Methods for isolating γδ T cells

Abstract

The present invention relates to methods for isolating non-hematopoietic tissue-resident lymphocytes, particularly γδT cells. Such γδT cells include non-Vδ2 cells, such as Vδ1, Vδ3, and Vδ5 cells, and such non-hematopoietic tissues include skin and intestines. It will be appreciated that such isolated non-hematopoietic tissue-resident lymphocytes have great utility in adoptive T cell therapy, chimeric receptor therapy, and the like. Also provided are methods for expanding isolated tissue-resident lymphocytes, particularly methods for isolating and expanding γδT cells. The present invention also relates to single cells and cell populations produced by the methods described herein.

Description

Technical Field

[0001] The present invention relates to methods for isolating non-hematopoietic tissue-resident lymphocytes, particularly γδT cells. Such γδT cells include non-Vδ2 cells, such as Vδ1, Vδ3, and Vδ5 cells, and such non-hematopoietic tissues include the skin and intestine. It will be appreciated that such isolated non-hematopoietic tissue-resident lymphocytes have significant utility in adoptive T cell therapy, chimeric receptor therapy, and the like. The present invention also relates to single cells and cell populations produced by the methods described herein. Background Art

[0002] Growing interest in T-cell immunotherapy for cancer has focused on the apparent ability of CD8+ and CD4+ αβ T-cell subsets to recognize cancer cells and mediate host-protective functions, particularly when inhibitory pathways exerted through PD-1, CTLA-4, and other receptors are disinhibited by clinically mediated antagonism. However, αβ T cells are MHC-restricted, which can lead to graft-versus-host disease.

[0003] γδ T cells represent a subset of T cells that express a unique, defined γδ T cell receptor (TCR) on their surface. This TCR consists of one γ chain and one δ chain. Human γδ TCR chains are selected from three major δ chains (Vδ1, Vδ2, and Vδ3) and six γ chains. Human γδ T cells can be broadly classified based on their TCR chains, as certain γ and δ types are more commonly present on cells in one or more tissue types, although not exclusively. For example, most blood-resident γδ T cells express a Vδ2 TCR, such as Vγ9Vδ2, while this is less common in tissue-resident γδ T cells, which more frequently use Vδ1 in the skin and Vγ4 in the intestine.

[0004] Most methods for isolating lymphocytes rely on isolating these cell types from the blood. Non-hematopoietic tissue-resident lymphocytes, such as γδ T cells, may have properties that are particularly suitable for certain applications, such as for targeting non-hematopoietic tumors and other targets. However, isolating such tissue-resident lymphocytes in clinically relevant numbers remains a challenge, particularly since many indications require numbers in the range of 10 8 Importantly, substantial cell loss during production means that more starting cells must be generated.

[0005] Because non-hematopoietic tissue-resident lymphocytes, particularly γδ T cells, are not readily available in large quantities, they have not been well characterized or studied for therapeutic applications. Therefore, there is a need in the art for methods of isolating non-hematopoietic tissue-resident lymphocytes, particularly γδ T cells, in sufficient quantities for further expansion and potential use as therapy, such as adoptive T cell therapy.

[0006] Clark et al. (2006) J. Invest. Dermatol. 126(5):1059-70 describe a method for isolating skin-resident T cells from normal and diseased skin. However, the method described therein is not suitable for clinical use because of the presence of animal products, and in particular because the yield of isolated cells is relatively low, i.e., 100 cells per cm 2 Less than 10 organizations 6 cells. The method described in Clark et al. uses chopped samples, resulting in the deliberate destruction of the structural integrity of tissue samples. WO2017072367 and WO2018 / 202808 relate to a method for amplifying non-hematopoietic tissue-resident γδT cells in vitro, which is carried out by culturing lymphocytes obtained from non-hematopoietic tissues in the presence of at least interleukin 2 (IL-2) and / or interleukin 15 (IL-15). WO2015189356 describes a composition for amplifying lymphocytes obtained from a sample obtained by a single collection, comprising at least two types of cytokines selected from IL-2, IL-15 and IL-21. Therefore, there is still a need for a method for separating tissue-resident non-hematopoietic lymphocytes (such as from skin), which produces a larger amount of cells suitable for clinical use. Summary of the Invention

[0007] According to a first aspect of the present invention, there is provided a method for isolating lymphocytes from a non-hematopoietic tissue sample, the method comprising the following steps:

[0008] (i) culturing a non-hematopoietic tissue sample in the presence of interleukin 1 beta (IL-1 beta); and

[0009] (ii) Collecting cultured lymphocyte populations from non-hematopoietic tissue samples.

[0010] According to another aspect, there is provided a method for isolating γδ T cells from a non-hematopoietic tissue sample, the method comprising the steps of:

[0011] (i) culturing a non-hematopoietic tissue sample in the presence of IL-1β; and

[0012] (ii) Collecting cultured γδ T cell populations from non-hematopoietic tissue samples.

[0013] According to another aspect, there is provided a method for isolating and expanding lymphocytes from a non-hematopoietic tissue sample, the method comprising the steps of:

[0014] (i) isolating a lymphocyte population from a non-hematopoietic tissue sample according to the methods described herein; and

[0015] (ii) further culturing the lymphocyte population for at least 5 days to produce an expanded lymphocyte population.

[0016] According to another aspect, there is provided a method for isolating and expanding γδ T cells from a non-hematopoietic tissue sample, the method comprising the steps of:

[0017] (i) isolating a γδ T cell population from a non-hematopoietic tissue sample according to the methods described herein; and

[0018] (ii) further culturing the γδ T cell population for at least 5 days to produce an expanded γδ T cell population. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 : Percentage (%) of viable γδ T cells (A) and total γδ T cells per grid across all conditions (B). Conditions were grouped by protein source (AB = human 10% AB serum; plasma = 2.5% human plasma; SR = 5% serum replacement) and subdivided into the presence (+) or absence (-) of IL-1β. Details of the different conditions are shown in Table 2. Bars represent experimental medians. Dashed lines represent the median of experimental controls (STD ISO).

[0020] Figure 2 Graph showing the % of Vδ1 T cells in viable cells (A) and total Vδ1 T cells per grid across all conditions (B). Conditions were grouped by protein source in the presence or absence of IL-1β. Bars represent experimental medians. Dashed lines represent the median of experimental controls.

[0021] Figure 3 : Graph showing live cell results for each grid grouped by protein source and broken down into the presence or absence of IL-4. Bars represent experimental medians. Dashed lines represent the median of experimental controls.

[0022] Figure 4 : Graph showing results grouped by protein source and broken down into the % of γδ T cells in viable cells in the presence or absence of IL-4. Bars represent experimental medians. Dashed lines represent the median of experimental controls.

[0023] Figure 5 : Graph showing total Vδ1T results for each grid grouped by protein source and subdivided into the presence or absence of IL-4. Bars represent experimental medians. Dashed lines represent the median of experimental controls.

[0024] Figure 6: Results are shown for % NKG2A expression (A) and % CD45RA expression (B) on Vδ1 T cells, grouped by protein source and broken down into the presence or absence of IL-4. Bars represent experimental medians. Dashed lines represent the median of experimental controls.

[0025] Figure 7 : Graph showing the results of % TIGIT expression on Vδ1 T cells grouped by protein source. Bars represent experimental medians. Dashed lines represent the median of experimental controls.

[0026] Figure 8 : Graph showing the results for total Vδ1 T cells grouped by protein source. Bars represent experimental medians. Dashed lines represent the median of experimental controls.

[0027] Figure 9 : Graph showing the results of total viable cells per grid grouped by protein source. Bars represent experimental medians. Dashed lines represent the median of experimental controls.

[0028] Figure 10 : Graph showing results for total TCR-negative cells grouped by protein source and subdivided into the presence or absence of IFN-γ. The dotted line represents the median using the previous isolation method.

[0029] Figure 11 : Total γδ T cells per grid under optimal conditions compared to the standard 2 cytokine isolation method.

[0030] Figure 12 : Graphs showing the effect of using freshly obtained isolated cells or isolated cells that had been frozen prior to expansion on the fold expansion (A) and % expansion of γδ T cells (B).

[0031] Figure 13 A) Comparison of total cell viability at the end of 21 days of isolated culture in the presence of IL-2, IL-4, IL-15, and IL-1β with or without the addition of 18.8 ng / ml IL-21. The dotted line represents the minimum acceptable viability. B) Graph showing the expansion of isolated γδ T cells (shown as % of viable cells) after 14 days of culture in the presence of IL-1β and IL-21 after thawing.

[0032] Figure 14 : Summary of cultures isolated with “IL-21” (IL-2, IL-4, IL-15, IL-1β, and IL-21) (IL-2, IL-4, IL-15, and IL-1β) or “no IL-21” (IL-2, IL-4, IL-15, and IL-1β) harvested after 19 or 21 days. DETAILED DESCRIPTION

[0033] According to a first aspect of the present invention, there is provided a method for isolating lymphocytes from a non-hematopoietic tissue sample, the method comprising the following steps:

[0034] (i) culturing a non-hematopoietic tissue sample in the presence of interleukin 1 beta (IL-1 beta); and

[0035] (ii) Collecting cultured lymphocyte populations from non-hematopoietic tissue samples.

[0036] According to another aspect of the present invention, there is provided a method for isolating γδ T cells from a non-hematopoietic tissue sample, the method comprising the following steps:

[0037] (i) culturing a non-hematopoietic tissue sample in the presence of IL-1β; and

[0038] (ii) Collecting cultured γδ T cell populations from non-hematopoietic tissue samples.

[0039] The results presented here show the detailed design of experiments conducted to establish an optimal protocol for isolating tissue-resident γδ T cells from non-hematopoietic tissue samples. Surprisingly, all conditions with optimal γδ T cell yields contained IL-1β, indicating that this cytokine facilitates the generation of high levels of γδ T cells during the isolation process.

[0040] As used herein, "IL-1β" refers to natural or recombinant IL-1β or variants thereof (e.g., mutants, muteins, analogs, subunits, receptor complexes, fragments, isomers, and peptide mimetics thereof) that act as agonists for one or more IL-1 receptor (IL-1R) subunits. IL-1 is a proinflammatory cytokine that plays a major role in a variety of diseases, including inflammatory diseases. IL-1 consists of two molecular species, IL-1α and IL-1β, which share only limited sequence identity but exert similar biological activities by binding to IL-1 receptors (type I and type II). Mature human IL-1β appears as a 153 amino acid sequence after cleavage of 116 amino acids from the N-terminus of the precursor polypeptide by CASP1, as described in Andrei et al. (2004) PNAS 101(26):9745-9750. IL-1β muteins are polypeptides in which specific substitutions have been made to the IL-1β protein while retaining the ability to bind to IL-1R. IL-1β mutant proteins can be characterized by amino acid insertions, deletions, substitutions, and modifications at one or more sites or other residues in the native IL-1β polypeptide chain. According to the present disclosure, any such insertions, deletions, substitutions, and modifications result in IL-1β mutant proteins that retain IL-1R binding activity. Exemplary mutant proteins can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions.

[0041] Nucleic acids encoding human IL-1β can be obtained by conventional procedures such as polymerase chain reaction (PCR). The amino acid sequence of human IL-1β (Gene ID 3553) is registered in Genbank as NP_000567 or in UniProt as P01584. The amino acid sequence of mouse (Mus musculus) IL-1β (Gene ID 16176) is registered in Genbank as NP_032387 or in UniProt as P10749.

[0042] IL-1β can also refer to IL-1β derived from a variety of mammalian species, including, for example, humans, monkeys, cattle, pigs, horses and mice. Variants can comprise conservatively substituted sequences, which means that a given amino acid residue is replaced by a residue with similar physicochemical characteristics. Examples of conservative substitutions include one aliphatic residue replacing another aliphatic residue, such as Ile, Val, Leu or Ala replacing each other, or one polar residue replacing another polar residue, such as between Lys and Arg; between Glu and Asp; or between Gln and Asn. Other such conservative substitutions (e.g., substitutions of entire regions with similar hydrophobic characteristics) are well known. The present invention also encompasses naturally occurring IL-1β variants. Examples of such variants are proteins produced by alternative mRNA splicing events or by proteolytic cleavage of IL-1β proteins, in which IL-1β binding properties are retained.

[0043] Separation method

[0044] "Isolation (isolation / isolating)" cells mentioned herein, particularly lymphocytes and / or γδT cells, refer to methods or processes for removing, separating, purifying, enriching or otherwise removing cells from a tissue or cell pool. It should be understood that such references include the terms "separated", "removed", "purified", "enriched", etc. Isolation of γδT cells includes separating or separating cells from a complete non-hematopoietic tissue sample or from stromal cells (e.g., fibroblasts or epithelial cells) of a non-hematopoietic tissue. Such separation may alternatively or additionally include separating or separating γδT cells from other hematopoietic cells (e.g., αβT cells or other lymphocytes). Isolation can continue for a defined period of time, for example, from the time a tissue explant or biopsy is placed in a separation culture to the time cells are collected from the culture, such as by centrifugation or other means for transferring the separated cell population to amplify culture or for other purposes, or removing the initial tissue explant or biopsy from the culture. The separation step can continue for at least about 3 days to about 45 days. In one embodiment, the separation step continues at least about 10 days to at least 28 days. In another embodiment, the separation step continues at least 14 days to at least 21 days. Therefore, the separation step can continue at least 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, about 35 days, about 40 days or about 45 days. In one embodiment, the separation step continues 19 days. In another embodiment, the separation step continues 21 days. It is to be understood that, although the cell proliferation separated during this separation step may not be significant, it is not necessarily absent. In fact, for those skilled in the art, it has been recognized that the separated cell also can begin to divide to produce a plurality of this type of cells in the separation container containing tissue and / or support.

[0045] Therefore, references herein to "isolated γδT cells," "isolated γδT cell population / isolated population of γδT cells," "separated γδT cells," "separated γδT cell population or separated population of γδT cells" will be understood to refer to hematopoietic cells or hematopoietic cell populations including γδ cells that have been isolated, separated, removed, purified or enriched from a sample of non-hematopoietic tissue origin, such that the cells have no substantial contact with non-hematopoietic cells or cells contained within intact non-hematopoietic tissue. Similarly, references herein to "isolated or separated Vδ1 T cell populations" refer to hematopoietic cells including Vδ1 T cells that have been isolated, separated, removed, purified or enriched from a sample of non-hematopoietic tissue origin, such that the cells have no substantial contact with non-hematopoietic cells or cells contained within intact non-hematopoietic tissue. Thus, separation or isolation refers to the separation, separation, removal, purification or enrichment of hematopoietic cells (eg, γδ T cells or other lymphocytes) from non-hematopoietic cells (eg, stromal cells, fibroblasts and / or epithelial cells).

[0046] The method for separating γδT cells as defined herein may include destroying tissue (e.g., chopping), and then separating γδT cells from other cell types. Preferably, the method for separating γδT cells as defined herein may include "crawl-out" γδT cells and other cell types from a complete non-hematopoietic tissue sample or explant or biopsy tissue matrix, wherein tissue-resident lymphocytes are physically separated from the tissue matrix without the need to destroy the tissue matrix. By maintaining the integrity of the tissue matrix, it has been found that tissue-resident lymphocytes are preferentially discharged from the tissue matrix, while suppressive cell types such as fibroblasts are rarely or not discharged, and these cell types are retained in the explant or biopsy and can then be easily removed at the end of separation. Therefore, in some embodiments, a small amount of fibroblasts are released from the tissue into the culture using a complete non-hematopoietic tissue sample or tissue matrix. This type of "climbing out" method utilizing complete non-hematopoietic tissue or tissue matrix has the advantage of reducing the need for excessive processing of non-hematopoietic tissue samples or tissue matrix, maintains the structural integrity of non-hematopoietic tissue or tissue matrix, and can have the unexpected advantage of providing a higher separation cell yield.

