing of the non-hematopoietic tissue sample is for a period sufficient for isolation of lymphocytes other than γδ T cells (e.g., αβ T cells and / or NK (natural killer) cells) from the non-hematopoietic tissue sample. In certain embodiments, the culturing period by the method defined herein is at least 14 days. In certain embodiments, the culturing period by the method defined herein is less than 45 days, e.g., less than 30 days, e.g., less than 25 days. In further embodiments, the culturing period by the method defined herein is from 14 days to 35 days, e.g., from 14 days to 21 days. In still further embodiments, the culturing period by the method defined herein is about 21 days. In certain embodiments, the culturing period by the method defined herein is at least 14 days. In certain embodiments, the culturing period by the method defined herein is less than 45 days, e.g., less than 30 days, e.g., less than 25 days. In further embodiments, the culturing period by the method defined herein is from 14 days to 35 days, e.g., from 14 days to 21 days. In still further embodiments, the culturing period by the method defined herein is about 21 days. In certain embodiments, the culturing period by the method defined herein is at least 14 days. In certain embodiments, the culturing period by the method defined herein is less than 45 days, e.g., less than 30 days, e.g., less than 25 days. In further embodiments, the culturing period by the method defined herein is from 14 days to 35 days, e.g., from 14 days to 21 days. In still further embodiments, the culturing period by the method defined herein is about 21 days. In certain embodiments, the culturing period by the method defined herein is about 21 days.

[0052] In certain embodiments of the present invention, the lymphocytes and / or γδ T cells isolated according to the methods defined herein are recovered from the culture of the non-hematopoietic tissue sample after culturing the non-hematopoietic tissue sample. The recovery of the lymphocytes and / or γδ T cells as defined herein may include physical recovery of the lymphocytes and / or γδ T cells from the culture, isolation of the lymphocytes and / or γδ T cells from other lymphocytes (e.g., αβ T cells, γδ T cells, and / or NK cells), or isolation and / or separation of the lymphocytes and / or γδ T cells from stromal cells (e.g., fibroblasts). In one embodiment, the lymphocytes and / or γδ T cells are recovered by mechanical means (e.g., pipetting). In a further embodiment, the lymphocytes and / or γδ T cells are recovered by magnetic separation and / or labeling. In yet a further embodiment, the lymphocytes and / or γδ T cells are recovered by flow cytometry techniques, e.g., by FACS. Thus, in certain embodiments, γδ T cells are recovered by specific labeling of γδ T cells. In a further embodiment, lymphocytes are recovered by specific labeling of lymphocytes to distinguish them from other cells in the culture. Such recovery of lymphocytes and / or γδ T cells may include physical removal from the culture of the non-hematopoietic tissue sample, transfer to separate culture vessels, or transfer to separate or different culture conditions, as will be understood. It will be understood that such recovery of lymphocytes and / or γδ T cells is performed after a period sufficient to obtain a population of lymphocytes and / or γδ T cells isolated from the non-hematopoietic tissue sample.

[0053] ​​​​​​​​​​​​​​​​would be. In certain embodiments, lymphocytes and / or γδ T cells are obtained from a non-hematopoietic tissue sample at least 1 week, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days after culturing. Preferably, lymphocytes and / or γδ T cells are obtained within 40 days, such as within 38 days, within 36 days, within 34 days, within 32 days, within 30 days, within 28 days within, within 26 days, or within 24 days. In one embodiment, lymphocytes and / or γδ T cells are obtained at least 14 days after culturing of the non-hematopoietic tissue sample. In a further embodiment lymphocytes and / or γδ T cells are obtained 14 to 21 days after culturing of the non-hematopoietic tissue sample.

[0054] In certain embodiments of the invention, the non-hematopoietic tissue sample is cultured in a medium substantially free of serum (e.g., a medium containing serum-free medium or serum replacement (SR)). Thus, in one embodiment, the non-hematopoietic tissue sample is cultured in serum-free medium. Such serum-free media may also include serum replacement media based on chemically defined components to avoid the use of human or animal-derived sera. In alternative embodiments, the non-hematopoietic tissue sample is cultured in a medium containing serum (e.g., human AB serum or fetal bovine serum (FBS)). In one embodiment, the non-hematopoietic tissue sample is cultured in a medium containing serum replacement. In one embodiment, the non-hematopoietic tissue sample is cultured in a medium free of animal-derived products.

[0055] Embodiments of the invention in which the non-hematopoietic tissue sample is cultured in serum-free medium are understood to have the advantage of avoiding problems related to serum filtration, precipitation, contamination, and supply. Furthermore, animal-derived products are not preferred for use in the manufacture of clinical-grade human therapeutics. As seen herein, the inventors have surprisingly found that the use of serum-free media for the isolation of cells, particularly Vδ1 γδ cells, substantially increases the number of cells obtained from non-hematopoietic tissue samples as compared to the use of media containing AB serum. In particular, the isolation of γδ T cells from non-hematopoietic tissue samples cultured in serum-free media

[0056] In one embodiment, the methods defined herein are performed in an isolation container. The reference to an "isolation container" refers to a container containing a non-hematopoietic tissue sample for the separation of lymphocytes and / or γδ T cells, optionally further comprising a synthetic scaffold. It should be noted that the isolation container can only be used for the isolation method and cannot be used for further amplification steps.

[0057] In one embodiment, the methods defined herein are performed in a container comprising a gas- permeable material (e.g., an isolation container). Such materials are permeable to gases such as oxygen, carbon dioxide, and / or nitrogen, allowing gas exchange between the contents of the container and the surrounding environment. The reference herein to a "container" is understood to include culture dishes, culture plates, single-well dishes, multi-well dishes, multi-well plates, flasks, multi-layer flasks, bottles (e.g., roller bottles), bioreactors, bags, tubes, and the like. Such containers are known in the art for use in methods involving the expansion of non-adherent cells and other lymphocytes. However, as shown herein , Containers containing gas-permeable materials surprisingly find utility even in the isolation of γδ T cells, which are normally considered adhesive. The use of such containers in culture has been found to greatly increase the yield of γδ T cells isolated from non-hematopoietic tissue samples. Such containers have also been found to preferentially support γδ T cells and other lymphocytes over fibroblasts and other stromal cells (e.g., epithelial cells) including adherent cell types. Thus, in one embodiment, a container containing a gas-permeable material as defined herein preferentially supports γδ T cells as well as other lymphocytes (e.g., αβ T cells and / or NK cells). In a further embodiment, fibroblasts and / or other stromal cells (e.g., epithelial cells) are not present in the cultures conducted in containers containing gas-permeable materials. Such containers containing gas-permeable materials may further comprise a non-porous gas-permeable material. Thus, in one embodiment, the gas-permeable material is non-porous. In some embodiments, the gas-permeable material is a membrane film such as silicone, fluorinated ethylene propylene, polyolefin, or ethylene vinyl acetate copolymer. Further, such containers may comprise only a portion of the gas-permeable material, the gas-permeable membrane film, or the non-porous gas-permeable material. Thus, according to a further embodiment, the container comprises a lid, a bottom, and at least one side wall, wherein at least a portion of the bottom of the container comprises a gas-permeable material in a substantially horizontal plane when the lid is above the bottom. In one embodiment, the container comprises a lid, a bottom, and at least one side wall, wherein at least a portion of the bottom has been found to greatly increase the yield of γδ T cells isolated from non-hematopoietic tissue samples. Such containers have also been found to preferentially support γδ T cells and other lymphocytes over fibroblasts and other stromal cells (e.g., epithelial cells) including adherent cell types. Thus, in one embodiment, a container containing a gas-permeable material as defined herein preferentially supports γδ T cells as well as other lymphocytes ( e.g., αβ T cells and / or NK cells). In a further embodiment, fibroblasts and / or other stromal cells (e.g., epithelial cells) are not present in the cultures conducted in containers containing gas-permeable materials. e.g., αβ T cells and / or NK cells) are preferentially supported. In a further embodiment, fibroblasts and / or other stromal cells (e.g., epithelial cells) are not present in the cultures conducted in containers containing gas-permeable materials. Such containers containing gas-permeable materials may further comprise a non-porous gas-permeable material. Thus, in one embodiment, the gas-permeable material is non-porous. In some embodiments, the gas-permeable material is a membrane film such as silicone, fluorinated ethylene propylene, polyolefin, or ethylene vinyl acetate copolymer. Further, such containers may comprise only a portion of the gas-permeable material, the gas-permeable membrane film, or the non-porous gas-permeable material. Thus, according to a further embodiment, the container comprises a lid, a bottom, and at least one side wall, wherein at least a portion of the bottom of the container comprises a gas-permeable material in a substantially horizontal plane when the lid is above the bottom. In one embodiment, the container comprises a lid, a bottom, and at least one side wall, wherein at least a portion of the bottom has been found to greatly increase the yield of γδ T cells isolated from non-hematopoietic tissue samples. Such containers

[0058] containing gas-permeable materials may further comprise a non-porous gas-permeable material. Thus, in one embodiment, the gas-permeable material is non-porous. In some embodiments, the gas-permeable material is a membrane film such as silicone, fluorinated ethylene propylene, polyolefin, or ethylene vinyl acetate copolymer. Further, such containers may comprise only a portion of the gas-permeable material, the gas-permeable membrane film, or the non-porous gas-permeable material. Thus, according to a further embodiment, the container comprises a lid, a bottom, and at least one side wall, wherein at least a portion of the bottom of the container comprises a gas-permeable material in a substantially horizontal plane when the lid is above the bottom. In one embodiment, the container comprises a lid, a bottom, and at least one side wall, wherein at least a portion of the bottom has been found to greatly increase the yield of γδ T cells isolated from non-hematopoietic tissue samples. Such containers containing gas-permeable materials may further comprise a non-porous gas-permeable material. Thus, in one embodiment, the gas-permeable material is non-porous. In some embodiments, the gas-permeable material is a membrane film such as silicone, fluorinated ethylene propylene, polyolefin, or ethylene vinyl acetate copolymer. Further, such containers may comprise only a portion of the gas-permeable material, the gas-permeable membrane film, or the non-porous gas-permeable material. Thus, according to a further embodiment, the container comprises a lid, a bottom, and at least one side wall, wherein at least a portion of the bottom of the container comprises a gas-permeable material in a substantially horizontal plane when the lid is above the bottom. In one embodiment, the container comprises a lid, a bottom, and at least one side wall, wherein at least a portion of the bottom has been found to greatly increase the yield of γδ T cells isolated from non-hematopoietic tissue samples. Such containers containing gas-permeable materials may further comprise a non-porous gas-permeable material. Thus, in one embodiment, the gas-permeable material is non-porous. In some embodiments, the gas-permeable material is a membrane film such as silicone, fluorinated ethylene propylene, polyolefin, or ethylene vinyl acetate copolymer. Further, such containers may comprise only a portion of the gas-permeable material, the gas-permeable membrane film, or the non-porous gas-permeable material. Thus, according to a further embodiment, the container comprises a lid, a bottom, and at least one side wall, wherein at least a portion of the bottom of the container comprises a gas-permeable material in a substantially horizontal plane when the lid is above the bottom. In one embodiment, the container comprises a lid, a bottom, and at least one side wall, wherein at least a portion of the bottom has been found to greatly increase the yield of γδ T cells isolated from non-hematopoietic tissue samples. Such containers containing gas-permeable materials may further comprise a non-porous gas-permeable material. Thus, in one embodiment, the gas-permeable material is non-porous. In some embodiments, the gas-permeable material is a membrane film such as silicone, fluorinated ethylene propylene, polyolefin, or ethylene vinyl acetate copolymer. Further, such containers may comprise only a portion of the gas-permeable material, the gas-permeable membrane film, or the non-porous gas-permeable material. Thus, according to a further embodiment, the container comprises a lid, a bottom, and at least one side wall, wherein at least a portion of the bottom of the container comprises a gas-permeable material in a substantially horizontal plane when the lid is above the bottom. In one embodiment, the container comprises a lid, a bottom, and at least one side wall, wherein at least a portion of the bottom has been found to greatly increase the yield of γδ T cells isolated from non-hematopoietic tissue samples. Such containers containing gas-permeable materials may further comprise a non-porous gas-permeable material. Thus, in one embodiment, the gas-permeable material is non-porous. In some embodiments, the gas-permeable material is a membrane film such as silicone, fluorinated ethylene propylene, polyolefin, or ethylene vinyl acetate copolymer. Further, such containers may comprise only a portion of the gas-permeable material, the gas-permeable membrane film, or the non-porous gas-permeable material. Thus, according to a further embodiment, the container comprises a lid, a bottom, and at least one side wall, wherein at least a portion of the bottom of the container comprises a gas-permeable material in a substantially horizontal plane when the lid is above the bottom. In one embodiment, the container comprises a lid, a bottom, and at least one side wall, wherein at least a portion of the bottom When the lid is above the bottom, it includes a gas-permeable material on a horizontal surface. Further In an embodiment, the container includes a lid, a bottom, and at least one side wall, where the at least one side wall can be on a vertical surface when the lid is above the bottom, or can be on a horizontal surface when the lid is not above the bottom, and includes a gas-permeable material. In such embodiments, it will be understood that only a part of the bottom or the side wall may include the gas-permeable material. Alternatively, the entire bottom or the entire side wall may include the gas-permeable material. In a further embodiment, the lid of the container including the gas-permeable material may be sealed, for example, by the use of an O-ring. It will be understood that such embodiments prevent leakage of the contents of the container or reduce its evaporation. Thus, in one embodiment, the container includes a liquid-tight container including a gas-permeable material to allow gas exchange. In an alternative embodiment, the lid of the container including the gas-permeable material is on a horizontal surface and above the bottom and is not sealed. Thus, in one embodiment, the lid is configured to allow gas exchange from the lid of the container. In a further embodiment, the bottom of the gas-permeable container is configured to allow gas exchange from the bottom of the container. In yet a further embodiment, the container including the gas-permeable material is a liquid-tight container and may further include an inlet and an outlet or a discharge pipe. Thus, in one embodiment, the container including the gas-permeable material includes a lid, a bottom, and optionally at least one side wall, where at least a part of the lid and the bottom includes the gas-permeable material, and when present, at least a part of It is described in WO2005035728 and US9255243 incorporated herein. These containers are also commercially available and are provided, for example, by Wilson Wolf Manufacturing's G-R EX (registered trademark) cell culture devices, such as G-REX6 well plates, G-REX24 well plates, and G-REX10 containers.

