Method for isolating and expanding cells

Abstract

The present invention relates to a method for isolating lymphocytes (particularly γδ T cells) from a non-hematopoietic tissue sample, comprising culturing the non-hematopoietic tissue sample in the presence of (a) interleukin-2 (IL-2) or interleukin-9 (IL-9); (b) interleukin-15 (IL-15); and (c) interleukin-21 (IL-21); and recovering the cultured population of lymphocytes from the non-hematopoietic tissue sample. Subsequent expansion methods, as well as the isolated cell populations obtained by the methods and uses thereof, are also provided.

Description

Technical Field

[0001] (Field of the Invention) The present invention relates to methods 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, and such non-hematopoietic tissues include skin and gastrointestinal tract. It will be understood that such isolated and / or expanded non-hematopoietic tissue resident lymphocytes are very useful in adoptive T cell therapy, chimeric receptor therapy, and the like. The present invention also relates to both individual cells and populations of 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 derepressed 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.

[0003] 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. Since specific γ and δ types are found in cells in one or more tissue types, although not exclusively, human γδ T cells can be broadly classified based on their TCR chains. For example, most blood resident γδ T cells express the Vδ2 TCR, such as Vγ9Vδ2, which is less common among tissue resident γδ T cells, which 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 blood. Non-hematopoietic tissue-resident lymphocytes such as αβ T cells, γδ T cells, and NK cells can have properties that are particularly suitable for certain applications, for example, 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 8 cells or more are required for many indications. Importantly, significant cell loss during production means that even more starting cells must be generated.

[0005] Non-hematopoietic tissue-resident lymphocytes, particularly αβ T cells, γδ T cells, and NK cells, are not easily obtained in large numbers and have therefore not been fully characterized or studied for therapeutic applications. Therefore, there is a need in the art for methods to isolate and expand non-hematopoietic tissue-resident lymphocytes, particularly γδ T cells, to a sufficient amount for study and potentially for use as a therapy, such as adoptive T cell therapy.

[0006] Clark et al. (2006) J. Invest. Dermatol. 126(5): 1059-70 describes a method for isolating skin-resident T cells from normal and diseased skin. However, the method described therein has a relatively low yield of isolated cells, particularly due to the presence of animal products, i.e., 10 2 cells per cm of tissue 6Since it is less than this amount, it is not suitable for clinical use. In the method described in the literature of Clark et al., a crushed sample is used, which results in an intentional destruction of the structural integrity of the tissue sample. WO2017072367 and WO2018 / 202808 relate to methods 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, which contains 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) culturing the non-hematopoietic tissue sample (a) in the presence of interleukin-2 (IL-2) or interleukin-9 (IL-9); (b) interleukin-15 (IL-15); and (c) interleukin-21 (IL-21) ; and (ii) recovering a population of lymphocytes cultured from the non-hematopoietic tissue sample : is provided.

[0008] 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 the non-hematopoietic tissue sample (a) in the presence of IL-2 or IL-9; (b) IL-15; and (c) IL-21 ; and (ii) Recovering a population of γδ T cells cultured from the non-hematopoietic tissue sample : A method is provided that includes

[0009] According to a further aspect of the present invention, 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 for at least 5 days to produce an expanded population of lymphocytes : A method is provided that includes

[0010] 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 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

[0011] (Brief Description of the Drawings)

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Mode for Carrying Out the Invention

[0012] (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) subjecting the non-hematopoietic tissue sample to (a) interleukin-2 (IL-2) or interleukin-9 (IL-9); (b) Interleukin-15 (IL-15); and (c) Interleukin-21 (IL-21) : a step of culturing in the presence of; and (ii) a step of recovering a population of lymphocytes cultured from the non-hematopoietic tissue sample : A method is provided that includes.

[0013] According to a further aspect of the present invention, a method for isolating γδ T cells from a non-hematopoietic tissue sample, comprising: (i) the non-hematopoietic tissue sample is (a) IL-2 or IL-9; (b) IL-15; and (c) IL-21 : a step of culturing in the presence of; and (ii) a step of recovering a population of γδ T cells cultured from the non-hematopoietic tissue sample : A method is provided that includes.

[0014] References herein to "isolating" or "isolation" of cells, particularly lymphocytes and / or γδ T cells, refer to methods or processes by which cells are removed, separated, purified, concentrated, or otherwise removed from a tissue or pool of cells. Such references will be understood to include the terms "separated," "removed," "purified," "concentrated," and like terms. Isolation of γδ T cells includes isolation or separation of cells from intact non-hematopoietic tissue samples or from stromal cells of non-hematopoietic tissue (e.g., fibroblasts or epithelial cells). Such isolation may alternatively or additionally include isolation or separation of γδ T cells from other hematopoietic cells (e.g., αβ T cells or other lymphocytes). Isolation can begin when an explant or biopsy of tissue is placed into an isolation culture and end when the cells are recovered from the culture, e.g., by centrifugation or other means for expanding the isolated cell population into a culture, or used for other purposes, or when the original tissue explant or biopsy is removed from the culture. The isolation process may be at least about 3 days to about 45 days. In one embodiment, the isolation process is at least about 10 days to at least 28 days. In a further embodiment, the isolation process is at least 14 days to at least 21 days. Thus, the isolation process may be at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, about 35, about 40, or about 45 days. It can be understood that during this isolation process, the proliferation of the isolated cells may not be significant, but it is not necessarily the case that there is no proliferation of the cells. Indeed, some skilled artisans will recognize that the isolated cells can begin to divide and generate multiple such cells within an isolation vessel containing tissue and / or a scaffold.

[0015] Accordingly, references herein to "isolated γδ T cells", "isolated γδ T cell population", "population of isolated γδ T cells", "separated γδ T cells", "separated γδ T cell population", or "population of separated γδ T cells" are understood to refer to hematopoietic cells or a population of hematopoietic cells containing γδ cells that have been isolated, separated, removed, purified, or enriched 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 "population of isolated or separated Vδ1 T cells" refer to hematopoietic cells containing Vδ1 T cells that have been isolated, separated, removed, purified, or enriched 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. Thus, isolation or separation refers to the isolation, separation, removal, purification, or enrichment of hematopoietic cells (e.g., γδ T cells or other lymphocytes) from non-hematopoietic cells (e.g., stromal cells, fibroblasts, and / or epithelial cells).

[0016] The method for isolating γδ T cells as defined herein can include tissue disruption (e.g., mincing) and subsequent separation of γδ T cells from other cell types. Preferably, the method for isolating γδ T cells as defined herein can include the “creeping out” of γδ T cells and other cell types from intact non-hematopoietic tissue samples or explants or biopsy tissue matrices, where tissue resident lymphocytes physically detach 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 preferentially escape from the tissue matrix with little or no escape of inhibitory cell types such as fibroblasts that are retained in the explant or biopsy and can be easily removed at the end of isolation later. 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 from the tissue into the culture. Such a “creeping out” method utilizing intact non-hematopoietic tissue or tissue matrices has the advantage of reducing the need for excessive processing of non-hematopoietic tissue samples or tissue matrices, maintaining the structural integrity of the non-hematopoietic tissue or tissue matrix, and providing the unexpected advantage of resulting in a higher isolated cell yield.

[0017] Accordingly, 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 an intact biopsy or explant is one in which the structural integrity of the biopsy or explant has not been intentionally disrupted within the perimeter of the excision that removes the biopsy or explant from the tissue sample. Such an intact biopsy or explant has a three-dimensional structure that is mostly maintained except for minor disruptions caused by manipulation. Therefore, this intact biopsy or explant has not been mechanically disrupted, for example, by crushing or mincing, 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.

[0018] The method for isolating γδ T cells using "crawling out" or, for example, the method as defined herein can include culturing cells and / or non-hematopoietic tissue samples in the presence of cytokines and / or chemokines sufficient to induce the isolation or separation of γδ T cells and / or other lymphocytes as defined herein. Accordingly, in one embodiment of the present invention, the isolation of γδ T cells from a non-hematopoietic tissue sample includes culturing the non-hematopoietic tissue sample in the presence of IL-2, IL-15, and IL-21. In an alternative embodiment, the isolation of γδ T cells from a non-hematopoietic tissue sample includes culturing the non-hematopoietic tissue sample in the presence of IL-9, IL-15, and IL-21.

[0019] In one embodiment, the isolation of γδ T cells according to the first aspect of the present invention further comprises culturing a non-hematopoietic tissue sample in the presence of interleukin-4 (IL-4). Thus, in a further embodiment, the non-hematopoietic tissue sample is cultured in the presence of IL-2, IL-15, IL-21, and IL-4. In an alternative further embodiment, the non-hematopoietic tissue sample is cultured in the presence of IL-9, IL-15, IL-21, and IL-4.

[0020] As used herein, "IL-2" refers to native or recombinant IL-2 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 interleukin-2 receptor (IL-2R) subunits. Such an agent can support the growth of CTLL-2 (33; American Type Culture Collection (ATCC®) TIB 214), an IL-2-dependent cell line. Mature human IL-2 results in a 133 amino acid sequence (minus a signal peptide consisting of an additional 20 N-terminal amino acids) as described in Fujita et al., Cell 1986. 46.3:401-407. An IL-2 mutein is a polypeptide that has undergone specific substitutions to the interleukin-2 protein while retaining the ability to bind to IL-2Rβ, such as the polypeptides described in US 2014 / 0046026. An IL-2 mutein can be characterized by one or more 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 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions.

[0021] 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_000577.2 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.

[0022] 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 with similar physicochemical characteristics. Examples of conservative substitutions include the substitution of one aliphatic residue for another, such as the interchange of Ile, Val, Leu, or Ala, 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 an entire region with similar hydrophobic characteristics, are well known. Native 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 properties are retained. Alternative splicing of mRNA can yield a biologically active IL-2 protein that is 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-2 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).

[0023] As used herein, "IL-15" refers to native or recombinant IL-15 or variants thereof that act as agonists (e.g., mutants, muteins, analogs, subunits, receptor complexes, fragments, isoforms, and peptidomimetics) of one or more IL-15 receptor (IL-15R) subunits. IL-15, like IL-2, is a known T cell growth factor that can support the growth of the IL-2-dependent cell line CTLL-2. IL-15 was first reported as a 114-amino acid mature protein by Grabstein et al. (Grabstein et al., Science 1994. 264.5161: 965-969). The term "IL-15" as used herein means native or recombinant IL-15 and its mutants, analogs, subunits, or complexes thereof (e.g., receptor complexes, e.g., the sushi peptide described in WO 2007 / 046006), each of which can stimulate the growth of CTLL-2 cells. In the CTLL-2 proliferation assay, the supernatant of cells transfected with an in-frame fusion of recombinantly expressed precursor and mature forms of IL-15 can induce CTLL-2 cell proliferation.

[0024] 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 at the ATCC® on February 19, 1993, and was assigned accession number 69245.

[0025] The amino acid sequence of human IL-15 (Gene ID 3600) is found in Genbank under the accession locators NP000576.1 GI: 10835153 (isoform 1) and NP_751915.1 GI: 26787986 (isoform 2). The murine (Mus musculus) IL-15 amino acid sequence (Gene ID 16168) is found in Genbank under the accession locator NP_001241676.1 GI: 363000984.

[0026] IL-15 can also refer to IL-15 derived from various mammalian species, including, for example, humans, monkeys, cows, pigs, horses, and mice. As used herein, an IL-15 "mutant" or "variant" is a polypeptide that is substantially homologous to the sequence of native mammalian IL-15 but has an amino acid sequence that differs from the native mammalian IL-15 polypeptide due to amino acid deletions, insertions, or substitutions. 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 replacement of one aliphatic residue with another, such as the substitution of Ile, Val, Leu, or Ala for one another, or the substitution of one polar residue with another, such as the substitution between Lys and Arg; between Glu and Asp; or between Gln and Asn. 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 mRNA can yield 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 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 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).

[0027] As used herein, "IL-4" refers to native or recombinant IL-4 or variants thereof that act as agonists (e.g., mutants, muteins, analogs, subunits, receptor complexes, fragments, isoforms, and peptidomimetics) of one or more IL-4 receptor (IL-4R) subunits. Such agents can support the differentiation of naive helper T cells (Th0 cells) into Th2 cells. Mature human IL-4 results as a 129 amino acid sequence (minus the signal peptide consisting of an additional 24 N-terminal amino acids). An IL-4 mutein is a polypeptide that has specific substitutions made to the interleukin-4 protein while retaining the ability to bind to IL-4Rα, such as the polypeptides described in U.S. Patent No. 6,313,272. An IL-4 mutein can be characterized by amino acid insertions, deletions, substitutions, and modifications at one or more sites of other residues of the native IL-4 polypeptide chain. According to the present disclosure, any such insertions, deletions, substitutions, and modifications result in an IL-4 mutein that retains IL-2Rα binding activity. Exemplary muteins can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions.