[0047] Therefore, the method for separating the lymphocyte of non-hematopoietic tissue origin as defined herein includes a method for separating the lymphocyte of non-hematopoietic tissue origin from a complete biopsy or explant of non-hematopoietic tissue. This complete biopsy or explant is a biopsy or explant in which the structural integrity of the biopsy or explant is not intentionally destroyed within the cutting perimeter of the biopsy or explant taken out from a tissue sample. This complete biopsy or explant will have a three-dimensional structure that is substantially maintained except for the slight damage caused by processing. Therefore, for example, this complete biopsy or explant is not mechanically destroyed, such as by chopping or cutting off destruction, nor by chemical enzymatic destruction. However, destroyed tissue can be used in the separation method of the present invention. In one embodiment, the lymphocyte separated is an αβT cell. In an alternative embodiment, the lymphocyte separated is a γδT cell. In another embodiment, the lymphocyte separated is a TCR negative cell (i.e., a cell that is negative for αβTCR and γδTCR expression). TCR negative cells are good indicators of the presence of natural killer (NK) cells. Thus, in another embodiment, the isolated lymphocytes are NK cells.It will be appreciated that more than one type of lymphocyte may be isolated from the same isolation step.

[0048] Methods for isolating γδ T cells using "climbing out" or methods such as those defined herein comprise culturing cells and / or non-hematopoietic tissue samples in the presence of cytokines and / or chemokines sufficient to induce the separation or isolation of γδ T cells and / or other lymphocytes as defined herein.

[0049] In one embodiment, step (i) further comprises culturing the non-hematopoietic tissue sample in the presence of interleukin 2 (IL-2). In another embodiment, step (i) further comprises culturing the non-hematopoietic tissue sample in the presence of interleukin 15 (IL-15). In yet another embodiment, step (i) further comprises culturing the non-hematopoietic tissue sample in the presence of IL-2 and IL-15.

[0050] As used herein, "IL-2" refers to natural or recombinant IL-2 or variants thereof (e.g., mutants, muteins, analogs, subunits, receptor complexes, fragments, isomers, and peptide mimetics thereof) that act as agonists for one or more IL-2 receptor (IL-2R) subunits. Such agents can support the proliferation of the IL-2-dependent cell line CTLL-2 (33; American Type Culture Collection TIB 214). Mature human IL-2 appears in the form of 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. IL-2 mutant proteins are polypeptides in which specific substitutions have been made to interleukin-2 proteins while retaining the ability to bind to IL-2Rβ, such as those described in US 2014 / 0046026. IL-2 mutant proteins may be characterized by amino acid insertions, deletions, substitutions, and modifications at one or more sites or other residues in the native IL-2 polypeptide chain. According to the present disclosure, any such insertions, deletions, substitutions, and modifications result in IL-2 mutant proteins that retain IL-2Rβ binding activity. Exemplary mutant proteins may comprise substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids.

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

[0052] IL-2 can also refer to IL-2 derived from a variety of mammalian species, including, for example, humans, monkeys, cattle, pigs, horses and mice. Variants can include sequences with conservative substitutions, which means that a given amino acid residue is replaced by a residue with similar physicochemical characteristics. Examples of conservative substitutions include one aliphatic residue replacing another aliphatic residue, such as Ile, Val, Leu or Ala replacing each other, or one polar residue replacing another polar residue, such as between Lys and Arg; between Glu and Asp; or between Gln and Asn. Other such conservative substitutions (e.g., substitutions of entire regions with similar hydrophobic characteristics) are well known. The present invention also encompasses naturally occurring IL-2 variants. Examples of such variants are proteins produced by alternative mRNA splicing events or by proteolytic cleavage of IL-2 proteins, in which IL-2 binding properties are retained. Alternative splicing of mRNA can produce truncated but biologically active IL-2 proteins. Changes attributable to proteolysis include, for example, differences in the N-terminus or C-terminus when expressed in different types of host cells, due to the proteolytic removal of one or more terminal amino acids (typically 1-10 amino acids) from the IL-2 protein. In some embodiments, the ends or interior of the protein can be modified, for example, with chemical groups such as polyethylene glycol 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).

[0053] As used herein, "IL-15" refers to natural or recombinant IL-15 or its variants (e.g., mutants, mutant proteins, analogs, subunits, receptor complexes, fragments, isomers, and peptide mimetics thereof) that act as agonists for 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 proliferation of the IL-2-dependent cell line CTLL-2. IL-15 was first reported by Grabstein et al. (Grabstein et al. Science 1994.264.5161:965-969) as a mature protein of 114 amino acids. As used herein, the term "IL-15" refers to natural or recombinant IL-15 and its mutant proteins, analogs, subunits, or complexes thereof (e.g., receptor complexes, such as sushi peptides, as described in WO 2007 / 046006), and each of which can stimulate the proliferation of CTLL-2 cells. In the CTLL-2 proliferation assay, supernatants from cells transfected with an in-frame fusion of recombinantly expressed IL-15 precursor and mature forms can induce CTLL-2 cell proliferation.

[0054] Human IL-15 can be obtained according to the procedure described by Grabstein et al. (Grabstein et al. Science 1994.264.5161:965-969) or by conventional procedures such as PCR. Human IL-15 cDNA was deposited on February 19, 1993 at And the registration number is 69245.

[0055] The amino acid sequence of human IL-15 (Gene ID 3600) is accessed in Genbank as NP_000576.1 GI:10835153 (isoform 1) and NP_751915.1 GI:26787986 (isoform 2). The amino acid sequence of mouse (Mus musculus) IL-15 (Gene ID 16168) is accessed in Genbank as NP_001241676.1 GI:363000984.

[0056] IL-15 can also refer to IL-15 derived from various mammalian species, including, for example, humans, monkeys, cows, pigs, horses, and mice. As referred to 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 may comprise sequences with conservative substitutions, meaning that a given amino acid residue is replaced with a residue having similar physicochemical characteristics. Examples of conservative substitutions include substitutions of one aliphatic residue for another aliphatic residue, such as Ile, Val, Leu, or Ala for each other, or substitutions of one polar residue for another polar residue, such as between Lys and Arg; between Glu and Asp; or between Gln and Asn. Other such conservative substitutions (e.g., substitutions of entire regions with similar hydrophobic characteristics) are well known. The present invention also encompasses naturally occurring IL-15 variants. Examples of such variants are proteins produced by alternative mRNA splicing events or by proteolytic cleavage of IL-15 proteins, in which IL-15 binding properties are retained. Alternative splicing of mRNA can produce truncated but biologically active IL-15 proteins. Changes attributable to proteolysis include, for example, differences in the N-terminus or C-terminus when expressed in different types of host cells, due to proteolytic removal of one or more terminal amino acids (typically 1-10 amino acids) from the IL-15 protein. In some embodiments, for example, chemical groups such as polyethylene glycol can be used to modify the ends or interior of the protein to change 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).

[0057] In one embodiment, the isolation of lymphocytes or γδT cells according to the present invention further comprises culturing the non-hematopoietic tissue sample in the presence of interleukin 4 (IL-4). Thus, in another embodiment, the non-hematopoietic tissue sample is cultured in the presence of IL-1β and IL-4.

[0058] As used herein, "IL-4" refers to natural or recombinant IL-4 or variants thereof (e.g., mutants, mutant proteins, analogs, subunits, receptor complexes, fragments, isomers, and peptide mimetics thereof) that act as agonists for one or more IL-4 receptor (IL-4R) subunits. Such agents can support the differentiation of naive helper T cells (Th0 cells) to Th2 cells. Mature human IL-4 appears in the form of a sequence of 129 amino acids (minus the signal peptide, which consists of an additional 24 N-terminal amino acids). IL-4 muteins are polypeptides in which specific substitutions have been made to interleukin-4 proteins while retaining the ability to bind to IL-4Rα, such as those described in U.S. Patent No. 6,313,272. IL-4 muteins can be characterized by amino acid insertions, deletions, substitutions, and modifications at one or more sites or other residues in the native IL-4 polypeptide chain. According to the present disclosure, any such insertions, deletions, substitutions, and modifications result in IL-4 muteins that retain IL-4Rα binding activity. Exemplary muteins can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions.

[0059] Nucleic acid encoding human IL-4 can be obtained by conventional procedures such as PCR. The amino acid sequence of human IL-4 (Gene ID 3565) is registered in Genbank as NG_023252. The amino acid sequence of mouse (Mus musculus) IL-4 (Gene ID 16189) is registered in Genbank as NC_000077.6.

[0060] IL-4 can also refer to IL-4 derived from a variety of mammalian species, including, for example, humans, monkeys, cattle, pigs, horses and mice. Variants can include sequences with conservative substitutions, which means that a given amino acid residue is replaced by a residue with similar physicochemical characteristics. Examples of conservative substitutions include one aliphatic residue replacing another aliphatic residue, such as Ile, Val, Leu or Ala replacing each other, or one polar residue replacing another polar residue, such as between Lys and Arg; between Glu and Asp; or between Gln and Asn. Other such conservative substitutions (e.g., substitutions of entire regions with similar hydrophobic characteristics) are well known. The present invention also encompasses naturally occurring IL-4 variants. Examples of such variants are proteins produced by alternative mRNA splicing events or by proteolytic cleavage of IL-4 proteins, in which IL-4 binding properties are retained. Alternative splicing of mRNA can produce truncated but biologically active IL-4 proteins. Changes attributable to proteolysis include, for example, differences in the N-terminus or C-terminus when expressed in different types of host cells, due to the proteolytic removal of one or more terminal amino acids (usually 1-10 amino acids) from the IL-4 protein. In some embodiments, the ends or interior of the protein can be modified, for example with chemical groups such as polyethylene glycol, 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).

[0061] In one embodiment, the isolation of lymphocytes or γδT cells according to the present invention further comprises culturing a non-hematopoietic tissue sample in the presence of interferon gamma (IFN-γ). Thus, in another embodiment, the non-hematopoietic tissue sample is cultured in the presence of IL-1β and IFN-γ.

[0062] As used herein, "IFN-γ" refers to natural or recombinant IFN-γ or its variants (e.g., mutants, mutant proteins, analogs, subunits, receptor complexes, fragments, isomers, and peptide mimetics thereof) that act as one or more IFN-γ receptor (IFNGR) subunit agonists. In particular, IFN-γ interacts with a heterodimeric receptor consisting of interferon gamma receptor 1 (IFNGR1) and interferon gamma receptor 2 (IFNGR2). Mature human IFN-γ appears in the form of a 143 amino acid sequence (minus a signal peptide, which consists of another 23 N-terminal amino acids). IFN-γ mutants are polypeptides in which IFN-γ proteins have been specifically substituted while retaining the ability to bind to IFNGR, such as those described in U.S. Patent No. 9,296,804. IFN-γ mutants may be characterized by amino acid insertions, deletions, substitutions, and modifications at one or more sites or other residues in a natural IFN-γ polypeptide chain. According to the present disclosure, any such insertions, deletions, substitutions and modifications result in IFN-γ mutant proteins that retain IFN-γR binding activity. Exemplary mutant proteins may comprise substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids.

[0063] Nucleic acids encoding human IFN-γ can be obtained by conventional procedures such as PCR. The amino acid sequence of human IFN-γ (Gene ID 3458) is registered in Genbank as NG_015840.1 or in UniProt as P01579. The amino acid sequence of mouse (Mus musculus) IFN-γ (Gene ID 15978) is registered in Genbank as NC_000076.6 or in UniProt as P01580.

[0064] IFN-γ can also refer to IFN-γ derived from a variety of mammalian species, including, for example, humans, monkeys, cattle, pigs, horses and mice. Variants can include sequences of conservative substitutions, which means that a given amino acid residue is replaced by a residue with similar physicochemical characteristics. Examples of conservative substitutions include one aliphatic residue replacing another aliphatic residue, such as Ile, Val, Leu or Ala replacing each other, or one polar residue replacing another polar residue, such as between Lys and Arg; Between Glu and Asp; Or between Gln and Asn. Other such conservative substitutions (e.g., substitutions of entire regions with similar hydrophobic characteristics) are well known. The present invention also encompasses naturally occurring IFN-γ variants. Examples of such variants are proteins produced by alternative mRNA splicing events or by proteolytic cleavage of IFN-γ proteins, in which IFN-γ binding properties are retained.

[0065] In one embodiment, the isolation of lymphocytes or γδT cells according to the present invention further comprises culturing the non-hematopoietic tissue sample in the presence of interleukin 21 (IL-21). Thus, in another embodiment, the non-hematopoietic tissue sample is cultured in the presence of IL-1β and IL-21.

[0066] As used herein, "IL-21" refers to natural or recombinant IL-21 or its variants (e.g., mutants, mutant proteins, analogs, subunits, receptor complexes, fragments, isomers, and peptide mimetics thereof) that act as agonists for one or more IL-21 receptor (IL-21R) subunits. Such agents can support natural killer (NK) and cytotoxic (CD8 + ) T cell proliferation. Mature human IL-21 appears in the form of a 133 amino acid sequence (minus signal peptide, which is composed of other 22 N-terminal amino acids). IL-21 mutants are following polypeptides, wherein interleukin 21 protein has been specifically replaced, while retaining the ability in conjunction with IL-21R α, such as those described in U.S. Patent number 9,388,241. The feature of IL-21 mutants can be amino acid insertions, deletions, replacements and modifications at one or more sites or other residues in the natural IL-21 polypeptide chain. According to the present disclosure, any such insertion, deletion, replacement and modification all obtain retaining the IL-21 mutants of IL-21R binding activity. Exemplary mutants can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions.

[0067] Nucleic acids encoding human IL-21 can be obtained by conventional procedures such as PCR. The amino acid sequence of human IL-21 (Gene ID 59067) is registered in Genbank as NC_000004.12. The amino acid sequence of mouse (Mus musculus) IL-21 (Gene ID 60505) is registered in Genbank as NC_000069.6.

[0068] IL-21 can also refer to IL-21 derived from a variety of mammalian species, including, for example, humans, monkeys, cattle, pigs, horses and mice. Variants can include sequences of conservative substitutions, which means that a given amino acid residue is replaced by a residue with similar physicochemical characteristics. Examples of conservative substitutions include one aliphatic residue replacing another aliphatic residue, such as Ile, Val, Leu or Ala replacing each other, or one polar residue replacing another polar residue, such as between Lys and Arg; between Glu and Asp; or between Gln and Asn. Other such conservative substitutions (e.g., substitutions of entire regions with similar hydrophobic characteristics) are well known. The present invention also encompasses naturally occurring IL-21 variants. Examples of such variants are proteins produced by alternate mRNA splicing events or by proteolytic cleavage of IL-21 proteins, in which IL-21 binding properties are retained. Alternate splicing of mRNA can produce truncated but biologically active IL-21 proteins. Changes attributable to proteolysis include, for example, differences in the N-terminus or C-terminus when expressed in different host cell types, due to the proteolytic removal of one or more terminal amino acids (typically 1-10 amino acids) from the IL-21 protein. In some embodiments, the ends or interior of the protein can be modified, for example, with chemical groups such as polyethylene glycol 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).

[0069] In an alternative embodiment, culture is carried out in the absence of IL-21. In an alternative embodiment, culture is carried out in the absence of IFN-γ. After statistical analysis of the results obtained from the experiment as described herein, it seems that when at least there is no IFN-γ, such as when there is no IFN-γ and IL-21, it is more beneficial to have IL-1 β in the separation culture. Result described herein further demonstrates that, under certain conditions, when there is no IL-21 or when IL-21 is with a concentration between 15ng / mL and 25ng / mL, particularly 18 to 20ng / ml, such as 18,19 or 20ng / mL, for example, when the concentration of 18.8ng / mL exists, it is also possible that there is IL-1 β in the separation culture.