[0059] Thus, according to one aspect of the present invention, there is provided a method for isolating lymphocytes from a non-hematopoietic tissue sample, which (i) placing the non-hematopoietic tissue sample in a container containing a gas-permeable material; (ii) culturing the non-hematopoietic tissue sample in the presence of IL-2 and IL-15; and (iii) recovering a population of lymphocytes cultured from the non-hematopoietic tissue sample : is provided.

[0060] According to a further aspect of the present invention, there is provided a method for isolating γδ T cells from a non-hematopoietic tissue sample, which (i) placing the non-hematopoietic tissue sample in a container containing a gas-permeable material; (ii) culturing the non-hematopoietic tissue sample in the presence of IL-2 and IL-15; and (iii) recovering a population of γδ T cells cultured from the non-hematopoietic tissue sample : is provided.

[0061] In one embodiment, the non-hematopoietic tissue sample is placed on a synthetic scaffold. As used in this specification, "synthetic scaffold", "scaffold", and "grid" are used interchangeably and refer to a non-natural three-dimensional structure suitable for supporting cell growth. The non-hematopoietic tissue sample is placed on the synthetic scaffold or the synthetic scaffold is This allows lymphocytes to attach to the scaffold and promote lymphocyte egress from the explant onto the scaffold. Synthetic scaffolds can be made of polymers (e.g., natural or synthetic polymers, e.g., poly(ethylene glycol)). Vinylpyrrolidone, polymethyl methacrylate, methylcellulose, polystyrene, polypropylene, polyurethane), ceramics (e.g., tricalcium phosphate, calcium aluminate calcium, calcium hydroxyapatite), or metals (e.g., tantalum, titanium, Platinum and metals of the same element group as platinum, niobium, hafnium, tungsten, and The material may be constructed from natural and / or synthetic materials, such as alloys of the above. In an embodiment, the synthetic scaffold is tantalum coated. (e.g., collagen (e.g., collagen I or collagen II), fibronectin, laminin Nin, integrin, angiogenic factors, anti-inflammatory factors, glycosaminoglycans, vitrogens, Antibodies and fragments thereof, cytokines (e.g., IL-2, IL-15, and combinations thereof) are provided by the methods of the art. The scaffold surface may be coated or coated with a material according to methods known in the art. Encapsulated within the encapsulated material or added to the culture medium to enhance cell attachment, migration, survival, or proliferation This and other methods can be used to identify several other non-hematopoietic tissue types, e.g. For example, lymphocytes can be isolated from the skin, gastrointestinal tract, prostate, and breast.

[0062] In one embodiment, the non-hematopoietic tissue sample is subjected to a step of isolating lymphocytes from the non-hematopoietic tissue sample. In a further embodiment, the composite scaffold is placed inside a container used for the In this study, the synthetic scaffold was designed to transport lymphocytes and / or gamma-erythrocytes from a non-hematopoietic tissue sample to the bottom of the vessel. configured to promote δ T cell egress. Such embodiments enable the isolation and / or separation of lymphocytes (e.g., γδ T cells, αβ T cells, and / or NK cells) from non-hematopoietic tissue samples and / or stromal cells (e.g., fibroblasts and / or epithelial cells). Such embodiments have the advantage of enabling the isolation and / or separation of lymphocytes (e.g., γδ T cells, αβ T cells, and / or NK cells) from non-hematopoietic tissue samples and / or stromal cells (e.g., fibroblasts and / or epithelial cells). Furthermore, such embodiments enable the recovery of lymphocytes (e.g., γδ T cells, αβ T cells, and / or NK cells) from non-hematopoietic tissue samples to the bottom of a culture vessel. In certain embodiments, the synthetic scaffold is configured to promote the egress of γδ T cells from non-hematopoietic tissue samples. In further embodiments, the synthetic scaffold is configured to promote the egress of lymphocytes, e.g., αβ T cells and / or NK cells, from non-hematopoietic tissue samples. Furthermore, such embodiments enable the recovery of lymphocytes (e.g., γδ T cells, αβ T cells, and / or NK cells) from non-hematopoietic tissue samples to the bottom of a culture vessel. In certain embodiments, the synthetic scaffold is configured to promote the egress of γδ T cells from non-hematopoietic tissue samples. In further embodiments, the synthetic scaffold is configured to promote the egress of lymphocytes, e.g., αβ T cells and / or NK cells, from non-hematopoietic tissue samples. Furthermore, such embodiments enable the recovery of lymphocytes (e.g., γδ T cells, αβ T cells, and / or NK cells) from non-hematopoietic tissue samples to the bottom of a culture vessel. In certain embodiments, the synthetic scaffold is configured to promote the egress of γδ T cells from non-hematopoietic tissue samples. In further embodiments, the synthetic scaffold is configured to promote the egress of lymphocytes, e.g., αβ T cells and / or NK cells, from non-hematopoietic tissue samples. In certain embodiments, the synthetic scaffold is configured to promote the egress of γδ T cells from non-hematopoietic tissue samples. In further embodiments, the synthetic scaffold is configured to promote the egress of lymphocytes, e.g., αβ T cells and / or NK cells, from non-hematopoietic tissue samples. In further embodiments, the synthetic scaffold is configured to promote the egress of lymphocytes, e.g., αβ T cells and / or NK cells, from non-hematopoietic tissue samples. In further embodiments, the synthetic scaffold is configured to promote the egress of lymphocytes, e.g., αβ T cells and / or NK cells, from non-hematopoietic tissue samples. In further embodiments, the synthetic scaffold is configured to promote the egress of lymphocytes, e.g., αβ T cells and / or NK cells, from non-hematopoietic tissue samples.

[0063] Thus, in one aspect of the methods defined herein, the synthetic scaffold is configured to promote lymphocyte egress from non-hematopoietic tissue samples to the bottom of a culture vessel. In a further aspect of the methods defined herein, the synthetic scaffold is configured to promote γδ T cell egress from non-hematopoietic tissue samples to the bottom of a culture vessel. Thus, in one aspect of the methods defined herein, the synthetic scaffold is configured to promote lymphocyte egress from non-hematopoietic tissue samples to the bottom of a culture vessel. In a further aspect of the methods defined herein, the synthetic scaffold is configured to promote γδ T cell egress from non-hematopoietic tissue samples to the bottom of a culture vessel. In a further aspect of the methods defined herein, the synthetic scaffold is configured to promote γδ T cell egress from non-hematopoietic tissue samples to the bottom of a culture vessel. In a further aspect of the methods defined herein, the synthetic scaffold is configured to promote γδ T cell egress from non-hematopoietic tissue samples to the bottom of a culture vessel.

[0064] The methods of the present invention provide a much greater total cell yield than previously described. In one embodiment, the total number of isolated cells is at least 10 6 cells / cm 2 of tissue sample, at least 2 ×10 6 cells / cm 2 of tissue sample, at least 5 × 10 6 cells / cm 2 of tissue sample, at least 10 × 10 6 cells / cm 2 of tissue sample, at least 20 ×106 cells / cm 2 , at least 30×10 6 cells / cm 2 , at least 40×10 6 cells / cm 2 , at least 5 0×10 6 cells / cm 2 , at least 60×10 6 cells / cm 2 , at least 70×10 6 cells / cm 2 , at least 80×10 6 cells / cm 2 , at least 90×10 6 cells / cm 2 , at least 100×10 6 cells / cm 2 , at least also 150×10 6 cells / cm 2 , at least 200×10 6 cells / cm 2 is. In a specific embodiment, the total number of isolated cells is at least 50×10 6 cells / cm 2 is. In another embodiment, the total number of isolated cells is at least 100×10 6 cells / cm 2 is.

[0065] γδ T cells that are dominant in the blood are mainly Vδ2 T cells, while γδ T cells that are dominant in non-hematopoietic tissues are mainly Vδ1 T cells. As a result, Vδ1 T cells contain approximately 70 - 80% of the resident γδ T cell population in non-hematopoietic tissues. However, some Vδ2 T cells are also found in non-hematopoietic tissues, such as the gastrointestinal tract, where the Vδ2 T cells can contain approximately 10 - 20% of the γδ T cells. Some γδ T cells resident in non-hematopoietic tissues do not express either Vδ1 TCR or Vδ2 TCR, and this specification also includes obtained. Some γδ T cells resident in non-hematopoietic tissues do not express either Vδ1 TCR or Vδ2 TCR, and this specification In the specification, they are referred to as double-negative (DN) γδ T cells. These DN γδ T cells are likely to express Vδ3 in a large portion, and Vδ5-expressing T cells are in the minority. Therefore, the γδ T cells that reside routinely in non-hematopoietic tissues and the γδ T cells isolated by the method of the present invention are, preferably, non-Vδ2 T cells, such as Vδ1 T cells, and contain a smaller amount of DN γδ T cells.

[0066] Accordingly, in one preferred embodiment, the γδ T cells isolated by the method defined herein contain a population of Vδ1 T cells. In one embodiment, the γδ T cells isolated by the method defined herein contain a population of DN γδ T cells. In one embodiment the γδ T cells isolated by the method defined herein contain a population of Vδ3 T cells. In one embodiment the γδ T cells isolated by the method defined herein contain a population of Vδ5 T cells.

[0067] γδ T cells can also be defined by the type of γ chain they express. In a further embodiment, the γδ T cells isolated by the method defined herein contain a population of Vγ4 T cells. In most cases, Vγ4 T cells are obtained from gastrointestinal tissue samples.

[0068] The isolation method results in a greater number (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) of cells than the reference population. a population of isolated γδ T cells that is 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 1,0 00-fold, at least 5,000-fold, at least 10,000-fold). Provided.

[0069] In some embodiments, the population of γδ T cells isolated according to the method of the invention has a low percentage of cells expressing TIGIT. For example, the population of isolated γδ T cells is 90 % or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, or may have a frequency of TIGIT+ cells of less than 10%. Alternatively, the population of isolated γδ T cells is about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, or about 10% frequency of TIGIT+ cells. In certain embodiments, the population of isolated γδ T cells has a frequency of TIGIT+ cells of less than 80% Thus, in one embodiment, the population of isolated γδ T cells has a frequency of TIGIT+ cells of about 70%. In a further embodiment, the isolated population of γδ T cells has a frequency of TIGIT+ cells of less than 60%. In yet a further embodiment the isolated population of γδ T cells has a frequency of TIGIT+ cells of about 30%. Thus in one embodiment, the isolated γδ T cells do not substantially express TIGIT.

[0070] In some embodiments, a population of isolated Vδ1 T cells has a low frequency of TIGIT+ cells. For example, a population of isolated Vδ1 T cells can have less than 90%, less than 80%, less than 70%, less than 60% less than, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% frequency of TIGIT+ cells other than the population of Vδ1 T cells. Alternatively, a population of isolated Vδ1 T cells can have about 90%, about 80 %, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, or about 10% frequency of TIGIT+ cells. In one embodiment, a population of isolated Vδ1 T cells has a frequency of TIGIT+ cells of less than 80%. Thus, in one embodiment, a population of isolated Vδ1 T cells has about 70% frequency of TIGIT+ cells. In a further embodiment, a population of isolated Vδ1 T cells has a frequency of TIGIT+ cells of less than 60%. In yet a further embodiment, a population of isolated Vδ1 T cells has about 30% frequency of TIGIT+ cells. Thus, in one embodiment, isolated Vδ1 T cells substantially do not express TIGIT. In some embodiments, a population of γδ T cells isolated according to the methods of the invention expresses CD27.

[0071] For example, a population of isolated γδ T cells can have more than 10%, more than 20%, more than 30%, more than 40 %, more than 50%, more than 60%, more than 70%, more than 80%, or more than 90% frequency of CD27+ cells. Alternatively, a population of isolated γδ T cells can have about 10%, about 20%, about 30%, about 40%, about 50%, about 60 %, about 70%, about 80%, or about 90% frequency of CD27+ cells. In one embodiment In some embodiments, a population of isolated γδ T cells can have more than 10%, more than 20%, more than 30%, more than 40 %, more than 50%, more than 60%, more than 70%, more than 80%, or more than 90% frequency of CD27+ cells. Alternatively, a population of isolated γδ T cells can have about 10%, about 20%, about 30%, about 40%, about 50%, about 60 Thus, the isolated population of γδ T cells has a frequency of CD27+ cells of more than 10%. In an embodiment, the isolated population of γδ T cells has a frequency of about 20% CD27+ cells. In a further embodiment, the isolated population of γδ T cells comprises CD27+ cells with a frequency of greater than 20%. In one embodiment, the isolated population of γδ T cells has a frequency of about 20% CD27 + cells.

[0072] In some embodiments, the isolated population of V51 T cells expresses CD27. In some embodiments, the isolated γδ T cells express CD27. In the isolated Vδ1 T cell population, the proportion of Vδ1 T cells was greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, or having a frequency of CD27+ cells of greater than 60%, greater than 70%, greater than 80%, or greater than 90%. The population of γδ T cells is approximately 10%, approximately 20%, approximately 30%, approximately 40%, approximately 50%, approximately 60%, approximately 70%, approximately 80%, approximately 90%, approximately 100%, approximately 110%, approximately 120%, approximately 130%, approximately 140%, approximately 150%, approximately 160%, approximately 170%, approximately 180%, approximately 190%, approximately 200%, approximately 210%, approximately In one embodiment, the isolated V51 may have a frequency of CD27+ cells of about 90% or about 90%. The population of T cells has a frequency of CD27+ cells of greater than 10%. Thus, in one embodiment, The isolated population of V51 T cells has a frequency of CD27+ cells of about 20%. In one embodiment, the isolated population of V51 T cells has a frequency of CD27+ cells of more than 20%. In an embodiment, the population of isolated V51 T cells has a frequency of CD27+ cells of about 20%.

