Method for isolating gamma delta T cells

Abstract

The present invention relates to a method for isolating non-hematopoietic tissue resident lymphocytes, in particular gamma delta T cells. Such [gamma] [delta] T cells include non-V [delta] 2 cells, such as V [delta] 1, V [delta] 3 and V [delta] 5 cells, and such non-hematopoietic tissues include the skin and the intestinal tract. It should be understood that such isolated non-hematopoietic tissue resident lymphocytes have great uses in adoptive T cell therapy, chimeric receptor therapy, and the like. Also provided are methods for expanding isolated tissue resident lymphocytes, in particular for isolating and expanding gamma delta T cells. The invention also relates to individual cells and populations of cells produced by the methods described herein.

Description

[0001] This application is a divisional application of the invention patent application filed on May 12, 2021, with Chinese application number 202180035561.3 and entitled "Method for Isolating γδT Cells". Technical Field

[0002] This invention relates to a method for isolating resident lymphocytes, particularly γδT cells, from non-hematopoietic tissues. Such γδT cells include non-Vδ2 cells, such as Vδ1, Vδ3, and Vδ5 cells, and such non-hematopoietic tissues include skin and intestines. It should be understood that such isolated resident lymphocytes from non-hematopoietic tissues have significant applications in adoptive T-cell therapy, chimeric receptor therapy, and the like. The invention also relates to single cells and cell populations generated by the methods described herein. Background Technology

[0003] The growing interest in T-cell immunotherapy for cancer focuses 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 imposed through PD-1, CTLA-4, and other receptors are desuppressed by clinically mediated antagonism. However, αβ T cells are MHC-restricted, which can lead to graft-versus-host disease.

[0004] γδT cells represent a subset of T cells that express a unique, defining γδT cell receptor (TCR) on their surface. This TCR consists of one γ chain and one δ chain. Human γδTCR chains are selected from three major δ chains (Vδ1, Vδ2, and Vδ3) and six γ chains. Human γδT cells can be broadly classified according to their TCR chains because certain γ and δ types are more prevalent on cells in one or more tissue types, although not unique. For example, most blood-resident γδT cells express Vδ2 TCRs, such as Vγ9Vδ2, while this is less common in tissue-resident γδT cells, which more frequently use Vδ1 in the skin and Vγ4 in the intestine.

[0005] 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 possess properties particularly suitable for certain applications, such as targeting non-hematopoietic tumors and other targets. However, isolating such tissue-resident lymphocytes in clinically relevant quantities remains a challenge, especially since many indications require a range of 10-1. 8 More than one clinical dose per cell. Importantly, the significant cell loss during production means that even more starting cells must be generated.

[0006] Because non-hematopoietic tissue resident lymphocytes (especially γδT 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 to isolate non-hematopoietic tissue resident lymphocytes (especially γδT cells) in sufficient quantities for further expansion and potentially suitability as a therapy, such as adoptive T-cell therapy.

[0007] 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 particularly because the yield of isolated cells is relatively low, i.e., per cm 2 fewer than 10 organizations 6 The method described by Clark et al. uses shredded samples, resulting in intentional destruction of the structural integrity of the tissue sample. WO2017072367 and WO2018 / 202808 relate to methods for expanding non-hematopoietic tissue-resident γδT cells in vitro by culturing lymphocytes obtained from non-hematopoietic tissue in the presence of at least interleukin-2 (IL-2) and / or interleukin-15 (IL-15). WO2015189356 describes a composition for expanding lymphocytes obtained from samples acquired via apheresis, comprising at least two types of cytokines selected from IL-2, IL-15, and IL-21. Therefore, there remains a need for a method for isolating tissue-resident non-hematopoietic lymphocytes (such as from skin) that produces larger quantities of cells suitable for clinical use. Summary of the Invention

[0008] According to a first aspect of the present invention, a method for isolating lymphocytes from a non-hematopoietic tissue sample is provided, the method comprising the following steps:

[0009] (i) Culture of non-hematopoietic tissue samples in the presence of interleukin-1β (IL-1β); and

[0010] (ii) Collecting cultured lymphocyte populations from non-hematopoietic tissue samples.

[0011] According to another aspect, a method for isolating γδT cells from a non-hematopoietic tissue sample is provided, the method comprising the following steps:

[0012] (i) Culture non-hematopoietic tissue samples in the presence of IL-1β; and

[0013] (ii) Collect cultured γδT cell populations from non-hematopoietic tissue samples.

[0014] According to another aspect, a method for isolating and expanding lymphocytes from a non-hematopoietic tissue sample is provided, the method comprising the following steps:

[0015] (i) Isolating lymphocyte populations from non-hematopoietic tissue samples according to the methods described herein; and

[0016] (ii) Further culture the lymphocyte population for at least 5 days to generate an expanded lymphocyte population.

[0017] According to another aspect, a method for isolating and expanding γδT cells from a non-hematopoietic tissue sample is provided, the method comprising the following steps:

[0018] (i) Isolating a population of γδT cells from non-hematopoietic tissue samples according to the methods described herein; and

[0019] (ii) Further culture the γδT cell population for at least 5 days to generate an expanded γδT cell population. Attached Figure Description

[0020] Figure 1 Percentage of γδT cells in live cells (%) (A) and total γδT cells per grid for all conditions (B). Conditions were grouped by protein source (AB = 10% human AB serum; plasma = 2.5% human plasma; SR = 5% serum substitute) and further subdivided into presence (+) or absence (-) of IL-1β. See Table 2 for details of the different conditions. Bars represent experimental medians. Dashed lines represent the median of experimental controls (STDISO).

[0021] Figure 2 The graph shows the percentage of Vδ1T cells in live cells (A) and the total number of Vδ1T cells per grid under all conditions (B). Conditions are grouped by protein source with or without IL-1β. Bars represent experimental medians. Dashed lines represent the median of experimental controls.

[0022] Figure 3 This graph shows the live cell results grouped by protein source and subdivided into grids for the presence or absence of IL-4. Bars represent the experimental median. Dashed lines represent the median of the experimental controls.

[0023] Figure 4 This graph shows the results of γδT cells in live cells grouped by protein source and further subdivided into those with and without IL-4. Bars represent the experimental median. Dashed lines represent the median of the experimental controls.

[0024] Figure 5This graph shows the total Vδ1T results grouped by protein source and subdivided into grids for the presence or absence of IL-4. Bars represent the experimental median. Dashed lines represent the median of experimental controls.

[0025] Figure 6 The results show the percentage of NKG2A expression (A) and CD45RA expression (B) on Vδ1T cells grouped by protein source and further subdivided into those with or without IL-4. Bars represent experimental medians. Dashed lines represent the median of experimental controls.

[0026] Figure 7 This graph shows the results of TIGIT expression percentage in Vδ1T cells grouped by protein source. Bars represent the experimental median. Dashed lines represent the median of the experimental controls.

[0027] Figure 8 This graph shows the results of total Vδ1T cells grouped by protein source. The bars represent the experimental median. The dashed line represents the median of the experimental controls.

[0028] Figure 9 This graph shows the total viable cells for each grid grouped by protein source. Bars represent the experimental median. Dashed lines represent the median for experimental controls.

[0029] Figure 10 : This graph shows the results of total TCR-negative cells grouped by protein source and further subdivided into those with or without IFN-γ. The dashed line represents the median when using the previous separation method.

[0030] Figure 11 Total γδT cell results per grid under optimal conditions compared to the standard 2-cytokine isolation method.

[0031] Figure 12 : A graph showing the effect of using freshly obtained isolated cells or isolated cells frozen before expansion on expansion fold (A) and γδT cell expansion % (B).

[0032] Figure 13 A) Comparison of total cell viability at the end of 21 days of isolation culture with and without the addition of 18.8 ng / ml IL-21, in the presence of IL-2, IL-4, IL-15, and IL-1β. The dashed line represents the minimum acceptable viability. B) A graph showing the expansion of isolated γδT cells after 14 days of culture in the presence of IL-1β and IL-21 after thawing (shown as % of γδT cells in live cells).

[0033] Figure 14Summary of isolation cultures using “IL-21” (IL-2, IL-4, IL-15, IL-1β and IL-21) or “IL-21-free” (IL-2, IL-4, IL-15 and IL-1β) harvested after 19 or 21 days. Detailed Implementation

[0034] According to a first aspect of the present invention, a method for isolating lymphocytes from a non-hematopoietic tissue sample is provided, the method comprising the following steps:

[0035] (i) Culture of non-hematopoietic tissue samples in the presence of interleukin-1β (IL-1β); and

[0036] (ii) Collecting cultured lymphocyte populations from non-hematopoietic tissue samples.

[0037] 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:

[0038] (i) Culture non-hematopoietic tissue samples in the presence of IL-1β; and

[0039] (ii) Collect cultured γδT cell populations from non-hematopoietic tissue samples.

[0040] The results presented in this article demonstrate the detailed experimental design for establishing an optimal protocol for isolating tissue-resident γδT cells from non-hematopoietic tissue samples. Surprisingly, all conditions exhibiting optimal γδT cell yield were found to contain IL-1β, indicating that this cytokine promotes the generation of high levels of γδT cells during the isolation process.

[0041] As used herein, “IL-1β” refers to natural or recombinant IL-1β or its variants (e.g., mutants, mutant proteins, analogs, subunits, receptor complexes, fragments, isoforms, and peptide mimics) that act as agonists of one or more IL-1 receptor (IL-1R) subunits. IL-1 is a pro-inflammatory 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 the precursor polypeptide by 116 amino acids from the N-terminus by CASP1, as described by Andrei et al. (2004) PNAS 101(26):9745-9750. Mutant IL-1β proteins are polypeptides in which the IL-1β protein has been specifically substituted while retaining the ability to bind IL-1R. The IL-1β mutant protein may be characterized by the insertion, deletion, substitution, and modification of amino acids at one or more sites or other residues in the native IL-1β polypeptide chain. According to this disclosure, any such insertion, deletion, substitution, and modification yields an IL-1β mutant protein that retains IL-1R binding activity. Exemplary mutant proteins may contain substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids.

[0042] The nucleic acid encoding human IL-1β can be obtained using routine procedures such as polymerase chain reaction (PCR). The amino acid sequence of human IL-1β (gene ID 3553) is accessed in GenBank under accession number NP_000567 or in UniProt under accession number P01584. The amino acid sequence of mouse (Mus musculus) IL-1β (gene ID 16176) is accessed in GenBank under accession number NP_032387 or in UniProt under accession number P10749.

[0043] IL-1β can also refer to IL-1β derived from various mammalian species, including, for example, humans, apes, cattle, pigs, horses, and mice. Variants can contain conserved substitution sequences, meaning that a given amino acid residue is replaced by a residue with similar physicochemical characteristics. Examples of conserved 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 conserved substitutions (e.g., substitution of an entire region with similar hydrophobic characteristics) are well known. The present invention also covers naturally occurring IL-1β variants. Examples of such variants are proteins produced by alternating mRNA splicing events or by proteolytic cleavage of the IL-1β protein, in which IL-1β binding properties are preserved.

[0044] Separation methods

[0045] The term "isolation / isolating" of cells, particularly lymphocytes and / or γδT cells, as used herein refers to a method or process 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 isolating cells from intact non-hematopoietic tissue samples or from stromal cells (e.g., fibroblasts or epithelial cells) of non-hematopoietic tissue. Such isolation may alternatively or additionally include separating or isolating 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 the tissue explant or biopsy is placed in the isolation culture to the time the cells are collected from the culture, such collection being by centrifugation or other means for transferring the isolated cell population for expansion culture or for other purposes, or by removing the initial tissue explant or biopsy from the culture. The isolation step can last from at least about 3 days to about 45 days. In one embodiment, the separation step lasts from at least about 10 days to at least 28 days. In another embodiment, the separation step lasts from at least 14 days to at least 21 days. Therefore, the separation step can last for 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 lasts for 19 days. In another embodiment, the separation step lasts for 21 days. It is understood that although cell proliferation during this separation step may not be significant, it is not necessarily absent. In fact, it will be recognized by those skilled in the art that the separated cells may also begin to divide within a separation container containing tissue and / or scaffolds to produce multiple such cells.

[0046] Therefore, the terms "isolated γδT cells," "isolated γδT cell population," "separated γδT cells," and "separated γδT cell population" mentioned in this article will be understood to refer to hematopoietic cells or populations of hematopoietic cells, including γδ cells that have been isolated, separated, removed, purified, or enriched from non-hematopoietic tissue samples, such that these cells do not have extensive contact with non-hematopoietic cells or cells contained within intact non-hematopoietic tissue. Similarly, the term "isolated or separated Vδ1T cell population" mentioned in this article refers to hematopoietic cells, including Vδ1T cells that have been isolated, separated, removed, purified, or enriched from non-hematopoietic tissue samples, such that these cells do not have extensive contact with non-hematopoietic cells or cells contained within intact non-hematopoietic tissue. Therefore, separation or isolation refers to the separation, separation, removal, purification, or enrichment of hematopoietic cells (e.g., γδT cells or other lymphocytes) from non-hematopoietic cells (e.g., stromal cells, fibroblasts, and / or epithelial cells).

[0047] Methods for isolating γδT cells, as defined herein, may include tissue disruption (e.g., mincing) followed by separation of the γδT cells from other cell types. Preferably, methods for isolating γδT cells, as defined herein, may include “crawl-out” γδT cells and other cell types from an intact non-hematopoietic tissue sample or explant or biopsy tissue matrix, wherein tissue-resident lymphocytes are physically separated from the tissue matrix without requiring disruption of the tissue matrix. By maintaining the integrity of the tissue matrix, it has been found that tissue-resident lymphocytes preferentially exit from the tissue matrix, while suppressive cell types such as fibroblasts exit little or no, remaining in the explant or biopsy and then easily removed at the end of the isolation process. Thus, in some embodiments, using an intact non-hematopoietic tissue sample or tissue matrix results in the release of a small number of fibroblasts from the tissue into the culture. Such “crawl-out” methods utilizing intact non-hematopoietic tissue or tissue matrix have the advantage of reducing the need for excessive processing of the non-hematopoietic tissue sample or tissue matrix, maintaining the structural integrity of the non-hematopoietic tissue or tissue matrix, and may have the unexpected advantage of providing a higher yield of isolated cells.