[0070] In certain embodiments, the methods defined herein comprise a concentration of typically 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). / mL, for example, 10 IU / mL to 20 IU / mL, 20 IU / mL to 30 IU / mL, 30 IU / mL to 40 IU / mL, 40 IU / mL to 50 IU / mL, 50 IU / mL to 75 IU / mL, 75 IU / mL to 100 IU / mL, 100 IU / mL to 150 IU / mL, 150 IU / mL to 200 IU / mL, 200 IU / mL to 500 IU / mL or 500 IU / mL to 1,000 IU / mL). In certain embodiments, the methods defined herein include IL-2 at a concentration generally below 1,000 IU / mL, such as below 500 IU / mL. In some embodiments, the methods include IL-2 at a concentration of about 100 IU / mL, such as 138 IU / mL.

[0071] In other embodiments, the methods defined herein comprise a concentration of typically at least 10 IU / mL, such as at least 100 IU / mL, in particular at least 500 IU / mL (e.g. 10 IU / mL to 1,000 IU / mL, 20 IU / mL to 900 IU / mL, 25 IU / mL to 750 IU / mL, 30 IU / mL to 600 IU / mL, 40 IU / mL to 500 IU / mL, 50 IU / mL to 600 IU / mL, 70 IU / mL to 750 IU / mL, 80 IU / mL to 800 IU / mL, 90 IU / mL to 900 IU / mL, 100 IU / mL to 1,0 ... In some embodiments, the method comprises IL-15 at a concentration of about 600 IU / mL.

[0072] In some embodiments, isolating γδ T cells from a non-hematopoietic tissue sample comprises culturing in the presence of both IL-2 and IL-15 (each at any concentration listed above). In some cases, the concentration of IL-2 is about 138 IU / mL and the concentration of IL-15 is 600 IU / mL.

[0073] In other embodiments, methods defined herein include IL-21 at a concentration typically of at least 0.01 IU / mL, such as at least 0.1 IU / mL (e.g., 0.01 IU / mL to 100 IU / mL, 0.05 IU / mL to 50 IU / mL, 0.1 IU / mL to 10 IU / mL, 1 IU / mL to 5 IU / mL, e.g., 0.01 IU / mL to 0.05 IU / mL, 0.05 IU / mL to 0.1 IU / mL, 0.1 IU / mL to 1 IU / mL, 5 IU / mL to 10 IU / mL, 10 IU / mL to 50 IU / mL, 50 IU / mL to 100 IU / mL). In certain embodiments, methods defined herein include IL-21 at a concentration typically lower than 10 IU / mL, such as lower than 5 IU / mL. In some embodiments, the method includes an IL-21 concentration of about 1 IU / mL, such as 1.05 IU / mL. In other embodiments, the method includes an IL-21 concentration of 15 to 25 ng / mL. Therefore, in one embodiment, the method includes an IL-21 concentration between 15 ng / mL and 25 ng / mL. In another embodiment, the method includes an IL-21 concentration of 18 to 20 ng / ml, such as 18, 19 or 20 ng / mL, for example 18.8 ng / mL.

[0074] In other embodiments, the methods defined herein include IL-4 at a concentration of typically at least 1 IU / mL, such as at least 10 IU / mL (e.g., 1 IU / mL to 1,000 IU / mL, 5 IU / mL to 500 IU / mL, 10 IU / mL to 250 IU / mL, 50 IU / mL to 150 IU / mL, e.g., 1 IU / mL to 5 IU / mL, 5 IU / mL to 10 IU / mL, 10 IU / mL to 50 IU / mL, 50 IU / mL to 100 IU / mL, 100 IU / mL to 150 IU / mL). In certain embodiments, the methods defined herein include IL-4 at a concentration of typically less than 500 IU / mL, such as less than 100 IU / mL. In some embodiments, the methods include IL-4 at a concentration of about 100 IU / mL, such as 95 IU / mL. In other embodiments, the methods include IL-4 at a concentration of about 30 IU / mL, such as 31.6 IU / mL.

[0075] Therefore, in some embodiments, separation of γδT cells from non-hematopoietic tissue samples is included in the presence of IL-2, IL-15, IL-4 and IL-21 (each at any concentration listed above) and cultured. In other embodiments, the concentration of IL-2 is about 138IU / mL, the concentration of IL-15 is 600IU / mL, the concentration of IL-4 is 95IU / mL, and the concentration of IL-21 is between 15ng / mL and 25ng / mL, such as 18 to 20ng / ml, such as 18, 19 or 20ng / mL, for example, 18.8ng / mL. Therefore, in a specific embodiment, separation of γδT cells is included in the presence of IL-2 at a concentration of 138IU / mL, IL-15 at a concentration of 600IU / mL, IL-4 at a concentration of 95IU / mL, IL-1 β at a concentration of 4500IU / mL, and optionally IL-21 at a concentration of 18.8ng / mL and cultured.

[0076] " Non-hematopoietic tissue " or " non-hematopoietic tissue sample " mentioned herein include skin (such as human skin) and intestine (such as human intestine). Non-hematopoietic tissue is tissue other than blood, bone marrow or thymus tissue. In one embodiment, the non-hematopoietic tissue sample is skin (such as human skin). In another embodiment, the non-hematopoietic tissue sample is intestine or gastrointestinal tract (such as human intestine or human gastrointestinal tract). In some embodiments, lymphocytes and / or γδT cells are not obtained from a specific type of biological fluid sample (such as blood or joint fluid). In some embodiments, the non-hematopoietic tissue sample from which lymphocytes and / or γδT cells are separated according to the method defined herein is skin (such as human skin), which can be obtained by methods known in the art. Alternatively, the method for separating lymphocytes and / or γδT cells provided herein can be applied to gastrointestinal tract (such as colon or intestine), breast, lung, prostate, liver, spleen, pancreas, uterus, vagina and other skin, mucosa or serosal membrane. Lymphocytes and / or γδT cells can also reside in human cancer tissue samples (such as breast or prostate tumors). In some embodiments, lymphocytes and / or γδT cells can be from human cancer tissue samples (e.g., solid tumor tissue). In other embodiments, lymphocytes and / or γδT cells can be from non-hematopoietic tissue samples other than human cancer tissue (e.g., tissue without a large number of tumor cells). For example, lymphocytes and / or γδT cells can be from a skin area (e.g., healthy skin) separated from nearby or adjacent cancer tissue. Therefore, in some embodiments, γδT cells are not obtained from human cancer tissue. In other embodiments, lymphocytes are not obtained from human cancer tissue.

[0077] In one embodiment, the non-hematopoietic tissue sample of the methods defined herein has been obtained from a human. In alternative embodiments, the non-hematopoietic tissue sample of the methods defined herein has been obtained from a non-human animal subject.

[0078] Methods for obtaining such tissues are known in the art. Examples of such methods include surgical explants or punch biopsies, and depending on the method, size may vary. In some embodiments, non-hematopoietic tissue samples are obtained by punch biopsy.

[0079] In some embodiments of the present invention, the non-hematopoietic tissue sample is a complete biopsy. " complete " biopsy or " explant " mentioned herein comprise substantially undamaged or undamaged tissue and tissue sample, so that the structural integrity of the biopsy or explant in the cutting perimeter of taking out the biopsy or explant from the tissue sample is not intentionally destroyed. This complete biopsy or explant will have the three-dimensional structure that basically keeps except the slight destruction caused by processing. Therefore, for example, this complete biopsy or explant are not mechanically destroyed, such as by chopping or cutting off destruction, also not by chemical enzymatic destruction. Complete biopsy or complete tissue sample can comprise all the components of whole tissue, complete tissue, a part of tissue or described tissue. For example, in one embodiment, complete biopsy comprises all layers of skin. In another embodiment, biopsy comprises the epidermis and dermis of skin. It should be understood that in wherein biopsy is complete in this type of embodiment, keeps the separation and the distinction between these layers. Therefore, " complete " mentioned herein also comprises the biopsy of the full thickness of non-hematopoietic tissue sample.

[0080] Thus, in a specific embodiment of the invention, the non-hematopoietic tissue sample is not minced. In other embodiments, the intact biopsy is a punch biopsy. In yet another embodiment, the intact biopsy is obtained by punch biopsy.

[0081] In one embodiment, the non-hematopoietic tissue sample is a punch biopsy. The punch biopsy can be any shape, but is conveniently a circular cross-section and suitably has a diameter of at least 1mm. In other embodiments again, the non-hematopoietic tissue sample comprises a diameter of at least 2mm, such as a punch biopsy of at least 3mm, at least 4mm, at least 5mm, at least 6mm, at least 7mm or at least 8mm in diameter. In other embodiments, the non-hematopoietic tissue sample comprises a diameter of 8mm or less, such as a punch biopsy of 7mm or less, 6mm or less, 5mm or less or 3mm or less in diameter. In one embodiment, the non-hematopoietic tissue sample comprises a diameter between 1mm and 8mm, such as a punch biopsy of 2mm and 4mm in diameter. In a specific embodiment, the non-hematopoietic tissue sample comprises a diameter of 3mm.

[0082] In one embodiment, the biopsy is a skin biopsy and includes the epidermis and dermis. In another embodiment, the biopsy does not substantially include subcutaneous fat. Therefore, in one embodiment, the biopsy includes the epidermis and dermis and does not substantially include a subcutaneous fat layer. In another embodiment, the biopsy does not include subcutaneous fat. Alternatively, subcutaneous fat is not removed and therefore exists (or at least partially exists) in the biopsy. Therefore, in another embodiment, the biopsy is composed of the epidermis and dermis. In one embodiment, the biopsy includes the full thickness of a non-hematopoietic tissue sample.

[0083] Method of the present invention includes cultivating non-hematopoietic tissue sample as defined herein. " cultivating " mentioned herein includes adding cell and / or non-hematopoietic tissue sample to culture medium, and described cell includes the cell that separates, separates, takes out, purifies or enriches from non-hematopoietic tissue sample, and described culture medium comprises the required and / or preferred growth factor and / or nutrient substance of cell and / or non-hematopoietic tissue sample. Should be understood that such culture conditions can be adjusted according to the cell or cell colony to be separated from the non-hematopoietic tissue sample of the present invention, or can be adjusted according to the cell or cell colony to be separated and amplified from the non-hematopoietic tissue sample.

[0084] In certain embodiments, the duration of culturing non-hematopoietic tissue samples is sufficient to separate γδT cells from non-hematopoietic tissue samples. In alternative embodiments, the duration of culturing non-hematopoietic tissue samples is sufficient to separate lymphocytes (such as αβT cells and / or NK (natural killer) cells) other than γδT cells from non-hematopoietic tissue samples. In certain embodiments, the duration of culturing according to the method defined herein is at least 7 days. In certain embodiments, the duration of culturing according to the method defined herein is at least 14 days. In certain embodiments, the duration of culturing according to the method defined herein is less than 45 days, such as less than 40 days, such as less than 35 days, such as less than 30 days, such as less than 25 days. In another embodiment, the duration of culturing according to the method defined herein is between 14 days and 35 days, such as between 14 days and 21 days. In yet another embodiment, the duration of culturing according to the method defined herein is about 19 days, such as 19 days. In another embodiment, the duration of culturing according to the method defined herein is about 21 days, such as 21 days.

[0085] In a particular embodiment of the present invention, lymphocytes and / or γδT cells separated according to the method defined herein are collected from the culture of non-hematopoietic tissue samples after cultivating non-hematopoietic tissue samples. Collecting lymphocytes and / or γδT cells as defined herein can include physically collecting lymphocytes and / or γδT cells, separating lymphocytes and / or γδT cells and other lymphocytes (such as αβT cells, γδT cells and / or NK cells) from culture or separating and / or separating lymphocytes and / or γδT cells and stromal cells (such as fibroblasts). In one embodiment, lymphocytes and / or γδT cells are collected by mechanical means (such as pipetting). In another embodiment, lymphocytes and / or γδT cells are collected by magnetic separation and / or magnetic labeling. In yet another embodiment, lymphocytes and / or γδT cells are collected by flow cytometry techniques such as FACS. Therefore, in certain embodiments, γδT cells are collected by specific marker γδT cells. In other embodiments, lymphocytes are collected by specifically labeling them to distinguish them from other cells in the culture. It should be understood that such collection of lymphocytes and / or γδ T cells can include physical removal from the culture of the non-hematopoietic tissue sample, transfer to a separate culture container, or separate or different culture conditions.

[0086] It should be understood that this collection of lymphocytes and / or γδT cells is carried out after a duration sufficient to obtain separated lymphocyte colonies and / or γδT cell colonies from non-hematopoietic tissue samples. In certain embodiments, lymphocytes and / or γδT cells are collected after culturing non-hematopoietic tissue samples for at least one week, at least 10 days, at least 11 days, at least 12 days, at least 13 days or at least 14 days. Suitably, lymphocytes and / or γδT cells are collected after 40 days or less, such as 38 days or less, 36 days or less, 34 days or less, 32 days or less, 30 days or less, 28 days or less, 26 days or less or 24 days or less. In one embodiment, lymphocytes and / or γδT cells are collected after culturing non-hematopoietic tissue samples for at least 14 days. In another embodiment, lymphocytes and / or γδT cells are collected after culturing non-hematopoietic tissue samples for 14 to 21 days. In yet another embodiment, the lymphocytes and / or γδ T cells are collected after about 19 days in culture, such as after 19 days. In yet another embodiment, the lymphocytes and / or γδ T cells are collected after about 21 days in culture, such as after 21 days.

[0087] In certain embodiments of the present invention, a non-hematopoietic tissue sample is cultured in a culture medium containing serum, such as human AB serum or fetal bovine serum (FBS). In another embodiment, the non-hematopoietic tissue is cultured in a culture medium containing 10% human AB serum. In another embodiment, the non-hematopoietic tissue is cultured in a culture medium containing 5% human AB serum. According to this embodiment, a serum replacement as defined below may additionally be included in the culture medium. Thus, in yet another embodiment, the non-hematopoietic tissue is cultured in a culture medium containing 5% human AB serum and 5% serum replacement.

[0088] In certain embodiments of the invention, the non-hematopoietic tissue sample is cultured in a medium containing plasma (eg, human plasma). In another embodiment, the non-hematopoietic tissue is cultured in a medium containing 2.5% human plasma.

[0089] In an alternative embodiment of the present invention, the non-hematopoietic tissue sample is cultured in a medium that is substantially free of serum, such as a serum-free medium or a medium containing a serum replacement (SR). In another embodiment, the non-hematopoietic tissue is cultured in a medium containing a 5% serum replacement. Thus, in one embodiment, the non-hematopoietic tissue sample is cultured in a serum-free medium. Such serum-free medium may also include serum replacement medium, wherein the serum replacement is based on chemically defined components to avoid the use of sera from human or animal sources.

[0090] In one embodiment, the non-hematopoietic tissue sample is cultured in a culture medium that is free of animal-derived products.

[0091] In one embodiment, the method as defined herein is performed in a separation container. Reference to a "separation container" refers to a container comprising a non-hematopoietic tissue sample for separating lymphocytes and / or γδ T cells, optionally further comprising a synthetic scaffold. It should be noted that the separation container may be used solely for the separation method and not for further expansion steps.

[0092] In one embodiment, the method as defined herein is carried out in a container (e.g., a separation container) comprising a gas permeable material. Such materials can be permeable to gases such as oxygen, carbon dioxide, and / or nitrogen to allow gas exchange between the contents of the container and the surrounding atmosphere. It should be understood that the "container" mentioned herein includes culture dishes, culture plates, single-well dishes, multi-well dishes, multi-well plates, flasks, multi-layer flasks, bottles (such as spinner bottles), bioreactors, bags, tubes, etc. Such containers are known in the art for methods involving amplification of non-adherent cells and other lymphocytes. However, containers comprising gas permeable materials can also surprisingly be used to separate γδT cells that are generally considered to be adherent. It was found that culturing using such containers can greatly increase the yield of γδT cells isolated from non-hematopoietic tissue samples. It was also found that such containers preferentially support γδT cells and other lymphocytes including adherent cell types relative to fibroblasts and other stromal cells (e.g., epithelial cells). Therefore, in one embodiment, a container comprising a gas permeable material as defined herein preferentially supports γδT cells and other lymphocytes (e.g., αβT cells and / or NK cells). In another embodiment, fibroblasts and / or other stromal cells (eg, epithelial cells) are absent from cultures conducted in containers comprising gas permeable materials.