[0073] In some embodiments of any of the aforementioned aspects, the isolated population of γδ T cells comprises , compared to a reference population (e.g., compared to a population of γδ T cells isolated using an alternative method greater than CD124, CD215, CD360, CTLA4, CD1b, BTLA, CD39, CD45RA, Fas ligand, one or more surface expressions of a marker selected from the group consisting of CD25, ICAM-1, CD31, KLRG1, CD30, and CD2. Further or alternatively, the population of isolated γδ T cells may have cells expressing one or more of the 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, at a greater frequency compared to a reference population. In particular, the marker is selected from CD4 5RA and CD25. In some embodiments, the population of isolated γδ T cells may have one or more surface expressions of a marker selected from the group consisting of NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LA G3, CCR4, CD69, PD-1, and CD64, at a lower frequency compared to a reference population. Further or alternatively, the population of isolated γδ T cells may have cells expressing one or more of the markers selected from the group consisting of NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4 CD69, PD-1, and CD64, at a lower frequency compared to a reference population. In some embodiments, the population of isolated Vδ1 T cells has one or more of CD124, CD215, CD360, CTLA4, CD1b, BTLA, CD39, CD45RA, Fas ligand, CD25, ICA M-1, CD31, KLRG1, CD30, and CD2, at a greater frequency compared to a reference population. one or more of the markers selected from the group consisting of

[0074] In some embodiments, the population of isolated Vδ1 T cells has one or more of CD124, CD215, CD360, CTLA4, CD1b, BTLA, CD39, CD45RA, Fas ligand, CD25, ICA M-1, CD31, KLRG1, CD30, and CD2, at a greater frequency compared to a reference population. one or more of the markers selected from the group consisting of​ have surface expression of numbers. In some embodiments, a population of isolated γδ T cells has a higher frequency of CD124, CD215, CD360, CTLA4, CD1b, BTLA, CD39, CD45RA compared to a reference and has cells expressing one or more of the markers selected from the group consisting of Fas ligand, CD25, ICAM-1, CD31, KLRG1, CD30, and CD2. In some embodiments, a population of isolated γδ T cells has a lower surface expression of one or more of the 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 reference population. In other embodiments, a population of isolated γδ T cells has cells expressing one or more of the markers selected from the group consisting of NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1, and CD64 at a lower frequency compared to a reference

[0075] When isolated from non-hematopoietic tissue (e.g., skin), γδ T cells are typically part of a larger lymphocyte population containing, for example, αβ T cells, B cells, and natural killer (NK) cells. In some embodiments, 1% - 10% of the isolated lymphocyte population is γδ T cells (e.g., 1 - 10% of the isolated skin-derived lymphocyte population is γδ T cells). In most cases, the γδ T cell population (e.g., skin-derived γδ T cell population) contains a population of large Vδ1 T cells. In some embodiments, the isolated lymphocytes (e.g., skin 1-10% of the population of isolated lymphocytes are Vδ1 T cells (e.g., Vδ1 T cells are isolated The population of γδ T cells may represent more than 50%, more than 60%, more than 70%, more than 80%, or more than 90% of the population of γδ T cells. In some cases, less than 10% of the isolated population of γδ T cells are Vδ2 T cells (e.g., Less than 10% of the isolated skin-derived γδ T cell population are Vδ2 T cells).

[0076] Non-Vδ1 T cells or non-DN T cells, such as Vδ2 T cells, αβ T cells, B cells, or NK cells are removed from the isolated population of γδ T cells (e.g., before, during, or after the expansion step). It can be removed.

[0077] Isolated γδ T cells (e.g., γδ T cells isolated from the skin, e.g., isolated from the skin isolated Vδ1 T cells) can be expressed in response to corresponding hematopoietic tissue-derived cells (e.g., blood-derived γδ T cells and / or or blood-derived Vδ2 T cells). For example, a population of isolated γδ T cells The panel may be a reference population, e.g., a population of TCR-activated non-hematopoietic tissue-resident γδ T cells or a corresponding Hematopoietic tissue-derived cells (e.g., blood-derived γδ T cells and / or blood-derived Vδ2 T cells) Some populations may express higher levels of CCR3, CCR4, CCR7, CCR8, or CD103 than other populations. In one embodiment, the isolated population of γδ T cells is at least 5%, 10%, 15%, 20%, %, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of CCR3 + cell; 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 of CCR7 + Cells; at least 5%, 10% , 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of CCR8 + Cells; and / or at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60% , 70%, 80%, 90%, or more of CD103 + Cells. A population of isolated γδ T cells may express one or more, two or more, three or more, four or more, five or more , or all six of CCR3, CCR4, CCR7, CCR8, or CD103.

[0078] In some embodiments, a population of isolated γδ T cells (e.g., skin-derived γδ T cells and / or skin-derived Vδ1 T cells) expresses higher levels of NKGD2, CD56, CD69, and / or TIM3 than a reference population, e.g., a population of non-hematopoietic tissue-resident γδ T cells with activated TCR and / or the corresponding hematopoietic tissue-derived cells (e.g., blood-derived γδ T cells and / or blood-derived Vδ2 T cells). In some embodiments, a population of isolated γδ T cells expresses at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 9 0%, or more of NKGD2 cells; at least 5%, 10%, 15%, 20%, 25%, 30%, 4 + 0%, 50%, 60%, 70%, 80%, 90%, or more of CD56 cells; at least 5%, 10 + %, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more thereof. of CD69 + cells; and / or at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60 %, 70%, 80%, 90%, or more TIM3 + cells. A population of isolated γδ T cells may express one or more, two or more, three or more, four or more, or all five of NKGD2, CD56, CD69, and / or TIM3.

[0079] A population of isolated non-hematopoietic tissue-derived γδ T cells (e.g., skin-derived γδ T cells and / or skin-derived Vδ1 T cells) can also be characterized by function. Perform a function assay known in the art to assay any non-hematopoietic tissue-derived cell of the invention (e.g., an isolated γδ T cell, a population of skin-derived Vδ1 T cells, or an expanded population of γδ T cells and / or skin-derived Vδ1 T cells) and a reference cell (e.g., a population of non-hematopoietic tissue-resident γδ T cells with activated TCR or a corresponding hematopoietic tissue-derived cell, e.g., a population of blood-derived γδ T cells and / or blood-derived Vδ2 T cells) to determine the functional differences between them. Such assays include , proliferation assays, cytotoxicity assays, binding assays, assays that measure persistence and / or location and the like.

[0080] Thus, in one aspect of the invention, a method for isolating a lymphocyte and / or γδ T cell population as defined herein produces a population that includes a surface phenotype that is consistent with an un-depleted lymphocyte and / or γδ T cell population .

[0081] According to one aspect of the invention, it can be obtained by any of the methods defined herein A population of isolated lymphocytes (e.g., skin-derived αβ T cells and / or NK cells) is provided.

[0082] According to one aspect of the invention, a population of isolated lymphocytes (e.g., skin-derived αβ T cells and / or NK cells) obtained by any of the methods defined herein is provided.

[0083] According to a further aspect of the invention, a population of isolated γδ T cells obtained by any of the methods defined herein is provided.

[0084] According to a further aspect of the invention, a population of isolated γδ T cells obtainable by any of the methods defined herein is provided.

[0085] In one embodiment, the isolated population comprises more than 5% γδ T cells, e.g., 7% - 12% γδ T cells. In one embodiment, the isolated population comprises Vδ1 cells, wherein less than 50%, e.g., less than 40% of the Vδ1 cells express TIGIT. In one embodiment, the isolated population comprises Vδ1 cells, wherein more than 50%, e.g., more than 60% of the Vδ1 cells express CD27.

[0086] Isolated non-hematopoietic tissue-resident lymphocytes may be suitable for use without further expansion or may be expanded in a further step.

[0087] In certain embodiments, the invention features methods of expanding non-hematopoietic tissue-resident lymphocytes and / or γδ T cells (e.g., skin-derived αβ T cells, NK cells, γδ T cells, and / or non-Vδ2 T cells, e.g., Vδ1 T cells and / or DN T cells). These methods involve in vitro... ​​​​​​​​​​​ It can be carried out in vitro. In some embodiments, the γδ T cells are expanded from a population of γδ T cells isolated from a non-hematopoietic tissue sample according to the method defined herein. Generally, non-hematopoietic tissue resident γδ T cells can spontaneously expand when the physical contact with stromal cells (e.g., skin fibroblasts) is removed. The method defined herein can be used to induce such separation, resulting in the de-suppression of γδ T cells and inducing expansion. In certain embodiments, lymphocytes (e.g., skin-derived αβ T cells and / or NK cells, gastrointestinal-derived αβ T cells and / or NK cells) are expanded from a population of lymphocytes isolated from a non-hematopoietic tissue sample according to the method defined herein. As used herein, references to "expanded" or "an expanded population of lymphocytes and / or γδ T cells" include a population of cells that is larger or contains a greater number than the non-expanded population. Such a population may be large, small, or a mixed population with expansion of some or a particular cell type within the population. The term "expansion process" is understood to refer to a process that results in an expanded or an expansion of a population. Thus, an expanded or an expansion of a population may have a greater number or contain more cells compared to a population in which the expansion process has not been performed or prior to any expansion process. Any number shown herein to indicate expansion (e.g., fold increase or expansion multiple) is further understood to indicate the number or size of a population of cells or an increase in the number of cells and to indicate the amount of expansion.

[0088]

[0089] ​​​​​​​​​​​​ Thus, in one embodiment, lymphocytes and / or γδ T cells isolated according to the method of the present invention are expanded. Such expansion may include culturing the γδ T cells in the presence of IL-2 and IL-15. Alternatively, the expansion may include culturing the γδ T cells in the presence of IL-9 and IL-15. It will be understood that any expansion step is carried out for a period effective to produce a population of expanded lymphocytes and / or γδ T cells. In one embodiment, the period effective to produce a population of expanded lymphocytes and / or γδ T cells is at least 5 days. Thus, in one embodiment, the expansion is carried out in the presence of IL-2 and IL-15 for at least 5 days in an amount effective to produce a population of expanded γδ T cells, culturing the γδ T cells. In an alternative embodiment, the expansion is carried out in the presence of IL-9 and IL-15 for at least 5 days in an amount effective to produce a population of expanded γδ T cells, culturing the γδ T cells. In a further embodiment, the expansion is carried out for a period (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 days to 28 days, or about 21 days) in an amount effective to produce a population of expanded lymphocytes and / or γδ T cells, culturing the lymphocytes and / or γδ T cells. For several hours (e.g., about 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, 2 1, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 days). In one embodiment, the lymphocytes and / or γδ T cells are expanded for 14 to 21 days. Thus, including the isolation and expansion process (e.g., for 1 to 40 days, e.g., for 14 to 21 days), the process can last for 28 to 56 days, or about 41 days in some embodiments.

[0090] In a further embodiment, the expansion includes culturing the γδ T cells for 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 1 1 day, at least 12 days, at least 13 days, at least 14 days, at least 21 days, at least 28 days, or longer, e.g., for 5 to 40 days, for 7 to 35 days, for 14 to 28 days, or about 21 days. In one embodiment, the expansion process includes culturing the γδ T cells for at least 10 days, 15 days, or 20 days to produce an expanded population. In one embodiment, the expansion process includes culturing the γδ T cells for 5 to 25 days, e.g., for 14 to 21 days In a further embodiment, the expansion process includes culturing the γδ T cells for about 20 days .

[0091] In some embodiments, IL-2 effective to produce an expanded population of γδ T cells Typical amounts are 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 / m L 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 more). In some embodiments, the amount of IL-2 effective to produce an expanded population of γδ T cells is about 100 IU / mL.

[0092] In some embodiments, the typical amount of IL-15 effective to produce a population 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) is at least 0.1 ng / mL (e.g., from 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 250 ng / mL, 75 ng / mL to 200 ng / mL, 100 ng / mL to 175 ng / mL, 125 ng / mL to 150 ng / mL, 150 ng / mL to 175 ng / mL ​~400 ng / mL, or 100 ng / mL~250 ng / mL, for example, 0.1 ng / mL~1.0 ng / mL, 1.0 ng / mL~5.0 ng / mL, 5.0 ng / mL~10 ng / mL, 10 ng / mL~20 ng / mL, 20 ng / mL~50 ng / mL, 50 ng / mL~100 ng / mL, 10 0 ng / mL~200 ng / mL, 200 ng / mL~500 ng / mL, or 500 ng / mL~1,000 ng / mL). In some embodiments, the amount of IL-15 effective to produce an expanded population of γδ T cells is about 10 ng / mL.