[0048] Therefore, methods for isolating lymphocytes of non-hematopoietic tissue origin as defined herein include methods for isolating lymphocytes of non-hematopoietic tissue origin from intact biopsies or explants. Such intact biopsies or explants are those in which the structural integrity of the biopsies or explants is not intentionally compromised within the cutting perimeter of the biopsies or explants removed from the tissue sample. Such intact biopsies or explants will have a substantially preserved three-dimensional structure except for minor damage caused by processing. Thus, for example, such intact biopsies or explants are not mechanically destroyed, such as by chopping or cutting, nor are they chemically or enzymatically destroyed. However, destroyed tissue can be used in the isolation method of the present invention. In one embodiment, the isolated lymphocytes are αβT cells. In an alternative embodiment, the isolated lymphocytes are γδT cells. In yet another embodiment, the isolated lymphocytes are TCR-negative cells (i.e., cells that are negative for both αβTCR and γδTCR expression). TCR-negative cells are a good indicator of the presence of natural killer (NK) cells. Therefore, in another embodiment, the isolated lymphocytes are NK cells. It is understood that more than one type of lymphocyte can be isolated from the same separation step.

[0049] Methods for isolating γδT cells using “crawling” or, for example, as defined herein, include culturing cell and / or non-hematopoietic tissue samples in the presence of cytokines and / or chemokines sufficient to induce the isolation or separation of γδT cells and / or other lymphocytes as defined herein.

[0050] In one embodiment, step (i) further includes culturing the non-hematopoietic tissue sample in the presence of interleukin 2 (IL-2). In another embodiment, step (i) further includes culturing the non-hematopoietic tissue sample in the presence of interleukin 15 (IL-15). In yet another embodiment, step (i) further includes culturing the non-hematopoietic tissue sample in the presence of both IL-2 and IL-15.

[0051] As used herein, “IL-2” refers to natural or recombinant IL-2 or its variants (e.g., mutants, mutant proteins, analogs, subunits, receptor complexes, fragments, isoforms, and peptide mimics) that act as agonists of one or more IL-2 receptor (IL-2R) subunits. Such agents can support the IL-2-dependent cell line CTLL-2 (33; American Center for Type Culture Collection).

[0052] The proliferation of TIB 214. Mature human IL-2 occurs as a 133-amino acid sequence (minus the signal peptide, which consists of an additional 20 N-terminal amino acids), as described by Fujita et al., Cell 1986.46.3:401-407. IL-2 mutant proteins are polypeptides in which interleukin-2 protein has been specifically substituted while retaining the ability to bind IL-2Rβ, such as those described in US2014 / 0046026. IL-2 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-2 polypeptide chain. According to this disclosure, any such insertions, deletions, substitutions, and modifications result in an IL-2 mutant protein that retains IL-2Rβ binding activity. Exemplary mutant proteins may contain substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids.

[0053] The nucleic acid encoding human IL-2 can be obtained through routine procedures such as PCR. The amino acid sequence of human IL-2 (gene ID 3558) is accessed in GenBank with the accession number NP_000577.2GI:28178861. The amino acid sequence of mouse (house mouse) IL-2 (gene ID 16183) is accessed in GenBank with the accession number NP_032392.1GI:7110653.

[0054] IL-2 can also refer to IL-2 derived from various mammalian species, including, for example, humans, apes, cattle, pigs, horses, and mice. Variants can contain conserved substitution sequences, meaning that a given amino acid residue is replaced by a residue with similar physicochemical characteristics. Examples of conserved 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 conserved substitutions (e.g., substitution of an entire region with similar hydrophobic characteristics) are well known. The present invention also covers naturally occurring IL-2 variants. Examples of such variants are proteins produced by alternating mRNA splicing events or by proteolytic cleavage of the IL-2 protein, in which IL-2 binding properties are preserved. Alternating splicing of mRNA can produce truncated but biologically active IL-2 proteins. Variations 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 removal of one or more terminal amino acids (typically 1-10 amino acids) from the IL-2 protein through proteolysis. 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).

[0055] 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, isoforms, and peptide mimics) that act as agonists of one or more IL-15 receptor (IL-15R) subunits. Like IL-2, IL-15 is a known T-cell growth factor that supports 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 (e.g., receptor complexes, such as sushi peptide, as described in WO2007 / 046006), each of which can stimulate the proliferation of CTLL-2 cells. In the CTLL-2 proliferation assay, the supernatant of cells transfected with recombinant IL-15 precursor and mature in-frame fusion product induced CTLL-2 cell proliferation.

[0056] 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 through routine procedures such as PCR. Human IL-15 cDNA was stored on February 19, 1993 at [location missing]. And the registration number is 69245.

[0057] The amino acid sequence of human IL-15 (gene ID 3600) is mapped in GenBank as NP000576.1GI:10835153 (isomer 1) and NP_751915.1GI:26787986 (isomer 2). The amino acid sequence of mouse (house mouse) IL-15 (gene ID 16168) is mapped in GenBank as NP_001241676.1GI:363000984.

[0058] IL-15 can also refer to IL-15 derived from various mammalian species, including, for example, humans, apes, cattle, 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 natural mammalian IL-15 but has an amino acid sequence different from that of the natural mammalian IL-15 polypeptide due to amino acid deletions, insertions, or substitutions. Variants may contain conserved substitution sequences, meaning that a given amino acid residue is replaced by a residue having similar physicochemical characteristics. Examples of conserved 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 conserved substitutions (e.g., substitution of an entire region having similar hydrophobic characteristics) are well known. The present invention also covers naturally occurring IL-15 variants. Examples of such variants are proteins produced by alternating mRNA splicing events or by proteolytic cleavage of the IL-15 protein, in which the IL-15 binding properties are preserved. Alternating splicing of mRNA can produce truncated but biologically active IL-15 proteins. Variations 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 removal of one or more terminal amino acids (typically 1-10 amino acids) from the IL-15 protein via proteolysis. 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).

[0059] In one embodiment, the isolation of lymphocytes or γδT cells according to the invention further includes culturing non-hematopoietic tissue samples in the presence of interleukin-4 (IL-4). Therefore, in another embodiment, the non-hematopoietic tissue samples are cultured in the presence of IL-1β and IL-4.

[0060] 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, isoforms, and peptide mimics thereof) that act as agonists of one or more IL-4 receptor (IL-4R) subunits. Such agents can support the differentiation of naive helper T cells (Th0 cells) into Th2 cells. Mature human IL-4 occurs as a 129-amino acid sequence (minus the signal peptide, which consists of an additional 24 N-terminal amino acids). Mutant IL-4 proteins are polypeptides in which interleukin-4 protein has been specifically substituted while retaining the ability to bind IL-4Rα, such as those described in U.S. Patent No. 6,313,272. Mutant IL-4 proteins can be characterized by amino acid insertions, deletions, substitutions, and modifications at one or more sites or other residues in the natural IL-4 polypeptide chain. According to this disclosure, any such insertions, deletions, substitutions, and modifications result in mutant IL-4 proteins that retain IL-4Rα binding activity. Exemplary mutant proteins may contain substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids.

[0061] The nucleic acid encoding human IL-4 can be obtained through routine procedures such as PCR. The amino acid sequence of human IL-4 (gene ID 3565) is accessed in GenBank under the accession number NG_023252. The amino acid sequence of mouse (house mouse) IL-4 (gene ID 16189) is accessed in GenBank under the accession number NC_000077.6.

[0062] IL-4 can also refer to IL-4 derived from various mammalian species, including, for example, humans, apes, cattle, pigs, horses, and mice. Variants can contain conserved substitution sequences, meaning that a given amino acid residue is replaced by a residue with similar physicochemical characteristics. Examples of conserved 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 conserved substitutions (e.g., substitution of an entire region with similar hydrophobic characteristics) are well known. The present invention also covers naturally occurring IL-4 variants. Examples of such variants are proteins produced by alternating mRNA splicing events or by proteolytic cleavage of the IL-4 protein, in which IL-4 binding properties are preserved. Alternating splicing of mRNA can produce truncated but biologically active IL-4 proteins. Variations 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 removal of one or more terminal amino acids (typically 1-10 amino acids) from the IL-4 protein through proteolysis. 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).

[0063] In one embodiment, the isolation of lymphocytes or γδT cells according to the invention further includes culturing non-hematopoietic tissue samples in the presence of interferon-γ (IFN-γ). Therefore, in another embodiment, the non-hematopoietic tissue samples are cultured in the presence of IL-1β and IFN-γ.

[0064] 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, isoforms, and peptide mimics thereof) that act as agonists of one or more IFN-γ receptor (IFNGR) subunits. Specifically, IFN-γ interacts with a heterodimeric receptor composed of interferon-γ receptor 1 (IFNGR1) and interferon-γ receptor 2 (IFNGR2). Mature human IFN-γ occurs as a 143-amino acid sequence (minus the signal peptide, which consists of an additional 23 N-terminal amino acids). Mutant IFN-γ proteins are polypeptides in which specific substitutions have been made to the IFN-γ protein while retaining the ability to bind IFNGR, such as those described in U.S. Patent No. 9,296,804. Mutant IFN-γ proteins may be characterized by amino acid insertions, deletions, substitutions, and modifications at one or more sites or other residues in the natural IFN-γ polypeptide chain. According to this disclosure, any such insertion, deletion, substitution, and modification yields an IFN-γ mutant protein that retains IFN-γR binding activity. Exemplary mutant proteins may contain substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids.

[0065] The nucleic acid encoding human IFN-γ can be obtained through routine procedures such as PCR. The amino acid sequence of human IFN-γ (gene ID 3458) is accessed in GenBank under the accession number NG_015840.1 or in UniProt under the accession number P01579. The amino acid sequence of mouse (house mouse) IFN-γ (gene ID 15978) is accessed in GenBank under the accession number NC_000076.6 or in UniProt under the accession number P01580.

[0066] IFN-γ can also refer to IFN-γ derived from various mammalian species, including, for example, humans, apes, cattle, pigs, horses, and mice. Variants can contain conserved substitution sequences, meaning that a given amino acid residue is replaced by a residue with similar physicochemical characteristics. Examples of conserved 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 conserved substitutions (e.g., substitution of an entire region with similar hydrophobic characteristics) are well known. The invention also covers naturally occurring IFN-γ variants. Examples of such variants are proteins resulting from alternating mRNA splicing events or from proteolytic cleavage of IFN-γ proteins, in which IFN-γ binding properties are preserved.

[0067] In one embodiment, the isolation of lymphocytes or γδT cells according to the invention further includes culturing non-hematopoietic tissue samples in the presence of interleukin-21 (IL-21). Therefore, in another embodiment, the non-hematopoietic tissue samples are cultured in the presence of IL-1β and IL-21.

[0068] 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, isoforms, and peptide mimics) that act as agonists of one or more IL-21 receptor (IL-21R) subunits. Such agents can support natural killer (NK) and cytotoxic (CD8) responses. + T cell proliferation. Mature human IL-21 occurs as a 133-amino acid sequence (minus the signal peptide, which consists of an additional 22 N-terminal amino acids). IL-21 mutant proteins are polypeptides in which interleukin-21 protein has been specifically substituted while retaining the ability to bind IL-21Rα, such as those described in U.S. Patent No. 9,388,241. IL-21 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-21 polypeptide chain. According to this disclosure, any such insertions, deletions, substitutions, and modifications result in an IL-21 mutant protein that retains IL-21R binding activity. Exemplary mutant proteins may contain substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids.

[0069] The nucleic acid encoding human IL-21 can be obtained through routine procedures such as PCR. The amino acid sequence of human IL-21 (gene ID 59067) is accessed in GenBank under the accession number NC_000004.12. The amino acid sequence of mouse (house mouse) IL-21 (gene ID 60505) is accessed in GenBank under the accession number NC_000069.6.

[0070] IL-21 can also refer to IL-21 derived from various mammalian species, including, for example, humans, apes, cattle, pigs, horses, and mice. Variants can contain conserved substitution sequences, meaning that a given amino acid residue is replaced by a residue with similar physicochemical characteristics. Examples of conserved 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 conserved substitutions (e.g., substitution of an entire region with similar hydrophobic characteristics) are well known. The present invention also covers naturally occurring IL-21 variants. Examples of such variants are proteins produced by alternating mRNA splicing events or by proteolytic cleavage of the IL-21 protein, wherein the IL-21 binding properties are preserved. Alternating splicing of mRNA can produce truncated but biologically active IL-21 proteins. Variations 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 removal of one or more terminal amino acids (typically 1-10 amino acids) from the IL-21 protein through proteolysis. 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).

[0071] In alternative implementations, cultures were conducted in the absence of IL-21. In alternative implementations, cultures were conducted in the absence of IFN-γ. Statistical analysis of the results obtained from the experiments described herein suggests that the presence of IL-1β in the isolating culture may be more beneficial when at least IFN-γ is absent, such as when both IFN-γ and IL-21 are absent. The results described herein further demonstrate that, under certain conditions, the presence of IL-1β in the isolating culture may also be beneficial when IL-21 is absent or when IL-21 is present at concentrations between 15 ng / mL and 25 ng / mL, particularly 18 to 20 ng / mL, such as 18, 19, or 20 ng / mL, for example, 18.8 ng / mL.

[0072] In some embodiments, the methods defined herein include concentrations 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). IL-2 concentrations ranging from 10 IU / mL to 20 IU / mL, 20 IU / mL to 30 IU / mL, 30 IU / mL to 40 IU / mL, 40 IU / mL to 50 IU / mL, 50 IU / mL to 75 IU / mL, 75 IU / mL to 100 IU / mL, 100 IU / mL to 150 IU / mL, 150 IU / mL to 200 IU / mL, 200 IU / mL to 500 IU / mL, or 500 IU / mL to 1,000 IU / mL. In some embodiments, the methods defined herein include IL-2 concentrations typically below 1,000 IU / mL, such as below 500 IU / mL. In some embodiments, the methods include IL-2 concentrations of about 100 IU / mL, such as 138 IU / mL.