[0093] Such containers comprising breathable material can additionally comprise non-porous breathable material. Therefore, in one embodiment, breathable material is non-porous. In some embodiments, breathable material is a film, such as silicone, fluoroethylene polypropylene, polyolefin or ethylene vinyl acetate copolymer. In addition, such containers can only comprise a portion of breathable material, breathable film or non-porous breathable material. Therefore, according to another embodiment, the container comprises a top, a bottom and at least one sidewall, wherein at least a portion of the bottom of the container comprises breathable material, and when the top is above the bottom, the breathable material is on a substantially horizontal plane. In one embodiment, the container comprises a top, a bottom and at least one sidewall, wherein at least a portion of the bottom comprises breathable material, and when the top is above the bottom, the breathable material is on a horizontal plane. In another embodiment, the container comprises a top, a bottom and at least one sidewall, wherein at least a portion of the bottom comprises breathable material, and when the top is above the bottom, the breathable material can be on a vertical plane, or when the top is not above the bottom, the breathable material can be a horizontal plane. It should be understood that in such embodiments, only a portion of the bottom or the sidewall can comprise breathable material. Alternatively, the entire bottom or the entire sidewall may comprise a breathable material. In yet another embodiment, the top of the container comprising a breathable material may be sealed, for example, by using an O-ring. Such embodiments should be understood to prevent overflowing of the container contents or reduce their evaporation. Therefore, in certain embodiments, the container comprises a liquid-tight container comprising a breathable material that allows gas exchange. In alternative embodiments, the top of the container comprising a breathable material is on a horizontal plane and above the bottom and is not sealed. Therefore, in certain embodiments, the top is configured to allow gas exchange from the top of the container. In other embodiments, the bottom of the breathable container is configured to allow gas exchange from the bottom of the container. In yet another embodiment, the container comprising a breathable material may be a liquid-tight container and also include inlet and outlet ports or pipes. Therefore, in certain embodiments, the container comprising a breathable material comprises a top, a bottom and optionally at least one sidewall, wherein at least a portion of the top and the bottom comprises a breathable material, and if present, at least a portion of at least one sidewall comprises a breathable material. Example containers are described in WO2005035728 and US9255243, which are incorporated herein by reference. These containers are also commercially available, such as those provided by Wilson Wolf Manufacturing. Cell culture devices such as G-REX 6-well plates, G-REX 24-well plates, and G-REX 10 vessels.

[0094] In one embodiment, non-hematopoietic tissue sample is placed on synthetic support. As used herein, " synthetic support ", " support " and " grid " are used interchangeably, and refer to the non-natural three-dimensional structure that is suitable for supporting cell growth. Non-hematopoietic tissue sample can be placed on or attached to synthetic support, to promote lymphocyte to be discharged on support from explant. Synthetic support can be made of natural material and / or synthetic material, and described material is such as polymer (such as natural or synthetic polymer, such as polyvinyl pyrrolidone, polymethyl methacrylate, methylcellulose, polystyrene, polypropylene, polyurethane), ceramic (such as tricalcium phosphate, calcium aluminate, calcium hydroxyapatite) or metal (such as tantalum, titanium, platinum and the metal of the same element group with the combination of platinum, niobium, hafnium, tungsten and its alloy). In one embodiment of the invention, synthetic support is coated with tantalum. Biological factors (e.g., collagen (such as collagen I or collagen II), fibronectin, laminin, integrin, angiogenic factors, anti-inflammatory factors, glycosaminoglycans, vitrogen, antibodies and fragments thereof, cytokines (e.g., IL-2, IL-15, IL-4, IL-21, IL-1β and combinations thereof, such as IL-2, IL-15, IL-4, IL-21 and combinations thereof) can be coated onto the scaffold surface, encapsulated within the scaffold material, or added to the culture medium to enhance cell adhesion, migration, survival, or proliferation according to methods known in the art. This method and other methods can be used to isolate lymphocytes from many other non-hematopoietic tissue types (e.g., skin, intestine, prostate, and breast).

[0095] In one embodiment, a non-hematopoietic tissue sample is placed on a synthetic support in a container for separating lymphocytes from a non-hematopoietic tissue sample. In another embodiment, the synthetic support is configured to promote lymphocytes and / or γδT cells to be discharged to the bottom of the container from the non-hematopoietic tissue sample. This embodiment has the advantage of allowing separation and / or separation of lymphocytes (such as γδT cells, αβT cells and / or NK cells) from non-hematopoietic tissue samples and / or stromal cells (such as fibroblasts and / or epithelial cells). In addition, such embodiments allow lymphocytes (such as γδT cells, αβT cells and / or NK cells) to be collected to the bottom of the culture container from the non-hematopoietic tissue sample. In a specific embodiment, the synthetic support is configured to promote γδT cells to be discharged from the non-hematopoietic tissue sample. In another embodiment, the synthetic support is configured to promote lymphocytes such as αβT cells and / or NK cells to be discharged from the non-hematopoietic tissue sample.

[0096] Thus, in one aspect of the methods defined herein, the synthetic scaffold is configured to promote the egress of lymphocytes from a non-hematopoietic tissue sample to the bottom of a culture vessel. In another aspect of the methods defined herein, the synthetic scaffold is configured to promote the egress of γδ T cells from a non-hematopoietic tissue sample to the bottom of a vessel.

[0097] The methods of the present invention provide a greater total cell yield than previously described methods. In one embodiment, the total number of isolated cells from a tissue sample is at least 10 of the total number of cells isolated from the tissue sample. 6 cells / cm 2 , at least 2x10 6 cells / cm 2 , at least 5x10 6 cells / cm 2 , at least 10x10 6 cells / cm 2 , at least 20x10 6 cells / cm 2 , at least 30x10 6 cells / cm 2 , at least 40x10 6 cells / cm 2 , at least 50x10 6 cells / cm 2 , at least 60x10 6 cells / cm 2 , at least 70x10 6 cells / cm 2 , at least 80x10 6 cells / cm 2 , at least 90x10 6 cells / cm 2 , at least 100x10 6 cells / cm 2 , at least 150x10 6 cells / cm 2 , at least 200x10 6 cells / cm 2 In one embodiment, the total number of isolated cells is at least 50 x 10 6 cells / cm 2 In another embodiment, the total number of isolated cells is at least 100 x 10 6 cells / cm 2 .

[0098] The γδT cells that dominate in the blood are mainly Vδ2T cells, while the γδT cells that dominate in non-hematopoietic tissues are mainly Vδ1T cells, so Vδ1T cells account for about 70-80% of the γδT cell populations resident in non-hematopoietic tissues. However, some Vδ2T cells are also present in non-hematopoietic tissues, such as in the intestine, where they can account for about 10-20% of γδT cells. Some γδT cells residing in non-hematopoietic tissues express neither Vδ1TCR nor Vδ2TCR and are referred to herein as double negative (DN) γδT cells. These DNγδT cells may be mostly T cells expressing Vδ3 and a few T cells expressing Vδ5. Therefore, the γδT cells that typically reside in non-hematopoietic tissues and are separated by the method of the present invention are preferably non-Vδ2T cells, such as Vδ1T cells, which contain a smaller amount of DNγδT cells.

[0099] Thus, in a preferred embodiment, the γδ T cells isolated by the method defined herein comprise a Vδ1 T cell population. In one embodiment, the γδ T cells isolated by the method defined herein comprise a DN γδ T cell population. In one embodiment, the γδ T cells isolated by the method defined herein comprise a Vδ3 T cell population. In one embodiment, the γδ T cells isolated by the method defined herein comprise a Vδ5 T cell population.

[0100] γδT cells can also be defined by the type of γ chain they express. In another embodiment, the γδT cells isolated by the method defined herein comprise a population of Vγ4T cells. In most cases, the Vγ4T cells are obtained from intestinal tissue samples.

[0101] The isolation method provides a population of isolated γδ T cells that is greater in number than a reference population (e.g., at least 2-fold greater in number, at least 3-fold greater in number, at least 4-fold greater in number, at least 5-fold greater in number, at least 6-fold greater in number, at least 7-fold greater in number, at least 8-fold greater in number, at least 9-fold greater in number, at least 10-fold greater in number, at least 15-fold greater in number, at least 20-fold greater in number, at least 25-fold greater in number, at least 30-fold greater in number, at least 35-fold greater in number, at least 40-fold greater in number, at least 50-fold greater in number, at least 60-fold greater in number, at least 70-fold greater in number, at least 80-fold greater in number, at least 90-fold greater in number, at least 100-fold greater in number, at least 200-fold greater in number, at least 300-fold greater in number, at least 400-fold greater in number, at least 500-fold greater in number, at least 600-fold greater in number, at least 700-fold greater in number, at least 800-fold greater in number, at least 900-fold greater in number, at least 1,000-fold greater in number, at least 5,000-fold greater in number, at least 10,000-fold greater in number).

[0102] In some embodiments, the γδ T cell population isolated according to the methods of the present invention has a low proportion of cells expressing NKG2A. For example, the isolated γδ T cell population may have a frequency of NKG2A+ cells of less than 40%, less than 35%, less than 30%, less than 20%, or less than 10%. Alternatively, the isolated γδ T cell population may have a frequency of NKG2A+ cells of about 40%, about 30%, about 20%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1%. In certain embodiments, the isolated γδ T cell population has a frequency of NKG2A+ cells of less than 10%. Thus, in one embodiment, the isolated γδ T cell population has a frequency of NKG2A+ cells of about 8%. Thus, in one embodiment, the isolated γδ T cells do not substantially express NKG2A.

[0103] In some embodiments, the Vδ1 T cell population isolated according to the method of the present invention has a low proportion of cells expressing NKG2A. For example, the isolated Vδ1 T cell population may have a NKG2A+ cell frequency of less than 40%, less than 35%, less than 30%, less than 20% or less than 10%. Alternatively, the isolated Vδ1 T cell population may have an NKG2A+ cell frequency of about 40%, about 30%, about 20%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2% or about 1%. In certain embodiments, the isolated Vδ1 T cell population has an NKG2A+ cell frequency of less than 10%. Therefore, in one embodiment, the isolated Vδ1 cell population has an NKG2A+ cell frequency of about 9%. In one embodiment, the isolated Vδ1 cell population has an NKG2A+ cell frequency of about 7%. Therefore, in one embodiment, the isolated Vδ1 cells do not substantially express NKG2A. In one embodiment, less than 10% of the isolated Vδ1 T cell population express NKG2A.

[0104] In some embodiments, the γδ T cell population isolated according to the methods of the present invention has a low proportion of cells expressing CD45RA. CD45RA is a marker associated with terminal differentiation, so it is desirable to reduce the expression of this marker in the isolated cell population. For example, the isolated γδ T cell population may have a CD45RA+ cell frequency of less than 80%, less than 70%, less than 60%, less than 50%, less than 40% or less than 30%. Alternatively, the isolated γδ T cell population may have a CD45RA+ cell frequency of about 50%, about 40%, about 30%, about 20% or about 10%. In certain embodiments, the isolated γδ T cell population has a CD45RA+ cell frequency of less than 30%. Thus, in one embodiment, the isolated γδ T cell population has a CD45RA+ cell frequency of about 10%.

[0105] In some embodiments, the Vδ1 T cell population isolated according to the method of the present invention has a low proportion of cells expressing CD45RA. For example, the isolated Vδ1 T cell population may have a CD45RA+ cell frequency of less than 80%, less than 70%, less than 60%, less than 50%, less than 40% or less than 30%. Alternatively, the isolated Vδ1 T cell population may have a CD45RA+ cell frequency of about 50%, about 40%, about 30%, about 20% or about 10%. In certain embodiments, the isolated Vδ1 T cell population has a CD45RA+ cell frequency of less than 30%. Therefore, in one embodiment, the isolated Vδ1 cell population has a CD45RA+ cell frequency of about 10%. In one embodiment, less than 80% of the isolated Vδ1 T cell population expresses CD45RA, such as less than 30% of the isolated Vδ1 T cell population expresses CD45RA.

[0106] After separation from non-hematopoietic tissue (e.g., skin), γδT cells will generally become part of a larger lymphocyte population, which contains, for example, αβT cells, B cells, and natural killer (NK) cells. In some embodiments, 1%-10% of the separated lymphocyte populations are γδT cells (e.g., 1-10% of the separated skin-derived lymphocyte populations are γδT cells). In most cases, γδT cell populations (e.g., skin-derived γδT cell populations) will include large Vδ1T cell populations. In some embodiments, 1-10% of the separated lymphocyte populations (e.g., skin-derived lymphocytes) are Vδ1T cells (e.g., Vδ1T cells can represent more than 50%, more than 60%, more than 70%, more than 80%, or more than 90% of the separated γδT cell populations). In some cases, less than 10% of the separated γδT cell populations are Vδ2T cells (e.g., less than 10% of the separated skin-derived γδT cell populations are Vδ2T cells).

[0107] 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 (eg, before, during or after the expansion step).

[0108] The isolated γδT cells (e.g., γδT cells isolated from the skin, e.g., Vδ1T cells isolated from the skin) have a phenotype different from that of cells of corresponding hematopoietic tissue origin (e.g., blood-derived γδT cells and / or blood-derived Vδ2T cells). For example, the isolated γδT cell population may express higher levels of CCR3, CCR4, CCR7, CCR8, or CD103 compared to a reference population (e.g., a TCR-activated non-hematopoietic tissue-resident γδT cell population or a corresponding hematopoietic tissue-derived cell population (e.g., blood-derived γδT cells and / or blood-derived Vδ2T cells). In some embodiments, the isolated γδT cell population comprises 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 + The isolated γδ T cell population 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.

[0109] The isolated non-hematopoietic tissue-derived γδT cell (e.g., skin-derived γδT cell and / or skin-derived Vδ1T cell) population can also be characterized by function. Functional assays known in the art can be performed to determine the functional differences between any non-hematopoietic tissue-derived cells of the present invention (e.g., isolated γδT cell populations, isolated skin-derived Vδ1T cell populations, or expanded γδT cell populations and / or expanded skin-derived Vδ1T cell populations) and reference cells (e.g., TCR-activated non-hematopoietic tissue-resident γδT cell populations or corresponding hematopoietic tissue-derived cells (e.g., blood-derived γδT cells and / or blood-derived Vδ2T cells) populations). Such assays may include proliferation assays, cytotoxicity assays, binding assays, assays that measure persistence and / or location, and the like.

[0110] Thus, in one aspect of the invention, the method for isolating a population of lymphocytes and / or γδ T cells as defined herein results in a population comprising a surface phenotype consistent with a non-exhausted lymphocyte and / or γδ T cell population.

[0111] The method of the present invention has also been shown to improve the separation of other lymphocytes such as TCR negative cells. Therefore, according to another aspect of the present invention, there is provided a method for isolating lymphocytes from a non-hematopoietic tissue sample, the method comprising the steps of:

[0112] (i) culturing a non-hematopoietic tissue sample in a medium comprising serum replacement, human plasma and / or human AB serum; and

[0113] (ii) collecting a cultured lymphocyte population from a non-haematopoietic tissue sample. Suitably, the culture medium comprises serum replacement.

[0114] As shown in the examples provided herein, protein sources can have an impact on the number of TCR negative cells isolated from non-hematopoietic tissue samples. Therefore, in one embodiment, the isolated lymphocyte population includes a TCR negative cell population. It is known in the art that TCR negative cell populations typically contain a large number of NK cells. The isolated TCR negative cell population may contain NK cells. In one embodiment, at least 50%, such as at least 60%, 70% or 80% of the isolated TCR negative cell population is NK cells.