[0028] Nucleic acids encoding human IL-4 can be obtained by conventional procedures such as polymerase chain reaction (PCR). The amino acid sequence of human IL-4 (Gene ID 3565) is found in Genbank under the accession locator NG_023252. The murine (Mus musculus) IL-4 amino acid sequence (Gene ID 16189) is found in Genbank under the accession locator NC_000077.6.

[0029] IL-4 can also refer to IL-4 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 with a residue having similar physicochemical characteristics. Examples of conservative substitutions include the substitution of one aliphatic residue for another, for example, the interchange of Ile, Val, Leu, or Ala, 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 an entire region having similar hydrophobic characteristics, are well known. Native IL-4 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-4 protein, where the IL-4 binding characteristics are retained. Alternative splicing of mRNA can result in an IL-4 protein that is truncated but biologically active. 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-4 protein. In some embodiments, the termini of the protein can be modified with a chemical group, 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).

[0030] As used herein, "IL-21" refers to native or recombinant IL-21 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-21 receptor (IL-21R) subunits. Such agents include natural killer (NK) and cytotoxic (CD8 +) It can support the proliferation of T cells. Mature human IL-21 results as a 133-amino acid sequence (subtracting the signal peptide consisting of an additional 22 N-terminal amino acids). An IL-21 mutein is a polypeptide that has specific substitutions made to the interleukin-21 protein while retaining the ability to bind to IL-21Rα, for example, the polypeptide described in U.S. Patent No. 9,388,241. An IL-21 mutein can be characterized by amino acid insertions, deletions, substitutions, and modifications at one or more sites of other residues of the native IL-21 polypeptide chain. According to the present disclosure, any such insertions, deletions, substitutions, and modifications result in an IL-21 mutein that retains IL-21R binding activity. Exemplary muteins can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions.

[0031] Nucleic acids encoding human IL-21 can be obtained by conventional procedures such as polymerase chain reaction (PCR). The amino acid sequence of human IL-21 (Gene ID 59067) is found in Genbank under the accession locator NC_000004.12. The murine (Mus musculus) IL-21 amino acid sequence (Gene ID 60505) is found in Genbank under the accession locator NC_000069.6.

[0032] IL-21 can also refer to IL-21 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, 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, such as the substitution of entire regions having similar hydrophobic characteristics, are well known. Native IL-21 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-21 protein, where the IL-21 binding properties are retained. Alternative splicing of mRNA can result in an IL-21 protein that is truncated but biologically active. Mutations resulting from proteolysis can include, for example, differences in the N- or C-terminus upon expression in various host cells due to proteolytic removal of one or more terminal amino acids (usually 1 to 10 amino acids) from the IL-21 protein. In some embodiments, the ends of the protein can be modified with chemical groups, such as polyethylene glycol, for example, to alter its physical properties (Yang et al., Cancer 1995. 76: 687-694). In some embodiments, the ends or interior of the protein can be modified with additional amino acids (Clark-Lewis et al., PNAS 1993. 90:3574-3577).

[0033] As used herein, "IL-9" refers to native or recombinant IL-9 or variants thereof that act as agonists (e.g., mutants, muteins, analogs, subunits, receptor complexes, fragments, isoforms, and peptidomimetics) of one or more IL-9 receptor (IL-9R) subunits. Mature human IL-9 results in an amino acid sequence of 144 amino acids. An IL-9 mutein is a polypeptide that has specific substitutions made to the interleukin-9 protein while retaining the ability to bind to IL-9R. An IL-9 mutein can be characterized by amino acid insertions, deletions, substitutions, and modifications at one or more sites of other residues of the native IL-9 polypeptide chain. According to the present disclosure, any such insertions, deletions, substitutions, and modifications result in an IL-9 mutein that retains IL-9R binding activity. Exemplary muteins can include substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids.

[0034] Nucleic acids encoding human IL-9 can be obtained by conventional procedures such as polymerase chain reaction (PCR). The amino acid sequence of human IL-9 is provided by UniProtKB P15248.

[0035] IL-9 can also refer to IL-9 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, 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. Native IL-9 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-9 protein, where the IL-9 binding properties are retained. Alternative splicing of mRNA can result in truncated but biologically active IL-9 proteins. Mutations resulting from proteolysis can 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-9 protein. In some embodiments, the ends of the protein can be modified with chemical groups, such as polyethylene glycol, for example, to alter its physical properties (Yang et al., Cancer 1995. 76: 687-694). In some embodiments, the ends or interior of the protein can be modified with additional amino acids (Clark-Lewis et al., PNAS 1993. 90:3574-3577).

[0036] In certain embodiments, the methods defined herein typically include IL-2 at a concentration of at least 10 IU / mL, such as 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 / mL to 250 IU / mL, or 100 IU / mL to 200 IU / mL, e.g., 10 IU / mL to 20 IU / mL, 20 IU / mL to 30 IU / mL, 30 IU / mL to 40 IU / mL, 40 IU / mL to 50 IU / mL, 50 IU / mL to 75 IU / mL, 75 IU / mL to 100 IU / mL, 100 IU / mL to 150 IU / mL, 150 IU / mL to 200 IU / mL, 200 IU / mL to 500 IU / mL, or 500 IU / mL to 1,000 IU / mL). In certain embodiments, the methods defined herein typically include IL-2 at a concentration of less than 1,000 IU / mL, such as less than 500 IU / mL. In some embodiments, the method includes IL-2 at a concentration of about 100 IU / mL.

[0037] In further embodiments, the methods defined herein typically include IL-15 at a concentration of at least 0.1 ng / mL, such as 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, e.g., 0.1 ng / mL to 1.0 ng / mL, 1.0 ng / mL to 5.0 ng / mL, 5.0 ng / mL to 10 ng / mL, 10 ng / mL to 20 ng / mL, 20 ng / mL to 100 ng / mL, 20 ng / mL to 50 ng / mL, 40 ng / mL to 70 ng / 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 / mL, or 500 ng / mL to 1,000 ng / mL). In further embodiments, the methods defined herein typically include IL-15 at a concentration less than 500 ng / mL, such as less than 100 ng / mL. In some embodiments, the method includes IL-15 at a concentration of about 50 ng / mL.

[0038] In some embodiments, the isolation of γδ T cells from non-hematopoietic tissue samples includes culturing in the presence of both IL-2 and IL-15, each 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.

[0039] In a further embodiment, the method defined herein typically comprises IL-21 at a concentration of at least 0.1 ng / mL, such as at least 1.0 ng / mL (e.g., 0.1 ng / mL to 1,000 ng / mL, 1.0 ng / mL to 100 ng / mL, 1.0 ng / mL to 50 ng / mL, 2 ng / mL to 50 ng / mL, 3 ng / mL to 10 ng / mL, 4 ng / mL to 8 ng / mL, 5 ng / mL to 10 ng / mL, 6 ng / mL to 8 ng / mL, e.g., 0.1 ng / mL to 10 ng / mL, 1.0 ng / mL to 5 ng / mL, 1.0 ng / mL to 10 ng / mL, 1.0 ng / mL to 20 ng / mL). In a further embodiment, the method defined herein typically comprises IL-21 at a concentration of less than 100 ng / mL, such as less than 50 ng / mL. In some embodiments, the method comprises IL-21 at a concentration of about 6 ng / mL, such as about 6.25 ng / mL.

[0040] In a further embodiment, the method defined herein typically comprises IL-4 at a concentration of at least 0.1 ng / mL, such as at least 10 ng / mL (e.g., 0.1 ng / mL to 1,000 ng / mL, 1.0 ng / mL to 100 ng / mL, 1.0 ng / mL to 50 ng / mL, 2 ng / mL to 50 ng / mL, 3 ng / mL to 40 ng / mL, 4 ng / mL to 30 ng / mL, 5 ng / mL to 20 ng / mL, 10 ng / mL to 20 ng / mL, e.g., 0.1 ng / mL to 50 ng / mL, 1.0 ng / mL to 25 ng / mL, 5 ng / mL to 25 ng / mL). In a further embodiment, the method defined herein typically comprises IL-4 at a concentration of less than 100 ng / mL, such as less than 50 ng / mL, particularly less than 20 ng / mL. In some embodiments, the method comprises IL-4 at a concentration of about 15 ng / mL.

[0041] 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 obtained according to the methods defined herein is skin (e.g., human skin), which can be obtained by methods known in the art. Alternatively, the methods of isolating lymphocytes and / or γδ T cells provided herein can be applied to the digestive tract (e.g., colon or gastrointestinal tract), mammary gland, lung, prostate, liver, spleen, pancreas, uterus, vagina, and other cutaneous membranes, mucosae, or serosae. Lymphocytes and / or γδ T cells can reside in human cancer tissue samples, such as tumors of the breast or prostate. In some embodiments, lymphocytes and / or γδ T cells can be from a human cancer tissue sample (e.g., solid tumor tissue). In other embodiments, lymphocytes and / or γδ T cells can be from a non-hematopoietic tissue sample other than human cancer tissue (e.g., tissue that does not contain a significant number of tumor cells). For example, lymphocytes and / or γδ T cells can be from an area of skin (e.g., healthy skin) that is distant from or adjacent to 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.

[0042] In one embodiment, the non-hematopoietic tissue sample of the methods defined herein is obtained from a human. In an alternative embodiment, the non-hematopoietic tissue sample of the methods defined herein is obtained from a non-human animal subject.

[0043] Methods for obtaining such tissue are known in the art. Examples of such methods include scalpel explants or punch biopsies, which can vary in size. In some embodiments, the non-hematopoietic tissue sample is obtained by punch biopsy.

[0044] In some embodiments of the present invention, the non-hematopoietic tissue sample is a non-invasive biopsy. As used herein, the reference to a "non-invasive" biopsy or "explant" includes tissue and tissue samples that are not substantially disrupted or disrupted such that the structural integrity of the biopsy or explant is 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 the procedure. 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. It will be understood that in such embodiments where the biopsy is non-invasive, the separation and distinction of such layers are maintained. Thus, the reference to "non-invasive" herein further includes biopsies of all layers of the non-hematopoietic tissue sample.

[0045] Thus, in one 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-minced and / or non-invasive non-hematopoietic tissue sample. Further, as shown herein, cells obtained from a non-minced and / or non-invasive non-hematopoietic tissue sample according to the methods defined herein can retain phenotypes useful for subsequent expansion and / or manipulation methods known in the art.

[0046] In a further embodiment, the non-invasive biopsy is skin (e.g., human skin), or the non-invasive biopsy is gastrointestinal (e.g., human gastrointestinal). In one embodiment, the non-hematopoietic tissue sample has a minimum cross-sectional dimension of at least 1 mm. It will be understood that the "minimum cross-sectional dimension" refers to the minimum or shortest length measured through the centroid of the tissue sample. It will further be understood that the "maximum cross-sectional dimension" refers to the maximum or longest length measured through the centroid of the tissue sample. As used herein, the term "centroid" is the average or mean position of all points of the tissue sample. According to a further embodiment, it will be understood that the non-hematopoietic tissue sample has a minimum cross-sectional dimension 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. In a further embodiment, the non-hematopoietic tissue sample has a minimum cross-sectional dimension 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 minimum cross-sectional dimension of from 1 mm to 8 mm (inclusive), for example, from 2 mm to 4 mm. In a particular embodiment, the non-hematopoietic tissue sample has a minimum cross-sectional dimension of about 3 mm. In a particular embodiment, the non-hematopoietic tissue sample has a cross-sectional dimension of about 3 mm. According to a further embodiment, it will be understood that the non-hematopoietic tissue sample has a maximum cross-sectional dimension 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. In a further embodiment, the non-hematopoietic tissue sample has a maximum cross-sectional dimension 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-sectional dimension of from 1 mm to 8 mm (inclusive), for example, from 2 mm to 4 mm. In a particular embodiment, the non-hematopoietic tissue sample has a maximum cross-sectional dimension of about 3 mm.

[0047] According to a further embodiment, the non-hematopoietic tissue sample is at least 1 mm 2has a minimum cross-sectional area. It will be understood that "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 "maximum cross-sectional area" refers to the area of the largest cross-section measured around the centroid of the tissue sample. As used herein, the term "centroid" is the average or mean position of all points of the tissue sample. In a further embodiment, the non-hematopoietic tissue sample is at least 2 mm 2 at least 3 mm 2 at least 4 mm 2 at least 5 mm 2 at least 6 mm 2 at least 7 mm 2 at least 8 mm 2 at least 9 mm 2 or at least 10 mm 2 has a minimum cross-sectional area. 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 mm 2 or less, 10 mm 2 or less, or 8 mm 2 has a minimum 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 has a minimum cross-sectional area. In a particular embodiment, the non-hematopoietic tissue sample has a minimum cross-sectional area of about 7 mm 2 In a further embodiment, the non-hematopoietic tissue sample is at least 2 mm 2 at least 3 mm 2 at least 4 mm 2 at least 5 mm 2 at least 6 mm 2 at least 7 mm 2 at least 8 mm 2 at least 9 mm 2 or at least 10 mm 2has a maximum cross-sectional area. 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 mm 2 or less, 10 mm 2 or less, or 8 mm 2 or less in 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 in maximum cross-sectional area. In a particular embodiment, the non-hematopoietic tissue sample is about 7 mm 2 in maximum cross-sectional area.