[0073] In other embodiments, the methods defined herein include concentrations typically at least 10 IU / mL, such as at least 100 IU / mL, particularly 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, 5...). IL-15 concentrations ranging from 0 IU / mL to 400 IU / mL, 75 IU / mL to 250 IU / mL, or 100 IU / mL to 200 IU / mL, such as 100 IU / mL to 900 IU / mL, 200 IU / mL to 800 IU / mL, 300 IU / mL to 700 IU / mL, 400 IU / mL to 600 IU / mL, or 500 IU / mL to 1,000 IU / mL. In some embodiments, the methods defined herein include IL-15 concentrations typically below 1,000 IU / mL, such as below 700 IU / mL. In some embodiments, the methods include IL-15 concentrations of about 600 IU / mL.

[0074] In some implementations, isolating γδT cells from non-hematopoietic tissue samples involves culturing them in the presence of both IL-2 and IL-15 (each at any of the concentrations listed above). In some cases, the concentration of IL-2 is approximately 138 IU / mL, and the concentration of IL-15 is 600 IU / mL.

[0075] In other embodiments, the methods defined herein include IL-21 at concentrations typically 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, such as 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 some embodiments, the methods defined herein include IL-21 at concentrations typically below 10 IU / mL, such as below 5 IU / mL. In some embodiments, the method includes IL-21 at a concentration of about 1 IU / mL, such as 1.05 IU / mL. In other embodiments, the method includes IL-21 at a concentration of 15 to 25 ng / mL. Thus, in one embodiment, the method includes IL-21 at a concentration between 15 ng / mL and 25 ng / mL. In another embodiment, the method includes IL-21 at a concentration of 18 to 20 ng / mL, such as 18, 19, or 20 ng / mL, for example, 18.8 ng / mL.

[0076] In other embodiments, the methods defined herein include IL-4 at concentrations 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, such as 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 some embodiments, the methods defined herein include IL-4 at concentrations typically below 500 IU / mL, such as below 100 IU / mL. In some embodiments, the methods include IL-4 at concentrations of about 100 IU / mL, such as 95 IU / mL. In other embodiments, the method includes IL-4 at a concentration of about 30 IU / mL, such as 31.6 IU / mL.

[0077] Therefore, in some embodiments, isolating γδT cells from non-hematopoietic tissue samples involves culturing them in the presence of IL-2, IL-15, IL-4, and IL-21 (each at any concentration listed above). In other embodiments, the concentration of IL-2 is about 138 IU / mL, the concentration of IL-15 is 600 IU / mL, the concentration of IL-4 is 95 IU / mL, and the concentration of IL-21 is between 15 ng / mL and 25 ng / mL, such as 18 to 20 ng / mL, such as 18, 19, or 20 ng / mL, for example, 18.8 ng / mL. Thus, in one specific embodiment, isolating γδT cells involves culturing them in the presence of IL-2 at a concentration of 138 IU / mL, IL-15 at a concentration of 600 IU / mL, IL-4 at a concentration of 95 IU / mL, IL-1β at a concentration of 4500 IU / mL, and optionally IL-21 at a concentration of 18.8 ng / mL.

[0078] The term "non-hematopoietic tissue" or "non-hematopoietic tissue sample" as used herein includes skin (e.g., human skin) and intestines (e.g., human intestines). Non-hematopoietic tissue is tissue other than blood, bone marrow, or thymus tissue. In one embodiment, the non-hematopoietic tissue sample is skin (e.g., human skin). In another embodiment, the non-hematopoietic tissue sample is intestines or the gastrointestinal tract (e.g., human intestines 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 synovial fluid). In some embodiments, the non-hematopoietic tissue sample from which lymphocytes and / or γδT cells are isolated according to the methods defined herein is skin (e.g., human skin), which can be obtained by methods known in the art. Alternatively, the methods for isolating lymphocytes and / or γδT cells provided herein can be applied to the gastrointestinal tract (e.g., colon or intestines), breast, lung, prostate, liver, spleen, pancreas, uterus, vagina, and other skin, mucous membranes, or serous membranes. Lymphocytes and / or γδT cells may also reside in human cancer tissue samples (e.g., breast or prostate tumors). In some embodiments, lymphocytes and / or γδT cells may be derived from human cancer tissue samples (e.g., solid tumor tissue). In other embodiments, lymphocytes and / or γδT cells may be derived 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 may be derived from a skin region separated from nearby or adjacent cancer tissue (e.g., healthy skin). Therefore, in some embodiments, γδT cells are not obtained from human cancer tissue. In other embodiments, lymphocytes are not obtained from human cancer tissue.

[0079] In one embodiment, the non-hematopoietic tissue sample for the method defined herein has been obtained from a human body. In an alternative embodiment, the non-hematopoietic tissue sample for the method defined herein has been obtained from a non-human animal subject.

[0080] Methods for obtaining such tissue are known in the art. Examples of such methods include scalpel explants or punch biopsy, and the size may vary depending on the method. In some embodiments, non-hematopoietic tissue samples are obtained via punch biopsy.

[0081] In some embodiments of the invention, the non-hematopoietic tissue sample is a complete biopsy. The term "complete" biopsy or "explant" as used herein includes substantially undamaged or uninjured tissue and tissue samples, such that the structural integrity of the biopsy or explant is not intentionally compromised within the cutting perimeter from which it is removed from the tissue sample. Such a complete biopsy or explant will have a three-dimensional structure substantially preserved except for minor damage caused by processing. Thus, for example, such a complete biopsy or explant is not mechanically destroyed, such as by chopping or cutting, nor is it chemically or enzymatically destroyed. A complete biopsy or complete tissue sample may include whole tissue, complete tissue, a portion of tissue, or all components of said tissue. For example, in one embodiment, a complete biopsy includes all layers of skin. In another embodiment, the biopsy includes the epidermis and dermis of the skin. It should be understood that in such embodiments where the biopsy is complete, the separation and distinction between these layers are maintained. Therefore, "complete" as used herein also includes a biopsy of the full thickness of a non-hematopoietic tissue sample.

[0082] Therefore, in one specific embodiment of the invention, the non-hematopoietic tissue sample is not shredded. In other embodiments, the intact biopsy sample is a drill-through biopsy sample. In yet another embodiment, the intact biopsy sample is obtained through a drill-through biopsy.

[0083] In one embodiment, the non-hematopoietic tissue sample is a drill-through biopsy. The drill-through biopsy can be of any shape, but conveniently has a circular cross-section and suitably a diameter of at least 1 mm. In yet another embodiment, the non-hematopoietic tissue sample includes a drill-through biopsy with a diameter of at least 2 mm, such as at least 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, or 8 mm. In still other embodiments, the non-hematopoietic tissue sample includes a drill-through biopsy with a diameter of 8 mm or less, such as 7 mm or less, 6 mm or less, 5 mm or less, or 3 mm or less. In one embodiment, the non-hematopoietic tissue sample includes a drill-through biopsy with a diameter between 1 mm and 8 mm, such as between 2 mm and 4 mm. In a specific embodiment, the non-hematopoietic tissue sample includes a drill-through biopsy with a diameter of 3 mm.

[0084] In one embodiment, the biopsy is a skin biopsy and comprises the epidermis and dermis. In another embodiment, the biopsy substantially excludes subcutaneous fat. Thus, in one embodiment, the biopsy comprises the epidermis and dermis and substantially excludes the subcutaneous fat layer. In another embodiment, the biopsy excludes subcutaneous fat. Alternatively, subcutaneous fat is not removed and is therefore present (or at least partially present) in the biopsy. Thus, in yet another embodiment, the biopsy consists of the epidermis and dermis. In one embodiment, the biopsy comprises the full thickness of a non-hematopoietic tissue sample.

[0085] The method of the present invention includes culturing a non-hematopoietic tissue sample as defined herein. "Culturing" as used herein includes adding cells and / or a non-hematopoietic tissue sample to a culture medium, said cells including cells isolated, separated, removed, purified, or enriched from the non-hematopoietic tissue sample, said culture medium containing growth factors and / or nutrients required and / or preferred by the cells and / or the non-hematopoietic tissue sample. It should be understood that such culture conditions can be adjusted according to the cells or cell populations to be isolated from the non-hematopoietic tissue sample according to the invention, or according to the cells or cell populations to be isolated and expanded from the non-hematopoietic tissue sample.

[0086] In some embodiments, the culture time of the non-hematopoietic tissue sample is sufficient to isolate γδT cells from the non-hematopoietic tissue sample. In alternative embodiments, the culture time of the non-hematopoietic tissue sample is sufficient to isolate lymphocytes other than γδT cells (e.g., αβT cells and / or NK (natural killer) cells) from the non-hematopoietic tissue sample. In some embodiments, the culture time according to the method defined herein is at least 7 days. In some embodiments, the culture time according to the method defined herein is at least 14 days. In some embodiments, the culture time 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 culture time according to the method defined herein is between 14 and 35 days, such as between 14 and 21 days. In yet another embodiment, the culture time according to the method defined herein is about 19 days, such as 19 days. In yet another embodiment, the culture time according to the method defined herein is about 21 days, such as 21 days.

[0087] In certain embodiments of the invention, lymphocytes and / or γδT cells isolated according to methods as defined herein are collected from cultures of non-hematopoietic tissue samples after culturing the samples. Collection of lymphocytes and / or γδT cells as defined herein may include physically collecting lymphocytes and / or γδT cells from the culture, separating lymphocytes and / or γδT cells from other lymphocytes (e.g., αβT cells, γδT cells, and / or NK cells), or separating and / or separating lymphocytes and / or γδT cells from stromal cells (e.g., fibroblasts). In one embodiment, lymphocytes and / or γδT cells are collected mechanically (e.g., by 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 using flow cytometry techniques such as FACS. Thus, in some embodiments, γδT cells are collected by specifically labeling 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 γδT cells may include physically removing them from cultures of non-hematopoietic tissue samples, transferring them to separate culture containers, or using separate or different culture conditions.

[0088] It should be understood that this collection of lymphocytes and / or γδT cells is performed after a duration sufficient to obtain a population of isolated lymphocytes and / or γδT cells from the non-hematopoietic tissue sample. In some embodiments, lymphocytes and / or γδT cells are collected after culturing the non-hematopoietic tissue sample 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. Suitablely, lymphocytes and / or γδT cells are collected after a period of 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 the non-hematopoietic tissue sample for at least 14 days. In another embodiment, lymphocytes and / or γδT cells are collected after culturing the non-hematopoietic tissue sample for 14 to 21 days. In yet another embodiment, lymphocytes and / or γδT cells are collected after approximately 19 days of culture, such as after 19 days. In yet another embodiment, lymphocytes and / or γδT cells are collected after approximately 21 days of culture, such as after 21 days.

[0089] In some embodiments of the invention, non-hematopoietic tissue samples are cultured in a medium containing serum (e.g., human AB serum or fetal bovine serum (FBS)). In another embodiment, the non-hematopoietic tissue is cultured in a medium containing 10% human AB serum. In yet another embodiment, the non-hematopoietic tissue is cultured in a medium containing 5% human AB serum. According to this embodiment, a serum substitute as defined below may be additionally included in the medium. Thus, in yet another embodiment, the non-hematopoietic tissue is cultured in a medium containing 5% human AB serum and 5% serum substitute.

[0090] In some embodiments of the invention, non-hematopoietic tissue samples are cultured in a culture medium containing plasma (e.g., human plasma). In another embodiment, the non-hematopoietic tissue is cultured in a culture medium containing 2.5% human plasma.

[0091] In an alternative embodiment of the invention, non-hematopoietic tissue samples are cultured in a substantially serum-free medium (e.g., serum-free medium or medium containing serum substitutes (SR)). In another embodiment, the non-hematopoietic tissues are cultured in a medium containing 5% serum substitute. Thus, in one embodiment, the non-hematopoietic tissue samples are cultured in a serum-free medium. Such serum-free mediums may also include serum substitute mediums, wherein the serum substitute is based on chemically defined components to avoid the use of serum of human or animal origin.

[0092] In one implementation, non-hematopoietic tissue samples are cultured in a culture medium that does not contain products of animal origin.

[0093] In one implementation, the method as defined herein is performed in a separation container. The term "separation container" refers to a container comprising a non-hematopoietic tissue sample for separating lymphocytes and / or γδT cells, said container optionally also comprising a synthetic scaffold. It should be noted that the separation container may be used only for the separation method and not for further amplification steps.

[0094] In one embodiment, the method as defined herein is carried out in a container comprising a breathable material (e.g., an isolation container). Such a material is permeable to gases such as oxygen, carbon dioxide, and / or nitrogen to allow gas exchange between the container contents and the surrounding atmosphere. It should be understood that the term "container" as used herein includes culture dishes, culture plates, single-well dishes, multi-well dishes, multi-well plates, flasks, multilayer flasks, bottles (such as roller bottles), bioreactors, bags, tubes, etc. Such containers are known in the art for methods involving the amplification of non-adherent cells and other lymphocytes. However, containers comprising breathable materials can also surprisingly be used to isolate γδT cells, which are generally considered adherent. It has been found that culturing using such containers can significantly increase the yield of γδT cells isolated from non-hematopoietic tissue samples. It has also been 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 breathable 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 (e.g., epithelial cells) are not present in the culture taken in a container containing breathable material.

[0095] Such containers comprising breathable materials may additionally include non-porous breathable materials. Thus, in one embodiment, the breathable material is non-porous. In some embodiments, the breathable material is a film, such as silicone, fluoropolymer polypropylene, polyolefin, or ethylene vinyl acetate copolymer. Furthermore, such containers may include only a portion of the breathable material, a breathable membrane, or a non-porous breathable material. Thus, according to yet another embodiment, the container includes a top, a bottom, and at least one sidewall, wherein at least a portion of the bottom of the container includes breathable material, and the breathable material is in a substantially horizontal plane when the top is above the bottom. In one embodiment, the container includes a top, a bottom, and at least one sidewall, wherein at least a portion of the bottom includes breathable material, and the breathable material is in a horizontal plane when the top is above the bottom. In another embodiment, the container includes a top, a bottom, and at least one sidewall, wherein the at least one sidewall includes breathable material, and the breathable material may be in a vertical plane when the top is above the bottom, or may be in a horizontal plane when the top is not above the bottom. It should be understood that in such embodiments, only a portion of the bottom or the sidewall may include 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 the breathable material may be sealed, for example, by using an O-ring. Such embodiments should be understood to prevent spillage of the container contents or to reduce their evaporation. Thus, in some embodiments, the container comprises a liquid-sealed container comprising a breathable material that allows gas exchange. In alternative embodiments, the top of the container comprising the breathable material is on a horizontal plane and above the bottom and is not sealed. Thus, in some 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 the breathable material may be a liquid-sealed container and also includes inlet and outlet ports or pipes. Thus, in some embodiments, the container comprising the 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 the breathable material, and, if present, at least a portion of at least one sidewall comprises the 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 supplied by Wilson Wolf Manufacturing. Cell culture devices, such as G-REX 6-well plates, G-REX 24-well plates, and G-REX 10 containers.