[0115] The culture method used in this aspect of the invention may contain the other conditions described herein. In one embodiment, step (i) further comprises culturing the non-hematopoietic tissue sample in the presence of IL-2 and IL-15.

[0116] In one embodiment, step (i) further comprises culturing the non-hematopoietic tissue sample in the presence of IL-4, IL-1β, IL-21 and / or IFN-γ. In another embodiment, step (i) further comprises culturing the non-hematopoietic tissue sample in the presence of IL-4 and / or IL-1β.

[0117] In one embodiment, culture is carried out in the absence of IFN-γ. After statistical analysis of the results obtained from the experiment described herein, it seems that in the absence of IFN-γ, the number of TCR negative cells obtained during separation may increase. In one embodiment, culture is carried out in the absence of IL-21. In alternative embodiments, step (i) is also included in the presence of concentration between 15ng / mL and 25ng / mL, such as 18 to 20ng / ml, such as 18,19 or 20ng / mL IL-21, in the case of cultivating non-hematopoietic tissue samples. In another embodiment, culture is carried out in the presence of concentration of 18.8ng / mL IL-21.

[0118] In one embodiment, the method includes freezing the lymphocyte colony or γδT cell colony separated. The cells can be frozen, for example, in Cryostor10 cell freezing solution. Many freezing solutions and parameters are known in the art and can be used for this aspect of the present invention. Frozen cells can be stored, for example, between -80°C and -200°C, optionally in liquid nitrogen (vapor phase), until needed for use.

[0119] According to one aspect of the present invention, there is provided an isolated lymphocyte (eg skin-derived αβ T cell and / or NK cell) population obtained by any method defined herein. In another embodiment, the isolated lymphocyte population is frozen.

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

[0121] According to another aspect of the present invention there is provided an isolated population of γδ T cells obtained by any of the methods defined herein. In another embodiment, the isolated population of γδ T cells is frozen.

[0122] According to another aspect of the present invention there is provided an isolated population of γδ T cells obtainable by any of the methods defined herein.

[0123] According to another aspect of the present invention, a method for isolating γδT cells from a non-hematopoietic tissue sample is provided, the method comprising the following steps:

[0124] (i) isolating a γδ T cell population from a non-hematopoietic tissue sample; and

[0125] (ii) The isolated γδ T cell population is frozen.

[0126] Previous methods involved freezing γδ T cells after expansion, but the inventors found that cells frozen after isolation were at least as enriched and expanded as fresh equivalents. Freezing after isolation allows time to perform small-scale quality control expansion validation on the donor before proceeding to large-scale expansion, which is resource-intensive and operator-intensive. It also enables the generation of multiple batches of cells from a single donor (i.e., to generate one batch of expanded cells and potentially generate more batches from the same donor later as needed).

[0127] In this aspect of the invention, separation methods may include methods described herein or alternative separation methods, such as in the presence of IL-2, IL-4, IL-9, IL-15, IL-21, or a combination thereof (such as IL-2 and IL-15, particularly IL-2, IL-15 optionally in combination with IL-4 and / or IL-21) and culturing non-hematopoietic tissue samples. In one embodiment, γδ T cells are separated by culturing non-hematopoietic tissue samples in the presence of IL-2 and IL-15, optionally in combination with IL-1β, IL-4, and / or IL-21. According to this embodiment, when IL-21 is present, its concentration is between 15ng / mL and 25ng / mL, such as 18, 19, or 20ng / mL. Therefore, in another embodiment, when IL-21 is present, its concentration is 18.8ng / mL.

[0128] In one embodiment, the persistent period of separation step (i) is at least 14 days. In other embodiments, the persistent period of separation step (i) is less than 21 days. In other embodiments again, the persistent period of separation step (i) is between 14 days and 35 days, such as about or between 19 days and 21 days. Therefore, in one embodiment, the persistent period of separation step (i) is about 19 days, such as 19 days. In another embodiment, the persistent period of separation step (i) is about 21 days, such as 21 days.

[0129] Cells can be frozen in a suitable freezing solution (such as Cryostor10 cell freezing solution). Many freezing / cryopreservation solutions and parameters are known in the art and can be used for this aspect of the present invention. Suitable freezing solutions can contain DMSO and other suitable culture medium supplements, such as human serum albumin, dextran, dextrose, NaCl, hydroxyethyl starch (Hespan) or PlasmaLyte A. The cells are then frozen to a temperature of approximately -80 ℃ to approximately -200 ℃, such as approximately -80 ℃ to approximately -135 ℃.

[0130] Cryopreservation can be accomplished by placing the vials into a freezing container and storing in a -80°C freezer for, for example, 1-3 days, and then transferring to the vapor phase of a liquid nitrogen storage system. In an alternative embodiment, the isolated γδ T cells are frozen in a controlled rate freezer.

[0131] It will be appreciated that frozen cells are suitable for long-term storage, and thus isolated cells can be kept frozen for a sustained period of time, followed by subsequent thawing and expansion. Frozen cells can be stored, for example, at between -80°C and -200°C, optionally in liquid nitrogen (vapor phase), until required for use.

[0132] After cryopreservation, the cells can be thawed (i.e., thawed), for example, in a 37°C water bath. The thawed cells can then be used in an amplification method. The amplification method can include any method described herein or as described in the art, for example, see WO2017072367 and WO2018202808.

[0133] Thus, the method may further comprise thawing the frozen γδ T cell population.Furthermore, the method may comprise culturing the thawed γδ T cell population for at least 5 days to produce an expanded γδ T cell population.

[0134] Therefore, according to another aspect of the present invention, there is provided a method for isolating and expanding γδ T cells from a non-hematopoietic tissue sample, the method comprising the following steps:

[0135] (i) isolating γδ T cells from non-hematopoietic tissue samples;

[0136] (ii) cryo-isolated γδ T cells;

[0137] (iii) thawing γδ T cells; and

[0138] (iv) culturing the thawed γδ T cells for at least 5 days to generate an expanded γδ T cell population.

[0139] In a specific embodiment, separation step (i) is included in the presence of a concentration between 15ng / mL and 25ng / mL, such as 18 to 20ng / ml, such as 18, 19 or 20ng / mL, for example, 18.8ng / mL of IL-21 and IL-1 β, and culture non-hematopoietic tissue sample for about 19 days, such as the duration of 19 days. In another embodiment, separation step (i) is included in the presence of IL-1 β and in the absence of IL-21, and culture non-hematopoietic tissue sample for about 19 days, such as 19 days, or about 21 days, such as the duration of 21 days. The data provided herein demonstrate that such separation conditions produce viable γ δ T cells after thawing in step (iii), and produce the ability to expand during subsequent amplification culture, i.e., effectively produce amplified γ δ T cell colonies in step (iv).

[0140] According to another aspect of the present invention, there is provided a frozen isolated γδ T cell population (ie, the frozen γδ T cells obtained in step (ii)), which is obtained by the method described herein.

[0141] According to another aspect of the present invention, there is provided a frozen isolated γδ T cell population (ie the frozen γδ T cells obtained in step (ii)), which is obtainable by the method described herein.

[0142] Amplification method

[0143] In certain embodiments, the present invention is characterized in that a method for amplifying non-hematopoietic tissue resident lymphocytes and / or γδT cells (e.g., αβT cells, NK cells, γδT cells and / or non-Vδ2T cells of skin origin, such as Vδ1T cells and / or DNT cells). These methods can be performed in vitro. In some embodiments, γδT cells are amplified from γδT cell colonies separated from non-hematopoietic tissue samples according to the methods defined herein. Typically, non-hematopoietic tissue resident γδT cells are able to spontaneously amplify after removing physical contact with stromal cells (e.g., skin fibroblasts). The method defined herein can be used to induce this separation, thereby causing γδT cells to de-suppress to trigger amplification. In certain embodiments, lymphocytes (e.g., αβT cells and / or NK cells of skin origin, αβT cells and / or NK cells of intestinal origin) are amplified from lymphocyte colonies separated from non-hematopoietic tissue samples according to the methods defined herein.

[0144] As used herein, reference to "expanded" or "expanded lymphocyte populations and / or γδT cell populations" includes cell populations that are larger or contain a greater number of cells than a non-expanded population. Such populations can be high in number, low in number, or mixed in number with amplification of a certain proportion or specific cell type within the population. It will be understood that the term "amplification step" refers to a process that results in amplification or the generation of an amplified population. Thus, an amplified or expanded population may be larger in number or contain a greater number of cells than a population that has not undergone an amplification step or prior to any amplification step. It will also be understood that any number indicating amplification (e.g., a fold increase or fold amplification) herein describes an increase in the number or size of a cell population or the number of cells, and indicates the amount of amplification.

[0145] Thus, in one embodiment, lymphocytes or γδ T cells isolated according to the methods of the invention are expanded.In one embodiment, the isolated lymphocyte or γδ T cell population is frozen and then thawed prior to expansion.

[0146] Such amplification may include culturing γδT cells in the presence of IL-2, IL-15 and IL-21, optionally including IL-4. Alternatively, such amplification may include culturing γδT cells in the presence of IL-9, IL-15 and IL-21, optionally including IL-4. It should be understood that any amplification step is performed for a duration effective to produce amplified lymphocyte populations and / or γδT cell populations. In one embodiment, the duration of effectively producing amplified lymphocyte populations and / or γδT cell populations is at least 5 days. Therefore, in one embodiment, amplification is included in the presence of IL-2, IL-15 and IL-21, culturing γδT cells for at least 5 days in an amount effective to produce amplified γδT cell populations. In another embodiment, amplification is included in the presence of IL-2, IL-15, IL-21 and IL-4, culturing γδT cells for at least 5 days in an amount effective to produce amplified γδT cell populations. In yet another embodiment, expanding comprises culturing the γδ T cells for at least 5 days in the presence of IL-9, IL-15, and IL-21 in an amount effective to produce an expanded γδ T cell population. In one embodiment, expanding comprises culturing the γδ T cells for at least 5 days in the presence of IL-9, IL-15, IL-21, and IL-4 in an amount effective to produce an expanded γδ T cell population.

[0147] In other embodiments, expanding comprises culturing lymphocytes and / or γδ T cells for a duration (e.g., at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 21 days, at least 28 days, or longer, e.g., 5 to 40 days, 7 to 35 days, 14 to 28 days, or about 21 days) in an amount effective to produce an expanded γδ T cell population. In some embodiments, lymphocytes and / or γδ T cells are expanded in culture for a period of 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 one embodiment, lymphocytes and / or γδ T cells are expanded for a period of 14 to 21 days. Thus, in some embodiments, including the isolation and expansion period (e.g., 1 to 40 days, such as 14 to 21 days), the isolation and expansion steps can last between 28 and 56 days or about 41 days.

[0148] In other embodiments, the expansion comprises culturing the γδ T cells for at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 21 days, at least 28 days or longer, for example, 5 to 40 days, 7 to 35 days, 14 to 28 days, or about 21 days. In one embodiment, the expansion step comprises culturing the γδ T cells for at least 10, 15, or 20 days to produce an expanded population. In one embodiment, the expansion step comprises culturing the γδ T cells for between 5 and 25 days, such as between 14 and 21 days. In another embodiment, the expansion step comprises culturing the γδ T cells for about 20 days.

[0149] In some embodiments, a typical amount of IL-2 effective to produce an expanded population of γδ T cells is 1 IU / mL to 2,000 IU / mL (e.g., 5 IU / mL to 1,000 IU / mL, 10 IU / mL to 500 IU / mL, 20 IU / mL to 400 IU / mL, 50 IU / mL to 250 IU / mL, or about 100 IU / mL, e.g., 5 IU / mL to 10 IU / mL, 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 60 IU / mL, 60 IU / mL to 70 IU / mL, 70 IU / mL to / mL, 80 IU / mL to 90 IU / mL, 90 IU / mL to 100 IU / mL, 100 IU / mL to 120 IU / mL, 120 IU / mL to 140 IU / mL, 140 IU / mL to 150 IU / mL, 150 IU / mL to 175 IU / mL, 175 IU / mL to 200 IU / mL, 200 IU / mL to 300 IU / mL, 300 IU / mL to 400 IU / mL, 400 IU / mL to 500 IU / mL, 500 IU / mL to 1,000 IU / mL, 1,000 IU / mL to 1,500 IU / mL, 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.

[0150] In some embodiments, the expanded γδ T cells (eg, skin-derived γδ T cells and / or non-Vδ2 T cells, such as Vδ1 T cells and / or DN) are effectively generated. A typical amount of IL-15 for a population of 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, such as 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 an expanded population of γδ T cells is about 10 ng / mL.

[0151] In some embodiments, a typical amount of IL-21 effective to generate an expanded population of γδ T cells (e.g., skin-derived γδ T cells and / or non-Vδ2 T cells, such as Vδ1 T cells and / or DN T cells) is 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 other embodiments, the amount of IL-21 is generally a concentration lower than 200ng / mL, such as 188.8ng / mL. In other embodiments, the amount of IL-21 is generally a concentration lower than 100ng / mL, such as less than 50ng / mL, such as 37.5ng / mL. In other embodiments, the amount of IL-21 is generally a concentration of 20ng / mL or lower, such as 18 to 20ng / ml, such as 18, 19 or 20ng / mL, for example 18.8ng / mL. In some embodiments, the method includes an IL-21 having a concentration of about 6ng / mL (such as about 6.25ng / mL).

[0152] In other embodiments, the methods defined herein include IL-4 at a concentration of typically 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, for example 0.1 ng / mL to 50 ng / mL, 1.0 ng / mL to 25 ng / mL, 5 ng / mL to 25 ng / mL). In other embodiments, the methods defined herein include IL-4 at a concentration of typically less than 100 ng / mL (such as less than 50 ng / mL, particularly less than 20 ng / mL). In some embodiments, the methods include IL-4 at a concentration of about 15 ng / mL.

[0153] Also provided herein is the replacement or addition of other factors in the amplification culture of non-hematopoietic tissue resident lymphocytes and / or γδT cells.For example, in some embodiments, in addition to any one of IL-2 and IL-15 or replacing any one of IL-2 and IL-15, further comprising 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). These additional or alternative factors for amplifying lymphocytes such as αβT cells or NK cells are known in the art. In one embodiment, these factors are used for amplification, and they selectively promote the amplification of γδT cells. In another embodiment, these factors are used for amplification, and they selectively promote the amplification of lymphocytes such as αβT cells and / or NK cells.

[0154] It will be appreciated that the amount of each of the above cytokines required to produce an expanded population of γδ T cells will depend on the concentration of one or more other cytokines. For example, if the concentration of IL-2 is increased or decreased, the concentration of IL-15 can be correspondingly decreased or increased, respectively. As described above, the amount effective to produce an expanded population herein refers to the combined effect of all factors on cell expansion.

[0155] The expansion method provides an expanded γδ T cell population that is greater in number than a reference population. In some embodiments, the expanded population of γδ T cells is greater in number than the isolated population of γδ T cells prior to the expanding step (e.g., at least 2-fold in number, at least 3-fold in number, at least 4-fold in number, at least 5-fold in number, at least 6-fold in number, at least 7-fold in number, at least 8-fold in number, at least 9-fold in number, at least 10-fold in number, at least 15-fold in number, at least 20-fold in number, at least 25-fold in number, at least 30-fold in number, at least 35-fold in number, at least 40-fold in number, at least 50-fold in number, at least 60-fold in number, at least 70-fold in number, at least 80-fold in number, at least 90-fold in number, at least 100-fold in number, at least 200-fold in number, at least 300-fold in number, at least 400-fold in number, at least 500-fold in number, at least 600-fold in number, at least 700-fold in number, at least 800-fold in number, at least 900-fold in number, at least 1,000-fold in number, at least 5,000-fold in number, at least 10,000-fold in number, or more relative to the isolated population of γδ T cells prior to the expanding step).