[0093] Substitution or addition of other factors in the expansion culture of non-hematopoietic tissue resident γδ T cells can also be used. Such additional or substitute factors for the expansion of lymphocytes such as αβ T cells or NK cells are known in the art. In one embodiment, such factors are used in an expansion that selectively promotes the expansion of γδ T cells. In a further embodiment, such factors are used in an expansion that selectively promotes the expansion of lymphocytes

[0094] such as αβ T cells and / or NK cells. It will be understood that the amount of each of the above cytokines required to produce an expanded population of γδ T cells is determined by the concentration of one or more other cytokines. For example, if the concentration of IL-2 increases or decreases, the concentration of IL-15 can decrease or increase, respectively, accordingly. As described above, the effective

[0095] amount to produce an expanded population refers to the combined effect of all factors on cell In that embodiment, the expanded population of γδ T cells is greater in number than the population of isolated γδ T cells prior to the expansion 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 than that compared to the population of isolated γδ T cells prior to the expansion step). In one embodiment, the expansion step comprises culturing the isolated γδ T cells in the absence of substantial stromal cell contact. In a further embodiment, the expansion step comprises culturing the isolated γδ T cells in the absence of substantial fibroblast contact. It will be understood that the expansion methods provided herein are also applicable to the expansion of other lymphocytes (e.g., αβ T cells and / or NK cells

[0096] ). In such embodiments, the expansion step comprises culturing the isolated lymphocytes in the presence of relevant growth factors and / or nutrients (e.g., cytokines ). ).

[0097] It will be understood that the expansion methods provided herein are also applicable to the expansion of other lymphocytes (e.g., αβ T cells and / or NK cells ). In such embodiments, the expansion step comprises culturing the isolated lymphocytes in the presence of relevant growth factors and / or nutrients (e.g., cytokines ). Culturing in the presence of a cytokine and / or chemokine to produce a population of expanded lymphocytes (e.g., αβ T cells and / or NK cells).

[0098] In one embodiment, a method of expanding a population of γδ T cells as defined herein comprises culturing γδ T cells or other lymphocytes in a serum-free medium. In a further embodiment, a method of expanding a population of γδ T cells as defined herein comprises culturing γδ T cells in a medium containing a serum substitute. Thus, it is understood that expansion of such γδ T cells in a serum-free medium or a medium containing a serum substitute obtains advantages similar to those described above.

[0099] In some embodiments, substantial TCR pathway activation is absent during the expansion process (e.g., no exogenous TCR pathway activator is included in the culture). In one embodiment, the expansion process comprises the absence of an exogenous TCR pathway agonist. Further provided herein is a method of expanding γδ T cells isolated according to the methods defined herein, wherein the expansion method does not involve contact with feeder cells, tumor cells, and / or antigen-presenting cells. Thus, in a further embodiment of the methods defined herein, expansion of γδ T cells comprises culturing γδ T cells in the substantial absence of stromal cell contact.

[0100] Also provided is a population of large non-hematopoietic tissue-derived 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) (e.g., by removing stromal cell contact and / or TCR stimulation, or by culturing in the presence of an effective amount of a factor​ By culturing, it is a means of producing at a high rate. In some embodiments, as described herein The expansion step described is to expand γδ T cells with a low population doubling time given by the following formula as follows:

Number

[0101] Considering the information provided herein, those skilled in the art will recognize that the present invention relates to γδ T cells derived from non-hematopoietic tissues (e.g., skin-derived γδ T cells and / or non-Vδ2 T cells, e.g., Vδ1 T cells and / or DN T cells) being expanded with a population doubling time of less than 5 days (e.g., 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, 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, 32 hours).

[0102] In some embodiments, within 7 days from the start of culture, the expanded population of γδ T cells (e.g., the expanded population of Vδ1 T cells and / or DN T cells) has at least 10 times the number of γδ T cells compared to the population of isolated γδ T cells before expansion (e.g., at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, at least 10 0 times, at least 150 times, at least 200 times, at least 300 times, at least 400 times, at least as compared to the population of isolated γδ T cells before expansion). 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 number of γδ T cells). In some embodiments, within 14 days from the start of culture, the expanded population of γδ T cells (e.g., the population of expanded Vδ1 T cells and / or DN T cells) contains at least 20-fold number of γδ T cells (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 number of γδ T cells) compared to the population of isolated γδ T cells before expansion. In some embodiments, within 21 days from the start of culture, the expanded population of γδ T cells (e.g., the population of expanded Vδ1 T cells and / or DN T cells) contains at least 50-fold number of γδ T cells (e.g., at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100 fold, compared to the population of isolated γδ T cells before expansion) times, at least 150 times, at least 200 times, at least 300 times, at least 400 times, at least also 500 times, at least 600 times, at least 700 times, at least 800 times, at least 900 times, at least 1,000 times, at least 2,000 times, at least 3,000 times, at least 4,000 times, at least 5,000 times, at least 6,000 times, at least 7,000 times, at least 8,000 times, at least 9,000 times, or at least 10,000 times the number of γδ T cells). In some embodiments within 28 days from the culture, the expanded population of γδ T cells (e.g., the expanded Vδ1 T cells and / or the population of DN T cells) is, compared to the population of isolated γδ T cells before expansion, at least 100 times the number of γδ T cells (e.g., compared to the population of isolated γδ T cells before expansion, at least 110 times, at least 120 times, at least 130 times, at least 140 times, at least 150 times , at least 200 times, at least 300 times, at least 400 times, at least 500 times, at least 600 times, at least 700 times, at least 800 times, at least 900 times, at least 1,000 times, at least 2,000 times, at least 3,000 times, at least 4,000 times, at least 5,000 times, at least 6,000 times, at least 7,000 times, at least 8,000 times, at least 9,000 times, at least 10,000 times, at least 12,000 times, or at least 15,000 times the number of γδ T cells).

[0103] The non-hematopoietic tissue-derived 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) expanded by the methods provided herein are ​It can have a phenotype well-suited for anti-tumor efficacy. In some embodiments, the expanded population of γδ T cells (e.g., skin-derived Vδ1 T cells) has a higher mean expression of CD27 than a reference population (e.g., the population of isolated γδ T cells prior to the expansion process). In some embodiments, the expanded population of γδ T cells has at least a two-fold increase (e.g., at least three-fold, at least four-fold, at least five-fold, at least six-fold, at least seven-fold, at least eight-fold, at least nine-fold, at least ten-fold, at least fifteen-fold, at least twenty-fold, at least twenty-five-fold, at least thirty-fold, at least forty-fold, at least fifty-fold, at least sixty-fold, at least seventy-fold, at least eighty-fold, at least ninety-fold, at least one hundred-fold, at least one hundred and fifty-fold, at least two hundred fold, at least three hundred-fold, at least four hundred-fold, at least five hundred-fold, at least six hundred-fold, at least seven hundred-fold, at least eight hundred-fold, at least nine hundred-fold, at least one thousand-fold, at least five thousand fold, at least ten thousand-fold, at least twenty thousand-fold, or more) in mean expression of CD27 compared to the population of isolated γδ T cells.

[0104] While a distinct subset of the expanded population of 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) can upregulate CD27, another subset is CD27 low or CD27 negative . In this case, the frequency of CD27 cells in the expanded population compared to the population of isolated γδ T cells is greater. positive It is possible. For example, the expanded population of γδ T cells has a frequency of CD27 that is at least 5% higher compared to the frequency of the isolated population of γδ T cells before expansion. cells (e.g., at least 10%, at least 15%, at least 20% positive compared to the frequency of the isolated population of γδ T cells before expansion, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% compared to the frequency of the isolated population of γδ T cells before expansion, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% compared to the frequency of the isolated population of γδ T cells before expansion, at least 100% higher frequency of CD27 cells). In some embodiments, the number of CD27 positive cells in the expanded population compared to the isolated population of γδ T cells can increase. For example, the expanded population of γδ T cells can have at least twice the number of CD27 cells compared to the isolated population of γδ T cells before expansion. The expanded positive population of γδ T cells can have a frequency of CD27+ cells greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80% greater than 90%. Alternatively, the expanded population of γδ T cells can have a frequency of CD27+ cells of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%. In certain embodiments, the expanded population of γδ T cells has a frequency of CD27+ cells greater than 50%. cells compared to the isolated population of γδ T cells before expansion. The expanded positive population of γδ T cells can have a frequency of CD27+ cells greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80% greater than 90%. Alternatively, the expanded population of γδ T cells can have a frequency of CD27+ cells of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%. In certain embodiments, the expanded population of γδ T cells has a frequency of CD27+ cells greater than 50%. greater than 90%. Alternatively, the expanded population of γδ T cells can have a frequency of CD27+ cells of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%. In certain embodiments, the expanded population of γδ T cells has a frequency of CD27+ cells greater than 50%. In some embodiments, the expansion method provided herein results in expanded non-hematopoietic tissue-derived γδ T cells (e.g., skin-derived γδ T cells and / or non-Vδ2 T cells, such as compared to a reference population (e.g., the isolated population of γδ T cells before the expansion step) having lower TIGIT expression. % higher frequency of CD27+ cells.

[0105] In some embodiments, the expansion method provided herein results in expanded non-hematopoietic tissue-derived γδ T cells (e.g., skin-derived γδ T cells and / or non-Vδ2 T cells, such as compared to a reference population (e.g., the isolated population of γδ T cells before the expansion step) having lower TIGIT expression. skin-derived γδ T cells and / or non-Vδ2 T cells) with lower TIGIT expression compared to a reference population (e.g., the isolated population of γδ T cells before the expansion step). then produces a population of γδ T cells (e.g., skin-derived γδ T cells that are not Vδ2 T cells, such as Vδ1 T cells and / or DN T cells). In some embodiments, the expanded population of γδ T cells has a lower average expression of TIGIT than a reference population (e.g., the population of isolated γδ T cells before the expansion step). In some embodiments, the expanded population of γδ T cells has at least 10% less (e.g., at least 20% less, at least 30% less, at least 40% less, at least 50% less, at least 60% less, at least 70% less, at least 80% less, at least 90% less, or up to 100% less) average expression of TIGIT than the population of isolated γδ T cells. The expanded population of γδ T cells has less than 90%, less than 80%, less than 70%, less than 60% less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% frequency of TIGIT+ cells. Alternatively, the expanded population of γδ T cells can have a frequency of TIGIT+ cells of about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, or about 10%. In certain embodiments, the population of isolated γδ T cells has a frequency of TIGIT+ cells of less than 80%. In some embodiments, the expanded population of 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 or frequent number of CD 27 cells and a low frequency of TIGIT cells. In some embodiments, the expanded population of γδ T cells has a higher frequency of CD27 compared to a reference population (e.g., compared to the population of isolated γδ T cells before expansion). In some embodiments, the expanded population of 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 or frequent number of CD 27

[0106] In some embodiments, the expanded population of 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 or frequent number of CD 27 cells and a low frequency of TIGIT + cells. In some embodiments, the expanded population of γδ T cells has a higher frequency of CD27 compared to a reference population (e.g., compared to the population of isolated γδ T cells before expansion). + In some embodiments, the expanded population of γδ T cells has a higher frequency of CD27 compared to a reference population (e.g., compared to the population of isolated γδ T cells before expansion). In some embodiments, the expanded population of γδ T cells has a higher frequency of CD27 compared to a reference population (e.g., compared to the population of isolated γδ T cells before expansion). cells and a low frequency of TIGIT+ TIGIT - cells. For example, an expanded population of γδ T cells has, a frequency of CD27 that is at least 5% higher compared to the frequency of the population of isolated γδ T cells before expansion + 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). It may have. In some embodiments, the number of CD27 TIGIT + TIGIT - cells in the expanded population may increase compared to the population of isolated γδ T cells. For example, the expanded population of γδ T cells has 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 expansion. It may have.

[0107] Optionally, the expanded γδ T cells (e.g., skin-derived γδ T cells and / or non-Vδ2 T CD27 within a population of cells, such as, Vδ1 T cells and / or DN T cells + in a population of γδ T cells The average expression of TIGIT is low compared to a reference population. In some embodiments, the expanded C D27 + The population of γδ T cells has a lower average expression of TIGIT than a reference population (e.g., an isolated CD27 + γδ T cells population prior to the expansion step). In some embodiments, the expanded CD27 + The population of γδ T cells is at least 10% less + than the population of isolated CD27 γδ T cells (e.g., at least 20% less than the population of isolated CD27 + γδ T cells, at least 30% less, at least 40% less, at least 50% less, at least 60% less , at least 70% less, at least 80% less, at least 90% less, or up to 100 % less) in average expression of TIGIT.

[0108] Furthermore or alternatively, the median expression of CD27 in a population of expanded 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 high compared to a reference population. For example, the population of expanded TIGIT - γδ T cell population has a frequency of CD27 that is at least 5% higher compared to the frequency of the population of isolated TIGIT - γδ T cells prior to expansion. For example, the population of expanded TIGIT - γδ T cells has a frequency of CD27 that is at least 10%, at least 15%, at least 20%, at least 25%, at least as high as the frequency of the population of isolated TIGIT + γδ T cells prior to expansion (e.g., at least - γδ T cell frequency of the population of isolated TIGIT 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% greater frequency of CD27 + cells). In some embodiments, isolated TIGIT - γδ T cells compared to a population of cells, the number of CD27 + cells in the expanded population can increase. For example, expanded TIGIT - γδ T cell population is the isolated TIGIT before expansion - γδ T cell population and compared to, at least twice the number of CD27 + cells (e.g., the isolated TIGIT before expansion - γδ T cells population), 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 10 0% greater frequency of CD27 + cells).