[0096] In one embodiment, a non-hematopoietic tissue sample is placed on a synthetic scaffold. As used herein, the terms "synthetic scaffold," "scaffold," and "mesh" are used interchangeably and refer to a non-natural three-dimensional structure suitable for supporting cell growth. The non-hematopoietic tissue sample may be placed on or attached to the synthetic scaffold to facilitate the release of lymphocytes from the explant onto the scaffold. The synthetic scaffold may be constructed from natural and / or synthetic materials, such as polymers (e.g., natural or synthetic polymers such as polyvinylpyrrolidone, polymethyl methacrylate, methylcellulose, polystyrene, polypropylene, polyurethane), ceramics (e.g., tricalcium phosphate, calcium aluminate, calcium hydroxyapatite), or metals (e.g., tantalum, titanium, platinum, and metals in the same element group as platinum, niobium, hafnium, tungsten, and their alloys). In one embodiment of the invention, the synthetic scaffold is coated with tantalum. Biological factors (such as collagen (e.g., collagen I or collagen II), fibronectin, laminin, integrins, angiogenic factors, anti-inflammatory factors, glycosaminoglycans, vitrogens, antibodies and fragments thereof), and 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 surface of a scaffold, encapsulated within a scaffold material, or added to a culture medium according to methods known in the art to enhance cell adhesion, migration, survival, or proliferation. This and other methods can be used to isolate lymphocytes from many other non-hematopoietic tissue types, such as skin, intestine, prostate, and breast.

[0097] In one embodiment, a non-hematopoietic tissue sample is placed on a synthetic scaffold within a container for isolating lymphocytes from the non-hematopoietic tissue sample. In another embodiment, the synthetic scaffold is configured to facilitate the expulsion of lymphocytes and / or γδT cells from the non-hematopoietic tissue sample to the bottom of the container. This embodiment has the advantage of allowing the isolation and / or separation of lymphocytes (e.g., γδT cells, αβT cells, and / or NK cells) from the non-hematopoietic tissue sample and / or stromal cells (e.g., fibroblasts and / or epithelial cells). Furthermore, such embodiments allow the collection of lymphocytes (e.g., γδT cells, αβT cells, and / or NK cells) from the non-hematopoietic tissue sample to the bottom of the culture container. In one specific embodiment, the synthetic scaffold is configured to facilitate the expulsion of γδT cells from the non-hematopoietic tissue sample. In another embodiment, the synthetic scaffold is configured to facilitate the expulsion of lymphocytes such as αβT cells and / or NK cells from the non-hematopoietic tissue sample.

[0098] Therefore, in one aspect of the method defined herein, the synthetic scaffold is configured to facilitate the removal of lymphocytes from a non-hematopoietic tissue sample to the bottom of the culture vessel. In another aspect of the method defined herein, the synthetic scaffold is configured to facilitate the removal of γδT cells from a non-hematopoietic tissue sample to the bottom of the vessel.

[0099] The method of the present invention provides a greater total cell yield than previously described methods. In one embodiment, the total number of isolated cells from the tissue sample is at least 10 times that of 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 specific implementation, the total number of isolated cells is at least 50 x 10⁻⁶. 6 cells / cm 2 In another specific implementation, the total number of isolated cells is at least 100 x 10⁻⁶. 6 cells / cm 2 .

[0100] The dominant γδT cells in the blood are primarily Vδ2 T cells, while in non-hematopoietic tissues, the dominant γδT cells are primarily Vδ1 T cells. Therefore, Vδ1 T cells comprise approximately 70-80% of the γδT cell population residing in non-hematopoietic tissues. However, some Vδ2 T cells are also present in non-hematopoietic tissues, such as the intestine, where they can account for approximately 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 DN γδT cells may consist mostly of Vδ3-expressing T cells and a minority of Vδ5-expressing T cells. Therefore, γδT cells typically residing in non-hematopoietic tissues and isolated by the method of this invention are preferably non-Vδ2 T cells, such as Vδ1 T cells, containing a smaller number of DN γδT cells.

[0101] Therefore, in a preferred embodiment, the γδT cells isolated by the methods defined herein comprise a Vδ1T cell population. In one embodiment, the γδT cells isolated by the methods defined herein comprise a DNγδT cell population. In one embodiment, the γδT cells isolated by the methods defined herein comprise a Vδ3T cell population. In one embodiment, the γδT cells isolated by the methods defined herein comprise a Vδ5T cell population.

[0102] γδT cells can also be defined by the type of γ chain they express. In another embodiment, γδT cells isolated by the methods defined herein comprise a population of Vγ4T cells. In most cases, Vγ4T cells are obtained from intestinal tissue samples.

[0103] The isolation method provides isolated γδT cell populations in greater numbers than the reference population (e.g., at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1,000-fold, at least 5,000-fold, at least 10,000-fold).

[0104] In some embodiments, the γδT cell population isolated according to the method of the invention has a low proportion of cells expressing NKG2A. For example, the isolated γδT cell population may have an NKG2A+ cell frequency of less than 40%, less than 35%, less than 30%, less than 20%, or less than 10%. Alternatively, the isolated γδT 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 some embodiments, the isolated γδT cell population has an NKG2A+ cell frequency of less than 10%. Thus, in one embodiment, the isolated γδT cell population has an NKG2A+ cell frequency of about 8%. Thus, in one embodiment, the isolated γδT cells substantially do not express NKG2A.

[0105] In some embodiments, the Vδ1T cell population isolated according to the method of the invention has a low proportion of cells expressing NKG2A. For example, the isolated Vδ1T cell population may have an NKG2A+ cell frequency of less than 40%, less than 35%, less than 30%, less than 20%, or less than 10%. Alternatively, the isolated Vδ1T 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 some embodiments, the isolated Vδ1T cell population has an NKG2A+ cell frequency of less than 10%. Thus, 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%. Thus, in one embodiment, the isolated Vδ1 cells substantially do not express NKG2A. In one implementation, less than 10% of the isolated Vδ1T cell population expresses NKG2A.

[0106] In some embodiments, the γδT cell population isolated according to the method of the invention has a low proportion of cells expressing CD45RA. CD45RA is a marker associated with terminal differentiation, and therefore it is desirable to reduce the expression of this marker in the isolated cell population. For example, the isolated γδT 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 γδT cell population may have a CD45RA+ cell frequency of about 50%, about 40%, about 30%, about 20%, or about 10%. In some embodiments, the isolated γδT cell population has a CD45RA+ cell frequency of less than 30%. Thus, in one embodiment, the isolated γδT cell population has a CD45RA+ cell frequency of about 10%.

[0107] In some embodiments, the Vδ1T cell population isolated according to the method of the invention has a low proportion of cells expressing CD45RA. For example, the isolated Vδ1T 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 Vδ1T cell population may have a CD45RA+ cell frequency of about 50%, about 40%, about 30%, about 20%, or about 10%. In some embodiments, the isolated Vδ1T cell population has a CD45RA+ cell frequency of less than 30%. Thus, in one embodiment, the isolated Vδ1T cell population has a CD45RA+ cell frequency of about 10%. In one embodiment, less than 80% of the isolated Vδ1T cell population expresses CD45RA, such as less than 30% of the isolated Vδ1T cell population expressing CD45RA.

[0108] After separation from non-hematopoietic tissue (e.g., skin), γδT cells typically become part of a larger lymphocyte population containing, for example, αβT cells, B cells, and natural killer (NK) cells. In some embodiments, 1%–10% of the separated lymphocyte population is γδT cells (e.g., 1–10% of the separated skin-derived lymphocyte population is γδT cells). In most cases, the γδT cell population (e.g., skin-derived γδT cell population) will include a large Vδ1T cell population. In some embodiments, 1–10% of the separated lymphocyte population (e.g., skin-derived lymphocytes) is Vδ1T cells (e.g., Vδ1T cells may represent more than 50%, more than 60%, more than 70%, more than 80%, or more than 90% of the separated γδT cell population). In some cases, less than 10% of the separated γδT cell population is Vδ2T cells (e.g., less than 10% of the separated skin-derived γδT cell population is Vδ2T cells).

[0109] Non-Vδ1T cells or non-DN T cells, such as Vδ2T cells, αβT cells, B cells, or NK cells, can be removed from the isolated γδT cell population (e.g., before, during, or after the expansion step).

[0110] Isolated γδT cells (e.g., γδT cells isolated from skin, such as Vδ1T cells isolated from skin) have a different phenotype than cells derived from corresponding hematopoietic tissues (e.g., blood-derived γδT cells and / or blood-derived Vδ2T cells). For example, isolated γδT cell populations 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 includes 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 of CCR4 + Cells; at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of CCR7 + Cells; at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of CCR8 + Cells; and / or at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of CD103 + Cells. Isolated γδT cell populations 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.

[0111] Isolated populations of non-hematopoietic tissue-derived γδT cells (e.g., skin-derived γδT cells and / or skin-derived Vδ1T cells) can also be characterized functionally. Functional assays known in the art can be performed to determine 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 cell populations (e.g., blood-derived γδT cells and / or blood-derived Vδ2T cell populations)). Such assays may include proliferation assays, cytotoxicity assays, binding assays, measurements of persistence and / or location, etc.

[0112] Therefore, in one aspect of the invention, the method for isolating lymphocyte and / or γδT cell populations as defined herein produces a population comprising a surface phenotype consistent with that of a non-depleted lymphocyte and / or γδT cell population.

[0113] The method of the present invention has also been shown to improve the isolation of other lymphocytes, such as TCR-negative cells. Therefore, according to another aspect of the present invention, a method for isolating lymphocytes from a non-hematopoietic tissue sample is provided, the method comprising the steps of:

[0114] (i) culturing non-hematopoietic tissue samples in a culture medium containing serum substitutes, human plasma, and / or human AB serum; and

[0115] (ii) Collect cultured lymphocyte populations from non-hematopoietic tissue samples. Suitablely, the culture medium contains a serum substitute.

[0116] As illustrated in the embodiments provided herein, the protein source can influence 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.

[0117] The culture method used in this aspect of the invention may include other conditions described herein. In one embodiment, step (i) further includes culturing non-hematopoietic tissue samples in the presence of IL-2 and IL-15.

[0118] In one embodiment, step (i) further includes 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 includes culturing the non-hematopoietic tissue sample in the presence of IL-4 and / or IL-1β.

[0119] In one embodiment, culturing is performed in the absence of IFN-γ. Statistical analysis of the results obtained from the experiments described herein suggests that the number of TCR-negative cells obtained during isolation may be increased in the absence of IFN-γ. In one embodiment, culturing is performed in the absence of IL-21. In an alternative embodiment, step (i) further includes culturing non-hematopoietic tissue samples in the presence of IL-21 at concentrations between 15 ng / mL and 25 ng / mL, such as 18 to 20 ng / mL, such as 18, 19, or 20 ng / mL. In another embodiment, culturing is performed in the presence of IL-21 at a concentration of 18.8 ng / mL.

[0120] In one embodiment, the method includes freezing a separated population of lymphocytes or γδT cells. Cells can be frozen, for example, in a Cryptotor10 cell freezing solution. Many freezing solutions and parameters are known in the art and are applicable to this aspect of the invention. Frozen cells can be stored, for example, between -80°C and -200°C, optionally in liquid nitrogen (gas phase), until needed.

[0121] According to one aspect of the invention, an isolated population of lymphocytes (e.g., skin-derived αβT cells and / or NK cells) is provided, obtained by any method defined herein. In another embodiment, the isolated population of lymphocytes is frozen.

[0122] According to one aspect of the invention, a population of isolated lymphocytes (e.g., skin-derived αβT cells and / or NK cells) is provided, which is obtainable by any method defined herein.

[0123] According to another aspect of the invention, an isolated population of γδT cells is provided, which is obtained by any method defined herein. In another embodiment, the isolated population of γδT cells is frozen.

[0124] According to another aspect of the invention, a separate population of γδT cells is provided, which can be obtained by any method defined herein.

[0125] 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:

[0126] (i) Isolation of γδT cell populations from non-hematopoietic tissue samples; and

[0127] (ii) Freeze the isolated γδT cell population.

[0128] Previous methods involved freezing γδT cells after expansion, but the inventors have found that cells frozen after isolation are at least as enriched and expanded as fresh equivalents. Freezing after isolation allows time for small-scale, quality-controlled expansion validation of the donor before large-scale, resource-intensive, and operator-intensive expansion. 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 more batches from the same donor later as needed).

[0129] In this aspect of the invention, the isolation method may include the method described herein or alternative isolation methods, such as culturing non-hematopoietic tissue samples in the presence of IL-2, IL-4, IL-9, IL-15, IL-21, or combinations thereof (such as IL-2 and IL-15, particularly IL-2 and IL-15 optionally in combination with IL-4 and / or IL-21). In one embodiment, γδT cells are isolated 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 15 ng / mL and 25 ng / mL, such as 18, 19, or 20 ng / mL. Thus, in another embodiment, when IL-21 is present, its concentration is 18.8 ng / mL.

[0130] In one embodiment, the duration of separation step (i) is at least 14 days. In other embodiments, the duration of separation step (i) is less than 21 days. In yet another embodiment, the duration of separation step (i) is between 14 and 35 days, such as about or between 19 and 21 days. Thus, in one embodiment, the duration of separation step (i) is about 19 days, such as 19 days. In another embodiment, the duration of separation step (i) is about 21 days, such as 21 days.

[0131] Cells can be frozen in a suitable freezing solution (such as Cryptotor 10 cell freezing solution). Many freezing / cryopreservation solutions and parameters are known in the art and are applicable to this aspect of the invention. A suitable freezing solution may 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 approximately -80°C to approximately -200°C, such as temperatures from approximately -80°C to approximately -135°C.