[0048] According to a further embodiment, the non-hematopoietic tissue sample has a volume of at least 5 mm 3 . In a further embodiment, the non-hematopoietic tissue sample has a volume of at least 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 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 volume. In a further embodiment, the non-hematopoietic tissue sample is 250 mm 3 or less, 200 mm 3 or less, for example, 180 mm 3 or less, 1600 mm 3 or less, 140 mm 3 or less, 120 mm 3 or less, 100 mm 3 or less, 80 mm 3 or less, 60 mm 3 or less, 50 mm 3 or less, or 40 mm 3 or less in volume. In one embodiment, the non-hematopoietic tissue sample is 5 mm3 ~250 mm 3 、 for example, 15 mm 3 ~65 mm 3 and has a volume of. In certain embodiments, the non-hematopoietic tissue sample is about 35 mm 3 in volume.

[0049] In one embodiment, the non-hematopoietic tissue sample is a punch biopsy. The punch biopsy can be of any shape, but advantageously has a circular cross-section and preferably has a diameter of at least 1 mm. In a further embodiment, the non-hematopoietic tissue sample includes a punch biopsy with a diameter of at least 2 mm, for example, 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. In a further embodiment, the non-hematopoietic tissue sample includes a punch biopsy with a diameter of 8 mm or less, for example, 7 mm or less, 6 mm or less, 5 mm or less, or 3 mm or less. In one embodiment, the non-hematopoietic tissue sample includes a punch biopsy with a diameter of 1 mm to 8 mm, for example, 2 mm to 4 mm. In certain embodiments, the non-hematopoietic tissue sample includes a punch biopsy with a diameter of 3 mm.

[0050] In certain embodiments, the non-hematopoietic tissue sample includes a biopsy (e.g., a punch biopsy, particularly a punch biopsy with a circular cross-section) according to the size, area, volume, and / or diameter defined above, 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 thus is 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.

[0051] The method of the present invention involves culturing a non-hematopoietic tissue sample as defined herein. References herein to "culturing" include adding the cells and / or non-hematopoietic tissue sample to a medium containing growth factors and / or essential nutrients required and / or preferred by the cells and / or non-hematopoietic tissue sample, including cells isolated, separated, removed, purified, or concentrated from the non-hematopoietic tissue sample. Such culture conditions can be adapted in light of the cells or cell populations that will be isolated from the non-hematopoietic tissue sample according to the present invention, or in light of the cells or cell populations that will be isolated and expanded from the non-hematopoietic tissue sample, as will be understood.

[0052] In certain embodiments, culturing of the non-hematopoietic tissue sample is for a period sufficient to isolate γδ T cells from the non-hematopoietic tissue sample. In alternative embodiments, culturing of the non-hematopoietic tissue sample is for a period sufficient to isolate 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 as defined herein is at least 14 days. In certain embodiments, the culturing period by the method as 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 as 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 as defined herein is about 21 days.

[0053] In certain embodiments of the present invention, 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. Recovery of lymphocytes and / or γδ T cells as defined herein can include physical recovery of lymphocytes and / or γδ T cells from the culture, isolation of 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 lymphocytes and / or γδ T cells from stromal cells (e.g., fibroblasts). In one embodiment, lymphocytes and / or γδ T cells are recovered by mechanical means (e.g., pipetting). In a further embodiment, lymphocytes and / or γδ T cells are recovered by magnetic separation and / or labeling. In yet a further embodiment, lymphocytes and / or γδ T cells are recovered by flow cytometry techniques, e.g., 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. It will be understood that such recovery of lymphocytes and / or γδ T cells can 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.

[0054] 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 a non-hematopoietic tissue sample. In certain embodiments, the lymphocytes and / or γδ T cells are recovered 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 of the non-hematopoietic tissue sample. Preferably, the lymphocytes and / or γδ T cells are recovered within 40 days, such as within 38 days, within 36 days, within 34 days, within 32 days, within 30 days, within 28 days, within 26 days, or within 24 days. In one embodiment, the lymphocytes and / or γδ T cells are recovered at least 14 days after culturing of the non-hematopoietic tissue sample. In a further embodiment, the lymphocytes and / or γδ T cells are recovered 14 to 21 days after culturing of the non-hematopoietic tissue sample.

[0055] In certain embodiments of the invention, the non-hematopoietic tissue sample is cultured in a medium substantially free of serum (e.g., a serum-free medium or a medium containing a serum replacement (SR)). Thus, in one embodiment, the non-hematopoietic tissue sample is cultured in a serum-free medium. Such serum-free media can also include serum replacement media based on chemically defined components to avoid the use of human or animal-derived serum. 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 a serum replacement. In one embodiment, the non-hematopoietic tissue sample is cultured in a medium free of animal-derived products.

[0056] Embodiments according to the invention in which non-hematopoietic tissue samples are cultured in serum-free medium will be understood to have the advantage of avoiding problems associated with 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 also found that the use of serum-free medium 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 medium containing AB serum. In particular, the isolation of γδ T cells from non-hematopoietic tissue samples cultured in serum-free medium increases the yield of Vδ1 cells.

[0057] In one embodiment, the methods defined herein are performed in an isolation container. References to an "isolation container" refer 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.

[0058] In one embodiment, the methods defined herein are performed in a container (e.g., an isolation container) that includes a gas-permeable material. 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. References herein to "container" are 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 that include a gas-permeable material surprisingly find utility even in the isolation of γδ T cells, which are normally thought to be adhesive. Use of such containers in culture has been found to greatly increase the yield of isolated γδ T cells from non-hematopoietic tissue samples. Such containers have also been found to preferentially support γδ T cells and other lymphocytes over fibroblast and other stromal cell types (e.g., epithelial cells) that include adherent cell types. Thus, in one embodiment, a container that includes 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 cell types (e.g., epithelial cells) are not present in the cultures performed in a container that includes a gas-permeable material.

[0059] Such a container containing a gas-permeable material may further include 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 a container may include only a part of the gas-permeable material, a gas-permeable membrane film, or a non-porous gas-permeable material. Thus, according to yet a further embodiment, the container includes a lid, a bottom, and at least one side wall, wherein at least a part of the bottom of the container includes a gas-permeable material on a substantially horizontal plane when the lid is above the bottom. In one embodiment, the container includes a lid, a bottom, and at least one side wall, wherein at least a part of the bottom includes a gas-permeable material on a horizontal plane when the lid is above the bottom. In a further embodiment, the container includes a lid, a bottom, and at least one side wall, wherein the at least one side wall may be on a vertical plane when the lid is above the bottom, or may be on a horizontal plane when the lid is not above the bottom, and includes a gas-permeable material. In such an embodiment, 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 yet a further embodiment, the lid of the container containing the gas-permeable material may be sealed, for example, by the use of an O-ring. It will be understood that such an embodiment prevents leakage of the contents of the container or reduces its evaporation. Thus, in one embodiment, the container includes a liquid-tight container containing a gas-permeable material for enabling gas exchange. In an alternative embodiment, the lid of the container containing the gas-permeable material is on a horizontal plane, above the bottom, and is not sealed. Thus, in one embodiment, the lid is configured to enable gas exchange from the lid of the container. In a further embodiment, the bottom of the gas-permeable container is configured to enable gas exchange from the bottom of the container. In yet a further embodiment, the container containing 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, a container comprising a gas-permeable material includes a lid, a bottom, and optionally at least one sidewall, where at least a portion of the lid and the bottom comprises the gas-permeable material and, if present, at least a portion of at least one sidewall comprises the gas-permeable material. Examples of containers are described in WO2005035728 and US9255243, which are incorporated herein by reference. These containers are also commercially available and include, for example, the G-REX® cell culture devices provided by Wilson Wolf Manufacturing, such as the G-REX6 well plate, the G-REX24 well plate, and the G-REX10 container.

[0060] In one embodiment, the non-hematopoietic tissue sample is placed on a synthetic scaffold. As used herein, "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 can be placed on or attached to the synthetic scaffold to facilitate lymphocyte escape from the explant onto the scaffold. The synthetic scaffold can be constructed from natural and / or synthetic materials such as polymers (e.g., natural or synthetic polymers such as polyvinylpyrrolidone, polymethylmethacrylate, methylcellulose, polystyrene, polypropylene, polyurethane), ceramics (e.g., tricalcium phosphate, calcium aluminate, calcium hydroxyapatite), or metals (e.g., tantalum, titanium, platinum, and metals of the same elemental family as platinum, niobium, hafnium, tungsten, and combinations of these alloys). In one embodiment of the invention, the synthetic scaffold is tantalum-coated. Biological factors (e.g., collagen (e.g., collagen I or collagen II), fibronectin, laminin, integrin, angiogenic factors, anti-inflammatory factors, glycosaminoglycans, vitronectin, antibodies and fragments thereof, cytokines (e.g., IL-2, IL-15, IL-4, IL-21, IL9, and combinations thereof)) can be coated on the scaffold surface, encapsulated within the scaffold material, or added to the medium according to methods known in the art to enhance cell adhesion, migration, survival, or proliferation. Using this and other methods, lymphocytes can be isolated from several other non-hematopoietic tissue types, such as skin, gastrointestinal, prostate, and breast.

[0061] In one embodiment, the non-hematopoietic tissue sample is placed on a synthetic scaffold inside a container used to isolate lymphocytes from the non-hematopoietic tissue sample. In a further embodiment, the synthetic scaffold is configured to facilitate lymphocyte and / or γδ T cell escape from the non-hematopoietic tissue sample to the bottom of the container. 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 the non-hematopoietic tissue sample 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 the non-hematopoietic tissue sample to the bottom of the culture container. In certain embodiments, the synthetic scaffold is configured to facilitate the escape of γδ T cells from the non-hematopoietic tissue sample. In a further embodiment, the synthetic scaffold is configured to facilitate the escape of lymphocytes, e.g., αβ T cells and / or NK cells, from the non-hematopoietic tissue sample.

[0062] Thus, in one aspect of the method defined herein, the synthetic scaffold is configured to facilitate lymphocyte escape from the non-hematopoietic tissue sample to the bottom of the culture container. In a further aspect of the method defined herein, the synthetic scaffold is configured to facilitate γδ T cell escape from the non-hematopoietic tissue sample to the bottom of the culture container.

[0063] The method of the present invention provides 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 , at least 2×10 6 cells / cm 2 , at least 5×10 6 cells / cm 2 , at least 10×10 6 cells / cm 2 , at least 20×10 6 cells / cm 2 , at least 30×10 6 cells / cm2 , at least 40×10 6 cells / cm 2 , at least 50×10 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 150×10 6 cells / cm 2 , at least 200×10 6 cells / cm 2 . In a specific embodiment, the total number of isolated cells is at least 50×10 6 cells / cm 2 . In another embodiment, the total number of isolated cells is at least 100×10 6 cells / cm 2 .

[0064] 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 about 70-80% of the non-hematopoietic tissue-resident γδ T cell population. However, some Vδ2 T cells are also found in non-hematopoietic tissues, such as the gastrointestinal tract, where they can contain about 10-20% of the γδ T cells. Some of the γδ T cells resident in non-hematopoietic tissues do not express either Vδ1 TCR or Vδ2 TCR and are referred to herein as double-negative (DN) γδ T cells. Most of these DN γδ T cells are likely to express Vδ3, and Vδ5-expressing T cells are in the minority. Therefore, γδ T cells that are routinely resident in non-hematopoietic tissues and 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.

[0065] Thus, in one preferred embodiment, the γδ T cells isolated by the methods defined herein include a population of Vδ1 T cells. In one embodiment, the γδ T cells isolated by the methods defined herein include a population of DN γδ T cells. In one embodiment, the γδ T cells isolated by the methods defined herein include a population of Vδ3 T cells. In one embodiment, the γδ T cells isolated by the methods defined herein include a population of Vδ5 T cells.

[0066] 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 methods defined herein include a population of Vγ4 T cells. In most cases, Vγ4 T cells are obtained from gastrointestinal tissue samples.

[0067] The isolation method provides a population of isolated γδ T cells that is greater in 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, 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) than the reference population.