[0156] In one embodiment, the expanding step comprises culturing the isolated γδ T cells in the absence of contact with a plurality of stromal cells. In another embodiment, the expanding step comprises culturing the isolated γδ T cells in the absence of contact with a plurality of fibroblasts.

[0157] In other embodiments, the expansion step further comprises culturing the isolated γδ T cells in the presence of IL-4. Thus, in one embodiment, the expansion step comprises culturing the isolated γδ T cells in the presence of IL-2, IL-15, IL-4, and IL-21. Alternatively, the expansion step may comprise culturing the isolated γδ T cells in the presence of IL-9, IL-15, IL-4, and IL-21.

[0158] It should be understood that the expansion methods defined herein are also applicable to the expansion of other lymphocytes (e.g., αβT cells and / or NK cells). In such embodiments, the expansion step includes culturing the isolated lymphocytes in the presence of relevant growth factors and / or nutrients (e.g., cytokines and / or chemokines) to produce an expanded lymphocyte (e.g., αβT cells and / or NK cell) population.

[0159] In one embodiment, the method of expanding a γδT cell population as defined herein comprises culturing γδT cells or other lymphocytes in a medium containing serum or plasma. In an alternative embodiment, the method of expanding a γδT cell or other lymphocyte population as defined herein comprises culturing γδT cells in a serum-free medium. In another embodiment, the method of expanding a γδT cell or other lymphocyte population as defined herein comprises culturing γδT cells in a medium containing serum replacement.

[0160] In some embodiments, there is no substantial TCR pathway activation during the expansion step (e.g., the culture does not contain an exogenous TCR pathway activator). In one embodiment, the expansion step includes the absence of an exogenous TCR pathway agonist. In addition, provided herein is a method for expanding γδT 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. Therefore, in another embodiment of the methods defined herein, the expansion of γδT cells includes culturing γδT cells in the absence of substantial stromal cell contact.

[0161] Also provided is a method for generating a large population of non-hematopoietic tissue-derived γδT cells (e.g., skin-derived γδT cells and / or non-Vδ2 T cells, such as Vδ1 T cells and / or DN T cells) at a high rate (e.g., by removing stromal cell contact and / or TCR stimulation, or by culturing in the presence of an effective amount of factors). In some embodiments, the expansion step described herein expands γδT cells with a low population doubling time, the doubling time being given by the following equation:

[0162]

[0163] In view of the information provided herein, one skilled in the art will recognize that the present invention provides methods for expanding 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) 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).

[0164] In some embodiments, within 7 days of culture, the expanded γδ T cell population (e.g., an expanded Vδ1 T cell population and / or a DN T cell population) includes at least a 10-fold greater number of γδ T cells relative to the isolated γδ T cell population prior to expansion (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 greater number of γδ T cells relative to the isolated γδ T cell population prior to expansion). In some embodiments, within 14 days of culture, the expanded γδT cell population (e.g., the expanded Vδ1 T cell population and / or DN In some embodiments, the isolated γδ T cell population comprises at least a 20-fold greater number of γδ T cells relative to the isolated γδ T cell population prior to expansion (e.g., at least a 30-fold, at least a 40-fold, at least a 50-fold, at least a 60-fold, at least a 70-fold, at least a 80-fold, at least a 90-fold, at least a 100-fold, at least a 150-fold, at least a 200-fold, at least a 300-fold, at least a 400-fold, at least a 500-fold, at least a 600-fold, at least a 700-fold, at least a 800-fold, at least a 900-fold, at least a 1,000-fold, at least a 2,000-fold, at least a 3,000-fold, at least a 4,000-fold, at least a 5,000-fold, at least a 6,000-fold, at least a 7,000-fold, at least a 8,000-fold, at least a 9,000-fold, or at least a 10,000-fold greater number of γδ T cells relative to the isolated γδ T cell population prior to expansion). In some embodiments, within 21 days of culture, the expanded γδ T cell population (e.g., an expanded Vδ1 T cell population and / or a DN T cell population) includes at least 50-fold more γδ T cells relative to the isolated γδ T cell population before expansion (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 more γδ T cells relative to the isolated γδ T cell population before expansion).In some embodiments, within 28 days of culture, the expanded γδT cell population (e.g., the expanded Vδ1 T cell population and / or DN The isolated γδ T cell population) includes at least a 100-fold greater number of γδ T cells relative to the isolated γδ T cell population prior to expansion (e.g., at least a 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 greater number of γδ T cells relative to the isolated γδ T cell population prior to expansion).

[0165] Non-hematopoietic tissue-derived γδT cells (e.g., skin-derived γδT cells and / or non-Vδ2T cells, such as Vδ1T cells and / or DN T cells) expanded by the methods provided herein can have a phenotype that is well suited for anti-tumor efficacy. In some embodiments, the expanded γδT cell population (e.g., skin-derived Vδ1T cells) has a higher average expression of CD27 than a reference population (e.g., an isolated γδT cell population before the expansion step). In some embodiments, the expanded population of γδ T cells has an average expression of CD27 that is at least 2-fold greater than that of the isolated population of γδ T cells (e.g., at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1,000-fold, at least 5,000-fold, at least 10,000-fold, at least 20,000-fold, or more relative to that of the isolated population of γδ T cells).

[0166] Different fractions of the expanded γδ T cell population (e.g., skin-derived γδ T cells and / or non-Vδ2 T cells, such as Vδ1 T cells and / or DN T cells) may upregulate CD27, while another fraction may be CD27 低 or CD27 阴性 In this case, CD27 阳性For example, the expanded γδ T cell population may have at least 5% higher CD27 expression relative to the isolated γδ T cell population prior to expansion. 阳性 The cell frequency (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) of CD27 T cells relative to the isolated γδ T cell population before expansion. 阳性 In some embodiments, the expanded population has a CD27 阳性 The number of cells may be increased. For example, the expanded γδ T cell population may have at least twice the number of CD27 cells as the isolated γδ T cell population before expansion. 阳性 Cells. The expanded γδ T cell population may have a CD27+ cell 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, the expanded γδ T cell population may have a CD27+ cell frequency of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%. In certain embodiments, the expanded γδ T cell population has a CD27+ cell frequency of greater than 50%.

[0167] In some embodiments, the amplification methods as provided herein produce amplified non-hematopoietic tissue-derived γδT cells (e.g., skin-derived γδT cells and / or non-Vδ2T cells, such as Vδ1T cells and / or DN T cells) populations that have low TIGIT expression relative to a reference population (e.g., the isolated γδT cell population before the amplification step). In some embodiments, the amplified γδT cell population has a lower average expression of TIGIT than a reference population (e.g., the isolated γδT cell population before the amplification step). In some embodiments, the amplified γδT cell population has an average expression of TIGIT that is at least 10% lower than the isolated γδT cell population (e.g., at least 20% lower than the isolated γδT cell population, at least 30% lower, at least 40% lower, at least 50% lower, at least 60% lower, at least 70% lower, at least 80% lower, at least 90% lower, or as much as 100% lower). The expanded γδT cell population may have a TIGIT+ cell 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%. Alternatively, the expanded γδT cell population may have a TIGIT+ cell frequency of about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20% or about 10%. In certain embodiments, the isolated γδT cell population has a TIGIT+ cell frequency of less than 80%.

[0168] In some embodiments, the expanded γδ T cell (e.g., skin-derived γδ T cell or non-Vδ2 T cell, such as Vδ1 T cell and / or DN T cell) population has a high number or frequency of CD27 + Cells and low-frequency TIGIT + In some embodiments, the expanded γδ T cell population has a high frequency of CD27 relative to a reference population (e.g., relative to an isolated γδ T cell population prior to expansion). + TIGIT - For example, the expanded γδ T cell population may have at least 5% higher CD27 relative to the isolated γδ T cell population prior to expansion. + TIGIT - The cell frequency (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) of CD27 T cells relative to the isolated γδ T cell population before expansion. + TIGIT - In some embodiments, the expanded population has a CD27 +TIGIT - The number of cells may be increased. For example, the expanded γδ T cell population may have at least twice the number of CD27 cells as compared to the isolated γδ T cell population before expansion. + TIGIT - cells (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or up to 100% greater CD27 expression relative to the isolated γδ T cell population prior to expansion). + TIGIT - cell frequency).

[0169] In some cases, CD27 in a population of expanded γδ T cells (e.g., skin-derived γδ T cells and / or non-Vδ2 T cells, such as Vδ1 T cells and / or DN T cells) is expressed. + The average expression of TIGIT on the γδ T cell population is lower relative to the reference population. In some embodiments, the expanded CD27 + The γδ T cell population has a higher CD27 T cell number than the reference population (e.g., isolated CD27 T cell population before the expansion step). + In some embodiments, the expanded CD27 + The γδ T cell population has a higher CD27 + The γδ T cell population is at least 10% lower (e.g., lower than that of isolated CD27 + The average expression of TIGIT in a γδ T cell population is at least 20% lower, at least 30% lower, at least 40% lower, at least 50% lower, at least 60% lower, at least 70% lower, at least 80% lower, at least 90% lower, or up to 100% lower).

[0170] Additionally or alternatively, TIGIT in an expanded population of γδ T cells (eg, skin-derived γδ T cells and / or non-Vδ2 T cells, such as Vδ1 T cells and / or DN T cells) - The median expression of CD27 on the γδ T cell population was higher relative to the reference population. - γδ T cell population relative to isolated TIGIT before expansion - The γδ T cell population may have at least 5% higher CD27 + Cell frequency (e.g., relative to isolated TIGIT cells before expansion) -The γδ T cell population has at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or up to 100% higher CD27 + In some embodiments, relative to isolated TIGIT - γδT cell population, CD27 in the expanded population + The number of cells may increase. For example, the expansion of TIGIT - γδ T cell population relative to isolated TIGIT before expansion - The γδ T cell population can have at least twice the number of CD27 + cells (e.g., relative to isolated TIGIT cells before expansion) - The γδ T cell population has at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or up to 100% higher CD27 + cell frequency).

[0171] Increased or decreased expression of other markers including CD124, CD215, CD360, CTLA4, CD1b, BTLA, CD39, CD45RA, Fas ligand, CD25, ICAM-1, CD31, KLRG1, CD30, CD2, NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1, and CD64 can additionally or alternatively be used to characterize one or more expanded populations of 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). In some cases, relative to an isolated γδT cell population (e.g., before expansion), the expanded γδT cell population (e.g., skin-derived γδT cells and / or non-Vδ2 T cells, such as Vδ1 T cells and / or DN T cells) has a higher 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. Additionally or alternatively, relative to an isolated γδT cell population, the expanded γδT cell population may have a higher frequency of cells expressing one or more markers selected from the group consisting of CD124, CD215, CD360, CTLA4, CD1b, BTLA, CD39, CD45RA, Fas ligand, CD25, ICAM-1, CD31, KLRG1, CD30, and CD2. In some embodiments, the expanded γδ T cell population has a lower 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 relative to the isolated γδ T cell population. The expanded population can similarly have a lower frequency of cells expressing one or more markers selected from the group consisting of NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1, and CD64 relative to the isolated γδ T cell population.

[0172] A variety of basal culture media suitable for culturing and / or proliferating γδT cells are available, particularly culture media such as AIM-V, Iscoves culture media and RPMI-1640 (Life Technologies). Culture media can be supplemented with other culture media factors as defined herein, such as serum, serum proteins and selection agents, such as antibiotics. For example, in some embodiments, RPMI-1640 culture media contains 2mM glutamine, 10% FBS, 10mM HEPES pH7.2, 1% penicillin-streptomycin, sodium pyruvate (1mM; Life Technologies), non-essential amino acids (e.g., 100 μM Gly, Ala, Asn, Asp, Glu, Pro and Ser; 1X MEM non-essential amino acids (Life Technologies)) and 10 μl / L β-mercaptoethanol. In alternative embodiments, AIM-V culture media can be supplemented with CTS immune serum replacement and amphotericin B. In certain embodiments as defined herein, culture media can also be supplemented with IL-2 and IL-15. Conveniently, cells are cultured in an appropriate culture medium at 37° C. in a humidified atmosphere containing 5% CO 2 during isolation and / or expansion.

[0173] According to another aspect of the present invention, there is provided a method for isolating and expanding lymphocytes from a non-hematopoietic tissue sample, the method comprising the following steps:

[0174] (i) isolating a lymphocyte population from a non-hematopoietic tissue sample according to the method defined herein; and

[0175] (ii) further culturing the lymphocyte population (such as for at least 5 days) to produce an expanded lymphocyte population.

[0176] In one embodiment, the lymphocytes include αβT cells. Therefore, according to another aspect of the present invention, there is provided a method for isolating and expanding αβT cells from a non-hematopoietic tissue sample, the method comprising the following steps:

[0177] (i) isolating a population of αβ T cells from a non-hematopoietic tissue sample according to the method defined herein; and

[0178] (ii) further culturing the αβ T cell population (such as for at least 5 days) to produce an expanded αβ T cell population.

[0179] The culture in step (ii) can be performed by selective expansion, such as by selecting culture conditions in which αβ T cells are expanded in preference to other cell types present in the isolated population in step (i). Alternatively, the expansion conditions are not selective, and non-target cells (e.g., cells other than αβ T cells) can be depleted after the culture in step (ii). Alternatively, the expansion conditions are not selective, and non-target cells (e.g., cells other than αβ T cells) are depleted before the culture in step (ii). It should be noted that the purpose of these embodiments is to expand the total number of αβ T cells while also increasing their proportion in the population.

[0180] In one embodiment, the lymphocytes include NK cells. Therefore, according to another aspect of the present invention, there is provided a method for isolating and amplifying NK cells from a non-hematopoietic tissue sample, the method comprising the following steps:

[0181] (i) isolating a population of NK cells from a non-hematopoietic tissue sample according to the method defined herein; and

[0182] (ii) further culturing the NK cell population (such as for at least 5 days) to produce an expanded NK cell population.

[0183] The culture in step (ii) can be carried out by selective amplification, such as by selecting culture conditions in which NK cells are preferentially amplified over other cell types present in the isolated population in step (i). Alternatively, the amplification conditions are not selective and non-target cells (e.g., cells other than NK cells) can be exhausted after the culture in step (ii). Alternatively, the amplification conditions are not selective and non-target cells (e.g., cells other than NK cells) are exhausted before the culture in step (ii). It should be noted that the purpose of these embodiments is to amplify the total number of NK cells while also increasing their proportion in the population.

[0184] According to another aspect of the present invention, there is provided a method for isolating and expanding γδ T cells from a non-hematopoietic tissue sample, the method comprising the following steps:

[0185] (i) isolating a population of γδ T cells from a non-hematopoietic tissue sample according to the method defined herein; and

[0186] (ii) further culturing the γδ T cell population (such as for at least 5 days) to produce an expanded γδ T cell population.

[0187] The culture in step (ii) can be performed by selective expansion, such as by selecting culture conditions in which γδ T cells are preferentially expanded over other cell types present in the isolated population in step (i). Alternatively, the expansion conditions are not selective and non-target cells (e.g., cells other than γδ T cells) can be depleted after the culture in step (ii). Alternatively, the expansion conditions are not selective and non-target cells (e.g., cells other than γδ T cells) are depleted before the culture in step (ii). It should be noted that the purpose of these embodiments is to expand the total number of γδ T cells while also increasing their proportion in the population.

[0188] In one embodiment, the lymphocyte or γ δ T cell group of separation is frozen, then thawed before step (ii).It is surprising to find that freezing isolated cell colony is at least as enriched and amplified as fresh equivalent.Especially, data proof provided herein, obtain after thawing, have good vigor and have the ability (i.e., the ability of the γ δ T cell colony of amplification effectively produced in step (ii)) of separation during subsequent amplification culture, wherein separation step (i) is included in the presence of concentration between 15ng / mL and 25ng / mL, such as 18 to 20ng / ml, such as 18,19 or 20ng / mL, for example, 18.8ng / mL IL-21 and IL-1 β in the case of cultivating non-hematopoietic tissue sample about 19 days, such as the duration of 19 days, or alternatively in the presence of IL-1 β and in the absence of IL-21, cultivate about 19 days, such as 19 days, or about 21 days, such as the duration of 21 days.