[0109] 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 including, CCR4, CD69, PD-1, and CD64, an increase or decrease in the expression of other markers, further or alternatively used to characterize a population of one or more expanded non-hematopoietic tissue-derived 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) can be. Optionally, the expanded γδ T cell population (e.g., skin-derived γδ T cells and or non-Vδ2 T cells, e.g., Vδ1 T cells and / or DN T cells) has, for example, a greater average expression of one or more of the 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 the population of isolated γδ T cells before expansion. Additionally or alternatively, the expanded γδ T cell population has a higher frequency of cells expressing one or more of the 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 the population of isolated γδ T cells. In some embodiments, the expanded γδ T cell population has a lower average expression of one or more of the markers selected from the group consisting of NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1, and CD64 compared to the population of isolated γδ T cells. The expanded population can similarly have a lower frequency of cells expressing one or more of the markers selected from the group consisting of NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CC R4, CD69, PD-1, and CD64 compared to the population of isolated γδ T cells. Therefore, the non-hematopoietic tissue-resident γδ T cells produced by the method of the present invention have the following characteristics 1, CD31, KLRG1, CD30, and CD2. In some embodiments, the expanded γδ T cell population has a lower average expression of one or more of the markers selected from the group consisting of NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1, and CD64 compared to the population of isolated γδ T cells. The expanded population can similarly have a lower frequency of cells expressing one or more of the markers selected from the group consisting of NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CC R4, CD69, PD-1, and CD64 compared to the population of isolated γδ T cells. CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1, and CD64. The expanded population can similarly have a lower frequency of cells expressing one or more of the markers selected from the group consisting of NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CC R4, CD69, PD-1, and CD64 compared to the population of isolated γδ T cells. Therefore, the non-hematopoietic tissue-resident γδ T cells produced by the method of the present invention have the following characteristics 1, CD31, KLRG1, CD30, and CD2. In some embodiments, the expanded γδ T cell population has a lower average expression of one or more of the markers selected from the group consisting of NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1, and CD64 compared to the population of isolated γδ T cells. The expanded population can similarly have a lower frequency of cells expressing one or more of the markers selected from the group consisting of NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CC

[0110] Therefore, the non-hematopoietic tissue-resident γδ T cells produced by the method of the present invention have the following characteristics (i) CD69 high , TIM3 high , and CD28 low / absent Representation of (ii) upregulates one or more of CCR3, CD39, CD11b, and CD9; i) produce IFN-γ in response to NKG2D ligands in the absence of a TCR agonist; (iv) produce IFN-γ in response to a TCR agonist (v) in response to TCR activation, they produce IFN-γ, TNF-α, and GM (vi) produce one or more of IL-17 or IL-21 in response to TCR activation; (vii) grow in culture medium containing IL-2 without additional growth factors; (viii) exhibits a cytotoxic T cell response in the absence of a TCR agonist; and / or (ix) is normal It exhibits selective cytotoxicity against tumor cells rather than normal cells.

[0111] Optionally, the non-hematopoietic tissue resident γδ T cells produced by the methods of the invention are TCR antagonists. and / or produces IL-13 in the absence of a TCR agonist and / or produces NKG2D ligands in the absence of a TCR agonist. In response to this, they produce IFN-γ.

[0112] A number of basal culture media are suitable for use in the expansion of γδ T cells, in particular AIM-V, Iscoves medium, Media such as RPMI-1640 (Life Technologies) and RPMI-1640 (Life Technologies) are available. The medium may be supplemented with other media factors as defined in, for example, serum, serum proteins, and selective agents, antibiotics. For example, in some embodiments, 2 mM glutamine, 10% FBS, 10 mM H EPES, pH 7.2, 1% penicillin-streptomycin, sodium pyruvate (1 mM; Life Technologies Technologies), non-essential amino acids (e.g., 100 μM Gly, Ala, Asn, Asp, Glu, Pro, and Ser; 1×MEM non-essential amino acids (Life Technologies)), and RPMI-1640 medium containing 10 μl / L β-mercaptoethanol. In an alternative embodiment, the AIM-V medium may be supplemented with a serum replacement and amphotericin B manufactured by CTS Immune. In certain embodiments defined herein, the medium may further be supplemented with IL-2 and IL-15. Advantageously, the cells are cultured at 37 °C in a humidified atmosphere containing 5% CO2 in a suitable culture medium during isolation and / or expansion. r; 1×MEM non-essential amino acids (Life Technologies)), and RPMI-1640 medium containing 10 μl / L β-mercaptoethanol. In an alternative embodiment, the AIM-V medium may be supplemented with a serum replacement and amphotericin B manufactured by CTS Immune. In certain embodiments defined herein, the medium may further be supplemented with IL-2 and IL-15. Advantageously, the cells are cultured at 37 °C in a humidified atmosphere containing 5% CO2 in a suitable culture medium during isolation and / or expansion. r; 1×MEM non-essential amino acids (Life Technologies)), and RPMI-1640 medium containing 10 μl / L β-mercaptoethanol. In an alternative embodiment, the AIM-V medium may be supplemented with a serum replacement and amphotericin B manufactured by CTS Immune. In certain embodiments defined herein, the medium may further be supplemented with IL-2 and IL-15. Advantageously, the cells are cultured at 37 °C in a humidified atmosphere containing 5% CO2 in a suitable culture medium during isolation and / or expansion. r; 1×MEM non-essential amino acids (Life Technologies)), and RPMI-1640 medium containing 10 μl / L β-mercaptoethanol. In an alternative embodiment, the AIM-V medium may be supplemented with a serum replacement and amphotericin B manufactured by CTS Immune. In certain embodiments defined herein, the medium may further be supplemented with IL-2 and IL-15. Advantageously, the cells are cultured at 37 °C in a humidified atmosphere containing 5% CO2 in a suitable culture medium during isolation and / or expansion. r; 1×MEM non-essential amino acids (Life Technologies)), and RPMI-1640 medium containing 10 μl / L β-mercaptoethanol. In an alternative embodiment, the AIM-V medium may be supplemented with a serum replacement and amphotericin B manufactured by CTS Immune. In certain embodiments defined herein, the medium may further be supplemented with IL-2 and IL-15. Advantageously, the cells are cultured at 37 °C in a humidified atmosphere containing 5% CO2 in a suitable culture medium during isolation and / or expansion. r; 1×MEM non-essential amino acids (Life Technologies)), and RPMI-1640 medium containing 10 μl / L β-mercaptoethanol. In an alternative embodiment, the AIM-V medium may be supplemented with a serum replacement and amphotericin B manufactured by CTS Immune. In certain embodiments defined herein, the medium may further be supplemented with IL-2 and IL-15. Advantageously, the cells are cultured at 37 °C in a humidified atmosphere containing 5% CO2 in a suitable culture medium during isolation and / or expansion. r; 1×MEM non-essential amino acids (Life Technologies)), and RPMI-1640 medium containing 10 μl / L β-mercaptoethanol. In an alternative embodiment, the AIM-V medium may be supplemented with a serum replacement and amphotericin B manufactured by CTS Immune. In certain embodiments defined herein, the medium may further be supplemented with IL-2 and IL-15. Advantageously, the cells are cultured at 37 °C in a humidified atmosphere containing 5% CO2 in a suitable culture medium during isolation and / or expansion.

[0113] According to a further aspect of the invention, there is provided a method for isolating and expanding lymphocytes from a non-hematopoietic tissue sample, comprising: (i) isolating a population of lymphocytes from the non-hematopoietic tissue sample according to the method defined herein; and (ii) further culturing the population of lymphocytes (e.g., for at least 5 days) to produce an expanded population of lymphocytes. (ii) further culturing the population of lymphocytes (e.g., for at least 5 days) to produce an expanded population of lymphocytes.

[0114] In one embodiment, the lymphocytes include αβ T cells. Accordingly, according to a further aspect of the invention, there is provided a method for isolating and expanding αβ T cells from a non-hematopoietic tissue sample, comprising: (i) isolating a population of αβ T cells from the non-hematopoietic tissue sample according to the method defined herein; and (ii) further culturing the population of αβ T cells (e.g., for at least 5 days) to produce an expanded population of αβ T cells. (ii) further culturing the population of αβ T cells (e.g., for at least 5 days) to produce an expanded population of αβ T cells.

[0115] ​​​​​​ The culture in step (ii) may be by selective proliferation, for example, by selecting culture conditions under which NK cells expand preferentially over other cell types present in the isolated population of step (i). Alternatively, the expansion conditions may not be selective, and non-target cells (e.g., cells other than NK cells) may be removed after the culture of step (ii). Alternatively, the expansion conditions may not be selective, and the removal of non-target cells (e.g., cells other than NK cells) is performed prior to the culture of step (ii). It should be noted that the purpose of these embodiments is to expand the total number of NK cells while also increasing their proportion in the population.

[0116] In one embodiment, the lymphocytes include NK cells. Accordingly, in a further aspect of the invention, a method for isolating and expanding NK cells from a non-hematopoietic tissue sample, comprising: (i) isolating a population of NK cells from a non-hematopoietic tissue sample according to the method defined herein; and (ii) further culturing the population of NK cells (e.g., for at least 5 days) to produce an expanded population of NK cells : is provided.

[0117] The culture in step (ii) may be by selective expansion, for example, by selecting culture conditions under which NK cells expand preferentially over other cell types present in the isolated population of step (i). Alternatively, the expansion conditions may not be selective, and non-target cells (e.g., cells other than NK cells) may be removed after the culture of step (ii). Alternatively, the expansion conditions may not be selective, and the removal of non-target cells (e.g., cells other than NK cells) is performed prior to the culture of step (ii). ​​​​​​​​​​​​​The aim of these embodiments is to expand the total number of NK cells while also increasing their proportion in the population. It should be noted that this is the case.

[0118] In one embodiment, the lymphocytes include γδ T cells. Therefore, according to a further aspect of the present invention, a method for isolating and expanding γδ T cells from a non-hematopoietic tissue sample, comprising: (i) isolating a population of γδ T cells from a non-hematopoietic tissue sample according to the method defined herein; and (ii) further culturing the population of γδ T cells (for example, for at least 5 days) to produce an expanded population of γδ T cells. : is provided. (ii) The culturing in step (ii) may be by selective expansion, for example, by selecting culture conditions under which the γδ T cells are preferentially expanded over other cell types present in the isolated population of step (i). Alternatively, the expansion conditions may not be selective, and non-target cells (for example, cells other than γδ T cells) may be removed after the culturing in step (ii). Alternatively, the expansion conditions may not be selective, and the removal of non-target cells (for example, cells other than γδ T cells) may be performed before the culturing in step (ii). It should be noted that the aim of these embodiments is to expand the total number of γδ T cells while also increasing their proportion in the population.

[0119] The culturing in step (ii) may be by selective expansion, for example, by selecting culture conditions under which the γδ T cells are preferentially expanded over other cell types present in the isolated population of step (i). Alternatively, the expansion conditions may not be selective, and non-target cells (for example, cells other than γδ T cells) may be removed after the culturing in step (ii). Alternatively, the expansion conditions may not be selective, and the removal of non-target cells (for example, cells other than γδ T cells) may be performed before the culturing in step (ii). It should be noted that the aim of these embodiments is to expand the total number of γδ T cells while also increasing their proportion in the population. The aim of these embodiments is to expand the total number of γδ T cells while also increasing their proportion in the population. It should be noted that this is the case.

[0120] According to one aspect of the present invention, there is provided a population of expanded isolated lymphocytes (for example, skin-derived αβ T cells and / or NK cells) obtained by any of the methods defined herein.

[0121] According to a further aspect of the present invention, there is provided a population of expanded isolated lymphocyte cells obtainable by any of the methods defined herein.

[0122] According to yet a further aspect of the present invention, there is provided a population of expanded isolated γδ T cells obtainable by any of the methods defined herein.

[0123] According to yet a further aspect of the present invention, there is provided a population of isolated expanded γδ T cells obtainable by any of the methods defined herein.

[0124] In one embodiment, the isolated population comprises more than 50% γδ T cells, for example, more than 75% γδ T cells, particularly more than 85% γδ T cells. In one embodiment, the isolated population comprises Vδ1 cells, where less than 50%, for example, less than 25% of the Vδ1 cells express TIGIT. In one embodiment, the isolated population comprises Vδ1 cells, where more than 50%, for example, more than 60% of the Vδ1 cells express CD27.

[0125] The lymphocytes and / or γδ T cells obtained by the method of the present invention can be used, for example, as a medicament for adoptive T cell therapy. This involves transplantation of the lymphocytes and / or γδ T cells obtained by the method of the present invention into a patient. The therapy may be autologous, i.e., the γδ T cells may be transplanted back into the same patient from whom they were obtained, or the therapy may be allogeneic, i.e., γδ T cells from one person may be transplanted into a different patient. In the case of allogeneic transplantation, the γδ T cells are αβ T cells. ​​​​​​​​​​​​It may also be substantially free. For example, αβ T cells can be removed from the γδ T cell population using any suitable means known in the art (e.g., using magnetic beads, e.g., by negative selection) after expansion, for example. The treatment method may include: providing a sample of non-hematopoietic tissue obtained from a donor individual; culturing γδ T cells derived from the above sample to produce an expanded population; and administering the expanded population of γδ T cells to a recipient individual. It may be removed from the γδ T cell population using any suitable means known in the art (e.g., using magnetic beads, e.g., by negative selection) after expansion, for example. The treatment method may include: providing a sample of non-hematopoietic tissue obtained from a donor individual; culturing γδ T cells derived from the above sample to produce an expanded population; and administering the expanded population of γδ T cells to a recipient individual. It may be included.

[0126] 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 infection or HIV infection patient). Optionally, the patient has a solid tumor and / or is undergoing treatment for a solid tumor. It may be 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 infection or HIV infection patient). Optionally, the patient has a solid tumor and / or is undergoing treatment for a solid tumor. 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 infection or HIV infection patient). Optionally, the patient has a solid tumor and / or is undergoing treatment for a solid tumor. It may be included.

[0127] Since tissue-resident Vδ1 T and DN γδ T cells usually reside in non-hematopoietic tissues, these cells may also home to and be retained within the tumor mass, with a higher likelihood than their systemic blood-resident counterparts. Adoptive transfer of these cells may be more effective in targeting solid tumors and potentially other non-hematopoietic tissue-related immunopathology. Since tissue-resident Vδ1 T and DN γδ T cells usually reside in non-hematopoietic tissues, these cells may also home to and be retained within the tumor mass, with a higher likelihood than their systemic blood-resident counterparts. Adoptive transfer of these cells may be more effective in targeting solid tumors and potentially other non-hematopoietic tissue-related immunopathology. Since tissue-resident Vδ1 T and DN γδ T cells usually reside in non-hematopoietic tissues, these cells may also home to and be retained within the tumor mass, with a higher likelihood than their systemic blood-resident counterparts. Adoptive transfer of these cells may be more effective in targeting solid tumors and potentially other non-hematopoietic tissue-related immunopathology. It may be included.