[0068] In some embodiments, the population of γδ T cells isolated according to the methods of the invention has a low percentage of cells that express TIGIT. For example, the isolated population of γδ T cells can have a frequency of TIGIT+ cells that is 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%. Alternatively, the isolated population of γδ T cells can have a frequency of TIGIT+ cells that is about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, or about 10%. In one embodiment, the isolated population of γδ T cells has a frequency of TIGIT+ cells that is less than 80%. Thus, in one embodiment, the isolated population of γδ T cells has a frequency of TIGIT+ cells that is about 70%. In a further embodiment, the isolated population of γδ T cells has a frequency of TIGIT+ cells that is less than 60%. In yet a further embodiment, the isolated population of γδ T cells has a frequency of TIGIT+ cells that is about 30%. Thus, in one embodiment, the isolated γδ T cells do not substantially express TIGIT.

[0069] 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 TIGIT+ cells at a frequency of 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% of the population of Vδ1 T cells. Alternatively, a population of isolated Vδ1 T cells can have TIGIT+ cells at a frequency of about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, or about 10%. In one embodiment, a population of isolated Vδ1 T cells has TIGIT+ cells at a frequency of less than 80%. Thus, in one embodiment, a population of isolated Vδ1 T cells has TIGIT+ cells at a frequency of about 70%. In a further embodiment, a population of isolated Vδ1 T cells has TIGIT+ cells at a frequency of less than 60%. In yet a further embodiment, a population of isolated Vδ1 T cells has TIGIT+ cells at a frequency of about 30%. Thus, in one embodiment, isolated Vδ1 T cells do not substantially express TIGIT.

[0070] In some embodiments, a population of γδ T cells isolated according to the methods of the invention expresses CD27. For example, a population of isolated γδ T cells can have CD27+ cells at a frequency of greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90%. Alternatively, a population of isolated γδ T cells can have CD27+ cells at a frequency of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%. In one embodiment, a population of isolated γδ T cells has CD27+ cells at a frequency of greater than 10%. Thus, in one embodiment, a population of isolated γδ T cells has CD27+ cells at a frequency of about 20%. In a further embodiment, a population of isolated γδ T cells has CD27+ cells at a frequency of greater than 20%. In one embodiment, a population of isolated γδ T cells has CD27+ cells at a frequency of about 20%.

[0071] In some embodiments, a population of isolated Vδ1 T cells expresses CD27. In further embodiments, isolated γδ T cells express CD27. In some embodiments, a population of isolated Vδ1 T cells has CD27+ cells at a frequency greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90%. Alternatively, a population of isolated γδ T cells can have CD27+ cells at a frequency of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%. In certain embodiments, a population of isolated Vδ1 T cells has CD27+ cells at a frequency greater than 10%. Thus, in one embodiment, a population of isolated Vδ1 T cells has CD27+ cells at a frequency of about 20%. In further embodiments, a population of isolated Vδ1 T cells has CD27+ cells at a frequency greater than 20%. In one embodiment, a population of isolated Vδ1 T cells has CD27+ cells at a frequency of about 20%.

[0072] In some embodiments of any of the foregoing aspects, the isolated population of γδ T cells has a greater surface expression of one or more markers selected from the group consisting of CD124, CD215, CD360, CTLA4, CD1b, BTLA, CD39, CD45RA, Fas ligand, CD25, ICAM-1, CD31, KLRG1, CD30, and CD2 as compared to a reference population (e.g., as compared to a population of γδ T cells isolated using an alternative method). Additionally or alternatively, the isolated population of γδ T cells may have cells that express 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 as compared to the reference population. In particular, the marker is selected from CD45RA and CD25. In some embodiments, the isolated population of γδ T cells has a lower surface expression of one or more markers selected from the group consisting of NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1, and CD64 as compared to a reference population. Additionally or alternatively, the isolated population of γδ T cells may have cells that express 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 as compared to the reference population.

[0073] In some embodiments, a population of isolated Vδ1 T cells has surface 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, as compared to a reference population. In some embodiments, a population of isolated γδ T cells has cells that express 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 as compared to a reference. In some embodiments, a population of isolated γδ T cells has 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 at a lower level as compared to a reference population. In other embodiments, a population of isolated γδ T cells has cells that express 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 as compared to a reference population.

[0074] When isolated from non-hematopoietic tissue (e.g., skin), γδ T cells are typically part of a larger lymphocyte population that contains, for example, αβ T cells, B cells, and natural killer (NK) cells. In some embodiments, 1% to 10% of the isolated lymphocyte population is γδ T cells (e.g., 1 to 10% of the isolated skin-derived lymphocyte population is γδ T cells). In most cases, the γδ T cell population (e.g., the skin-derived γδ T cell population) contains a large population of Vδ1 T cells. In some embodiments, 1 to 10% of the isolated lymphocyte (e.g., skin-derived lymphocyte) population is Vδ1 T cells (e.g., Vδ1 T cells can represent more than 50%, more than 60%, more than 70%, more than 80%, or more than 90% of the isolated population of γδ T cells). Optionally, less than 10% of the isolated γδ T cell population is Vδ2 T cells (e.g., less than 10% of the isolated skin-derived γδ T cell population is Vδ2 T cells).

[0075] Non-Vδ1 T cells or non-DN T cells, such as Vδ2 T cells, αβ T cells, B cells, or NK cells, can be removed from the isolated γδ T cell population (e.g., before, during, or after the amplification step).

[0076] Isolated γδ T cells (e.g., γδ T cells isolated from skin, e.g., Vδ1 T cells isolated from skin) have a different phenotype from the corresponding hematopoietic tissue-derived cells (e.g., blood-derived γδ T cells and / or blood-derived Vδ2 T cells). For example, the isolated γδ T cell population can express higher levels of CCR3, CCR4, CCR7, CCR8, or CD103 than a reference population, such as a population of non-hematopoietic tissue-resident γδ T cells with activated TCR or a population of the corresponding hematopoietic tissue-derived cells (e.g., blood-derived γδ T cells and / or blood-derived Vδ2 T cells). In some embodiments, the isolated γδ T cell population expresses at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of CCR3 +Cells; at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more CCR4 + Cells; at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more CCR7 + Cells; at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more CCR8 + Cells; and / or at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more CD103 + Comprising 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.

[0077] 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 a population of 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 comprises at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more NKGD2 + Cells; at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more CD56 + Cells; at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more CD69 + Cells; and / or at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more TIM3+ It contains cells. A population of isolated γδ T cells can express one or more, two or more, three or more, four or more, or all five of NKGD2, CD56, CD69, and / or TIM3.

[0078] 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 functional assays known in the art to determine the functional differences between any non-hematopoietic tissue-derived cells of the present invention (e.g., a population of isolated γδ T cells, a population of skin-derived Vδ1 T cells, or an expanded population of γδ T cells and / or skin-derived Vδ1 T cells) and reference cells (e.g., a population of non-hematopoietic tissue-resident γδ T cells with activated TCR or corresponding hematopoietic tissue-derived cells, e.g., a population of blood-derived γδ T cells and / or blood-derived Vδ2 T cells). Such assays can include proliferation assays, cytotoxicity assays, binding assays, assays for measuring persistence and / or location, etc.

[0079] Thus, in one aspect of the present 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.

[0080] According to one aspect of the present invention, there is provided a population of isolated lymphocytes (e.g., skin-derived αβ T cells and / or NK cells) obtained by any of the methods defined herein.

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

[0082] According to a further aspect of the present invention, there is provided a population of isolated γδ T cells obtained by any of the methods defined herein.

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

[0084] In one embodiment, the isolated population comprises more than 5% γδ T cells, for example 7% - 12% γδ T cells. In one embodiment, the isolated population comprises Vδ1 cells, wherein less than 50%, for example less than 40%, of the Vδ1 cells express TIGIT. In one embodiment, the isolated population comprises Vδ1 cells, wherein more than 50%, for example more than 60%, of the Vδ1 cells express CD27.

[0085] The isolated non-hematopoietic tissue-resident lymphocytes may be suitable for use without further expansion or may be expanded in a further step.

[0086] 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 can be performed 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 methods defined herein. Generally, non-hematopoietic tissue-resident γδ T cells can spontaneously expand when physical contact with stromal cells (e.g., skin fibroblasts) is removed. The methods 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 methods defined herein.

[0087] 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 an unexpanded population. Such a population may be numerically greater, numerically less, or a mixed population with expansion of some or a particular cell type within the population. It will be understood that the term "expansion process" refers to a process that results in an expanded or an expansion of a population. Thus, an expanded or an expansion of a population may be numerically greater or contain more cells as compared to a population in which no expansion process has been performed or prior to any expansion process. It will further be understood that any numbers presented herein to indicate expansion (e.g., fold increase or expansion factor) are indicative of an increase in the number or size of a population of cells or an increase in the number of cells and of the amount of expansion.

[0088] Thus, in one embodiment, γδ T cells isolated according to the method of the present invention are expanded. Such expansion can optionally include culturing the γδ T cells in the presence of IL-2, IL-15, and IL-21, optionally including IL-4. Alternatively, the expansion can optionally include culturing the γδ T cells in the presence of IL-9, IL-15, and IL-21, optionally including IL-4. 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 includes culturing the γδ T cells for at least 5 days in an amount effective to produce a population of expanded γδ T cells in the presence of IL-2, IL-15, and IL-21. In a further embodiment, the expansion includes culturing the γδ T cells for at least 5 days in an amount effective to produce a population of expanded γδ T cells in the presence of IL-2, IL-15, IL-21, and IL-4. In yet a further embodiment, the expansion includes culturing the γδ T cells for at least 5 days in an amount effective to produce a population of expanded γδ T cells in the presence of IL-9, IL-15, and IL-21. In one embodiment, the expansion includes culturing the γδ T cells for at least 5 days in an amount effective to produce a population of expanded γδ T cells in the presence of IL-9, IL-15, IL-21, and IL-4.

[0089] In a further embodiment, the expansion comprises culturing lymphocytes and / or γδ T cells for a period of time (e.g., at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 21 days, at least 28 days, or a longer period, e.g., 5 to 40 days, 7 to 35 days, 14 to 28 days, or about 21 days) in an amount effective to produce an expanded population of γδ T cells. In some embodiments, the lymphocytes and / or γδ T cells are expanded 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, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 days) in culture. In one embodiment, the lymphocytes and / or γδ T cells are expanded for 14 to 21 days. Thus, including the isolation and expansion steps, including the isolation culture period (e.g., 1 to 40 days, e.g., 14 to 21 days), in some embodiments, can last 28 to 56 days, or about 41 days.

[0090] In a further embodiment, the expansion comprises culturing 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 11 days, at least 12 days, at least 13 days, at least 14 days, at least 21 days, at least 28 days, or a longer period, e.g., 5 to 40 days, 7 to 35 days, 14 to 28 days, or about 21 days. In one embodiment, the expansion step comprises culturing γδ T cells for at least 10 days, 15 days, or 20 days to produce an expanded population. In one embodiment, the expansion step comprises culturing γδ T cells for 5 to 25 days, e.g., 14 to 21 days. In a further embodiment, the expansion step comprises culturing γδ T cells for about 20 days.

[0091] In some embodiments, a typical amount of IL-2 effective to produce an expanded population of γδ T cells is from 1 IU / mL to 2,000 IU / mL (e.g., from 5 IU / mL to 1,000 IU / mL, from 10 IU / mL to 500 IU / mL, from 20 IU / mL to 400 IU / mL, from 50 IU / mL to 250 IU / mL, or about 100 IU / mL, e.g., from 5 IU / mL to 10 IU / mL, from 10 IU / mL to 20 IU / mL, from 20 IU / mL to 30 IU / mL, from 30 IU / mL to 40 IU / mL, from 40 IU / mL to 50 IU / mL, from 50 IU / mL to 60 IU / mL, from 60 IU / mL to 70 IU / mL, from 70 IU / mL to 80 IU / mL, from 80 IU / mL to 90 IU / mL, from 90 IU / mL to 100 IU / mL, from 100 IU / mL to 120 IU / mL, from 120 IU / mL to 140 IU / mL, from 140 IU / mL to 150 IU / mL, from 150 IU / mL to 175 IU / mL, from 175 IU / mL to 200 IU / mL, from 200 IU / mL to 300 IU / mL, from 300 IU / mL to 400 IU / mL, from 400 IU / mL to 500 IU / mL, from 500 IU / mL to 1,000 IU / mL, from 1,000 IU / mL to 1,500 IU / mL, from 1,500 IU / mL to 2,000 IU / mL, or 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, a 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., 0.1 ng / mL to 10,000 ng / mL, 1.0 ng / mL to 1,000 ng / mL, 5 ng / mL to 800 ng / mL, 10 ng / mL to 750 ng / mL, 20 ng / mL to 500 ng / mL, 50 ng / mL to 400 ng / mL, or 100 ng / mL to 250 ng / mL, e.g., 0.1 ng / mL to 1.0 ng / mL, 1.0 ng / mL to 5.0 ng / mL, 5.0 ng / mL to 10 ng / mL, 10 ng / mL to 20 ng / mL, 20 ng / mL to 50 ng / mL, 50 ng / mL to 100 ng / mL, 100 ng / mL to 200 ng / mL, 200 ng / mL to 500 ng / mL, or 500 ng / mL to 1,000 ng / mL). In some embodiments, the amount of IL-15 effective to produce a population of expanded γδ T cells is about 10 ng / mL.