[0132] Cryopreservation can be accomplished by placing vials in a cryogenic container, storing them at -80°C for, for example, 1-3 days, and then transferring them to the gas phase of a liquid nitrogen storage system. In an alternative embodiment, isolated γδT cells are frozen in a controlled-rate cryostat.

[0133] It should be understood that frozen cells are suitable for long-term storage, so isolated cells can remain frozen for a period of time before subsequent thawing and expansion. Frozen cells can be stored, for example, between -80°C and -200°C, optionally in liquid nitrogen (gas phase), until needed.

[0134] After cryopreservation, the cells can be thawed (i.e., defrost), for example, in a 37°C water bath. Thawed cells can then be used for amplification methods. Amplification methods can include any of the methods described herein or as described in the art, for example see WO2017072367 and WO2018202808.

[0135] Therefore, the method may additionally include thawing a frozen γδT cell population. Furthermore, the method may include culturing the thawed γδT cell population for at least 5 days to generate an expanded γδT cell population.

[0136] Therefore, according to another aspect of the present invention, a method for isolating and amplifying γδT cells from a non-hematopoietic tissue sample is provided, the method comprising the following steps:

[0137] (i) Isolating γδT cells from non-hematopoietic tissue samples;

[0138] (ii) Cryoisolated γδT cells;

[0139] (iii) Thawing γδT cells; and

[0140] (iv) Culture the thawed γδT cells for at least 5 days to generate an expanded γδT cell population.

[0141] In one specific embodiment, the isolation step (i) includes culturing a non-hematopoietic tissue sample for approximately 19 days, such as a duration of 19 days, in the presence of IL-21 and IL-1β at concentrations between 15 ng / mL and 25 ng / mL, such as 18 to 20 ng / mL, such as 18, 19, or 20 ng / mL, for example 18.8 ng / mL. In another embodiment, the isolation step (i) includes culturing a non-hematopoietic tissue sample for approximately 19 days, such as 19 days, or approximately 21 days, such as a duration of 21 days, in the presence of IL-1β and in the absence of IL-21. The data provided herein demonstrate that such isolation conditions produce viable γδT cells upon thawing in step (iii) and generate the ability to expand during subsequent expansion cultures, i.e., effectively producing an expanded γδT cell population in step (iv).

[0142] According to another aspect of the invention, a frozen isolated population of γδT cells (i.e., the frozen γδT cells obtained in step (ii)) is provided, which is obtained by the method described herein.

[0143] According to another aspect of the invention, a frozen isolated population of γδT cells (i.e., the frozen γδT cells obtained in step (ii)) is provided, which can be obtained by the method described herein.

[0144] Amplification methods

[0145] In some embodiments, the invention is characterized by methods for amplifying non-hematopoietic tissue-resident lymphocytes and / or γδT cells (e.g., skin-derived αβT cells, NK cells, γδT cells, and / or non-Vδ2T cells, such as Vδ1T cells and / or DNT cells). These methods can be performed in vitro. In some embodiments, γδT cells are amplified from a population of γδT cells isolated from a non-hematopoietic tissue sample according to methods defined herein. Typically, non-hematopoietic tissue-resident γδT cells are capable of spontaneous amplification after physical contact with stromal cells (e.g., skin fibroblasts). The methods defined herein can be used to induce this separation, resulting in deinhibition of γδT cells to trigger amplification. In some embodiments, lymphocytes (e.g., skin-derived αβT cells and / or NK cells, intestinal-derived αβT cells and / or NK cells) are amplified from a population of lymphocytes isolated from a non-hematopoietic tissue sample according to methods defined herein.

[0146] As used herein, references to “amplified” or “amplified lymphocyte and / or γδT cell population” include cell populations that are larger or contain a greater number of cells than non-amplified populations. Such populations can be large, small, or mixed populations with a particular proportion or specific cell type of amplification. It should be understood that the term “amplification step” refers to a process that results in or produces an amplified population. Therefore, an amplified or expanded population may be larger or contain a greater number of cells than a population that has not undergone an amplification step or has not undergone any amplification step. It should also be understood that any quantity indicating amplification (e.g., fold increase or fold expansion) herein refers to the number or size of the cell population or an increase in the number of cells and indicates the amount of amplification.

[0147] Therefore, in one embodiment, lymphocytes or γδT cells isolated according to the method of the present invention are amplified. In one embodiment, the isolated lymphocyte or γδT cell population is frozen and then thawed before amplification.

[0148] Such expansion may include culturing γδT cells in the presence of IL-2, IL-15, and IL-21, optionally including IL-4. Alternatively, such expansion 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 expansion step is performed for a duration in which an expanded lymphocyte population and / or γδT cell population is effectively generated. In one embodiment, the duration in which an expanded lymphocyte population and / or γδT cell population is effectively generated is at least 5 days. Therefore, in one embodiment, expansion includes culturing γδT cells for at least 5 days in the presence of IL-2, IL-15, and IL-21 to an amount that effectively generates an expanded γδT cell population. In another embodiment, expansion includes culturing γδT cells for at least 5 days in the presence of IL-2, IL-15, IL-21, and IL-4 to an amount that effectively generates an expanded γδT cell population. In yet another embodiment, expansion includes culturing γδT cells for at least 5 days in the presence of IL-9, IL-15, and IL-21 to efficiently generate an expanded γδT cell population. In one embodiment, expansion includes culturing γδT cells for at least 5 days in the presence of IL-9, IL-15, IL-21, and IL-4 to efficiently generate an expanded γδT cell population.

[0149] In other embodiments, expansion includes culturing lymphocytes and / or γδT cells in an amount that effectively produces an expanded population of γδT cells for a duration of time (e.g., at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 21 days, at least 28 days or longer, such as 5 to 40 days, 7 to 35 days, 14 to 28 days or about 21 days). In some embodiments, lymphocytes and / or γδT cells are expanded in the culture medium for a period of time from 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 γδT cells are expanded for a period of time from 14 to 21 days. Therefore, in some embodiments, including the isolation and culture 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.

[0150] In other embodiments, expansion includes culturing γδT 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, such as 5 to 40 days, 7 to 35 days, 14 to 28 days, or about 21 days. In one embodiment, the expansion step includes culturing γδT cells for at least 10, 15, or 20 days to generate an expanded population. In one embodiment, the expansion step includes culturing γδT cells for between 5 and 25 days, such as between 14 and 21 days. In another embodiment, the expansion step includes culturing γδT cells for about 20 days.

[0151] In some implementations, typical amounts of IL-2 that effectively generate an expanded population of γδT cells are 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...). 80 IU / 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 implementations, the amount of IL-2 that effectively generates an expanded population of γδT cells is approximately 100 IU / mL.

[0152] In some implementations, the efficient generation of expanded γδT cells (e.g., skin-derived γδT cells and / or non-Vδ2T cells, such as Vδ1T cells and / or DNA) Typical levels of IL-15 in the T cell population are at least 0.1 ng / mL (e.g., 0.1 ng / mL to 10,000 ng / mL, 1.0 ng / mL to 1,000 ng / mL, 5 ng / mL to 800 ng / mL, 10 ng / mL to 750 ng / mL, 20 ng / mL to 500 ng / mL, 50 ng / mL to 400 ng / mL or 100 ng / mL to 250 ng / mL, e.g., 0.1 ng / mL to 1.0 ng / mL, 1.0 ng / mL to 5.0 ng / mL, 5.0 ng / mL to 10 ng / mL, 10 ng / mL to 20 ng / mL, 20 ng / mL to 50 ng / mL, 50 ng / mL to 100 ng / mL, 100 ng / mL to 200 ng / mL, 200 ng / mL to 500 ng / mL or 500 ng / mL to 1,000 ng / mL). In some implementations, the amount of IL-15 that effectively generates an expanded population of γδT cells is approximately 10 ng / mL.

[0153] In some embodiments, the typical amount of IL-21 that effectively generates an expanded population of γδT cells (e.g., skin-derived γδT cells and / or non-Vδ2T cells, such as Vδ1T cells and / or DNT 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, such as 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 typically below 200 ng / mL, such as 188.8 ng / mL. In other embodiments, the amount of IL-21 is typically below 100 ng / mL, such as below 50 ng / mL, such as 37.5 ng / mL. In other embodiments, the amount of IL-21 is typically 20 ng / mL or lower, such as 18 to 20 ng / mL, such as 18, 19, or 20 ng / mL, for example, 18.8 ng / mL. In some embodiments, the method includes IL-21 at a concentration of about 6 ng / mL (such as about 6.25 ng / mL).

[0154] In other embodiments, the methods defined herein include IL-4 at concentrations 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, such as 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 concentrations typically below 100 ng / mL (e.g., below 50 ng / mL, particularly below 20 ng / mL). In some embodiments, the methods include IL-4 at a concentration of about 15 ng / mL.

[0155] This document also provides for the substitution or addition of other factors in the expansion culture of non-hematopoietic resident lymphocytes and / or γδT cells. For example, in some embodiments, in addition to or replacing any one of IL-2 and IL-15, one or more factors selected from the group consisting of IL-4, IL-6, IL-7, IL-8, IL-9, IL-12, IL-18, IL-33, IGF-1, IL-1β, human platelet lysate (HPL), and stromal cell-derived factor-1 (SDF-1) are included. These additional or alternative factors for expanding lymphocytes such as αβT cells or NK cells are known in the art. In one embodiment, these factors are used in the expansion process to selectively promote the expansion of γδT cells. In another embodiment, these factors are used in the expansion process to selectively promote the expansion of lymphocytes such as αβT cells and / or NK cells.

[0156] It should be understood that the amount of each of the aforementioned cytokines required to generate an expanded γδT cell population will depend on the concentration of one or more other cytokines. For example, if the concentration of IL-2 increases or decreases, the concentration of IL-15 may decrease or increase accordingly. As stated above, the amount required to effectively generate an expanded population in this document refers to the combined effect of all factors on cell expansion.

[0157] The amplification method provides an expanded population of γδT cells in greater numbers than the reference population. In some embodiments, the number of amplified γδT cells is greater than the number of isolated γδT cells before the amplification step (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 10,000, or more times the number of isolated γδT cells before the amplification step).

[0158] In one embodiment, the expansion step includes culturing isolated γδT cells in the absence of extensive stromal cell contact. In another embodiment, the expansion step includes culturing isolated γδT cells in the absence of extensive fibroblast contact.

[0159] In other embodiments, the amplification step further includes culturing isolated γδT cells in the presence of IL-4. Therefore, in one embodiment, amplification includes culturing isolated γδT cells in the presence of IL-2, IL-15, IL-4, and IL-21. Alternatively, amplification may include culturing isolated γδT cells in the presence of IL-9, IL-15, IL-4, and IL-21.

[0160] It should be understood that the amplification methods defined herein are also applicable to the amplification of other lymphocytes (e.g., αβT cells and / or NK cells). In such embodiments, the amplification step includes culturing isolated lymphocytes in the presence of relevant growth factors and / or nutrients (e.g., cytokines and / or chemokines) to generate an expanded population of lymphocytes (e.g., αβT cells and / or NK cells).

[0161] In one embodiment, a method for expanding a population of γδT cells as defined herein includes culturing γδT cells or other lymphocytes in a culture medium containing serum or plasma. In an alternative embodiment, a method for expanding a population of γδT cells or other lymphocytes as defined herein includes culturing γδT cells in a serum-free culture medium. In another embodiment, a method for expanding a population of γδT cells or other lymphocytes as defined herein includes culturing γδT cells in a culture medium containing a serum substitute.

[0162] In some embodiments, there is no significant TCR pathway activation during the amplification step (e.g., the culture does not contain exogenous TCR pathway activators). In one embodiment, the amplification step includes the absence of exogenous TCR pathway agonists. Furthermore, this document provides methods for amplifying γδT cells isolated according to the methods defined herein, wherein said amplification methods do not involve contact with feeder cells, tumor cells, and / or antigen-presenting cells. Therefore, in another embodiment of the methods defined herein, the amplification of γδT cells includes culturing γδT cells without significant stromal cell contact.

[0163] A method is also provided for generating large populations of non-hematopoietic tissue-derived γδT cells (e.g., skin-derived γδT cells and / or non-Vδ2T cells, such as Vδ1T cells and / or DNT cells) at high rates (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 steps described herein expand γδT cells with a low population doubling time, said doubling time being given by the following equation:

[0164]

[0165] Based on the information provided herein, those skilled in the art will recognize that the present invention provides a method for expanding non-hematopoietic tissue-derived γδT cells (e.g., skin-derived γδT cells and / or non-Vδ2T cells, such as Vδ1T cells and / or DNT 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).

[0166] In some embodiments, within 7 days of culture, the expanded γδT cell population (e.g., the expanded Vδ1T cell population and / or DNT cell population) includes at least 10 times the number of γδT cells relative to the isolated γδT cell population before expansion (e.g., at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, at least 100 times, at least 150 times, at least 200 times, at least 300 times, at least 400 times, at least 500 times, at least 600 times, at least 700 times, at least 800 times, at least 900 times, at least 1,000 times, at least 2,000 times, at least 3,000 times, at least 4,000 times, at least 5,000 times, at least 6,000 times, at least 7,000 times, or at least 8,000 times the number of γδT cells relative to the isolated γδT cell population before expansion). In some implementations, within 14 days of culture, the expanded γδT cell population (e.g., expanded Vδ1T cell population and / or DN) The T cell population includes at least 20 times the number of γδT cells relative to the pre-expansion isolated γδT cell population (e.g., at least 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000 times the number of γδT cells relative to the pre-expansion isolated γδT cell population). In some embodiments, within 21 days of culture, the expanded γδT cell population (e.g., the expanded Vδ1T cell population and / or DNT cell population) includes at least 50 times the number of γδT cells relative to the isolated γδT cell population before expansion (e.g., at least 60 times, at least 70 times, at least 80 times, at least 90 times, at least 100 times, at least 150 times, at least 200 times, at least 300 times, at least 400 times, at least 500 times, at least 600 times, at least 700 times, at least 800 times, at least 900 times, at least 1,000 times, at least 2,000 times, at least 3,000 times, at least 4,000 times, at least 5,000 times, at least 6,000 times, at least 7,000 times, at least 8,000 times, at least 9,000 times, or at least 10,000 times the number of γδT cells relative to the isolated γδT cell population before expansion).In some implementations, within 28 days of culture, the expanded γδT cell population (e.g., expanded Vδ1T cell population and / or DN) The T cell population includes at least 100 times the number of γδT cells relative to the pre-expansion isolated γδT cell population (e.g., at least 110 times, at least 120 times, at least 130 times, at least 140 times, at least 150 times, at least 200 times, at least 300 times, at least 400 times, at least 500 times, at least 600 times, at least 700 times, at least 800 times, at least 900 times, at least 1,000 times, at least 2,000 times, at least 3,000 times, at least 4,000 times, at least 5,000 times, at least 6,000 times, at least 7,000 times, at least 8,000 times, at least 9,000 times, at least 10,000 times, at least 12,000 times, or at least 15,000 times the number of γδT cells relative to the pre-expansion isolated γδT cell population).