[0189] In one embodiment, the lymphocytes include γδT cells. Therefore, according to another aspect of the present invention, there is provided a method for isolating and expanding γδT cells from a non-hematopoietic tissue sample, the method comprising the following steps:

[0190] (i) isolating a population of γδ T cells from a non-hematopoietic tissue sample according to the method defined herein; and

[0191] (ii) culturing the γδ T cell population in the presence of:

[0192] (a) IL-2 or IL-9;

[0193] (b) IL15; and

[0194] (c)(IL-21),

[0195] The cells are cultured for at least 5 days in an amount effective to produce an expanded γδ T cell population.

[0196] In certain embodiments of this aspect of the invention, culturing the γδT cell population further comprises culturing the γδT cell population in the presence of IL-4. Thus, in another aspect of the invention, a method for isolating and expanding γδT cells from a non-hematopoietic tissue sample is provided, the method comprising the steps of:

[0197] (i) isolating a population of γδ T cells from a non-hematopoietic tissue sample according to the method defined herein; and

[0198] (ii) culturing the γδ T cell population in the presence of:

[0199] (a) IL-2 or IL-9;

[0200] (b) IL15; and

[0201] (c) IL-21; and

[0202] (d) IL-4,

[0203] The cells are cultured for at least 5 days in an amount effective to produce an expanded γδ T cell population.

[0204] According to one aspect of the present invention, there is provided an expanded isolated lymphocyte (eg skin-derived αβ T cell and / or NK cell) population obtained by any of the methods defined herein.

[0205] According to another aspect of the present invention there is provided an expanded isolated lymphocyte population obtainable by any of the methods defined herein.

[0206] According to a further aspect of the present invention there is provided an expanded isolated γδ T cell population obtained by any of the methods defined herein.

[0207] According to a further aspect of the present invention there is provided an expanded isolated γδ T cell population obtainable by any of the methods defined herein.

[0208] Cell Applications

[0209] The lymphocytes and / or γδT cells obtained by the method of the present invention can be used as a medicine, for example, for adoptive T cell therapy. This involves transferring the lymphocytes and / or γδT cells obtained by the method of the present invention into a patient. The therapy can be autologous, i.e., the γδT cells can be transferred back into the same patient from whom they were obtained, or the therapy can be allogeneic, i.e., the γδT cells from one person can be transferred into a different patient. In the case of allogeneic transfer, the γδT cells may be substantially free of αβT cells. For example, αβT cells can be removed from a γδT cell population using any suitable means known in the art (e.g., by negative selection, e.g., using magnetic beads), for example, after amplification. The treatment method may include: providing a non-hematopoietic tissue sample obtained from a donor individual; isolating γδT cells from a non-hematopoietic tissue sample as described herein; culturing the separated γδT cells to produce an amplified population; and administering the amplified γδT cell population to a recipient individual.

[0210] The patient or subject to be treated is preferably a human cancer patient (e.g., a human cancer patient being treated for a solid tumor) or a viral infection patient (e.g., a CMV-infected or HIV-infected patient). In some cases, the patient has a solid tumor and / or is being treated for a solid tumor.

[0211] Because they typically reside in non-hematopoietic tissues, tissue-resident Vδ1 T cells and DNγδ T cells are also more likely to home to and remain within the tumor mass 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-associated immunopathologies.

[0212] Because γδ T cells are non-MHC restricted, they cannot be recognized as foreign by the host into which they are transferred, meaning they are unlikely to cause graft-versus-host disease. This means they can be used “off the shelf” and transferred into any recipient, for example for allogeneic adoptive T cell therapy.

[0213] The non-hematopoietic tissue-resident γδ T cells obtained by the method of the present invention express NKG2D and respond to NKG2D ligands (e.g., MICA) that are closely related to malignant tumors. They also express cytotoxic characteristics in the absence of any activation and are therefore likely to effectively kill tumor cells. For example, the non-hematopoietic tissue-resident γδ T cells obtained as described herein may express one or more of IFN-γ, TNF-α, GM-CSF, CCL4, IL-13, granulysin, granzymes A and B, and perforin in the absence of any activation, preferably all. IL-17A may not be expressed.

[0214] There is compelling evidence demonstrating the utility and suitability of non-hematopoietic tissue-resident γδ T cells obtained by the methods of the present invention for clinical use as "off-the-shelf" immunotherapeutic agents. These cells possess innate killing capacity, are not MHC-restricted, and exhibit improved homing and / or retention within tumors compared to other T cells.

[0215] In some embodiments, a method of treating an individual having a tumor in a 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 as described above to produce an expanded population, and administering the expanded γδ T cell population to the individual having the tumor.

[0216] The pharmaceutical composition may include a combination of expanded non-hematopoietic tissue-resident γδT cells as described herein and one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. 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. Cryopreservative solutions that can be used for the pharmaceutical compositions of the present invention include, for example, DMSO. The composition can be formulated, for example, for intravenous administration.

[0217] In one embodiment, the pharmaceutical composition is substantially free, eg, has no detectable levels of contaminants, such as endotoxins or mycoplasmas.

[0218] In some cases, a therapeutically effective amount of expanded γδ T cells obtained by any of the methods described above can be administered to a subject in a therapeutically effective amount (e.g., for treating cancer, such as for treating a solid tumor). In some cases, 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 DNT cells) is less than 10x10 12 cells (e.g., fewer than 9 x 10 12 cells, less than 8x10 per dose 12 cells, less than 7x10 per dose 12 cells, less than 6x10 per dose 12 cells, less than 5x10 per dose 12 cells, less than 4x10 per dose 12 cells, less than 3x10 per dose 12 cells, less than 2x10 per dose 12 cells, less than 1x10 per dose 12 cells, less than 9x10 per dose11 cells, less than 8x10 per dose 11 cells, less than 7x10 per dose 11 cells, less than 6x10 per dose 11 cells, less than 5x10 per dose 11 cells, less than 4x10 per dose 11 cells, less than 3x10 per dose 11 cells, less than 2x10 per dose 11 cells, less than 1x10 per dose 11 cells, less than 9x10 per dose 10 cells, less than 7.5x10 per dose 10 cells, less than 5x10 per dose 10 cells, less than 2.5x10 per dose 10 cells, less than 1x10 per dose 10 cells, less than 7.5x10 per dose 9 cells, less than 5x10 per dose 9 cells, less than 2.5x10 per dose 9 cells, less than 1x10 per dose 9 cells, less than 7.5x10 per dose 8 cells, less than 5x10 per dose 8 cells, less than 2.5x10 per dose 8 cells, less than 1x10 per dose 8 cells, less than 7.5x10 per dose 7 cells, less than 5x10 per dose 7 cells, less than 2.5x10 per dose 7 cells, less than 1x10 per dose 7 cells, less than 7.5x10 per dose 6 cells, less than 5x10 per dose 6 cells, less than 2.5x10 per dose 6 cells, less than 1x10 per dose 6 cells, less than 7.5x10 per dose 5 cells, less than 5x10 per dose 5 cells, less than 2.5x10 per dose 5 cells or less than 1x10 per dose 5 cells).

[0219] In some embodiments, the therapeutically effective amount of expanded γδT cells (e.g., skin-derived γδT cells and / or non-Vδ2 T cells, such as Vδ1 T cells and / or DNT cells) is less than 10×10 12 cells (e.g., fewer than 9x10 12 cells, less than 8x10 12 cells, less than 7x10 12 cells, less than 6x10 12 cells, less than 5x10 12 cells, less than 4x10 12 cells, less than 3x10 12 cells, less than 2x10 12 cells, less than 1x10 12 cells, less than 9x10 11 cells, less than 8x10 11 cells, less than 7x10 11 cells, less than 6x10 11 cells, less than 5x10 11 cells, less than 4x10 11 cells, less than 3x10 11 cells, less than 2x10 11 cells, less than 1x10 11 cells, less than 9x10 10 cells, less than 7.5x10 10 cells, less than 5x10 10 cells, less than 2.5x10 10 cells, less than 1x10 10 cells, less than 7.5x10 9 cells, less than 5x10 9 cells, less than 2.5x10 9 cells, less than 1x10 9 cells, less than 7.5x10 8 cells, less than 5x10 8 cells, less than 2.5x10 8 cells, less than 1x10 8 cells, less than 7.5x10 7 cells, less than 5x10 7 cells, less than 2.5x10 7 cells, less than 1x10 7 cells, less than 7.5x10 6 cells, less than 5x10 6 cells, less than 2.5x10 6cells, less than 1x10 6 cells, less than 7.5x10 5 cells, less than 5x10 5 cells, less than 2.5x10 5 cells or less than 1x10 5 cells).

[0220] In some embodiments, a dose of expanded non-hematopoietic tissue-resident γδ T cells as described herein comprises about 1×10 6 , 1.1x10 6 , 2x10 6 , 3.6x10 6 , 5x10 6 , 1x10 7 , 1.8x10 7 , 2x10 7 , 5x10 7 , 1x10 8 , 2x10 8 or 5x10 8 In some embodiments, the dose of expanded non-hematopoietic tissue-resident γδT cells (e.g., skin-derived γδT cells and / or non-Vδ2 T cells, such as Vδ1 T cells and / or DNT cells) comprises at least about 1×10 6 , 1.1x10 6 , 2x10 6 , 3.6x10 6 , 5x10 6 , 1x10 7 , 1.8x10 7 , 2x10 7 , 5x10 7 , 1x10 8 , 2x10 8 or 5x10 8 In some embodiments, the dose of expanded non-hematopoietic tissue-resident γδT cells (e.g., skin-derived γδT cells and / or non-Vδ2 T cells, such as Vδ1 T cells and / or DNT cells) comprises up to about 1×10 6 , 1.1x10 6 , 2x10 6 , 3.6x10 6 , 5x10 6 , 1x10 7 , 1.8x10 7 , 2x10 7 , 5x10 7 , 1x10 8 , 2x10 8 or 5x108 In some embodiments, the dose of expanded non-hematopoietic tissue-resident γδT cells (e.g., skin-derived γδT cells and / or non-Vδ2 T cells, such as Vδ1 T cells and / or DNT cells) comprises about 1.1 x 10 6 -1.8x10 7 In some embodiments, the dose of expanded non-hematopoietic tissue-resident γδT cells (e.g., skin-derived γδT cells and / or non-Vδ2 T cells, such as Vδ1 T cells and / or DNT cells) comprises about 1×10 7 , 2x10 7 , 5x10 7 , 1x10 8 , 2x10 8 , 5x10 8 , 1x10 9 , 2x10 9 or 5x10 9 In some embodiments, the dose of expanded non-hematopoietic tissue-resident γδT cells (e.g., skin-derived γδT cells and / or non-Vδ2 T cells, such as Vδ1 T cells and / or DNT cells) comprises at least about 1×10 7 , 2x10 7 , 5x10 7 , 1x10 8 , 2x10 8 , 5x10 8 , 1x10 9 , 2x10 9 , or 5x10 9 In some embodiments, the dose of expanded non-hematopoietic tissue-resident γδT cells (e.g., skin-derived γδT cells and / or non-Vδ2 T cells, such as Vδ1 T cells and / or DNT cells) comprises up to about 1×10 7 , 2x10 7 , 5x10 7 , 1x10 8 , 2x10 8 , 5x10 8 , 1x10 9 , 2x10 9 or 5x10 9 cells.

[0221] In one embodiment, the subject is administered 10 4 to 10 6In one embodiment, the subject receives an initial administration of a population of non-hematopoietic tissue-resident γδT cells (e.g., 10 cells per kg of subject body weight). 4 to 10 6 γδT cells, for example, 10 per kg of subject body weight 4 to 10 5 An initial administration of 10 γδ T cells per kg of subject body weight), and one or more (e.g., 2, 3, 4, or 5) subsequent administrations of expanded non-hematopoietic tissue-resident γδ T cells (e.g., 10 4 to 10 6 Expanded non-hematopoietic tissue-resident γδ T cells, for example, 10 per kg of subject body weight 4 to 10 5 In one embodiment, the one or more subsequent administrations are less than 15 days, e.g., 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days after the previous administration, e.g., less than 4, 3, or 2 days after the previous administration. In one embodiment, the subject receives a total of about 10 γδ T cells per kg of the subject's body weight over the course of at least three administrations of the γδ T cell population. 6 γδT cells, for example, a subject receives 1x10 5 The initial dose of γδT cells is 3x10 5 The second administration of γδ T cells and 6x10 5 The invention provides a third administration of γδ T cells, and for example, each administration is administered less than 4, 3 or 2 days after the previous administration.

[0222] The non-hematopoietic tissue resident γδ T cells obtained by the method of the present invention can also be genetically engineered to enhance therapeutic properties, such as for CAR-T therapy. This involves generating engineered T cell receptors (TCRs) to reprogram T cells with new specificities (such as the specificity of monoclonal antibodies). Engineered TCRs can make T cells specific to malignant cells and can therefore be used for cancer immunotherapy. For example, T cells can recognize cancer cells expressing tumor antigens, such as tumor-associated antigens that are not expressed by normal somatic cells from subject tissues. Therefore, CAR-modified T cells can be used for adoptive T cell therapy, for example, in cancer patients.

[0223] The use of blood-resident γδT cells for CAR has been described. However, non-hematopoietic tissue-resident γδT cells obtained by the method of the present invention may be particularly good carriers for CAR-T methods because they can be transduced with chimeric antigen-specific TCRs while retaining their innate ability to recognize transformed cells and may have better tumor penetration and retention than blood-resident γδT cells or traditional systemic αβT cells. In addition, their lack of MHC-dependent antigen presentation reduces the likelihood of graft-versus-host disease and allows them to target tumors expressing low levels of MHC. Similarly, they do not rely on traditional co-stimulation, such as enhancing targeting to tumors expressing low levels of co-stimulatory receptor ligands through the participation of CD28.

[0224] In some embodiments, one or more additional therapeutic agents may be administered to the subject. The additional therapeutic agent may be selected from the group consisting of an immunotherapeutic agent, a cytotoxic agent, a growth inhibitory agent, a radiotherapeutic agent, an anti-angiogenic agent, or a combination of two or more thereof. The additional therapeutic agent may be administered simultaneously with, before, or after the administration of the expanded γδ T cells. The additional therapeutic agent may be an immunotherapeutic agent that acts on a target within the subject (e.g., the subject's own immune system) and / or the transferred γδ T cells.

[0225] Administration of the composition can be carried out in any convenient manner. The compositions described herein can be administered to the patient transarterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous injection, or intraperitoneally, for example, by intradermal or subcutaneous injection. Compositions of non-hematopoietic tissue-resident γδ T cells can be injected directly into a tumor, lymph node, or site of infection.

[0226] It should be understood that all embodiments described herein are applicable to all aspects of the invention.

[0227] As used herein, the term "about" includes up to 10% (and including 10%) above and down to 10% (and including 10%) above the specified value, suitably up to 5% (and including 5%) above and down to 5% (and including 5%) above the specified value, particularly the specified value. The term "between..." includes the values ​​of the specified boundaries.

[0228] Certain aspects and embodiments of the present invention will now be described by way of example and with reference to the above-identified figures.

[0229] Example

[0230] Example 1. Methods and Materials

[0231] Unless otherwise stated, the following methods were used to generate the results of the subsequent examples.

[0232] Material

[0233] The sources of materials used in the experimental conditions are summarized in Table 1 .