[0128] Since γδ T cells are MHC-unrestricted, they do not recognize the host into which they are transplanted as foreign, which means that γδ T cells are less likely to cause graft-versus-host disease. This means that γδ T cells can be used "off the shelf" and transplanted into any recipient for allogeneic adoptive T cell therapy, for example. Since γδ T cells are MHC-unrestricted, they do not recognize the host into which they are transplanted as foreign, which means that γδ T cells are less likely to cause graft-versus-host disease. This means that γδ T cells can be used "off the shelf" and transplanted into any recipient for allogeneic adoptive T cell therapy, for example. Since γδ T cells are MHC-unrestricted, they do not recognize the host into which they are transplanted as foreign, which means that γδ T cells are less likely to cause graft-versus-host disease. This means that γδ T cells can be used "off the shelf" and transplanted into any recipient for allogeneic adoptive T cell therapy, for example. It may be included.

[0129] The non-hematopoietic tissue-resident γδ T cells obtained by the method of the present invention express NKG2D and respond to NKG2D ligands (e.g., MICA) that are strongly associated with malignant tumors. The non-hematopoietic tissue-resident γδ T cells also express a cytotoxic profile in the absence of any activation and are therefore likely to be effective in killing tumor cells. For example, the non-hematopoietic tissue-resident γδ T cells obtained as 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 in the absence of any activation. IL-17A may not be expressed. Thus, the findings reported herein provide strong evidence for the utility and suitability of clinical application of the non-hematopoietic tissue-resident γδ T cells obtained by the method of the present invention as a "commercially available" immunotherapy reagent. These cells possess natural-like apoptosis, have no MHC restriction, and exhibit improved homing to tumors and / or retention within tumors compared to other T cells. In some embodiments, a method of treating an individual having a tumor in non-hematopoietic tissue may include: providing a sample of the non-hematopoietic tissue obtained from a donor individual, culturing γδ T cells from the sample to produce an expanded population, and administering the expanded population of γδ T cells to an individual having a tumor.

[0130] A pharmaceutical composition may include the expanded non-hematopoietic tissue-resident γδ T cells described herein, either as one or more medicaments or in combination with a physiologically acceptable carrier, diluent, or excipient. Thus, the findings reported herein provide strong evidence for the utility and suitability of clinical application of the non-hematopoietic tissue-resident γδ T cells obtained by the method of the present invention as a "commercially available" immunotherapy reagent. These cells possess natural-like apoptosis, have no MHC restriction, and exhibit improved homing to tumors and / or retention within tumors compared to other T cells. In some embodiments, a method of treating an individual having a tumor in non-hematopoietic tissue may include: providing a sample of the non-hematopoietic tissue obtained from a donor individual, culturing γδ T cells from the sample to produce an expanded population, and administering the expanded population of γδ T cells to an individual having a tumor.

[0131] In some embodiments, a method of treating an individual having a tumor in non-hematopoietic tissue may include: providing a sample of the non-hematopoietic tissue obtained from a donor individual, culturing γδ T cells from the sample to produce an expanded population, and administering the expanded population of γδ T cells to an individual having a tumor. A pharmaceutical composition may include the expanded non-hematopoietic tissue-resident γδ T cells described herein, either as one or more medicaments or in combination with a physiologically acceptable carrier, diluent, or excipient. In some embodiments, a method of treating an individual having a tumor in non-hematopoietic tissue may include: providing a sample of the non-hematopoietic tissue obtained from a donor individual, culturing γδ T cells from the sample to produce an expanded population, and administering the expanded population of γδ T cells to an individual having a tumor.

[0132] A pharmaceutical composition may include the expanded non-hematopoietic tissue-resident γδ T cells described herein, either as one or more medicaments or in combination with a physiologically acceptable carrier, diluent, or excipient. In some embodiments, a method of treating an individual having a tumor in non-hematopoietic tissue may include: providing a sample of the non-hematopoietic tissue obtained from a donor individual, culturing γδ T cells from the sample to produce an expanded population, and administering the expanded population of γδ T cells to an individual having a tumor. . Such compositions may include buffers such as neutral buffered saline, 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 or glutathione; adjuvants (such as aluminum hydroxide); and preservatives. Examples of cryoprotective solutions that can be used in the pharmaceutical compositions of the present invention include, for example, DMSO. The compositions can be formulated, for example, for intravenous administration. ; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (such as aluminum hydroxide); and preservatives. Examples of cryoprotective solutions that can be used in the pharmaceutical compositions of the present invention include, for example, DMSO. The compositions can be formulated, for example, for intravenous administration. nitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (such as aluminum hydroxide); and preservatives. Examples of cryoprotective solutions that can be used in the pharmaceutical compositions of the present invention include, for example, DMSO. The compositions can be formulated, for example, for intravenous administration. agents such as EDTA or glutathione; adjuvants (such as aluminum hydroxide); and preservatives. Examples of cryoprotective solutions that can be used in the pharmaceutical compositions of the present invention include, for example, DMSO. The compositions can be formulated, for example, for intravenous administration. can include preservatives. Examples of cryoprotective solutions that can be used in the pharmaceutical compositions of the present invention include, for example, DMSO. The compositions can be formulated, for example, for intravenous administration. is mentioned. The compositions can be formulated, for example, for intravenous administration.

[0133] In one embodiment, the pharmaceutical composition is substantially free of detectable levels of contaminants such as endotoxin or mycoplasma, for example, the contaminants are absent. is mycoplasma contaminants, for example, the contaminants are absent.

[0134] Optionally, a therapeutically effective amount of the expanded γδ T cells obtained by any of the above methods can be administered to a subject (for example, for the treatment of cancer, for example, for the treatment of solid tumors) at a therapeutically effective dose. Optionally, a therapeutically effective amount of the expanded 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) is less than 10×10 cells per dose (for example, less than 9×10 cells per dose, less than 8×10 cells per dose, less than 7×10 12 cells per dose (for example, 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 3×1 0 12 cells per dose, less than 2×10 12less than cells, 1×10 per dose 12 less than cells, 9×10 per dose 11 less than cells, 8×10 per dose 11 less than cells, 7×10 per dose 11 less than cells, 6×10 per dose 11 less than cells, 5×10 per dose 11 less than cells, dose 4×10 per dose 11 less than cells, 3×10 per dose 11 less than cells, 2×10 per dose 11 less than cells, 1×10 per dose 11 less than cells, 9×10 per dose 10 less than cells, per dose 7.5×10 per dose 10 less than cells, 5×10 per dose 10 less than cells, 2.5×10 per dose 10 less than cells, 1×10 per dose 10 less than cells, 7.5×10 per dose 9 less than cells, per dose 5×10 per dose 9 less than cells, 2.5×10 per dose 9 less than cells, 1×10 per dose 9 less than cells, 7.5×10 per dose 8 less than cells, 5×10 per dose 8 less than cells, per dose 2,5×10 per dose 8 less than cells, 1×10 per dose 8 less than cells, 7.5×10 per dose 7 less than cells , 5×10 per dose 7 less than cells, 2,5×10 per dose 7 less than cells, 1×10 per dose 7 less than cells, 7.5×10 per dose 6 less than cells, 5×10 per dose 6Less than [X] cells, dosage per 2.5×10 6 less than [X] cells, per dosage of 1×10 6 less than [X] cells, per dosage of 7.5×10 5 less than [X] cells per dosage of 5×10 5 less than [X] cells, per dosage of 2.5×10 5 less than [X] cells, or dosage per 1×10 5 cells (less than [X] cells). In some embodiments, the therapeutically effective amount 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) is, during the course of treatment, less than 10×10 12 cells (e.g., during the course of treatment, less than 9×10 12 cells, less than 8×10 12 cells, less than 7×10 12 cells, less than 6×10 12 cells less than [X] cells, 5×10 12 cells, less than 4×10 12 cells, less than 3×10 12 cells, less than 2×10 12 cells less than [X] cells, 1×10 12 cells, less than 9×10 11 cells, less than 8×10 11 cells, less than 7×10 11 cells less than [X] cells, 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 1×1010 less than cells, 7.5×10 9 less than cells, 5×10 9 less than cells, 2.5×10 9 less than cells, 1×10 9 cells less than cells, 7.5×10 8 less than cells, 5×10 8 less than cells, 2,5×10 8 less than cells, 1×1 0 8 less than cells, 7.5×10 7 less than cells, 5×10 7 less than cells, 2,5×10 7 less than cells, 1×10 7 less than cells, 7.5×10 6 less than cells, 5×10 6 less than cells, 2,5×10 6 less than cells 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).

[0135] In some embodiments, the expanded non-hematopoietic tissue resident γδ T cell doses described herein are 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 and the dose of expanded non-hematopoietic tissue-resident 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) is at least about 1×10 6 , 1.1×1 0 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, the dose of expanded non-hematopoietic tissue-resident γ δ 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) is up to 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, the dose of expanded non-hematopoietic tissue-resident 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) is about 1.1×10 6 ~1.8×10 7 cells / kg. In some embodiments, the dose of expanded non-hematopoietic tissue-resident γ δ T cells (e.g., skin-derived γδ T cells and / or non-Vδ2 T cells, e.g., Vδ1 T cells and / or the dose of 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, the expanded non-hematopoietic tissue resident γδ T cells (e.g., skin-derived γδ T cells and / or non-Vδ2 T cells, e.g., Vδ1 T cell s and / or DN T cells) dose 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, the expanded non-hematopoietic tissue resident 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) dose is up to about 1×10 7 , 2×10 7 , 5×10 7 , 1×1 0 8 , 2×10 8 , 5×10 8 , 1×10 9 , 2×10 9 , or 5×10 9 cells.

[0136] In one embodiment, the subject is 10 per kg body weight of the subject 4 ~10 6 expanded non-hematopoietic tissue resident γδ T cells (e.g., skin-derived γδ T cells and / or non-Vδ2 T cells, e.g., Vδ1 T cell cells and / or DNT cells) are administered. In one embodiment, the subject is a non-hematopoietic tissue resident γδ T cell population in a first administration (e.g., 10 4 ~10 6 γδ T cells in the first administration per kg body weight of the subject, e.g., 10 ~10 4 γδ T cells), and one or more subsequent administrations (e.g., 2, 3, 4, or 5 times) of the expanded non-hematopoietic tissue 5 resident γδ T cells (e.g., 10 ~10 expanded non-hematopoietic tissue resident γδ T cells per kg body weight of the subject, e.g., 10 4 ~10 6 expanded non-hematopoietic tissue resident γδ T cells). In one embodiment, the one or more subsequent administrations are less than 15 days after the previous administration, e.g., less than 14, 13, 12 10 4 ~10 5 days, e.g., less than 4, 3, or 2 days after the previous administration. In one embodiment, the subject receives a total of about 10 γδ T cells per kg body weight of the subject during at least 3 administrations of the γδ T cell population, e.g., the subject receives an initial administration of 1×10 γδ T cells, a second administration of 3×10 γδ T cells, and a third administration of 6×10 6 γδ T cells, e.g., each administration is less than 4, 3, or 2 days after the previous administration. 5 γδ T cells, a second administration of 3×10 5 γδ T cells, and a third administration of 6×10 5 γδ T cells, e.g., each administration is less than 4, 3, or 2 days after the previous administration. The non-hematopoietic tissue resident γδ T cells obtained by the method of the present invention have enhanced therapeutic properties, e.g.,

[0137] they can also be genetically modified for CAR-T therapy. This can provide new specificities, e.g., Generation of a modified T cell receptor (TCR) that reprograms T cells with specificity for a monoclonal antibody is involved. The modified TCR is specific for malignant cells and can thus create useful T cells for cancer immunotherapy. For example, T cells can recognize cancer cells that express tumor antigens not expressed by normal somatic cells from the target tissue, such as tumor-associated antigens. Therefore, CAR-modified T cells can be used, for example, in adoptive T cell therapy for cancer patients. Use of blood-resident γδ T cells for CAR is described. However, the non-hematopoietic tissue-resident γδ T cells obtained by the method of the present invention can, on the one hand, have a chimeric antigen-specific TCR transduced and, on the other hand, retain their natural-like ability to recognize transformed cells and have better tumor infiltration and retention ability than either blood-resident γδ T cells or conventional systemic αβ T cells. Therefore, this is likely to be a particularly excellent vehicle for the CAR-T approach. Furthermore, due to the lack of its MHC-dependent antigen presentation, the potential for graft-versus-host disease is reduced, making it possible to target tumors that express low levels of MHC. Similarly, for example, its independence from conventional co-stimulation by engagement of CD28 enhances the targeting of tumors that express low levels of ligands for co-stimulatory receptors. In some embodiments, one or more additional therapeutic agents can be administered to the subject. The additional therapeutic agent can be selected from the group consisting of an immunotherapeutic agent, a cytotoxic agent, a growth inhibitor, a radiotherapy agent, an anti-angiogenic agent, or a combination of two or more of these agents. The additional therapeutic agent can be expanded. For example, T cells can recognize cancer cells that express tumor antigens not expressed by normal somatic cells from the target tissue, such as tumor-associated antigens. Therefore, CAR-modified T cells can be used, for example, in adoptive T cell therapy for cancer patients.

[0138] Use of blood-resident γδ T cells for CAR is described. However, the non-hematopoietic tissue-resident γδ T cells obtained by the method of the present invention can, on the one hand, have a chimeric antigen-specific TCR transduced and, on the other hand, retain their natural-like ability to recognize transformed cells and have better tumor infiltration and retention ability than either blood-resident γδ T cells or conventional systemic αβ T cells. Therefore, this is likely to be a particularly excellent vehicle for the CAR-T approach. Furthermore, due to the lack of its MHC-dependent antigen presentation, the potential for graft-versus-host disease is reduced and it becomes possible to target tumors that express low levels of MHC. Similarly, for example, due to its independence from conventional co-stimulation by engagement of CD28, the targeting of tumors that express low levels of ligands for co-stimulatory receptors is enhanced.