[0093] In some embodiments, a typical amount of IL-21 effective to produce a 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) is at least 0.1 ng / mL, e.g., at least 1.0 ng / mL (e.g., 0.1 ng / mL to 1,000 ng / mL, 1.0 ng / mL to 100 ng / mL, 1.0 ng / mL to 50 ng / mL, 2 ng / mL to 50 ng / mL, 3 ng / mL to 10 ng / mL, 4 ng / mL to 8 ng / mL, 5 ng / mL to 10 ng / mL, 6 ng / mL to 8 ng / mL, e.g., 0.1 ng / mL to 10 ng / mL, 1.0 ng / mL to 5 ng / mL, 1.0 ng / mL to 10 ng / mL, 1.0 ng / mL to 20 ng / mL). In further embodiments, the amount of IL-21 is typically at a concentration of less than 100 ng / mL, e.g., less than 50 ng / mL. In some embodiments, the method comprises IL-21 at a concentration of about 6 ng / mL, e.g., about 6.25 ng / mL.

[0094] In a further embodiment, the methods defined herein typically include IL-4 at a concentration of at least 0.1 ng / mL, such as at least 10 ng / mL (e.g., from 0.1 ng / mL to 1,000 ng / mL, from 1.0 ng / mL to 100 ng / mL, from 1.0 ng / mL to 50 ng / mL, from 2 ng / mL to 50 ng / mL, from 3 ng / mL to 40 ng / mL, from 4 ng / mL to 30 ng / mL, from 5 ng / mL to 20 ng / mL, from 10 ng / mL to 20 ng / mL, e.g., from 0.1 ng / mL to 50 ng / mL, from 1.0 ng / mL to 25 ng / mL, from 5 ng / mL to 25 ng / mL). In a further embodiment, the methods defined herein typically include IL-4 at a concentration of less than 100 ng / mL, such as less than 50 ng / mL, particularly less than 20 ng / mL. In some embodiments, the method includes IL-4 at a concentration of about 15 ng / mL.

[0095] Substitution or addition of other factors in the expansion culture of non-hematopoietic tissue resident lymphocytes and / or γδ T cells is also provided herein. For example, in some embodiments, any one or more factors selected from the group consisting of IL-4, IL-6, IL-7, IL-8, IL-9, IL-12, IL-18, IL-33, IGF-1, IL-1β, human platelet lysate (HPL), and stromal cell-derived factor-1 (SDF-1) are included in addition to or instead of any one of IL-2 and IL-15. Such additional or alternative 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 such as αβ T cells and / or NK cells.

[0096] The amount of each of the above cytokines required to produce an expanded population of γδ T cells will be understood to be 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, respectively, decrease or increase accordingly. As described above, an effective amount to produce an expanded population refers herein to the combined effect of all factors on cell expansion.

[0097] The expansion method provides a population of expanded γδ T cells that is greater in number than a reference population. In some embodiments, the population of expanded 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 numerous than the population of isolated γδ T cells prior to the expansion step).

[0098] 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.

[0099] In a further embodiment, the expansion step further comprises culturing the isolated γδ T cells in the presence of IL-4. Thus, in one embodiment, the expansion comprises culturing the isolated γδ T cells in the presence of IL-2, IL-15, IL-4, and IL-21. Alternatively, the expansion may comprise culturing the isolated γδ T cells in the presence of IL-9, IL-15, IL-4, and IL-21.

[0100] 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 and / or chemokines) to produce a population of expanded lymphocytes (e.g., αβ T cells and / or NK cells).

[0101] In one embodiment, a method of expanding a population of γδ T cells as defined herein comprises culturing the 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 the γδ T cells in a medium containing a serum replacement. Thus, it will be understood that the expansion of such γδ T cells in a serum-free medium or a medium containing a serum replacement obtains similar advantages as those described above.

[0102] In some embodiments, substantial TCR pathway activation is absent during the expansion step (e.g., no exogenous TCR pathway activator is included in the culture). In one embodiment, the expansion step 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, the expansion of γδ T cells comprises culturing the γδ T cells in the absence of substantial stromal cell contact.

[0103] Also provided is a means for highly producing 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). In some embodiments, the expansion process described herein expands γδ T cells with a low population doubling time given by the following formula:

Number

[0104] In view of the information provided herein, one of ordinary skill in the art will recognize that the present invention provides a method for expanding 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) 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, less than 2.9 days, less than 2.8 days, less than 2.7 days, less than 2.6 days, less than 2.5 days, less than 2.4 days, less than 2.3 days, less than 2.2 days, less than 2.1 days, less than 2.0 days, less than 46 hours, less than 42 hours, less than 38 hours, less than 35 hours, less than 32 hours).

[0105] In some embodiments, within 7 days from the start of the culture, the expanded population of γδ T cells (e.g., the expanded population of Vδ1 T cells and / or DN T cells) contains at least 10-fold the number of γδ T cells (e.g., at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1,000-fold, at least 2,000-fold, at least 3,000-fold, at least 4,000-fold, at least 5,000-fold, at least 6,000-fold, at least 7,000-fold, or at least 8,000-fold the number of γδ T cells) compared to the population of isolated γδ T cells before expansion. In some embodiments, within 14 days from the start of the culture, the expanded population of γδ T cells (e.g., the expanded population of Vδ1 T cells and / or DN T cells) contains at least 20-fold the 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 the 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 the culture, the expanded population of γδ T cells (e.g., the expanded population of Vδ1 T cells and / or DN T cells) contains at least 50-fold the 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, at least 150-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1,000-fold, at least 2,000-fold, at least 3,000-fold, at least 4,000-fold, at least 5,000-fold, at least 6,000-fold, at least 7,000-fold, at least 8,000-fold, at least 9,000-fold, or at least 10,000-fold the number of γδ T cells) compared to the population of isolated γδ T cells before expansion. In some embodiments, within 28 days from the start of the culture, the expanded population of γδ T cells (e.g., the expanded population of Vδ1 T cells and / or DN T cells) contains at least 100-fold the number of γδ T cells (e.g., at least 110-fold, at least 120-fold, at least 130-fold, at least 140-fold, at least 150-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1,000-fold, at least 2,000-fold, at least 3,000-fold, at least 4,000-fold, at least 5,000-fold, at least 6,000-fold, at least 7,000-fold, at least 8,000-fold, at least 9,000-fold, at least 10,000-fold, at least 12,000-fold, or at least 15,000-fold the number of γδ T cells) compared to the population of isolated γδ T cells before expansion.

[0106] The non-hematopoietic tissue-derived γδ T cells expanded by the methods provided herein (e.g., skin-derived γδ T cells and / or non-Vδ2 T cells, such as Vδ1 T cells and / or DN T cells) can have a phenotype well-suited for anti-tumor efficacy. In some embodiments, a population of expanded γδ T cells (e.g., skin-derived Vδ1 T cells) has a greater mean expression of CD27 than a reference population (e.g., a population of isolated γδ T cells prior to the expansion step). In some embodiments, a population of expanded γδ T cells is at least 2-fold greater (e.g., at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1,000-fold, at least 5,000-fold, at least 10,000-fold, at least 20,000-fold, or greater) in mean expression of CD27 compared to a population of isolated γδ T cells.

[0107] While a distinct portion of the 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) can upregulate CD27, another portion is CD27 low or CD27 negative In this case, the frequency of CD27 positive cells in the expanded population compared to the population of isolated γδ T cells can be greater. For example, a population of expanded γδ T cells has a frequency of CD27 that is at least 5% greater compared to the frequency of the population of isolated γδ T cells prior to expansion. positivecells (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% greater frequency of CD27 compared to the frequency of a population of isolated γδ T cells before expansion) positive cells). In some embodiments, the number of CD27 positive cells in the expanded population compared to the population of isolated γδ T cells can increase. For example, the expanded population of γδ T cells can have at least twice the number of CD27 positive cells compared to the population of isolated γδ T cells before expansion. The expanded population of γδ T cells can have a frequency of CD27+ cells that is greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, or 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 one embodiment, the expanded population of γδ T cells has a frequency of CD27+ cells that is greater than 50%.

[0108] The expansion methods provided herein, in some embodiments, yield a population of expanded non-hematopoietic tissue-derived 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) having lower TIGIT expression compared to a reference population (e.g., a population of isolated γδ T cells prior to the expansion process). In some embodiments, the population of expanded γδ T cells has a lower average TIGIT expression than a reference population (e.g., a population of isolated γδ T cells prior to the expansion process). In some embodiments, the population of expanded 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 TIGIT expression than the population of isolated γδ T cells. The population of expanded γδ T cells can have a frequency of TIGIT+ cells of 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%. Alternatively, the population of expanded 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%.

[0109] In some embodiments, a population of expanded γδ T cells (e.g., skin-derived γδ T cells or non-Vδ2 T cells, such as Vδ1 T cells and / or DN T cells) has a high number or high frequency of CD27 + cells and a low frequency of TIGIT + cells. In some embodiments, the population of expanded γδ T cells has a higher frequency of CD27 + TIGIT -It has cells. For example, a population of expanded γδ T cells has a frequency of CD27 that is at least 5% greater compared to the frequency of the population of isolated γδ T cells before expansion. + TIGIT - cells (e.g., a frequency of CD27 that is 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% greater compared to the frequency of the population of isolated γδ T cells before expansion). + TIGIT - cells) may be present. In some embodiments, the number of CD27 + TIGIT - cells in the expanded population compared to the population of isolated γδ T cells can increase. For example, a population of expanded γδ T cells has at least twice the number of CD27 + TIGIT - cells (e.g., a frequency of CD27 that is 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% greater compared to the frequency of the population of isolated γδ T cells before expansion). + TIGIT - cells) may be present.

[0110] Optionally, the average expression of TIGIT in 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) within the population of CD27 + γδ T cells is lower compared to a reference population. In some embodiments, the expanded population of CD27 + γδ T cells has a lower average expression of TIGIT compared to a reference population (e.g., the population of isolated CD27 + γδ T cells before the expansion step). In some embodiments, the expanded population of CD27+ The population of γδ T cells has an average expression of TIGIT that is 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) than the population of isolated CD27 + γδ T cells. + Furthermore or alternatively, TIGIT within 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)

[0111] has an average expression of TIGIT that is 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) than the population of γδ T cells. - The median expression of CD27 in the population of γδ T cells is high compared to a reference population. For example, the expanded population of TIGIT - γδ T cells has CD27 - cells at a frequency that is at least 5% higher (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or up to 100% higher) compared to the frequency of the population of isolated TIGIT + γδ T cells before expansion. In some embodiments, the number of CD27 - cells in the expanded population compared to the population of isolated TIGIT + γδ T cells can be increased. For example, the expanded population of TIGIT - γδ T cells has at least twice the number of CD27 + cells (e.g., the population of isolated TIGIT - γδ T cells before expansion) compared to the population of isolated TIGIT - γδ T cells before expansion. + cells. -At least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or up to 100% greater frequency of CD27 compared to the frequency of the population of γδ T cells + cells).

[0112] In addition to or instead of an increase or decrease in the expression of other markers, including CD124, CD215, CD360, CTLA4, CD1b, BTLA, CD39, CD45RA, Fas ligand, CD25, ICAM-1, CD31, KLRG1, CD30, CD2, NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1, and CD64, the expression can be 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). Optionally, the 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) has an average expression of one or more markers selected from the group consisting of CD124, CD215, CD360, CTLA4, CD1b, BTLA, CD39, CD45RA, Fas ligand, CD25, ICAM-1, CD31, KLRG1, CD30, and CD2 that is greater, for example, than that of an isolated population of γδ T cells before expansion. In addition to or instead of this, the population of expanded γδ T cells can have a frequency of cells that express one or more markers selected from the group consisting of CD124, CD215, CD360, CTLA4, CD1b, BTLA, CD39, CD45RA, Fas ligand, CD25, ICAM-1, CD31, KLRG1, CD30, and CD2 that is greater than that of an isolated population of γδ T cells. In some embodiments, the population of expanded γδ T cells has an average expression of one or more markers selected from the group consisting of NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1, and CD64 that is lower than that of an isolated population of γδ T cells. The expanded population can similarly have a frequency of cells that express one or more markers selected from the group consisting of NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1, and CD64 that is lower than that of an isolated population of γδ T cells.