[0167] 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 using the methods provided herein can possess phenotypes highly suited to antitumor efficacy. In some embodiments, the expanded γδT cell population (e.g., skin-derived Vδ1T cells) exhibits higher average CD27 expression than a reference population (e.g., a population of γδT cells isolated prior to the expansion step). In some embodiments, the expanded γδT cell population has an average CD27 expression that is at least 2-fold higher than that of the isolated γδT cell population (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).

[0168] Different fractions of the expanded γδT cell population (e.g., skin-derived γδT cells and / or non-Vδ2T cells, such as Vδ1T cells and / or DN T cells) can upregulate CD27, while another fraction can upregulate CD27. 低 Or CD27 阴性 In this case, compared to the isolated γδT cell population, the expanded population had a higher concentration of CD27 cells. 阳性The cell frequency may be even greater. For example, an expanded population of γδT cells may have at least 5% higher CD27 count compared to a pre-expansion isolated population of γδT cells. 阳性 Cell frequencies (e.g., CD27 at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or up to 100% higher than the pre-expansion isolated γδT cell population) 阳性 (Cell frequency). In some implementations, the expanded population has a higher CD27 content compared to the isolated γδT cell population. 阳性 The number of cells may increase. For example, the expanded γδT cell population may have at least twice the number of CD27 cells compared to the isolated γδT cell population before expansion. 阳性 Cells. The expanded γδT 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 γδT cell population may have a CD27+ cell frequency of approximately 10%, approximately 20%, approximately 30%, approximately 40%, approximately 50%, approximately 60%, approximately 70%, approximately 80%, or approximately 90%. In some embodiments, the expanded γδT cell population has a CD27+ cell frequency of greater than 50%.

[0169] In some embodiments, the amplification methods provided herein generate an expanded population of 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) with low TIGIT expression relative to a reference population (e.g., a population of isolated γδT cells prior to the amplification step). In some embodiments, the expanded γδT cell population has a lower average TIGIT expression than the reference population (e.g., a population of isolated γδT cells prior to the amplification step). In some embodiments, the expanded γδT cell population has at least 10% lower average TIGIT expression than the isolated γδT cell population (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or up to 100% lower than the isolated γδT cell population). 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 approximately 90%, approximately 80%, approximately 70%, approximately 60%, approximately 50%, approximately 40%, approximately 30%, approximately 20%, or approximately 10%. In some embodiments, the isolated γδT cell population has a TIGIT+ cell frequency of less than 80%.

[0170] In some implementations, the expanded population of γδT cells (e.g., skin-derived γδT cells or non-Vδ2T cells, such as Vδ1T cells and / or DNT cells) has a high number or frequency of CD27. + Cells and low-frequency TIGIT + Cells. In some embodiments, the expanded γδT cell population has a high frequency of CD27 compared to a reference population (e.g., compared to a pre-expansion isolated γδT cell population). + TIGIT - Cells. For example, an expanded population of γδT cells may have at least 5% higher CD27 content compared to a pre-expansion isolated population of γδT cells. + TIGIT - Cell frequencies (e.g., CD27 at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or up to 100% higher than the pre-expansion isolated γδT cell population) + TIGIT - (Cell frequency). In some implementations, the expanded population has a higher CD27 content compared to the isolated γδT cell population. +TIGIT - The number of cells may increase. For example, the expanded γδT cell population may have at least twice the number of CD27 cells compared to the isolated γδT cell population before expansion. + TIGIT - Cells (e.g., with 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 content relative to the isolated γδT cell population before expansion) + TIGIT - Cell frequency).

[0171] In some cases, CD27 is present in expanded populations of γδT cells (e.g., skin-derived γδT cells and / or non-Vδ2T cells, such as Vδ1T cells and / or DN T cells). + The average expression of TIGIT in the γδT cell population was lower than that in the reference population. In some implementations, amplified CD27... + The γδT cell population had a higher concentration than the reference population (e.g., CD27 isolated before the amplification step). + Low average TIGIT expression in γδT cell populations. In some implementations, amplified CD27... + The γδT cell population has a higher concentration of isolated CD27 cells than the isolated CD27 cells. + The γδT cell population is at least 10% lower (e.g., lower than isolated CD27). + The average expression of TIGIT in γδT cell populations was at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or up to 100% lower.

[0172] Alternatively or alternatively, TIGIT in an expanded population of γδT cells (e.g., skin-derived γδT cells and / or non-Vδ2T cells, such as Vδ1T cells and / or DN T cells) - The median CD27 expression in the γδT cell population was higher than that in the reference population. For example, expanded TIGIT - γδT cell population relative to pre-expansion isolated TIGIT - The γδT cell population may have at least 5% high CD27 content. + Cell frequency (e.g., relative to TIGIT isolated before expansion) -A γδ T cell population with 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 more than 100% CD27 content. + Cell frequency). In some implementations, relative to isolated TIGIT - γδT cell population, CD27 in the expanded population + The number of cells may increase. For example, expanded TIGIT - γδT cell population relative to pre-expansion isolated TIGIT - The γδT cell population may have at least twice the number of CD27. + Cells (e.g., compared to TIGIT isolated before expansion) - A γδ T cell population with 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 more than 100% CD27 content. + Cell frequency).

[0173] Increases or decreases in the expression of other biomarkers may additionally or alternatively be used to characterize one or more expanded populations of 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), said biomarkers 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. In some cases, compared to an isolated γδT cell population (e.g., pre-expansion), an expanded γδT cell population (e.g., skin-derived γδT cells and / or non-Vδ2T cells, such as Vδ1T cells and / or DN T cells) exhibits 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) may have a higher frequency of 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) In some implementations, compared to an isolated γδT cell population, an expanded γδT 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. Similarly, compared to an isolated γδT cell population, the expanded population may 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.

[0174] A variety of basal media suitable for culturing and / or proliferating γδT cells are available, particularly those such as AIM-V, Iscoves medium, and RPMI-1640 (Life Technologies). The media may be supplemented with other culture factors as defined herein, such as serum, serum proteins, and selectants, such as antibiotics. For example, in some embodiments, RPMI-1640 medium contains 2 mM glutamine, 10% FBS, 10 mM HEPES pH 7.2, 1% penicillin-streptomycin, sodium pyruvate (1 mM; Life Technologies), non-essential amino acids (e.g., 100 μM Gly, Ala, Asn, Asp, Glu, Pro, and Ser; 1X MEM non-essential amino acids (Life Technologies)), and 10 μl / L β-mercaptoethanol. In alternative embodiments, AIM-V medium may be supplemented with a CTS immune serum substitute and amphotericin B. In some embodiments as defined herein, the medium may also be supplemented with IL-2 and IL-15. Conveniently, during isolation and / or expansion, cells are cultured at 37°C in a suitable medium in a humid environment containing 5% CO2.

[0175] According to another aspect of the present invention, a method for isolating and expanding lymphocytes from a non-hematopoietic tissue sample is provided, the method comprising the following steps:

[0176] (i) Isolating lymphocyte populations from non-hematopoietic tissue samples according to the methods defined herein; and

[0177] (ii) Further culture the lymphocyte population (e.g., for at least 5 days) to generate an expanded lymphocyte population.

[0178] In one embodiment, the lymphocytes include αβT cells. Therefore, according to another aspect of the invention, a method for isolating and expanding αβT cells from a non-hematopoietic tissue sample is provided, the method comprising the following steps:

[0179] (i) Isolate αβT cell populations from non-hematopoietic tissue samples according to the methods defined herein; and

[0180] (ii) Further culture the αβT cell population (e.g., for at least 5 days) to generate an expanded αβT cell population.

[0181] The culture in step (ii) can be performed by selective expansion, such as by selecting culture conditions in which αβT cells are expanded preferentially over other cell types present in the isolated population in step (i). Alternatively, the expansion conditions may be non-selective, and non-target cells (e.g., cells other than αβT cells) may be depleted after the culture in step (ii). Or, the expansion conditions may be non-selective, and non-target cells (e.g., cells other than αβT cells) may be depleted before the culture in step (ii). It should be noted that the aim of these embodiments is to expand the total number of αβT cells while also increasing their proportion in the population.

[0182] In one embodiment, the lymphocytes include NK cells. Therefore, according to another aspect of the invention, a method for isolating and expanding NK cells from a non-hematopoietic tissue sample is provided, the method comprising the following steps:

[0183] (i) Isolating NK cell populations from non-hematopoietic tissue samples according to the methods defined herein; and

[0184] (ii) Further culture the NK cell population (e.g., for at least 5 days) to generate an expanded NK cell population.

[0185] The culture in step (ii) can be performed by selective amplification, such as by selecting culture conditions in which NK cells are amplified preferentially 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) may be depleted after the culture in step (ii). Or, the amplification conditions are not selective, and non-target cells (e.g., cells other than NK cells) are depleted before the culture in step (ii). It should be noted that the aim of these embodiments is to amplify the total number of NK cells while also increasing their proportion in the population.

[0186] According to another aspect of the present invention, a method for isolating and expanding γδT cells from a non-hematopoietic tissue sample is provided, the method comprising the following steps:

[0187] (i) Isolating a population of γδT cells from non-hematopoietic tissue samples according to the methods defined herein; and

[0188] (ii) Further culture the γδT cell population (e.g., for at least 5 days) to generate an expanded γδT cell population.

[0189] The culture in step (ii) can be performed by selective expansion, such as by selecting culture conditions in which γδT cells are expanded preferentially 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 γδT cells) may be depleted after the culture in step (ii). Or, the expansion conditions are not selective, and non-target cells (e.g., cells other than γδT cells) are depleted before the culture in step (ii). It should be noted that the aim of these embodiments is to expand the total number of γδT cells while also increasing their proportion in the population.

[0190] In one embodiment, the isolated lymphocyte or γδT cell population is frozen and then thawed prior to step (ii). Surprisingly, the frozen isolated cell population was found to be at least as enriched and expanded as the fresh equivalent. In particular, the data provided herein demonstrate that isolated γδT cells were obtained with good viability after thawing and the ability to expand well during subsequent expansion culture (i.e., the ability to efficiently generate an expanded γδT cell population in step (ii)), wherein the isolation step (i) includes culturing non-hematopoietic tissue samples for approximately 19 days, such as a duration of 19 days, in the presence of IL-21 and IL-1β at concentrations between 15 ng / mL and 25 ng / mL, such as 18 to 20 ng / mL, such as 18, 19, or 20 ng / mL, for example 18.8 ng / mL, and IL-1β, or alternatively, culturing for approximately 19 days, such as 19 days, or approximately 21 days, such as a duration of 21 days, in the presence of IL-1β and the absence of IL-21.

[0191] In one embodiment, the lymphocytes include γδT cells. Therefore, according to 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 following steps:

[0192] (i) Isolating a population of γδT cells from non-hematopoietic tissue samples according to the methods defined herein; and

[0193] (ii) The γδT cell population is cultured in the presence of the following conditions:

[0194] (a) IL-2 or IL-9;

[0195] (b)IL15; and

[0196] (c)(IL-21),

[0197] Culture for at least 5 days to generate an effective population of expanded γδT cells.

[0198] In some embodiments of this aspect of the invention, culturing the γδT cell population further includes the presence of IL-4. Therefore, 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 following steps:

[0199] (i) Isolating a population of γδT cells from non-hematopoietic tissue samples according to the methods defined herein; and

[0200] (ii) The γδT cell population is cultured in the presence of the following conditions:

[0201] (a) IL-2 or IL-9;

[0202] (b)IL15; and

[0203] (c)IL-21; and

[0204] (d)IL-4,

[0205] Culture for at least 5 days to generate an effective population of expanded γδT cells.

[0206] According to one aspect of the invention, an expanded isolated population of lymphocytes (e.g., skin-derived αβT cells and / or NK cells) is provided, obtained by any method defined herein.

[0207] According to another aspect of the invention, an expanded isolated lymphocyte population is provided, which can be obtained by any method defined herein.

[0208] According to another aspect of the invention, an expanded isolated population of γδT cells is provided, which is obtained by any method defined herein.

[0209] According to another aspect of the invention, an expanded isolated population of γδT cells is provided, which can be obtained by any method defined herein.

[0210] Application of cells

[0211] Lymphocytes and / or γδT cells obtained by the methods of the present invention can be used as pharmaceuticals, for example, for adoptive T-cell therapy. This involves transferring lymphocytes and / or γδT cells obtained by the methods of the present invention into a patient. The therapy can be autologous, i.e., the γδT cells can be transferred back to the same patient from whom they were obtained, or the therapy can be allogeneic, i.e., γδT cells from one person can be transferred into a different patient. In cases involving allogeneic transfer, the γδT cells may be substantially free of αβT cells. For example, αβT cells can be removed from the γδT cell population, for example, after expansion, using any suitable method known in the art (e.g., by negative selection, e.g., using magnetic beads). Therapeutic methods may include: providing a non-hematopoietic tissue sample obtained from a donor individual; isolating γδT cells from the non-hematopoietic tissue sample as described herein; culturing the isolated γδT cells to generate an expanded population; and administering the expanded γδT cell population to a recipient individual.

[0212] The patients or subjects to be treated are preferably human cancer patients (e.g., human cancer patients undergoing treatment for solid tumors) or patients with viral infections (e.g., patients with CMV infection or HIV infection). In some cases, the patient has a solid tumor and / or is receiving treatment for a solid tumor.