[0139] In some embodiments, one or more additional therapeutic agents can be administered to the subject. The additional therapeutic agent can be selected from the group consisting of an immunotherapeutic agent, a cytotoxic agent, a growth inhibitor, a radiotherapy agent, an anti-angiogenic agent, or a combination of two or more of these agents. The additional therapeutic agent can be expanded. ​​It can be administered simultaneously with, before, or after the administration of the γδ T cells that have been administered. Additional The therapeutic agent may be an immunotherapeutic agent that can act on the target of the subject's body (e.g., the subject's own immune system) and / or on the transplanted γδ T cells.

[0140] The administration of the composition can be carried out by any convenient method. The composition can be administered to a patient by transarterial, subcutaneous, intradermal, intratumoral, intranodal, intramedullary, intramuscular, intravenous injection, or intraperitoneally, e.g., by intradermal or subcutaneous injection. A composition of non-hematopoietic tissue-resident γδ T cells can be directly injected into a tumor, lymph node, or site of infection.

[0141] It will be understood that all of the embodiments described herein can be applied to all aspects of the present invention.

[0142] As used herein, the term "about", when used herein, includes values that are 10% less than and up to values that are 10% greater than the specified value, preferably values that are 5% less than and up to values that are 5% greater than the specified value, and in particular includes the specified value. The term "between" includes values within the specified range.

[0143] Now, specific aspects and embodiments of the present invention will be described by way of example and with reference to the above drawings.

Examples

[0144] (Example) (Example 1. Analysis method) Unless otherwise specified, the results of the following examples were obtained using the following methods.

[0145] (Flow cytometry) Flow cytometry was performed using the following antibody-fluorophore 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 Milte nyi. The viability dye (near IR) was from eBioscience. Ki-67 staining was performed on cells fixed and permeabilized using the Foxp3 staining buffer set (eBioscience). After each experiment, the cell population was washed in PBS and split in half. The cells were stained for viability with eFluor770 NIR, washed, and then stained with TrueStain (Biolegend) to avoid non-specific binding of the stained antibodies. Half of the sample was stained for the displayed surface markers, and the other half was stained only for lineage markers (CD3, Vδ1) and for equivalent isotype controls of the surface markers. Matched mouse isotype antibodies conjugated to the same fluorophore were used at the same concentration. Isotype controls do not bind to known human antigens and thus show non-specific binding or false positives. Data summaries show the percentage of cells that were positive for the compared displayed markers and thus stained at a higher level than the isotype. Flow cytometry data analysis was performed using FLOWJO (version 10.1).

[0146] (Population analysis) Skin resident lymphocytes were isolated using the method described in this specification. Among CD45+ cells, T cells were stained with anti-CD3 respectively, and NK cells, CD3−CD56+, were identified using anti-CD56 antibody. Among CD3+ cells, skin resident γδ T cells were identified using an antibody against the pan-γδ T cell receptor, and the proportion of conventional CD4 and CD8 positive αβ T cells within the CD3+, pan-γδ TCR− gate was identified using anti-CD8α.

[0147] (Determination of total cell number) The total cell number was counted using an NC-250 Nucleocounter (Chemometec, Copenhagen, Denmark) and the manufacturer's instructions.

[0148] (Example 2. Isolation of lymphocytes from human skin samples) A three-dimensional skin explant protocol was established. This is described in this specification. A tantalum-coated reticulated vitreous carbon scaffold (also called a grid) with dimensions of 20 mm × 1.5 mm (Ultramet, California, USA) or an equivalent was autoclaved, then washed, and completely immersed in PBS before use.

[0149] A complete isolation medium containing 1 L of AIM-V medium (Gibco, Life Technologies), 50 mL of serum replacement from CTS Immune (Life Technologies), human recombinant IL-2 (Miltenyi Biotech, Cat no 130-097-746) and human recombinant IL-15 (Miltenyi Biotech, Cat no 130-095-766) at the following concentrations It was prepared. For the first 7 days from the start of the culture, complete isolation medium containing 10 mL of Amphotericin B (250 μg / mL, Life Technologies) was used (“+AMP”). The target final concentrations of cytokines in the complete isolation medium were as follows: Samples of adult skin were obtained, transported within 48 hours of collection, and processed. Excess subcutaneous fat and hair were removed from the samples using a scalpel and forceps. The skin samples were placed with the epidermal side facing up, and the skin around the biopsy was held with sterile forceps using a punch biopsy of an appropriate size, and the skin was cut. Three biopsies were placed equidistantly with the epidermal side facing up and attached to the surface of a single tantalum-coated carbon grid. Using sterile forceps, the grid was transferred into a tissue culture vessel with a gas-permeable membrane, such as a well of a G-REX6 well plate (Wilson Wolf Manufacturing) containing 30 mL of complete isolation medium (+AMP), or into a G-REX100 bioreactor (Wilson Wolf Manufacturing) containing 300 mL of complete isolation medium (+AMP). One grid was placed in each well of a G-REX6 well plate, or three grids were placed in a G-REX10 bioreactor, or ten grids were placed in a G-REX100 bioreactor. The cultures were incubated at 37 °C in a 5% CO2 incubator. Table 1: Final concentrations of cytokines in the complete isolation medium

Table 1

[0150] ... ... ... ...

[0151] ... ... ... ... ... ... ... ...

[0152] Unless otherwise noted, gently aspirate the upper medium and replace it with 2× complete isolation medium (without AMP). By doing so and without disturbing the cells at the bottom of the plate or bioreactor, the medium was exchanged every 7 days.

[0153] To isolate lymphocytes, the grid containing the skin was removed from the G-REX6 well plate or G-REX10 or G-REX100 bioreactor and discarded for disposal. The cells present at the bottom of the plate or bioreactor were resuspended and transferred to a 500 mL centrifuge tube and then centrifuged (e.g., at 300 g for 10 minutes).

[0154] If a cell count is required, lymphocytes were counted at this stage according to the protocol described in Example 1. The results of an exemplary test are shown in Table 2: Table 2. Yield of isolated lymphocytes per donor [Table 2]

[0155] (Example 3. Optimization of punch biopsy size) From initial tests, it was shown that 3 mm punch biopsies were superior to standard skin mincing methods (Figure 1).

[0156] Punch biopsy sizes of 1 mm, 2 mm, 3 mm, 4 mm, and 8 mm were tested, and by using 2 mm explants minced with a scalpel as a control, the optimal punch biopsy size was further investigated. Skin samples were prepared as described in Example 2. Each size was tested by attaching one biopsy, skin side up, to the surface of the carbon grid and placing it in a 24 well plate ​​Placed in wells of (Corning). Each well contained AIM-V 10% human AB serum + IL- 2 and IL-15 at the above concentrations and standard concentrations of β-mercaptoethanol (2ME) and penicillin / streptomycin (P / S).

[0157] The medium was changed three times a week (half-medium change), and the biopsies were incubated in a 5% CO2 incubator at 37 °C for 21 days before cell harvesting and cell yield analysis.

[0158] The total cell yield was determined as described in Example 1. The results are shown in Table 2. These results indicate that biopsies with a diameter of 2 - 4 mm result in the highest cell yield. Table 3: Total cell yields obtained by biopsy type [[Table 3]]

[0159] The percentage of γδ T cells present in the cell yield was determined as described in Example 1. The results are presented in Figure 2. These results indicate that biopsies with a diameter of 3 mm result in the highest yield of γδ T cells.

[0160] (Example 4. Optimization of isolation vessel) Isolation in a 24-well plate was compared to using a vessel containing a gas-permeable material, such as a G-REX6 well plate (Wilson Wolf Manufacturing). Skin samples were prepared as described in Example 2. The biopsies were prepared as described in Example 2. The biopsies were attached to the surface of the carbon grid with the epidermis facing up, and then this carbon grid was placed in the wells of a 24-well plate or a G-REX6 well plate. ​​A 9 mm grid was used for the 24-well plate and a 20 mm grid for the G-REX 6-well plate. All samples were in AIM-V 10% AB serum + P / S + 2ME + IL-2 + IL-15. For well plates, the medium was changed three times a week. For G-REX 6-well plates, The biopsies were incubated at 37°C in a 5% CO2 incubator until the cells were fully grown. The cells were incubated for 21 days before cell yield analysis.

[0161] Total cell yields per plate and per biopsy were determined as described in Example 1. Experiments have shown that the G-REX 6-well plate can hold up to 1000 samples per biopsy when compared to the 24-well plate. It was shown that G-REX6 resulted in increased cell yield per plate (Figure 3 and Table 4). Although well plates allowed for an increased amount of tissue to be cultured (compared to 24-well plates), By contrast, the plates yielded a remarkable 25-fold increase in cell numbers (2.5-fold more tissue per plate). Table 4. Total cell yields obtained with 24-well vs. G-REX 6-well [Table 4]

[0162] Example 5. Optimization of isolation protocol The use of 3 mm punch biopsies cultured in G-REX vessels was further tested to optimize the isolation protocol. Skin samples were prepared as described in Example 2 and taken using a 3 mm punch biopsy. The cells were obtained and cultured in G-REX 6-well plates or G-REX 10 bioreactors as described in Example 2. The biopsies were placed in a 5% serum replacement (SR), 5% human AB serum, or 5% SR / 5% AB medium. cultured in +2ME+P / S+IL2 / 15 containing "Blend".

[0163] First, the period of cell isolation was tested. Biopsies were incubated in a 5% CO2 incubator at 37 °C for 14 or 21 days prior to cell yield analysis. The total cell yield per grid was determined as described in Example 1. The results are shown in Figure 4. For all media types, isolation after 3 weeks improved cell yield when compared to isolation after 2 weeks. The use of serum replacements compared to human AB serum (5% or 10%) was also tested. Biopsies were incubated in a 5% CO2 incubator at 37 °C for 21 days prior to cell analysis. The total cell yield per grid and the percentage of Vδ1 cells were measured as described in Example 1. The results are shown in Figure 5. Improved cell yields and higher ratios of Vδ1 cells were obtained using media supplemented with 5% serum replacement compared to human AB serum.

[0164]