[0113] Thus, the non-hematopoietic tissue resident γδ T cells produced by the method of the present invention can have one or more of the following characteristics: (i) CD69 high , TIM3 high , and CD28 low / absent show the phenotype of; (ii) upregulate one or more of CCR3, CD39, CD11b, and CD9; (iii) produce IFN-γ in response to NKG2D ligands in the absence of TCR agonists; (iv) produce IL-13 in the absence of TCR agonists; (v) produce one or more of IFN-γ, TNF-α, and GM-CSF in response to TCR activation; (vi) produce no or substantially no IL-17 in response to TCR activation; (vii) grow in culture medium containing IL-2 without additional growth factors; (viii) show a cytotoxic T cell response in the absence of TCR agonists; and / or (ix) show selective cytotoxicity against tumor cells compared to normal cells.

[0114] Optionally, the non-hematopoietic tissue resident γδ T cells produced by the method of the present invention produce IL-13 in the absence of TCR agonists and / or produce IFN-γ in response to NKG2D ligands in the absence of TCR agonists.

[0115] Many basal culture media suitable for use in the proliferation of γδ T cells are available, particularly media such as AIM-V, Iscoves medium, and RPMI-1640 (Life Technologies). The medium may be supplemented with other medium factors as defined herein, such as serum, serum proteins, and selection agents, antibiotics. For example, in some embodiments, RPMI-1640 medium containing 2 mM glutamine, 10% FBS, 10 mM HEPES, pH 7.2, 1% penicillin-streptomycin, sodium pyruvate (1 mM; Life Technologies), non-essential amino acids (e.g., 100 μM Gly, Ala, Asn, Asp, Glu, Pro, and Ser; 1×MEM non-essential amino acids (Life Technologies)), and 10 μl / L β-mercaptoethanol. In alternative embodiments, AIM-V medium may be supplemented with a serum replacement and amphotericin B from CTS Immune. In certain embodiments as defined herein, the medium may further be supplemented with IL-2 and IL-15. Conveniently, the cells are cultured at 37°C in a humidified atmosphere containing 5% CO2 in a suitable culture medium during isolation and / or expansion.

[0116] 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. : A method is provided.

[0117] 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) culturing the population of αβ T cells (e.g., for at least 5 days) to produce an expanded population of αβ T cells : A method is provided that includes.

[0118] The culturing in step (ii) may be by selective expansion, for example, by selecting culture conditions under which the αβ T cells are expanded 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 αβ 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 (e.g., cells other than αβ T cells) is performed prior to the culturing in step (ii). It should be noted that the purpose of these embodiments is to expand the total number of αβ T cells and, at the same time, increase their proportion in the population.

[0119] In one embodiment, the lymphocytes include NK cells. Therefore, according to 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) culturing the population of NK cells (e.g., for at least 5 days) to produce an expanded population of NK cells : A method is provided that includes.

[0120] The culturing in step (ii) may be by selective expansion, for example, by selecting culture conditions under which the NK cells are expanded 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 culturing in 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 culturing in step (ii). It should be noted that the purpose of these embodiments is to expand the total number of NK cells and, at the same time, increase their proportion in the population.

[0121] In one embodiment, the lymphocytes include γδ T cells. Thus, according to a further aspect of the 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 (e.g., for at least 5 days) to produce an expanded population of γδ T cells : is provided.

[0122] The culturing in step (ii) may be by selective expansion, for example, by selecting culture conditions in 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 (e.g., cells other than γδ T cells) may be removed after the culturing of step (ii). Alternatively, the expansion conditions may not be selective, and the removal of non-target cells (e.g., cells other than γδ T cells) may be performed prior to the culturing of step (ii). It should be noted that the purpose of these embodiments is to expand the total number of γδ T cells and at the same time increase their proportion in the population.

[0123] Thus, according to a further aspect of the 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) culturing the population of γδ T cells (a) in the presence of IL-2 or IL-9; (b) IL-15; and (c) IL-21 : for at least 5 days in an amount effective to produce an expanded population of γδ T cells : is provided.

[0124] In certain embodiments of this aspect of the invention, the culturing of the population of γδ T cells further comprises the presence of IL-4. Thus, in a further aspect of the invention, a method for the isolation and expansion of 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) culturing the population of γδ T cells (a) in the presence of IL-2 or IL-9; (b) in the presence of IL15; and (c) in the presence of IL-21; and (d) in the presence of IL-4 for at least 5 days in an amount effective to produce an expanded population of γδ T cells. A method is provided that includes the step.

[0125] According to one aspect of the invention, there is provided a population of expanded isolated lymphocytes (e.g., skin-derived αβ T cells and / or NK cells) obtained by any of the methods defined herein.

[0126] According to a further aspect of the invention, there is provided a population of expanded isolated lymphocyte cells obtainable by any of the methods defined herein.

[0127] According to yet a further aspect of the invention, there is provided a population of expanded isolated γδ T cells obtained by any of the methods defined herein.

[0128] According to yet a further aspect of the invention, there is provided a population of isolated expanded γδ T cells obtainable by any of the methods defined herein.

[0129] In one embodiment, the isolated population comprises more than 50% γδ T cells, such as more than 75% γδ T cells, particularly more than 85% γδ T cells. In one embodiment, the isolated population comprises Vδ1 cells, wherein less than 50%, such as less than 25%, of the Vδ1 cells express TIGIT. In one embodiment, the isolated population comprises Vδ1 cells, wherein more than 50%, such as more than 60%, of the Vδ1 cells express CD27.

[0130] 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 may be substantially free of αβ T cells. For example, the αβ T cells may be removed from the γδ T cell population, for example, after expansion, using any suitable means known in the art (e.g., using magnetic beads, e.g., by negative selection). The treatment method may include: providing a sample of non-hematopoietic tissue obtained from a donor individual; culturing γδ T cells from the above sample to produce an expanded population; and administering the expanded population of γδ T cells to a recipient individual.

[0131] The patient or subject to be treated is preferably a human cancer patient (e.g., a human cancer patient undergoing treatment for a solid tumor) or a viral infection patient (e.g., a CMV-infected or HIV-infected patient). Optionally, the patient has a solid tumor and / or is undergoing treatment for a solid tumor.

[0132] Tissue-resident Vδ1 T and DN γδ T cells normally reside in non-hematopoietic tissues, so these cells are also more likely to home to and be retained within tumor masses than their systemic blood-resident counterparts, and adoptive transfer of these cells may be more effective in targeting solid tumors and potentially other non-hematopoietic tissue-related immunopathologies.

[0133] 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 example, for allogeneic adoptive T cell therapy.

[0134] Non-hematopoietic tissue-resident γδ T cells obtained by the methods of the present invention express NKG2D and respond to NKG2D ligands (e.g., MICA) that are strongly associated with malignant tumors. 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, non-hematopoietic tissue-resident γδ T cells obtained as described herein can express one or more, preferably all, of IFN-γ, TNF-α, GM-CSF, CCL4, IL-13, granulysin, granzyme A and B, and perforin in the absence of any activation. IL-17A may not be expressed.

[0135] Therefore, the findings reported herein provide strong evidence for the practicality and suitability of clinical application of non-hematopoietic tissue-resident γδ T cells obtained by the methods of the present invention as "off-the-shelf" immunotherapy reagents. These cells possess natural-like apoptosis, do not have MHC restriction, and exhibit improved tumor homing and / or retention within tumors compared to other T cells.

[0136] 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.

[0137] A pharmaceutical composition may include the expanded non-hematopoietic tissue-resident γδ T cells described herein, either as one or more pharmaceuticals or in combination with a physiologically acceptable carrier, diluent, or excipient. Such compositions may include buffers, such as neutral buffered saline, phosphate buffered saline, etc.; 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 (e.g., aluminum hydroxide); and preservatives. Cryoprotective solutions that may be used in the pharmaceutical compositions of the invention include, for example, DMSO. The composition can be formulated, for example, for intravenous administration.

[0138] In one embodiment, the pharmaceutical composition is substantially free of detectable levels of contaminants, such as endotoxin or mycoplasma, e.g., the contaminants are absent.

[0139] Optionally, a therapeutically effective amount of the expanded γδ T cells obtained by any of the above methods can be administered to a subject at a therapeutically effective amount (e.g., for the treatment of cancer, e.g., for the treatment of solid tumors). Optionally, the therapeutically effective amount of the 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 less than 10×10 12 cells per dose (e.g., less than 9×10 12 cells per dose, less than 8×10 12 cells per dose, 7×10 12 cells per dose, less than 6×10 12 cells per dose, less than 5×10 12less than cells, 4×10 per dose 12 less than cells, 3×10 per dose 12 less than cells, 2×10 per dose 12 less 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, 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, 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, 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, 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 6 less than cells, 2,5×10 per dose 6 less than cells, 1×10 per dose6 less than cells, 7.5×10 per dose 5 less than cells, 5×10 per dose 5 less than cells, 2,5×10 per dose 5 less than cells, or 1×10 per dose 5 less than cells)

[0140] In some embodiments, a therapeutically effective amount 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, during the course of treatment, 10×10 12 less than cells (e.g., during the course of treatment, 9×10 12 less than cells, 8×10 12 less than cells, 7×10 12 less than cells, 6×10 12 less than cells, 5×10 12 less than cells, 4×10 12 less than cells, 3×10 12 less than cells, 2×10 12 less than cells, 1×10 12 less than cells, 9×10 11 less than cells, 8×10 11 less than cells, 7×10 11 less than cells, 6×10 11 less than cells, 5×10 11 less than cells, 4×10 11 less than cells, 3×10 11 less than cells, 2×10 11 less than cells, 1×10 11 less than cells, 9×10 10 less than cells, 7.5×10 10 less than cells, 5×10 10 less than cells, 2.5×10 10 less than cells, 1×10 10 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 less than cells, 7.5×10 8 less than cells, 5×108 less than cells, 2.5×10 8 less than cells, 1×10 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, 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).

[0141] In some embodiments, the dosage of the expanded non-hematopoietic tissue-resident γδ T cells described herein is about 1×10 6 , 1.1×10 6 , 2×10 6 , 3.6×10 6 , 5×10 6 , 1×10 7 , 1.8×10 7 , 2×10 7 , 5×10 7 , 1×10 8 , 2×10 8 , or 5×10 8 cells / kg. In some embodiments, the dosage of 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) is at least about 1×10 6 , 1.1×10 6 , 2×10 6 , 3.6×10 6 , 5×10 6 , 1×10 7 , 1.8×10 7 , 2×10 7 , 5×10 7 , 1×10 8 , 2×10 8 , or 5×108 contains 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 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 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 1×10 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 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 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 9It contains cells. 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 up to 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.

[0142] In one embodiment, the subject is administered 10 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 cells and / or DN T cells) per kg body weight of the subject. In one embodiment, the subject is given a first dose of a population of non-hematopoietic tissue-resident γδ T cells (e.g., a first dose of 10 4 ~10 6 γδ T cells per kg body weight of the subject, e.g., a first dose of 10 4 ~10 5 γδ T cells), and one or more (e.g., 2, 3, 4, or 5) subsequent doses of expanded non-hematopoietic tissue-resident γδ T cells (e.g., 10 4 ~10 6 expanded non-hematopoietic tissue-resident γδ T cells per kg body weight of the subject, e.g., one or more subsequent doses of 10 4 ~10 5 expanded non-hematopoietic tissue-resident γδ T cells per kg body weight of the subject). In one embodiment, the one or more subsequent doses are administered less than 15 days, e.g., less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days, e.g., less than 4, 3, or 2 days, after the previous dose. In one embodiment, the subject receives a total of about 10 6 γδ T cells per kg body weight of the subject during at least three administrations of the population of γδ T cells, e.g., the subject receives 1×10 5Initial administration of 5 γδ T cells, a second administration of 5 γδ T cells, and a third administration of

[0143] Non-hematopoietic tissue resident γδ T cells obtained by the method of the present invention can also be genetically modified for enhanced therapeutic properties, e.g., for CAR-T therapy. This involves the generation of a modified T cell receptor (TCR) that reprograms T cells to have a new specificity, e.g., the specificity of a monoclonal antibody. The modified TCR can create T cells specific for malignant cells and thus useful for cancer immunotherapy. For example, T cells can recognize cancer cells expressing tumor antigens not expressed by normal somatic cells from the tissue of interest, e.g., tumor-associated antigens. Thus, CAR-modified T cells can be used, e.g., for adoptive T cell therapy in cancer patients.

[0144] The use of blood resident γδ T cells in CARs has been described. However, non-hematopoietic tissue resident γδ T cells obtained by the method of the present invention can retain their native-like ability to recognize transformed cells while having a chimeric antigen-specific TCR transduced, and are likely to have better tumor infiltration and retention capabilities than either blood resident γδ T cells or conventional systemic αβ T cells, so this is likely to be a particularly excellent vehicle for the CAR-T approach. Furthermore, the lack of MHC-dependent antigen presentation reduces the potential for graft-versus-host disease, enabling targeting of tumors that express low levels of MHC. Similarly, targeting of tumors that express low levels of co-stimulatory receptor ligands is enhanced by its independence from conventional co-stimulation, e.g., by engagement of CD28.