[0213] Because they typically reside in non-hematopoietic tissues, tissue-resident Vδ1T cells and DNγδT cells are also more likely to home to and remain within tumor masses than their systemic blood-resident counterparts, and adoptive transfer of these cells may be more effective in targeting solid tumors and potentially other non-hematopoietic tissue-related immunopathologies.

[0214] Because γδT cells are non-MHC-restricted, they cannot be recognized as foreign by the host to which they are transferred, meaning they are unlikely to cause graft-versus-host disease. This means they can be used "off the job" and transferred to any recipient, for example, for allogeneic adoptive T-cell therapy.

[0215] Non-hematopoietic resident γδT cells obtained by the method of this invention express NKG2D and respond to NKG2D ligands (e.g., MICA) closely associated with malignant tumors. They also express cytotoxic signatures in the absence of any activation, and therefore may be effective in killing tumor cells. For example, non-hematopoietic resident γδT cells obtained as described herein may express one or more of IFN-γ, TNF-α, GM-CSF, CCL4, IL-13, granzyme A and B, and perforin in the absence of any activation, preferably all of them. IL-17A may not be expressed.

[0216] There is compelling evidence of the practicality and suitability of non-hematopoietic tissue-resident γδ T cells obtained by the methods of this invention for clinical application 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.

[0217] In some implementations, a method of treating an individual with a tumor in non-hematopoietic tissue may include: providing a sample of the non-hematopoietic tissue obtained from a donor individual, culturing γδT cells derived from the sample as described above to generate an expanded population, and applying the expanded γδT cell population to the individual with the tumor.

[0218] Pharmaceutical compositions may include combinations of expanded non-hematopoietic tissue-resident γδT cells as described herein with 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; peptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Cryopreservation solutions that can be used for the pharmaceutical compositions of the present invention include, for example, DMSO. Compositions may be formulated, for example, for intravenous administration.

[0219] In one embodiment, the pharmaceutical composition is substantially free of, for example, contaminants such as endotoxins or mycoplasma, at detectable levels.

[0220] In some cases, a therapeutically effective amount of expanded γδT cells obtained by any of the methods described above may be administered to a subject (e.g., for the treatment of cancer, such as solid tumors). In some cases, the therapeutically effective amount of expanded γδT cells (e.g., skin-derived γδT cells and / or non-Vδ2T cells, such as Vδ1T cells and / or DNT cells) is less than 10 x 10^6 cells per dose. 12 Cells (e.g., less than 9 x 10⁻⁶ per dose) 12 Cells, less than 8 x 10 per dose 12 Cells, less than 7 x 10 per dose 12 Cells, less than 6 x 10 per dose 12 Cells, less than 5 x 10 per dose 12 Cells, less than 4 x 10 per dose 12 Cells, less than 3 x 10 per dose 12 Cells, less than 2x10 per dose 12 Cells, less than 1 x 10 per dose 12Cells, less than 9 x 10 per dose 11 Cells, less than 8 x 10 per dose 11 Cells, less than 7 x 10 per dose 11 Cells, less than 6 x 10 per dose 11 Cells, less than 5 x 10 per dose 11 Cells, less than 4 x 10 per dose 11 Cells, less than 3 x 10 per dose 11 Cells, less than 2 x 10 per dose 11 Cells, less than 1x10 per dose 11 Cells, less than 9 x 10 per dose 10 Cells, less than 7.5 x 10⁻⁶ per dose 10 Cells, less than 5 x 10 per dose 10 Cells, less than 2.5 x 10⁻⁶ per dose 10 Cells, less than 1 x 10 per dose 10 Cells, less than 7.5 x 10⁻⁶ per dose 9 Cells, less than 5 x 10 per dose 9 Cells, less than 2.5 x 10⁻⁶ per dose 9 Cells, less than 1x10 per dose 9 Cells, less than 7.5 x 10⁻⁶ per dose 8 Cells, less than 5 x 10 per dose 8 Cells, less than 2.5 x 10⁻⁶ per dose 8 Cells, less than 1 x 10 per dose 8 Cells, less than 7.5 x 10⁻⁶ per dose 7 Cells, less than 5 x 10 per dose 7 Cells, less than 2.5 x 10⁻⁶ per dose 7 Cells, less than 1 x 10 per dose 7 Cells, less than 7.5 x 10⁻⁶ per dose 6 Cells, less than 5 x 10 per dose 6 Cells, less than 2.5 x 10⁻⁶ per dose 6 Cells, less than 1 x 10 per dose 6 Cells, less than 7.5 x 10⁻⁶ per dose 5 Cells, less than 5 x 10 per dose 5 Cells, less than 2.5 x 10⁻⁶ per dose 5 One cell or less than 1 x 10 per dose 5 (cells).

[0221] In some implementations, the therapeutically effective amount of expanded γδT cells (e.g., skin-derived γδT cells and / or non-Vδ2T cells, such as Vδ1T cells and / or DNT cells) is less than 10 x 10^6 cells during treatment. 12 Cells (e.g., less than 9x10 during treatment) 12 1 cell, less than 8 x 10 12 1 cell, less than 7 x 10 12 1 cell, less than 6 x 10 12 1 cell, less than 5 x 10 12 1 cell, less than 4 x 10 12 1 cell, less than 3 x 10 12 1 cell, less than 2 x 10 12 1 cell, less than 1 x 10 12 1 cell, less than 9 x 10 11 1 cell, less than 8 x 10 11 1 cell, less than 7 x 10 11 1 cell, less than 6 x 10 11 1 cell, less than 5 x 10 11 1 cell, less than 4 x 10 11 1 cell, less than 3 x 10 11 1 cell, less than 2 x 10 11 1 cell, less than 1 x 10 11 1 cell, less than 9 x 10 10 1 cell, less than 7.5 x 10 10 1 cell, less than 5 x 10 10 1 cell, less than 2.5 x 10 10 1 cell, less than 1 x 10 10 1 cell, less than 7.5 x 10 9 1 cell, less than 5 x 10 9 1 cell, less than 2.5 x 10 9 1 cell, less than 1 x 10 9 1 cell, less than 7.5 x 10 8 1 cell, less than 5 x 10 8 1 cell, less than 2.5 x 10 8 1 cell, less than 1 x 10 8 1 cell, less than 7.5 x 10 7 1 cell, less than 5 x 10 7 1 cell, less than 2.5 x 10 7 1 cell, less than 1 x 10 7 1 cell, less than 7.5 x 10 6 1 cell, less than 5 x 106 1 cell, less than 2.5 x 10 6 1 cell, less than 1x10 6 1 cell, less than 7.5 x 10 5 1 cell, less than 5 x 10 5 1 cell, less than 2.5 x 10 5 1 cell or less than 1 x 10 5 (cells).

[0222] In some implementations, the dose of expanded non-hematopoietic tissue-resident γδT cells, as described herein, comprises approximately 1 x 10⁻⁶ cells. 6 1.1 x 10 6 2 x 10 6 3.6 x 10 6 5 x 10 6 1x10 7 1.8 x 10 7 2 x 10 7 5 x 10 7 1x10 8 2 x 10 8 Or 5 x 10 8 Cells / kg. In some embodiments, the dose of expanded non-hematopoietic tissue-resident γδT cells (e.g., skin-derived γδT cells and / or non-Vδ2T cells, such as Vδ1T cells and / or DNT cells) comprises at least about 1 x 10⁻⁶ cells / kg. 6 1.1 x 10 6 2 x 10 6 3.6 x 10 6 5 x 10 6 1x10 7 1.8 x 10 7 2 x 10 7 5 x 10 7 1x10 8 2 x 10 8 Or 5x10 8 Cells / kg. In some embodiments, the dose of expanded non-hematopoietic tissue-resident γδT cells (e.g., skin-derived γδT cells and / or non-Vδ2T cells, such as Vδ1T cells and / or DNT cells) includes up to about 1 x 10⁻⁶ cells / kg. 6 1.1x10 6 2 x 10 6 3.6 x 10 6 5 x 10 6 1x10 7 1.8 x 10 7 2 x 107 5 x 10 7 1x10 8 2 x 10 8 Or 5 x 10 8 Cells / kg. In some embodiments, the dose of expanded non-hematopoietic tissue-resident γδT cells (e.g., skin-derived γδT cells and / or non-Vδ2T cells, such as Vδ1T cells and / or DNT cells) comprises approximately 1.1 x 10⁻⁶ cells / kg. 6 -1.8x 10 7 Cells / kg. In some embodiments, the dose of expanded non-hematopoietic tissue-resident γδT cells (e.g., skin-derived γδT cells and / or non-Vδ2T cells, such as Vδ1T cells and / or DNT cells) comprises approximately 1 x 10⁻⁶ cells / kg. 7 2x10 7 5 x 10 7 1x10 8 2x10 8 5 x 10 8 1x10 9 2 x 10 9 Or 5 x 10 9 Cells. In some embodiments, the dose of expanded non-hematopoietic tissue-resident γδT cells (e.g., skin-derived γδT cells and / or non-Vδ2T cells, such as Vδ1T cells and / or DNT cells) comprises at least about 1 x 10⁻⁶ cells. 7 2 x 10 7 5 x 10 7 1x10 8 2 x 10 8 5 x 10 8 1x10 9 2 x 10 9 or 5 x 10 9 Cells. In some embodiments, the dose of expanded non-hematopoietic tissue-resident γδT cells (e.g., skin-derived γδT cells and / or non-Vδ2T cells, such as Vδ1T cells and / or DNT cells) comprises up to about 1 x 102 cells. 7 2 x 10 7 5 x 10 7 1x10 8 2 x 10 8 5 x 10 8 1x10 9 2 x 10 9 Or 5 x 10 9 Each cell.

[0223] In one implementation scheme, the subject is administered 10 mg / kg of subject body weight. 4 Up to 10 6 A population of expanded non-hematopoietic tissue-resident γδT cells (e.g., skin-derived γδT cells and / or non-Vδ2T cells, such as Vδ1T cells and / or DNT cells). In one embodiment, the subject receives an initial administration of the non-hematopoietic tissue-resident γδT cell population (e.g., 10 cells per kg of subject body weight). 4 Up to 10 6 γδT cells, for example, 10 per kg of subject body weight 4 Up to 10 5 An initial administration of γδT cells, and one or more subsequent administrations of expanded non-hematopoietic tissue-resident γδT cells (e.g., 2, 3, 4, or 5 times) (e.g., 10 per kg of subject body weight). 4 Up to 10 6 Amplified non-hematopoietic tissue resident γδT cells, for example, 10 per kg of subject body weight 4 Up to 10 5 One or more subsequent administrations of expanded non-hematopoietic tissue-resident γδT cells. In one embodiment, one or more subsequent administrations are administered less than 15 days after the previous administration, for example, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days after the previous administration, for example, less than 4, 3, or 2 days after the previous administration. In one embodiment, during at least three administrations of the γδT cell population, the subject receives approximately 10 [units of something] per kg of subject body weight. 6 One γδT cell, for example, the subject receives 1 x 102 5 An initial dose of 3 x 10 γδT cells 5 A second administration of γδT cells and 6 x 10 5 The third administration of γδT cells, and for example, each administration is given less than 4, 3 or 2 days after the previous administration.

[0224] Non-hematopoietic tissue-resident γδ T cells obtained by the methods of this invention can also be genetically engineered to enhance therapeutic properties, such as for use in CAR-T therapy. This involves generating engineered T cell receptors (TCRs) to reprogram T cells with novel specificities (e.g., the specificity of monoclonal antibodies). Engineered TCRs can make T cells specific for malignant cells and thus usable in cancer immunotherapy. For example, T cells can recognize cancer cells expressing tumor antigens, such as tumor-associated antigens not expressed by normal somatic cells from a subject's tissue. Therefore, CAR-modified T cells can be used, for example, in adoptive T-cell therapy for cancer patients.

[0225] The use of hematopoietic γδT cells for CAR has been described. However, non-hematopoietic γδT cells obtained by the methods of this 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 compared to hematopoietic γδT cells or conventional systemic αβT cells. Furthermore, the 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 conventional co-stimulation, such as enhanced targeting to tumors expressing low levels of co-stimulatory receptor ligands through CD28 involvement.

[0226] In some implementations, one or more additional therapeutic agents may be administered to the subject. The additional therapeutic agents may be selected from the group consisting of: immunotherapeutic agents, cytotoxic agents, growth inhibitors, radiotherapy agents, anti-angiogenic agents, or combinations of two or more of these. The additional therapeutic agents may be administered simultaneously with, before, or after the administration of expanded γδT cells. The additional therapeutic agents may be immunotherapeutic agents that act on targets in the subject's body (e.g., the subject's own immune system) and / or metastatic γδT cells.

[0227] The composition can be administered in any convenient manner. The composition described herein can be administered to the patient via artery, subcutaneous, intradermal, tumor, nodular, medullary, intramuscular, intravenous, or intraperitoneal injection, such as via intradermal or subcutaneous injection. The composition for non-hematopoietic resident γδT cells can be directly injected into tumors, lymph nodes, or sites of infection.

[0228] It should be understood that all embodiments described herein can be applied to all aspects of the present invention.

[0229] As used herein, the term “about” includes up to 10% (inclusive) and down to 10% (inclusive) above the specified value, and appropriately up to 5% (inclusive) and down to 5% (inclusive) above the specified value, particularly the specified value. The term “between” includes values ​​that specify boundaries.

[0230] Some aspects and embodiments of the invention will now be described by way of examples and with reference to the accompanying drawings.

[0231] Example

[0232] Example 1. Methods and Materials

[0233] Unless otherwise stated, the following methods are used to generate the results of subsequent embodiments.

[0234] Material

[0235] Table 1 summarizes the sources of the materials used in the experimental conditions.

[0236] Table 1. Materials used in the study

[0237] reagents supplier AIM-V Fisher CTS immune serum substitutes Life Technologies Human serum albumin Sigma-Aldrich Human blood 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

[0238] Experimental modeling design

[0239] Experimental designs (DoEs) were generated using JMP software 15.0. A custom design using DoE was applied to generate experiments analyzing the effects of protein source, IL-1β, IFN-γ, IL-21, and IL-4 on deterministic outcomes, such as γδT cell enrichment. Protein source was treated as a categorical variable, while IL-1β, IFN-γ, IL-21, and IL-4 were treated as continuous variables. Furthermore, JMP scripts were used to reject experimental conditions containing only IL-4.