[0165] (Example 6. Cell Expansion) Once the cells are isolated using the above protocol, they can be expanded using methods known in the art. For example, the selective expansion of γδ T cells can be achieved using the expansion methods described in WO2017072367. This application provides an invention in the following aspects. (Aspect 1) A method for isolating lymphocytes from a non-hematopoietic tissue sample, comprising: (i) culturing a non-hematopoietic tissue sample, which is an intact biopsy having a minimum cross-sectional area of at least 2 mm obtained from non-hematopoietic tissue, in the presence of interleukin-2 (IL-2) and interleukin-15 (IL-15); and ​​​​​​​​​​​(ii) Recovering a population of lymphocytes cultured from the non-hematopoietic tissue sample : The method as described above. (Aspect 2) A method for isolating γδ T cells from a non-hematopoietic tissue sample, comprising: (i) Culturing a non-hematopoietic tissue sample, which is an intact biopsy having a minimum cross-sectional area of at least 2 mm obtained from non-hematopoietic tissue, in the presence of IL-2 and IL-15; and (ii) Recovering a population of γδ T cells cultured from the non-hematopoietic tissue sample : The method as described above. (Aspect 3) (Aspect 3) The method according to any one of Aspects 1 to 2, wherein the non-hematopoietic tissue sample has a minimum cross-sectional area of about 3 mm. (Aspect 4) The method according to any one of Aspects 1 to 3, wherein the non-hematopoietic tissue sample has a maximum cross-sectional area of 8 mm or less. (Aspect 5) The method according to any one of Aspects 1 to 4, wherein the non-hematopoietic tissue sample has a maximum cross-sectional area of 4 mm or less. (Aspect 6) A method for isolating lymphocytes from a non-hematopoietic tissue sample, comprising: (i) Culturing a non-hematopoietic tissue sample, which is an intact biopsy having a minimum cross-sectional area of at least 2 mm obtained from non-hematopoietic tissue, in the presence of IL-2 and IL-15; and 2 (ii) Recovering a population of lymphocytes cultured from the non-hematopoietic tissue sample : The method as described above. (Aspect 7) (Aspect 7) A method for isolating γδ T cells from a non-hematopoietic tissue sample, comprising: (i) Culturing a non-hematopoietic tissue sample, which is an intact biopsy having a minimum cross-sectional area of at least 2 mm obtained from non-hematopoietic tissue, in the presence of IL-2 and IL-15; and 2 (ii) Recovering a population of γδ T cells cultured from the non-hematopoietic tissue sample (ii) Recovering a population of γδ T cells cultured from the non-hematopoietic tissue sample : The method as described above. (Aspect 8) The non-hematopoietic tissue sample has a cross-sectional area of about 7 mm 2 The method according to Aspect 6 or Aspect 7. (Aspect 9) The non-hematopoietic tissue sample has a maximum cross-sectional area of 64 mm 2 The method according to any one of Aspects 6 to 8 having the following maximum cross-sectional area. : as described above. (Aspect 10) The non-hematopoietic tissue sample has a maximum cross-sectional area of 50 mm 2 The method according to any one of Aspects 6 to 9 having the following maximum cross-sectional area. : as described above. (Aspect 11) The non-hematopoietic tissue sample has a maximum cross-sectional area of 16 mm 2 The method according to any one of Aspects 6 to 10 having the following maximum cross-sectional area. : as described above. (Aspect 12) A method for isolating lymphocytes from a non-hematopoietic tissue sample, comprising: (i) Culturing a non-hematopoietic tissue sample, which is an intact biopsy having a volume of at least 2 mm obtained from a non-hematopoietic tissue, in the presence of IL-2 and IL-15; and 3 : as described above. (ii) Recovering a population of lymphocytes cultured from the non-hematopoietic tissue sample : The method as described above. (Aspect 13) A method for isolating γδ T cells from a non-hematopoietic tissue sample, comprising: : as described above. (i) Culturing a non-hematopoietic tissue sample, which is an intact biopsy having a volume of at least 2 mm obtained from a non-hematopoietic tissue, in the presence of IL-2 and IL-15; and 3 : as described above. (ii) Recovering a population of γδ T cells cultured from the non-hematopoietic tissue sample : The method as described above. : as described above. (Aspect 14) The non-hematopoietic tissue sample is at least 7 mm 3The method according to embodiment 12 or embodiment 13, having a volume of (Embodiment 15) wherein the non-hematopoietic tissue sample has a volume of about 21 mm 3 The method according to any one of embodiments 12 to 14, having a volume of . (Embodiment 16) wherein the non-hematopoietic tissue sample has a volume of 250 mm 3 The method according to any one of embodiments 12 to 15, having the following volume method. (Embodiment 17) wherein the non-hematopoietic tissue sample has a volume of 65 mm 3 The method according to any one of embodiments 12 to 16, having the following volume method. (Embodiment 18) The method according to any one of embodiments 1 to 17, wherein the non-invasive biopsy is obtained by punch biopsy . (Embodiment 19) The method according to embodiment 18, wherein the punch biopsy has a diameter of at least 2 mm. (Embodiment 20) The method according to embodiment 18, wherein the punch biopsy has a diameter of about 3 mm. (Embodiment 21) The method according to embodiment 18, wherein the punch biopsy has a diameter of 8 mm or less. (Embodiment 22) The method according to embodiment 18, wherein the punch biopsy has a diameter of 4 mm or less. (Embodiment 23) A method for isolating lymphocytes from a non-hematopoietic tissue sample, comprising (i) placing the non-hematopoietic tissue sample in a container containing a gas-permeable material; (ii) culturing the non-hematopoietic tissue sample in the presence of IL-2 and IL-15; and (iii) recovering a population of lymphocytes cultured from the non-hematopoietic tissue sample : The method as described above. (Embodiment 24) A method for isolating γδ T cells from a non-hematopoietic tissue sample, comprising (i) placing the non-hematopoietic tissue sample in a container containing a gas-permeable material; (ii) culturing the non-hematopoietic tissue sample in the presence of IL-2 and IL-15; and (iii) recovering a population of γδ T cells cultured from the non-hematopoietic tissue sample : The method as described above. (Aspect 25) The method according to aspect 23 or aspect 24, wherein the container comprises a liquid-sealed container containing a gas-permeable material for enabling gas exchange. The method according to aspect 23 or aspect 24. (Aspect 26) The method according to any one of aspects 23 to 25, wherein the bottom of the container is configured to enable gas exchange from the bottom of the container. The method according to any one of aspects 23 to 25. (Aspect 27) The method according to any one of aspects 23 to 26, wherein the non-hematopoietic tissue sample is disposed on a synthetic scaffold inside the container. The method according to any one of aspects 23 to 26. (Aspect 28) The method according to aspect 27, wherein the synthetic scaffold is tantalum-coated. (Aspect 29) The method according to aspect 27 or aspect 28, wherein the synthetic scaffold is configured to facilitate lymphocyte escape from the non-hematopoietic tissue sample to the bottom of the container. The method according to aspect 27 or aspect 28. (Aspect 30) The method according to any one of aspects 27 to 29, wherein the synthetic scaffold is configured to facilitate γδ T cell escape from the non-hematopoietic tissue sample to the bottom of the container. The method according to any one of aspects 27 to 29. (Aspect 31) The method according to any one of aspects 1, 6, 12, and 23, wherein the population of lymphocytes recovered from the culture of the non-hematopoietic tissue sample is a population of αβ T cells. The method according to any one of aspects 1, 6, 12, and 23. (Aspect 32) The method according to any one of aspects 1, 6, 12, and 23, wherein the population of lymphocytes recovered from the culture of the non-hematopoietic tissue sample is a population of NK cells. The method according to any one of aspects 1, 6, 12, and 23. (Aspect 33) The method according to any one of aspects 1 to 32, wherein the non-hematopoietic tissue sample is not disrupted before culturing. (Aspect 34) The method according to any one of aspects 1 to 33, wherein the lymphocytes or γδ T cells are recovered at least 7 days after culturing. (Aspect 35) The method according to any one of aspects 1 to 34, wherein the lymphocytes or γδ T cells are recovered at least 14 days after culturing. (Aspect 36) The method according to any one of aspects 1 to 35, wherein the lymphocytes or γδ T cells are recovered before 35 days of culturing. (Aspect 37) The method according to any one of aspects 1 to 36, wherein the lymphocytes or γδ T cells are recovered before 21 days of culturing. (Aspect 38) The method according to any one of aspects 1 to 37, wherein the non-hematopoietic tissue sample is cultured in a serum-free medium. (Aspect 39) The method according to any one of aspects 1 to 37, wherein the non-hematopoietic tissue sample is cultured in a medium containing serum or a serum substitute. (Aspect 40) The method according to any one of aspects 1 to 39, wherein the non-hematopoietic tissue sample is skin. (Aspect 41) The method according to aspect 40, wherein the non-hematopoietic tissue sample includes an epithelium and a dermis layer. (Aspect 42) The method according to any one of aspects 1 to 41, wherein the non-hematopoietic tissue sample is gastrointestinal or digestive tract. (Aspect 43) The method according to any one of aspects 1 to 42, wherein the non-hematopoietic tissue sample is obtained from a human. (Aspect 44) The method according to any one of aspects 1 to 43, wherein the IL-2 is human IL-2 or a functional equivalent thereof. ​ (Aspect 45) The method according to any one of Aspects 1 to 44, wherein the IL15 is human IL-15 or a functional equivalent thereof. Method. (Aspect 46) The method according to any one of Aspects 1 to 45, wherein the isolated cell population comprises a population of Vδ1 T cells. Method. (Aspect 47) The method according to Aspect 46, wherein the population of Vδ1 T cells expresses CD27 and / or does not substantially express TIGIT. Method. (Aspect 48) The method according to Aspect 46 or 47, wherein the population of Vδ1 T cells has a frequency of TIGIT+ cells of less than 80%. Method. (Aspect 49) The method according to any one of Aspects 46 to 48, wherein the population of Vδ1 T cells has a frequency of TIGIT+ cells of less than 60%. Method. (Aspect 50) The method according to any one of Aspects 46 to 49, wherein the population of Vδ1 T cells has a frequency of TIGIT+ cells of about 40%. Method. (Aspect 51) The method according to any one of Aspects 46 to 50, wherein the population of Vδ1 T cells has a frequency of TIGIT+ cells of about 30%. Method. (Aspect 52) The method according to any one of Aspects 46 to 51, wherein the population of Vδ1 T cells has a frequency of TIGIT+ cells of about 20%. Method. (Aspect 53) The method according to any one of Aspects 46 to 52, wherein the population of Vδ1 T cells has a frequency of TIGIT+ cells of about 10%. Method. (Aspect 54) The method according to any one of Aspects 46 to 53, wherein the population of Vδ1 T cells does not substantially express TIGIT. Method. (Aspect 55) The method according to any one of aspects 46 to 54, wherein the population of the Vδ1 T cells has CD27+ cells at a frequency of more than 10%. The method according to the item described above. (Aspect 56) The method according to any one of aspects 46 to 55, wherein the population of the Vδ1 T cells has CD27+ cells at a frequency of more than 20%. The method according to the item described above. (Aspect 57) The method according to any one of aspects 46 to 56, wherein the population of the Vδ1 T cells has CD27+ cells at a frequency of about 40%. The method according to the item described above. (Aspect 58) The method according to any one of aspects 46 to 57, wherein the population of the Vδ1 T cells has CD27+ cells at a frequency of about 80%. The method according to the item described above. (Aspect 59) The method according to any one of aspects 46 to 58, wherein the population of the Vδ1 T cells has CD27+ cells at a frequency of more than 80%. The method according to the item described above. (Aspect 60) The method according to any one of aspects 46 to 59, wherein the population of the Vδ1 T cells expresses CD27. (Aspect 61) The method according to any one of aspects 1 to 60, further comprising expanding the population of the isolated lymphocytes or γδ T cells. The method according to any one of the above items. (Aspect 62) A method for isolating and expanding lymphocytes from a non-hematopoietic tissue sample, comprising: (i) isolating a population of lymphocytes from the non-hematopoietic tissue sample according to the method described in any one of aspects 1 to 61; and (ii) further culturing the population of lymphocytes for at least 5 days to produce an expanded population of lymphocytes. (ii) further culturing the population of lymphocytes for at least 5 days to produce an expanded population of lymphocytes. The method described above, which includes the step of : producing. (Aspect 63) A method for isolating and expanding γδ T cells from a non-hematopoietic tissue sample, comprising: (i) isolating a population of γδ T cells from the non-hematopoietic tissue sample according to the method described in any one of aspects 1 to 62; and The step of isolating the population; and (ii) further culturing the population of γδ T cells for at least 5 days to produce an expanded population of γδ T cells : the method as described above. (Aspect 64) The method according to any one of Aspects 61 to 63, wherein the expansion step includes culturing the lymphocytes or γδ T cells in a serum-free medium. (Aspect 65) The method according to any one of Aspects 61 to 63, wherein the expansion step includes culturing the lymphocytes or γδ T cells in a medium containing serum or a serum substitute. (Aspect 66) The method according to any one of Aspects 63 to 65, wherein the expansion step includes culturing the γδ T cells in the absence of substantial stromal cell contact. (Aspect 67) The method according to any one of Aspects 63 to 66, wherein the expansion step includes the absence of an exogenous TCR pathway agonist. (Aspect 68) An isolated population of lymphocytes obtained by the method according to any one of Aspects 1 to 60. (Aspect 69) An isolated population of lymphocytes obtainable by the method according to any one of Aspects 1 to 60. (Aspect 70) An isolated population of γδ T cells obtainable by the method according to any one of Aspects 1 to 60. (Aspect 71) An isolated and expanded population of lymphocytes obtained by the method according to any one of Aspects 61 to 65. (Aspect 72) An isolated and expanded population of lymphocytes obtainable by the method according to any one of Aspects 61 to 65. (Aspect 73) ​​​​​​​​​An isolated and expanded γδ T cell population obtained by the method according to any one of aspects 61 to 65 cell population. (Aspect 74) An isolated and expanded γδ T cell population that can be obtained by the method according to any one of aspects 61 to 65

Claims

1. A method for isolating lymphocytes from a non-hematopoietic tissue sample, comprising: (i) culturing a non-hematopoietic tissue sample, which is an intact biopsy having a minimum cross-sectional area of at least 2 mm obtained from non-hematopoietic tissue, in the presence of interleukin-2 (IL-2) and interleukin-15 (IL-15), wherein the intact biopsy is obtained by punch biopsy, the step; and (ii) recovering a population of lymphocytes cultured from the non-hematopoietic tissue sample The method as described above. (ii) recovering a population of lymphocytes cultured from the non-hematopoietic tissue sample The method as described above.

2. The method according to claim 1, wherein the population of lymphocytes recovered from the culture of the non-hematopoietic tissue sample contains a population of γδ T cells. The method according to claim 1, wherein the population of lymphocytes recovered from the culture of the non-hematopoietic tissue sample contains a population of γδ T cells.

3. (i) the non-hematopoietic tissue sample has a minimum cross-sectional area of about 3 mm; (ii) the non-hematopoietic tissue sample has a maximum cross-sectional area of 8 mm or less; or (iii) both (i) and (ii), The method according to any one of claims 1 or 2.

4. The method according to any one of claims 1 to 3, wherein the method is performed in a container containing a gas-permeable material. The method according to any one of claims 1 to 3, wherein the method is performed in a container containing a gas-permeable material.

5. The method according to claim 4, wherein the non-hematopoietic tissue sample is placed on a synthetic scaffold inside the container. The method according to claim 4, wherein the non-hematopoietic tissue sample is placed on a synthetic scaffold inside the container.

6. The method according to claim 5, wherein the synthetic scaffold is tantalum-coated. 。

7. The method according to any one of claims 1 to 6, wherein the lymphocytes or γδ T cells are recovered after culturing for at least 7 days. The method according to any one of claims 1 to 6, wherein the lymphocytes or γδ T cells are recovered after culturing for at least 7 days.

8. The method according to any one of claims 1 to 7, wherein the non-hematopoietic tissue sample is (i) skin, (ii) gastrointestinal tract, or (iii) digestive tract. The method according to any one of claims 1 to 7, wherein the non-hematopoietic tissue sample is (i) skin, (ii) gastrointestinal tract, or (iii) digestive tract.

9. (i) the IL-2 is human IL-2; (ii) the IL-15 is human IL-15; or (iii) both (i) and (ii), The method according to any one of claims 1 to 8.

10. The population of lymphocytes recovered from the culture of the non-hematopoietic tissue sample contains a population of Vδ1 T cells, and and (i) the population of Vδ1 T cells has a frequency of TIGIT+ cells of less than 80%; (ii) the population of Vδ1 T cells has a frequency of CD27+ cells of more than 10%; or (iii) both (i) and (ii), The method according to any one of claims 2 to 9.

11. The method according to claim 1, wherein the population of lymphocytes recovered from the culture of the non-hematopoietic tissue sample contains a population of NK cells. The method according to claim 1, wherein the population of lymphocytes recovered from the culture of the non-hematopoietic tissue sample contains a population of NK cells.

12. The method according to any one of claims 1 to 11, further comprising expanding the population of the isolated lymphocytes as described in one item

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