[0145] 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 immunotherapy agent, a cytotoxic agent, a growth inhibitor, a radiation therapy agent, an anti-angiogenesis agent, or a combination of two or more of these agents. The additional therapeutic agent can be administered simultaneously with, before, or after the administration of the expanded γδ T cells. The additional therapeutic agent can be an immunotherapy agent that acts on targets of the subject's body (e.g., the subject's own immune system) and / or the transplanted γδ T cells.

[0146] Administration of the composition can be carried out by any convenient method. The compositions described herein can be administered to a patient by transarterial, subcutaneous, intradermal, intratumoral, intranodular, intramedullary, intramuscular, intravenous injection, or intraperitoneally, e.g., by intradermal or subcutaneous injection. Compositions of non-hematopoietic tissue resident γδ T cells can be injected directly into tumors, lymph nodes, or sites of infection.

[0147] It will be understood that all of the embodiments described herein can be applied to all aspects of the present invention.

[0148] As used herein, the term "about", when used herein, includes values that are less than or equal to the value that is 10% greater than the specified value and greater than or equal to the value that is 10% less than the specified value, preferably values that are less than or equal to the value that is 5% greater than the specified value and greater than or equal to the value that is 5% less than the specified value, and in particular includes the specified value. The term "between" includes values within the specified range.

[0149] Hereinafter, specific aspects and embodiments of the present invention will be described by way of example and with reference to the above drawings.

Example

[0150] (Example) (Example 1. Analysis method) Unless otherwise specified, the following methods were used to obtain the results of the following examples.

[0151] (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 Miltenyi. 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). At the end of each experiment, cell populations were washed in PBS and split in half. Cells were stained for viability with eFluor770 NIR, washed, and then stained with TrueStain (Biolegend) to avoid non-specific binding of the staining antibodies. Half of the sample was stained for the indicated surface markers and the other half was stained for lineage markers only (CD3, Vδ1) and using an equivalent isotype control for 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. Histograms are shown when compared to their corresponding isotype controls or as indicated. Data summary shows the percentage of cells that stained positive for the indicated markers being compared and thus at a higher level than the isotype. Flow cytometry data analysis was performed with FLOWJO (version 10.1).

[0152] The initial and final phenotypes of each cell population, including the expression of CD27 and TIGIT, were also determined using the mean fluorescence intensity (MFI).

[0153] (Population analysis) Skin resident lymphocytes were isolated using the method described in this specification. Among CD45+ cells, T cells were stained with anti-CD3, and NK cells, CD3−CD56+, were identified using an 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 proportions of conventional CD4 and CD8 positive αβ T cells within the CD3+, pan-γδ TCR− gate were identified using anti-CD8α.

[0154] (Determination of total cell number) The total cell number was determined using an NC-250 Nucleocounter (Chemometec, Copenhagen, Denmark) and the manufacturer's instructions.

[0155] (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) (Ultramet, California, USA) or an equivalent having dimensions of 20 mm × 1.5 mm was autoclaved, washed, and then completely immersed in PBS before use.

[0156] 1 L of AIM-V medium (Gibco, Life Technologies), 50 mL of serum replacement from CTS Immune (Life Technologies), recombinant human IL-2 (Miltenyi Biotech, Cat no 130-097-746), and recombinant human IL-15 (Miltenyi Biotech, Cat no 130-095-766) were contained, and recombinant human IL-21 (Miltenyi Biotech, Cat no 130-095-784) for 3 cytokine (3CK) measurement and recombinant human IL-4 (Miltenyi Biotech, Cat no 130-093-922) at the following concentrations for 4 cytokine (4CK) measurement were also included to prepare a complete isolation medium. For the first 7 days from the culture, a 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: Table 1: Final Concentrations of Cytokines in Complete Isolation Medium

Table 1

[0157] Samples of adult skin were obtained, transported within 48 hours of collection, and processed. Excess subcutaneous fat and hair were removed from the samples with a scalpel and forceps. The skin samples were placed with the epidermal side facing up, and using a punch biopsy of appropriate size, the skin around the biopsy was held with sterile forceps and the skin was cut.

[0158] Three biopsies were placed at equal intervals with the epidermal side 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 the G-REX6 well plate, or three grids were placed in the G-REX10 bioreactor, or ten grids were placed in the G-REX100 bioreactor. Alternatively, the biopsies were cultured in a conventional 24-well plate. The cultures were incubated at 37 °C in a 5% CO2 incubator.

[0159] Unless otherwise noted, the medium was gently aspirated and replaced with 2× complete isolation medium (without AMP) so as not to disturb the cells at the bottom of the plate or bioreactor, and the medium was changed every 7 days.

[0160] 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).

[0161] If a cell count was required, the lymphocytes were counted at this stage as described in Example 1. The results of exemplary tests are shown in Table 2: Table 2. Yield of isolated lymphocytes per donor [Table 2]

[0162] (Example 3. Use of additional cytokines in the isolation process) The use of additional cytokines was tested at the isolation stage. Three cytokine isolation methods (i.e., IL-2, IL-15, and IL-21) and four cytokine isolation methods (i.e., IL-2, IL-15, IL-21, and IL-4) were tested and directly compared with the two cytokine (i.e., IL-2 and IL-15) isolation method. Skin samples were prepared as described in Example 2.

[0163] The total cell yield and the ratio of γδ T cells to Vδ1 cells were determined as described in Example 1. The results are shown in Figure 1. The use of four cytokines in isolation was shown to improve cell yield and increase the number of γδ T cells and Vδ1 cells isolated. The results presented in Figure 2 also show that the use of three cytokines can increase cell yield and the number of γδ T cells and Vδ1 cells isolated.

[0164] The phenotype of the isolated Vδ1 cells was analyzed by measuring TIGIT and CD27 expression using the method described in Example 1. Vδ1 cells with low TIGIT expression and high CD27 expression are considered to have a desirable phenotype. The results are shown in Figures 3 and 4. Overall, the cells isolated using four cytokines and three cytokines had lower TIGIT expression and higher CD27 expression compared to the cells isolated using two cytokines.

[0165] (Example 4. Optimization of Punch Biopsy Size) From initial tests, 3 mm punch biopsies were shown to be superior to standard skin mincing methods (Figure 5).

[0166] Punch biopsy sizes of 1 mm, 2 mm, 3 mm, 4 mm, and 8 mm were tested, and the optimal punch biopsy size was further investigated by using 2 mm explants minced with a scalpel as a control. Skin samples were prepared as described in Example 2. Each size was tested by attaching one biopsy, epidermis side up, to the surface of the carbon grid and placing it in a well of a 24-well plate (Corning). Each well contained AIM-V 10% human AB serum + the above concentrations of IL-2 and IL-15 and standard concentrations of β-mercaptoethanol (2ME) and penicillin / streptomycin (P / S).

[0167] The medium was changed three times a week (half-medium change), and the biopsies were incubated at 37 °C in a 5% CO2 incubator for 21 days before cell harvesting and cell yield analysis.

[0168] Total cell yield was determined as described in Example 1. The results are shown in Table 3. These results indicate that biopsies with a diameter of 2 - 4 mm result in the highest cell yield. Table 3: Total cell yield obtained by biopsy type

Table 3

[0169] The percentage of γδ T cells present in the cell yield was determined as described in Example 1. The results are presented in Figure 6. These results indicate that biopsies with a diameter of 3 mm result in the highest yield of γδ T cells.

[0170] (Example 5. Optimization of isolation container) Isolation in 24-well plates was compared to using containers containing gas-permeable materials such as G-REX6 well plates (Wilson Wolf Manufacturing). Skin samples were prepared as described in Example 2. Biopsies were attached to the surface of the carbon grid with the epidermis facing up and then this carbon grid was placed into 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 was used for the G-REX6 well plate. All samples were placed in AIM-V 10% AB serum + P / S + 2ME + IL-2 and IL-15. For the 24-well plate, the medium was changed three times a week. For the G-REX6 well plate, only a weekly medium change was necessary. Biopsies were incubated in a 5% CO2 incubator at 37 °C for 21 days prior to cell yield analysis.

[0171] The total cell yield per plate and per biopsy was determined as described in Example 1. Experiments showed that the G-REX6 well plate resulted in an increase in cell yield per biopsy and per plate when compared to the 24-well plate (Figure 7 and Table 4). The G-REX6 well plate allowed for an increase in the amount of tissue being cultured (2.5 times more tissue compared to the 24-well plate), and this plate resulted in a surprising increase in cell numbers by a factor of 25. Table 4. Total cell yields obtained by 24-well plates vs G-REX6 well plates

Table 4

[0172] The use of G-REX vessels was tested using 2-cytokine, 3-cytokine, and 4-cytokine isolation protocols. The phenotype of Vδ1 cells was analyzed by measuring TIGIT and CD27 expression using the method described in Example 1. PD-1 expression was measured in isolated αβ T cells (CD3+, pan-γδ-negative cells). The results are shown in Figures 8 and 9. From these results, it is confirmed that Vδ1 cells isolated using four cytokines with G-REX vessels have lower TIGIT expression and higher CD27 expression compared to Vδ1 cells isolated using two cytokines with G-REX vessels, while αβ T cells isolated using four cytokines have lower PD-1 expression compared to αβ T cells isolated using two cytokines.

[0173] (Example 6. Optimization of Isolation Protocol) The use of 3 mm punch biopsies was further tested to optimize the isolation protocol. Skin samples were prepared and obtained using 3 mm punch biopsies as described in Example 2.

[0174] Comparisons with different media were tested. Biopsies were placed on a grid: · AIM-V containing 5% human AB serum and IL-2 / IL-15 (2CK) or IL-2 / IL-15 / IL-21 / IL-4 (4CK); or · SKIN-T containing 10% fetal calf serum (FCS) and IL-2 / IL-15 (2CK) or IL-2 / IL-15 / IL-21 / IL-4 (4CK) and cultured in 24-well plates containing either of these.

[0175] Biopsies were incubated in a 5% CO2 incubator at 37 °C for 14 days (AIM-V) or 21 days (SKIN-T) before cell yield analysis. The total cell yield per grid was determined as described in Example 1. The results are shown in Figure 10. Isolation in AIM-V resulted in better cell yields and overall more Vδ1 cell numbers even in a shorter period.

[0176] The duration of cell isolation was also tested. A 3 mm punch biopsy was placed on a grid and placed into a G-REX6 well plate or G-REX10 bioreactor as described in Example 2. The biopsy was cultured in AIM-V (containing 5% serum replacement (SR), 5% human AB serum, or 5% SR / 5% AB “blend”) + 2ME + P / S + IL2 / 15 and incubated at 37 °C in a 5% CO2 incubator for 14 or 21 days before cell yield analysis. The total cell yield per grid was determined as described in Example 1. The results are shown in FIG. 11. For all media types, isolation after 3 weeks improved cell yield when compared to isolation after 2 weeks.

[0177] The use of serum replacement compared to human AB serum (5% or 10%) was also tested. The biopsy was incubated at 37 °C in a 5% CO2 incubator for 21 days before cell analysis. The total cell yield per grid and the % of Vδ1 cells were measured as described in Example 1. The results are shown in FIG. 12. Improved cell yields and a higher ratio of Vδ1 cells were obtained using a medium supplemented with 5% serum replacement compared to human AB serum.

[0178] (Example 7. Cell Expansion) Once the cells are isolated using the above protocol, they can be expanded using methods known in the art. For example, selective expansion of γδ T cells can be achieved using the expansion method described in WO2017072367.

[0179] Expansion of γδ T cells using additional cytokines was also tested. As described in Example 2, skin tissue lymphocytes isolated using two cytokines (2CK) or four cytokines (4CK) were harvested 21 days after culture. The harvested cells were cultured in TexMACs (Miltenyi Biotech) medium containing 5% serum replacement and human recombinant IL-2, IL-4, IL-15, and IL-21. Using FACS, the cell types were analyzed as described in Example 1. The results are shown in FIG. 13. The use of four cytokines at the time of isolation resulted in a larger population of γδ T cells after expansion compared to the use of two cytokines at the time of isolation.

[0180] Using the method described in Example 1, the phenotype of Vδ1 cells was analyzed by measuring the expression of various markers. The results are shown in FIG. 14. The use of four cytokines at the time of isolation and subsequent expansion resulted in cells with greater CD27 expression compared to cells isolated using two cytokines.