[0234] Table 1. Materials used in the study

[0235] Reagents supplier AIM-V Fisher CTS immune serum replacement Life Technologies Human serum albumin Sigma-Aldrich human plasma Merck Chemicals Ltd. Human IL-2, Premium Grade Miltenyi Human IL-15, premium grade Miltenyi Human IL-4, Premium Grade Miltenyi Human IL-21 Peprotech human IFN-γ Peprotech Human IL-1β Peprotech

[0236] Experimental modeling design

[0237] JMP software 15.0 was used to generate a design of experiment (DoE). A DoE custom design was applied to generate an experiment analyzing the effects of protein source, IL-1β, IFN-γ, IL-21, and IL-4 on the determination of results (such as γδT cell enrichment). Protein source was treated as a categorical variable, and IL-1β, IFN-γ, IL-21, and IL-4 were treated as continuous variables. In addition, a JMP script was used to reject experimental conditions containing only IL-4.

[0238] A 2-level factorial design was applied to enable the determination of straight lines and 2-level interactions (eg IL-21*IL-1β).A design of 21 conditions was experimentally tested.

[0239] After collecting experimental data, outlier removal was achieved by calculating the studentized residual based on the γδ T cell % as the response readout. Any row that produced a studentized residual of >4 or <4 for γδ T cell % was excluded from the analysis. A total of 7 rows out of 84 were excluded.

[0240] After removing outliers, a standard least squares fit was applied to the given response based on the values ​​from each donor for that response. To account for donor variation, donor ID was added as an independent variable to the least squares fit model. Protein source and cytokine interaction p-values ​​associated with the selected outcome (such as % γδ T cells or % TIGIT+γδ T cells) were eliminated in a stepwise approach until only interactions with p < 0.05 remained.

[0241] Separation setup and harvesting

[0242] Prior to sample arrival, relevant culture media were prepared and 20 mm x 1.5 mm carbon matrices ("grids") (Cytomatrix Pty Ltd, Australia or Ultramet, USA) were autoclaved. The grids were then rinsed in phosphate buffered saline (PBS) and kept submerged in PBS until use.

[0243] Preparation of organotypic skin cultures

[0244] Skin samples were prepared by removing subcutaneous fat using forceps, scissors, and a scalpel. Each piece of tissue was taken at a time and punched multiple times using a 3 mm biopsy punch. Three tissue punches were placed on each grid.

[0245] A grid is placed in each hole of G-REX6 (Wilson Wolf). Each hole is filled with 5ml relevant basal medium and 5ml 2X relevant conditioned medium (obtaining 10ml 1X relevant conditioned medium). 100 μ L amphotericin B (Life Technologies) is added in each hole (obtaining 1% amphotericin B). Plate is incubated at 37 ℃ and 5% CO 2 down for 21 days. Cultivate by adding 10ml 2X relevant culture medium gently in each hole at the 7th day and the 14th day.

[0246] Separation and harvest

[0247] Remove the matrix. Use a pipette to resuspend the cells in each well and transfer them to a centrifuge tube. Wash the wells with PBS, and transfer the wash solution to a centrifuge tube. The isolated cells can then be analyzed, for example, by cell counting or flow cytometry.

[0248] Freezing of isolated cells

[0249] Once the isolated cells have been analyzed, the cell suspension is spun down in a centrifuge. The supernatant is discarded and the cell pellet is resuspended in Cryostor10 Cell Freezing Solution (Sigma Aldrich) at a final concentration of 100 x 10 6 The cell suspension was then transferred to a cryovial and placed in a cryogenic chamber or Mr Frosty freezing apparatus (ThermoScientific) and placed in a -80°C freezer overnight. The next day, the cells were transferred to liquid nitrogen storage (vapor phase).

[0250] Flow cytometry

[0251] Flow cytometry was performed using the following antibody-fluorochrome conjugates:

[0252] a) Bulk release group: CD45-FITC, CD25-PE, PANαβ-PerCP Vio700, NKG2D-PEVio770, Vδ1-Vioblue, PANγδ-APC.

[0253] b) Functional group: CD45RA-FITC, TIGIT-PE, PANαβ-PerCP Vio700, NKG2A-PEVio770, Vδ1-Vioblue, PD1-BV510, PANγδ-APC.

[0254] Commercial antibodies were purchased from Biolegend or Miltenyi. Viability dye (eFluor780) was from ThermoFischer Scientific. Flow cytometry data were analyzed using FLOWJO (version 10.6.2).

[0255] Determine total cell number

[0256] Total cell counts were generated using an NC-250 Nucleocounter (Chemometec, Copenhagen Denmark) and the manufacturer's instructions.

[0257] Example 2. Separation optimization experiment

[0258] We investigated the optimization of protocols for isolating γδ T cells from non-hematopoietic tissue samples to increase the yield of γδ T cells and potentially improve the quality of Vδ1 T cells upon isolation. Improving the starting material will help improve both yield and quality in subsequent expansion steps.

[0259] A design of experiment (DoE) investigated the effects of different protein sources and cytokines on skin isolate cultures. To achieve a feasible design, the medium type, IL-15 concentration, and IL-2 concentration were kept constant. Different combinations of IL-21, IFN-γ, and IL-1β were investigated. The addition of IL-4 was also investigated, although only in the presence of IL-21, IFN-γ, or IL-1β.

[0260] All cells were isolated in AIM-V medium supplemented with either 5% serum replacement (SR), 2.5% allogeneic plasma, or 10% allogeneic AB male serum. All cultures were supplemented with IL-2 and IL-15 and with various combinations of IL-4, IL-21, IFN-γ, and IL-1β. The culture conditions tested are summarized in Table 2.

[0261] Table 2. Summary of experimental conditions tested

[0262]

[0263]

[0264] The results of the culture conditions are shown in Figures 1 to 10 middle.

[0265] cytokines

[0266] In particular, the use of IL-1β increased the total yield of γδT cells and the total yield of isolated Vδ1T cells in the case of plasma and AB serum separation. The results of γδT cell % and total γδT cell in all conditions are shown in Figure 1 The percentage of Vδ1 T cells and the total Vδ1 T cells are shown in Figure 2 middle.

[0267] Adding IL-4 to AB serum had the effect of increasing the total number of cells isolated per grid ( Figure 3 A more modest increase was observed in plasma fractionated cultures.

[0268] Addition of IL-4 to plasma, SR or AB serum samples had the effect of increasing the % γδ enrichment in the cultures ( Figure 4 )’s role.

[0269] Addition of IL-4 to the plasma cultures had the effect of increasing the total number of Vδ1 cells isolated per grid ( Figure 5 The same effect was not observed in cultures treated with SR or human AB serum; however, treatment with AB serum increased the total number of isolated Vδ1 cells per grid compared to controls.

[0270] Adding IL-21 to AB serum cultures increased the total number of isolated γδ T cells. However, no benefit of IL-21 was seen in cultures isolated with plasma or SR. DoE modeling of the percentage of γδ T cells per donor in plasma with IL-1β and IFN-γ showed that IL-1β was most beneficial in the absence of IFN-γ and IL-21.

[0271] Phenotypic analysis

[0272] Addition of IL-4 to AB serum and plasma cultures had the effect of reducing the expression of NKG2A and CD45RA on Vδ1 T cells ( Figure 6 ).

[0273] Compared with plasma and AB serum fractionation cultures, SR treatment resulted in increased expression of the checkpoint inhibitor marker TIGIT on Vδ1 T cells ( Figure 7 ).

[0274] The analysis also showed that SR administration resulted in increased expression of the terminal differentiation marker CD45RA and the inhibitory receptor NKp44 on Vδ1 T cells relative to plasma and AB serum (data not shown).

[0275] Protein sources

[0276] Compared with SR culture isolation, the use of plasma and AB serum in culture had the effect of increasing the total number of Vδ1 T cells per grid ( Figure 8 However, DoE modeling showed that the addition of IFN-γ to SR isolated samples increased the total number of isolated γδ T cells.

[0277] The use of SR also appears to result in a decrease in the overall viability of isolated cells compared to plasma or AB serum as a protein source ( Figure 9 ).

[0278] Other lymphocytes

[0279] Conditions for generating TCR-negative cells (i.e., cells negative for both αβ-TCR and γδ-TCR) were also investigated. Using SR as a protein source resulted in an increase in the number of TCR-negative cells compared to plasma or AB serum ( Figure 10 The absence of IFN-γ also increased the number of TCR-negative cells.

[0280] in conclusion

[0281] The data show that the total number of isolated γδ cells can be greatly increased, which provides the possibility of increasing the initial number of expansion sets. All conditions with the best γδ T cell % and total γδ T cells per grid contained IL-1β (conditions 6, 8, 9, 12 and 14). The results of the best conditions compared to the standard 2 cytokine isolation method are shown in Figure 11 middle.

[0282] Therefore, a skin lymphocyte isolation mixture, designated as Passage 2, was identified by a DoE approach titrating a combination of human cytokines and human AB serum, human pooled plasma, or serum replacement, using human pooled plasma (previous isolation cultures used serum replacement) and the cytokines IL-2, IL-4, IL-15, and IL-1β (previous isolation cultures used only IL-2 and IL-15). Compared to the initial method (now also designated Passage 1), this new growth mixture reduced the total number of isolated viable lymphocytes from 50-100 x 10 cells per isolation cell matrix (grid). 6 cells (previously) increased to 75-200x10 6 Furthermore, passage 2 showed up to 3-fold higher presence of γδ T cells at the end of isolation and generally presented a favorable phenotype (e.g., lower expression of CD45RA and NKG2A).

[0283] Example 3. Study of frozen isolates

[0284] Previous conditions produced large numbers of γδ T cells, but their viability after harvest and thawing was very low. A new isolation and expansion method was investigated in which the isolated cells were frozen before expansion. After isolation, the cells were frozen according to the method detailed in Example 1. The effect on the expansion fold and percentage of γδ T cells in the subsequent expansion step is shown in Figure 12 Isolated cells frozen before expansion were found to be at least as enriched and expanded as fresh equivalents.

[0285] Example 4. Inclusion of IL-21 in the isolated preparation can increase the production of γδ T cells

[0286] The use of IL-21 was investigated to examine whether this cytokine could increase the total γδ T cell yield after isolation and subsequent expansion. Increasing the total γδ T cell content would allow these cells to be genetically engineered on a larger scale during subsequent expansion and potentially increase the total effector cell yield.

[0287] Separate cultures were established in GREX 100M units with AIMV medium supplemented with 2.5% allogeneic plasma (see Example 1 for details). The cultures were then supplemented with cytokines as detailed in Table 3.

[0288] Table 3. Cytokine conditions used for isolation and culture

[0289]

[0290] The isolated cultures using 18.8 ng / ml IL-21 were harvested on day 21 and cryopreserved (frozen) for subsequent expansion. The cells were then thawed and cell viability was measured by NC250 automated cell counting, and the results are shown in Figure 13 In A. In the presence and absence of IL-21, the overall survival of cells at thaw was above the minimum acceptable survival, with an average survival rate of >80% in the "no IL-21" cultures and an average survival rate of nearly 90% in the "IL-21" cultures. Cells were then cultured in the presence of TexMACS supplemented with 5% allogeneic plasma and IL-15 (80 ng / mL). TM The isolated cells were expanded in the presence of IL-21 supplemented with different levels of IL-21 (12.53, 18.8, 37.5, and 188 ng / mL) in the presence of IL-21 medium (Miltenyi Biotec). After 14 days of expansion, the cells were harvested and the enrichment of γδ T cells was measured by flow cytometry, as shown in Figure 5. Figure 13 As shown in Figure 2B. After expansion, cells isolated in the presence of IL-21 showed enrichment of γδ T cells over a 14-day culture period, independent of the amount of IL-21 used for expansion (showing comparable performance from 12.5 to 188.8 ng / mL). Thus, the presence of IL-21 in the isolation culture generated viable cells and maintained the ability of γδ T cells to subsequently expand. The functionality of these expanded cells in cytotoxicity assays was also confirmed.

[0291] Example 5. Comparison of Isolation and Culture without IL-21 and with 18.8 ng / ml IL-21

[0292] Donor-matched skin samples were established in isolation cultures using either the "no IL-21" formulation or the "IL-21" (18.8 ng / ml) cytokine formulation as detailed in Table 3. All isolation cultures were set up with AIMV medium supplemented with 2.5% allogeneic plasma (see Example 1 for details). The "no IL-21" isolation cultures were harvested on day 19 or day 21, while the "IL-21" isolation cultures were harvested only on day 19. At harvest, the total number of viable cells per culture grid, the total number of isolated γδ T cells per isolation culture grid, and the total number of Vδ1 T cells per isolation culture grid were recorded and the results are shown in Table 3. Figure 14 middle.

Claims

1. A method for isolating γδ T cells from a non-hematopoietic tissue sample, the method comprising the following steps: (i) culturing the non-hematopoietic tissue sample in the presence of IL-1β, interleukin 2 (IL-2), and interleukin 15 (IL-15); and (ii) collecting the cultured γδ T cell population from the non-hematopoietic tissue sample; wherein the non-hematopoietic tissue sample is skin.

2. The method of claim 1, wherein step (i) further comprises culturing the non-hematopoietic tissue sample in the presence of interleukin 4 (IL-4) and / or interferon gamma (IFN-γ).

3. The method according to claim 1 or 2, wherein the culturing is performed in the absence of interleukin 21 (IL-21).

4. The method of claim 1 or 2, wherein the culturing is performed in the presence of interleukin 21 (IL-21) at a concentration between 15 ng / mL and 25 ng / mL. The method according to claim 1 , wherein the non-hematopoietic tissue sample is cultured in a culture medium containing serum or plasma.

6. The method according to claim 1 or 2, wherein the non-hematopoietic tissue is cultured in a culture medium containing 2.5% human plasma.

7. The method according to claim 1 or 2, wherein the non-hematopoietic tissue is cultured in a medium containing 10% human AB serum.

8. The method of claim 1 or 2, wherein the γδ T cells are collected after at least 7 days in culture.

9. The method of claim 1 or 2, wherein the γδ T cells are collected after at least 14 days in culture.

10. The method according to claim 1 or 2, wherein the γδ T cells are collected after 35 days of culture.

11. The method according to claim 1 or 2, wherein the γδ T cells are collected after 19 days of culture.

12. The method according to claim 1 or 2, wherein the γδ T cells are collected after 21 days of culture.

13. The method of claim 1 or 2, wherein the non-hematopoietic tissue sample is a whole biopsy. The method of claim 13 , wherein the skin comprises an epidermis layer and a dermis layer.

15. The method of claim 1 or 2, wherein the non-hematopoietic tissue sample has been obtained from a human.

16. The method of claim 1 or 2, wherein the method is performed in a container comprising a gas permeable material.

17. The method of claim 1 or 2, wherein the isolated γδ T cell population comprises a Vδ1 T cell population.

18. The method of claim 17, wherein less than 80% of the Vδ1 T cell population expresses CD45RA.

19. The method of claim 17, wherein less than 10% of the Vδ1 T cell population expresses NKG2A.

20. The method of claim 1 or 2, further comprising expanding the isolated γδ T cell population.

21. A method for isolating and expanding γδ T cells from a non-hematopoietic tissue sample, the method comprising the steps of: (i) isolating a γδ T cell population from the non-hematopoietic tissue sample according to the method of claim 1; and (ii) further culturing the γδ T cell population for at least 5 days to produce an expanded γδ T cell population.

22. The method of claim 21 , wherein the isolated γδ T cell population is frozen and then thawed prior to step (ii).

23. The method of claim 21 or 22, wherein the expanding step comprises culturing the γδ T cells in the presence of: (a) IL-2 or IL-9; (b) IL-15; and optionally (c) IL-21, The cells are cultured for at least 5 days in an amount effective to produce an expanded γδ T cell population.

24. The method of claim 23, further comprising culturing the γδ T cells in the presence of IL-4.

25. The method of claim 21 or 22, wherein the expanding step comprises culturing the γδ T cells in the absence of substantial stromal cell contact.

26. The method of claim 21 or 22, wherein the expanding step comprises the absence of exogenous TCR pathway agonists.

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