[0181] The total number of γδ cells and Vδ1 cells per grid was measured as described in Example 1. The results are shown in FIG. 15. The use of four cytokines at the time of isolation was shown to increase the overall yield of Vδ1 cells after expansion. The present application provides an invention in the following aspects. (Aspect 1) A method for isolating lymphocytes from a non-hematopoietic tissue sample, comprising: (i) culturing the non-hematopoietic tissue sample in the presence of (a) interleukin-2 (IL-2) or interleukin-9 (IL-9); (b) interleukin-15 (IL-15); and (c) interleukin-21 (IL-21); and (ii) recovering a population of lymphocytes cultured from the non-hematopoietic tissue sample. (Aspect 2) A method for isolating γδ T cells from a non-hematopoietic tissue sample, comprising: (i) culturing the non-hematopoietic tissue sample in the presence of (a) IL-2 or IL-9; (b) IL-15; and (c) IL-21; and (ii) recovering a population of γδ T cells cultured from the non-hematopoietic tissue sample. (Aspect 3) The method according to Aspect 1 or Aspect 2, wherein step (i) further comprises culturing the non-hematopoietic tissue sample in the presence of interleukin-4 (IL-4). (Aspect 4) The method according to Aspect 1, wherein the population of lymphocytes recovered from the culture of the non-hematopoietic tissue sample is a population of αβ T cells. (Aspect 5) The method according to Aspect 1, wherein the population of lymphocytes recovered from the culture of the non-hematopoietic tissue sample is a population of NK cells. (Aspect 6) The method according to any one of Aspects 1 to 5, wherein the lymphocytes or γδ T cells are recovered at least 7 days after the start of the culture. (Aspect 7) The method according to any one of Aspects 1 to 6, wherein the lymphocytes or γδ T cells are recovered at least 14 days after the start of the culture. (Aspect 8) The method according to any one of Aspects 1 to 7, wherein the lymphocytes or γδ T cells are recovered within 35 days of the start of the culture. (Aspect 9) The method according to any one of Aspects 1 to 8, wherein the lymphocytes or γδ T cells are recovered within 21 days of the start of the culture. (Aspect 10) The method according to any one of Aspects 1 to 9, wherein the non-hematopoietic tissue sample is cultured in a serum-free medium. (Aspect 11) The method according to any one of Aspects 1 to 9, wherein the non-hematopoietic tissue sample is cultured in a medium containing serum or a serum substitute. (Aspect 12) The method according to any one of Aspects 1 to 11, wherein the non-hematopoietic tissue sample is an intact biopsy. (Aspect 13) The method according to any one of aspects 1 to 12, wherein the non-hematopoietic tissue sample is not disrupted prior to step (i). (Aspect 14) The method according to any one of aspects 1 to 13, wherein the non-hematopoietic tissue sample has a minimum cross-sectional diameter of at least 1 mm. (Aspect 15) The method according to any one of aspects 1 to 14, wherein the non-hematopoietic tissue sample has a minimum cross-sectional diameter of at least 2 mm. (Aspect 16) The method according to any one of aspects 1 to 15, wherein the non-hematopoietic tissue sample has a minimum cross-sectional diameter of about 3 mm. (Aspect 17) The method according to any one of aspects 1 to 16, wherein the non-hematopoietic tissue sample has a maximum cross-sectional diameter of 8 mm or less. (Aspect 18) The method according to any one of aspects 1 to 17, wherein the non-hematopoietic tissue sample has a maximum cross-sectional diameter of 4 mm or less. (Aspect 19) The non-hematopoietic tissue sample has a minimum cross-sectional diameter of at least 1 mm 2 The method according to any one of aspects 1 to 18, wherein the non-hematopoietic tissue sample has a minimum cross-sectional area of at least 1 mm². (Aspect 20) The non-hematopoietic tissue sample has a minimum cross-sectional diameter of at least 4 mm 2 The method according to any one of aspects 1 to 19, wherein the non-hematopoietic tissue sample has a minimum cross-sectional area of at least 4 mm². (Aspect 21) The non-hematopoietic tissue sample has a cross-sectional area of about 7 mm 2 The method according to any one of aspects 1 to 20, wherein the non-hematopoietic tissue sample has a cross-sectional area of about 7 mm². (Aspect 22) The non-hematopoietic tissue sample has a maximum cross-sectional area of 64 mm 2 The method according to any one of aspects 1 to 21, wherein the non-hematopoietic tissue sample has a maximum cross-sectional area of 64 mm² or less. (Aspect 23) The non-hematopoietic tissue sample has a maximum cross-sectional area of 50 mm 2 The method according to any one of aspects 1 to 22, wherein the non-hematopoietic tissue sample has a maximum cross-sectional area of 50 mm² or less. (Aspect 24) The non-hematopoietic tissue sample has a maximum cross-sectional area of 16 mm 2 The method according to any one of aspects 1 to 23, wherein the non-hematopoietic tissue sample has a maximum cross-sectional area of 16 mm² or less. (Aspect 25) The method according to any one of aspects 1 to 24, wherein the non-hematopoietic tissue sample comprises a punch biopsy having a diameter of at least 1 mm. (Aspect 26) The method according to any one of aspects 1 to 25, wherein the non-hematopoietic tissue sample comprises a punch biopsy having a diameter of at least 2 mm. (Aspect 27) The method according to any one of aspects 1 to 26, wherein the non-hematopoietic tissue sample comprises a punch biopsy having a diameter of about 3 mm. (Aspect 28) The method according to any one of aspects 1 to 27, wherein the non-hematopoietic tissue sample comprises a punch biopsy having a diameter of 8 mm or less. (Aspect 29) The method according to any one of aspects 1 to 28, wherein the non-hematopoietic tissue sample comprises a punch biopsy having a diameter of 4 mm or less. (Aspect 30) The method according to any one of aspects 1 to 29, wherein the non-hematopoietic tissue sample is skin. (Aspect 31) The method according to aspect 30, wherein the non-hematopoietic tissue sample comprises an epithelial layer and a dermal layer. (Aspect 32) The method according to any one of aspects 1 to 31, wherein the non-hematopoietic tissue sample is gastrointestinal or digestive tract. (Aspect 33) The method according to any one of aspects 1 to 32, wherein the method is carried out in a container containing a gas-permeable material. (Aspect 34) The method according to aspect 33, wherein the container includes a liquid-tight container containing a gas-permeable material for enabling gas exchange. (Aspect 35) The method according to aspect 33 or aspect 34, wherein the bottom of the container is configured to enable gas exchange from the bottom of the container. (Aspect 36) The method according to any one of aspects 33 to 35, wherein the non-hematopoietic tissue sample is disposed on a synthetic scaffold inside the container. (Aspect 37) The method according to aspect 36, wherein the synthetic scaffold is tantalum-coated. (Aspect 38) The method according to aspect 36 or aspect 37, wherein the synthetic scaffold is configured to promote lymphocyte escape from the non-hematopoietic tissue sample to the bottom of the container. (Aspect 39) The method according to aspect 36 or aspect 37, wherein the synthetic scaffold is configured to promote γδ T cell escape from the non-hematopoietic tissue sample to the bottom of the container. (Aspect 40) The method according to any one of aspects 1 to 39, wherein the non-hematopoietic tissue sample is obtained from a human. (Aspect 41) The method according to any one of aspects 1 to 40, wherein the IL-2 is human IL-2 or a functional equivalent thereof. (Aspect 42) The method according to any one of aspects 1 to 41, wherein the IL-9 is human IL-9 or a functional equivalent thereof. (Aspect 43) The method according to any one of aspects 1 to 42, wherein the IL15 is human IL-15 or a functional equivalent thereof. (Aspect 44) The method according to any one of aspects 1 to 43, wherein the IL-21 is human IL-21 or a functional equivalent thereof. (Aspect 45) The method according to any one of aspects 1 to 44, wherein the IL-4 is human IL-4 or a functional equivalent thereof. (Aspect 46) The method according to any one of aspects 1 to 45, wherein the population of isolated cells includes a population of Vδ1 T cells. (Aspect 47) The method according to aspect 46, wherein the population of Vδ1 T cells expresses CD27 and / or does not substantially express TIGIT. (Aspect 48) The method according to aspect 46 or aspect 47, wherein the population of Vδ1 T cells has a frequency of TIGIT+ cells of less than 80%. (Aspect 49) The method according to any one of aspects 46 to 48, wherein the population of the Vδ1 T cells has TIGIT+ cells at a frequency of less than 60%. (Aspect 50) The method according to any one of aspects 46 to 49, wherein the population of the Vδ1 T cells has TIGIT+ cells at a frequency of about 40%. (Aspect 51) The method according to any one of aspects 46 to 50, wherein the population of the Vδ1 T cells has TIGIT+ cells at a frequency of about 30%. (Aspect 52) The method according to any one of aspects 46 to 51, wherein the population of the Vδ1 T cells has TIGIT+ cells at a frequency of about 20%. (Aspect 53) The method according to any one of aspects 46 to 52, wherein the population of the Vδ1 T cells has TIGIT+ cells at a frequency of about 10%. (Aspect 54) The method according to any one of aspects 46 to 53, wherein the population of the Vδ1 T cells substantially does not express TIGIT. (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%. (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%. (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%. (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%. (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%. (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. (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 according to 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 : The method as described above. (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 according to any one of aspects 1 to 62; and (ii) 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 expansion step involves culturing the γδ T cells (a) in the presence of IL-2 or IL-9; (b) in the presence of IL-15; and (c) in the presence of IL-21 The method according to Aspect 62 or Aspect 63, comprising culturing for at least 5 days in an amount effective to produce an expanded population of γδ T cells. (Aspect 65) The method according to Aspect 64, further comprising culturing the γδ T cells in the presence of IL-4. (Aspect 66) The method according to any one of Aspects 61 - 65, wherein the expansion step comprises culturing the lymphocytes or γδ T cells in a serum-free medium. (Aspect 67) The method according to any one of Aspects 61 - 65, wherein the expansion step comprises culturing the lymphocytes or γδ T cells in a medium containing serum or a serum substitute. (Aspect 68) The method according to any one of Aspects 63 - 67, wherein the expansion step comprises culturing the γδ T cells in the absence of substantial stromal cell contact. (Aspect 69) The method according to any one of Aspects 63 - 68, wherein the expansion step comprises the absence of an exogenous TCR pathway agonist. (Aspect 70) An isolated population of lymphocytes obtained by the method according to any one of Aspects 1 - 60. (Aspect 71) An isolated population of lymphocytes obtainable by the method according to any one of Aspects 1 - 60. (Aspect 72) An isolated population of γδ T cells obtained by the method according to any one of Aspects 1 - 60. (Aspect 73) An isolated population of γδ T cells obtainable by the method according to any one of Aspects 1 - 60. (Aspect 74) An isolated and expanded population of lymphocytes obtained by the method according to any one of Aspects 61 - 67. (Aspect 75) An isolated and expanded population of lymphocytes obtainable by the method according to any one of Aspects 61 - 67. (Aspect 76) An isolated and expanded population of γδ T cells obtained by the method according to any one of Aspects 61 - 69. (Aspect 77) An isolated and expanded population of γδ T cells obtainable by the method according to any one of Aspects 61 - 69.

Claims

**Claim 1** A method for isolating γδ T cells from a skin sample, comprising: (i) culturing the skin sample (a) in the presence of interleukin-2 (IL-2); (b) in the presence of interleukin-15 (IL-15); and (c) in the presence of interleukin-21 (IL-21) ; and (ii) recovering a population of γδ T cells cultured from the skin sample ; wherein the method is carried out in a container comprising a gas-permeable silicone membrane, and the bottom of the container is configured to allow gas exchange from the bottom of the container. **Claim 2** The method according to claim 1, wherein step (i) further comprises culturing the skin sample in the presence of interleukin-4 (IL-4). **Claim 3** The method according to claim 1 or 2, wherein the skin sample is cultured in a medium containing serum or a serum substitute. **Claim 4** The method according to any one of claims 1 to 3, wherein the skin sample is a non-invasive biopsy. **Claim 5** The population of the recovered γδ T cells comprises a population of Vδ1 T cells, and the population of Vδ1 T cells (i) has TIGIT+ cells with a frequency of less than 80%; (ii) has CD27+ cells with a frequency of more than 10%; or (iii) is both (i) and (ii) The method according to any one of claims 1 to 4. **Claim 6** The method according to claim 1, further comprising the step of expanding the population of the recovered γδ T cells. **Claim 7** The step of expanding comprises culturing the population of the recovered γδ T cells (a) in the presence of IL-2 or IL-9; (b) in the presence of IL-15; and (c) in the presence of IL-21 in an amount effective to produce an expanded population of γδ T cells for at least 5 days. **Claim 8** The method according to claim 7, further comprising culturing the population of the recovered γδ T cells in the presence of IL-4. **Claim 9** The step of expanding comprises culturing the population of the recovered γδ T cells (i) in a medium containing serum or a serum substitute; (ii) in the absence of substantial stromal cell contact; (iii) in the absence of an exogenous TCR pathway agonist; or (iv) in any combination of (i) to (iii) The method according to any one of claims 6 to 8.

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