[0240] Two-level factorial designs were applied to enable the determination of linear and second-level interactions (e.g., IL-21*IL-1β). The designs under 21 conditions were experimentally tested.

[0241] After collecting experimental data, outlier removal was performed by calculating studentized residuals based on the γδT cell percentage as a response reading. Any row with a studentized residual >4 or <4 that produced a γδT cell percentage was excluded from the analysis. A total of 7 out of 84 rows were excluded.

[0242] After removing outliers, a standard least-squares fit is applied to a given response based on the value from each donor. To illustrate donor variation, donor ID is added as an independent variable to the least-squares fit model. The p-values ​​of protein origin and cytokine interactions associated with selected outcomes (such as % of γδT cells or % of TIGIT+γδT cells) are eliminated in a stepwise manner until only interactions with p < 0.05 are retained.

[0243] Separate setup and harvest

[0244] Before the samples arrive, prepare the relevant culture media and autoclave the 20 mm x 1.5 mm carbon matrix (“grid”) (Cytomatrix Pty Ltd, Australia or Ultramet, USA). Then rinse the grid in phosphate-buffered saline (PBS) and immerse it in PBS until use.

[0245] Preparation of organoid skin cultures

[0246] Skin samples were prepared by removing subcutaneous fat using tweezers, scissors, and a scalpel. One tissue sample was taken at a time and punched multiple times using a 3mm biopsy punch. Three tissue punches were placed on each grid.

[0247] Place a grid in each well of a G-REX6 (Wilson Wolf) plate. Fill each well with 5 ml of the relevant basal medium and 5 ml of 2X relevant conditioned medium (to obtain 10 ml of 1X relevant conditioned medium). Add 100 μL of amphotericin B (Life Technologies) to each well (to obtain 1% amphotericin B). Incubate the plate at 37°C and 5% CO2 for 21 days. Incubate on days 7 and 14 by gently adding 10 ml of 2X relevant medium to each well.

[0248] Separate harvest

[0249] Remove the matrix. Use a pipette to resuspend the cells in each well and transfer them to centrifuge tubes. Wash the wells with PBS and transfer the wash solution to centrifuge tubes as well. The isolated cells can then be analyzed subsequently, for example, by cell counting or in flow cytometry analysis.

[0250] Cryoisolated cells

[0251] Once the isolated cells have been analyzed, the cell suspension is centrifuged to allow sedimentation. The supernatant is discarded, and the cell pellet is resuspended in Cryptotor 10 cell freezing solution (Sigma Aldrich) to a final concentration of 100 x 10⁻⁶. 6 Cells / ml. The cell suspension was then transferred to cryovials and placed in a low-temperature chamber or a Mr Frosty cryostat (Thermo Scientific) overnight at -80°C. The next day, the cells were transferred to liquid nitrogen storage (gas phase).

[0252] Flow cytometry

[0253] Flow cytometry was performed using the following antibody-fluorescent dye conjugates:

[0254] a) Bulk Release Group: CD45-FITC, CD25-PE, PANαβ-PerCP Vio700, NKG2D-PEVio770, Vδ1-Vioblue, PANγδ-APC.

[0255] b) Functional groups: CD45RA-FITC, TIGIT-PE, PANαβ-PerCP Vio700, NKG2A-PEVio770, Vδ1-Vioblue, PD1-BV510, PANγδ-APC.

[0256] Commercial antibodies were purchased from Biolegend or Miltenyi. The reactive dye (eFluor780) was from ThermoFischer Scientific. Flow cytometry data were analyzed using FLOWJO (version 10.6.2).

[0257] Determine the total number of cells

[0258] The total cell count was generated using the NC-250 Nucleocounter (Chemometec, Copenhagen Denmark) and the manufacturer's instructions.

[0259] Example 2. Separation Optimization Experiment

[0260] This study investigated the optimization of protocols for isolating γδT cells from non-hematopoietic tissue samples to increase γδT cell yield and potentially improve Vδ1T cell quality at isolation. Improved starting materials will contribute to increased yield and quality in subsequent amplification steps.

[0261] The design of experiments (DoE) investigated the effects of different protein sources and cytokines on skin isolates. To ensure a feasible design, the culture 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.

[0262] All cells were isolated in AIM-V medium supplemented with one of the following: 5% serum substitute (SR), 2.5% allogeneic plasma, or 10% allogeneic AB male serum. All cultures were prepared with IL-2 and IL-15, and supplemented with different combinations of IL-4, IL-21, IFN-γ, and IL-1β. The culture conditions used in the tests are summarized in Table 2.

[0263] Table 2. Summary of experimental conditions for the test

[0264]

[0265]

[0266] The results of the culture conditions showed Figures 1 to 10 middle.

[0267] Cytokines

[0268] Especially in cases of plasma and AB serum separation, the use of IL-1β increased the total yield of γδT cells and the total yield of isolated Vδ1T cells. γδT cell percentage and total γδT cell results under all conditions are shown in... Figure 1 In the middle, and Vδ1T cell % and total Vδ1T cells were shown in Figure 2 middle.

[0269] Adding IL-4 to AB serum has the effect of increasing the total number of cells isolated in each grid. Figure 3 A more moderate increase was observed in plasma-isolated cultures.

[0270] Adding IL-4 to plasma, SR, or AB serum samples increases the γδ enrichment % in cultures. Figure 4 The role of ).

[0271] Adding IL-4 to plasma cultures has the effect of increasing the total number of Vδ1 cells isolated in each grid. Figure 5 No similar effect was observed in SR or human AB serum cultures; however, the use of AB serum increased the total number of Vδ1 cells isolated per grid compared to the control.

[0272] Adding IL-21 to AB serum cultures increased the total number of isolated γδT cells. However, no benefit of IL-21 was observed in cultures isolated from plasma or SR. DoE modeling of the percentage of γδT cells per donor in plasma containing IL-1β and IFN-γ indicated that IL-1β was most beneficial in the absence of IFN-γ and IL-21.

[0273] Phenotypic analysis

[0274] Adding IL-4 to AB serum and plasma cultures reduced the expression of NKG2A and CD45RA on Vδ1T cells. Figure 6 ).

[0275] Compared to plasma and AB serum isolates, the use of SR resulted in increased expression of the checkpoint inhibitor marker TIGIT on Vδ1T cells. Figure 7 ).

[0276] The analysis also showed that, compared with plasma and AB serum, the use of SR led to increased expression of the terminal differentiation marker CD45RA and the inhibitory receptor NKp44 on Vδ1T cells (data not shown).

[0277] Protein sources

[0278] Compared to SR culture isolation, the use of plasma and AB serum in culture had the effect of increasing the total number of Vδ1T cells per grid. Figure 8 However, DoE modeling showed that adding IFN-γ to SR isolates increased the total number of isolated γδT cells.

[0279] Compared to plasma or AB serum as a protein source, the use of SR also appears to result in a decrease in the overall viability of isolated cells. Figure 9 ).

[0280] Other lymphocytes

[0281] The 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 increased number of TCR-negative cells compared to plasma or AB serum. Figure 10 The absence of IFN-γ also increases the number of TCR-negative cells.

[0282] in conclusion

[0283] Data show that the total number of isolated γδ cells can be greatly increased, which provides the possibility of increasing the initial quantity of the expansion setup. All conditions with optimal γδ T cell percentage and total γδ T cells in each grid contained IL-1β (conditions 6, 8, 9, 12, and 14). Results of the optimal conditions compared to the standard 2-cytokine isolation method are shown in... Figure 11 middle.

[0284] Therefore, the DoE method, which titrates human cytokines and combinations of human AB serum, human combined plasma, or serum substitutes, was used to identify a skin lymphocyte separation mixture known as Generation 2, employing human combined plasma (previous isolates used serum substitutes) and cytokines IL-2, IL-4, IL-15, and IL-1β (previous isolates used only IL-2 and IL-15). Compared to the initial method (now also referred to as Generation 1), this new growth mixture increased the total number of isolated live lymphocytes from 50-100 x 10^10 per separation unit matrix (grid). 6 The number of cells (previously) increased to 75-200 x 10⁻⁶. 6 100 lymphocytes. In addition, the second generation showed up to 3 times higher γδT cell presence at the end of isolation and generally exhibited a favorable phenotype (e.g., lower CD45RA and NKG2A expression).

[0285] Example 3. Study of frozen isolates

[0286] Previous conditions yielded a large number of γδT cells, but the viability after harvesting and thawing was low. A new method for isolation and expansion was investigated, in which the isolated cells were frozen prior to expansion. After isolation, the cells were frozen according to the method detailed in Example 1. The effect on the fold increase and percentage of γδT cells in subsequent expansion steps is shown in... Figure 12 In this study, isolated cells frozen prior to amplification were found to be at least as enriched and amplified as fresh equivalents.

[0287] Example 4. The isolation formulation containing IL-21 can increase γδT cell production.

[0288] The use of IL-21 was investigated to examine whether this cytokine could increase total γδT cell yield after isolation and subsequent expansion. Increased total γδT cell content would allow for larger-scale genetic engineering of these cells in subsequent expansions and potentially increase total effector cell yield.

[0289] Isolation cultures were established in GREX100M units, with AIMV medium supplemented with 2.5% allogeneic plasma (see Example 1 for details). Cytokines were then added to the cultures as detailed in Table 3.

[0290] Table 3. Conditions for Cytokine Isolation and Culture

[0291]

[0292] Cells isolated and cultured using 18.8 ng / ml IL-21 were harvested on day 21 and cryopreserved (frozen) for subsequent expansion. Cell viability was then measured using an NC250 automated cell counting system, and the results are shown below. Figure 13 In A, the overall cell viability at thawing was higher than the minimum acceptable viability in both the presence and absence of IL-21, with an average viability % >80% in the IL-21-free culture and an average viability % close to 90% in the IL-21-free culture. Then, TexMACS were cultured in the presence of 5% allogeneic plasma supplemented with IL-15 (80 ng / mL). TM Cells isolated were amplified using different levels of IL-21 supplements (12.53, 18.8, 37.5, and 188 ng / mL) on Miltenyi Biotec culture medium. After 14 days of amplification, cells were harvested and γδT cell enrichment was measured by flow cytometry. Figure 13 As shown in B, after expansion, cells isolated in the presence of IL-21 showed enrichment of γδT cells during 14 days of culture, independent of the amount of IL-21 used for expansion (showing performance comparable to 12.5 to 188.8 ng / mL). Therefore, the presence of IL-21 in the isolated culture generates viable cells and maintains the ability of γδT cells to subsequently expand. The function of these post-expansion cells in cytotoxicity assays was also confirmed.

[0293] Example 5. Comparison of isolation cultures with and without IL-21 versus those with 18.8 ng / ml IL-21

[0294] Donor-matched skin samples were established in isolation cultures using either the “IL-21-free” formulation or the “IL-21” (18.8 ng / ml) cytokine formulation detailed in Table 3. All isolation cultures were prepared in AIMV medium supplemented with 2.5% allogeneic plasma (see Example 1). “IL-21-free” isolation cultures were harvested on day 19 or day 21, while “IL-21” isolation cultures were harvested only on day 19. At harvest, the total number of viable cells, the total number of isolated γδT cells, and the total number of Vδ1T cells in each isolation culture grid were recorded, and the results are shown in Table 3. Figure 14 middle.

[0295] This disclosure relates to the following implementation plan.

[0296] 1. A method for isolating lymphocytes from a non-hematopoietic tissue sample, the method comprising the following steps:

[0297] (i) The non-hematopoietic tissue samples were cultured in the presence of interleukin-1β (IL-1β); and

[0298] (ii) Collect cultured lymphocyte populations from the non-hematopoietic tissue sample.

[0299] 2. A method for isolating γδT cells from a non-hematopoietic tissue sample, the method comprising the following steps:

[0300] (i) Culture the non-hematopoietic tissue samples in the presence of IL-1β; and

[0301] (ii) Collect cultured γδT cell populations from the non-hematopoietic tissue samples.

[0302] 3. The method according to any of the preceding claims, wherein step (i) further comprises culturing the non-hematopoietic tissue sample in the presence of interleukin 2 (IL-2) and interleukin 15 (IL-15).

[0303] 4. The method according to any of the preceding claims, wherein step (i) further comprises culturing the non-hematopoietic tissue sample in the presence of interleukin-4 (IL-4) and / or interferon-γ (IFN-γ).

[0304] 5. The method according to any of the preceding claims, wherein the culture is performed in the absence of interleukin-21 (IL-21).

[0305] 6. The method according to any one of items 1 to 4, wherein the culture is carried out in the presence of interleukin-21 (IL-21) at a concentration between 15 ng / mL and 25 ng / mL, such as 18 to 20 ng / mL.

[0306] 7. The method according to any of the preceding claims, wherein the non-hematopoietic tissue sample is cultured in a culture medium containing serum or plasma.

[0307] 8. The method according to item 7, wherein the non-hematopoietic tissue is cultured in a culture medium containing 2.5% human plasma.

[0308] 9. The method according to item 7, wherein the non-hematopoietic tissue is cultured in a culture medium containing 10% human AB serum.

[0309] 10. The method according to any of the preceding claims, wherein the lymphocytes or γδT cells are collected after culturing for at least 7 days.

[0310] 11. The method according to any of the preceding claims, wherein the lymphocytes or γδT cells are collected after culturing for at least 14 days.

[0311] 12. The method according to any of the preceding claims, wherein the lymphocytes or γδT cells are collected 35 days before culture.

[0312] 13. The method according to any of the preceding claims, wherein the lymphocytes or γδT cells are collected after 19 days of culture.

[0313] 14. The method according to any of the preceding claims, wherein the lymphocytes or γδT cells are collected after 21 days of culture.

[0314] 15. The method according to any of the preceding claims, wherein the non-hematopoietic tissue sample is an intact biopsy.

[0315] 16. The method according to any of the preceding claims, wherein the non-hematopoietic tissue sample is skin.

[0316] 17. The method of claim 16, wherein the skin sample comprises the epidermis and the dermis.

[0317] 18. The method according to any of the preceding claims, wherein the non-hematopoietic tissue sample is the intestine or gastrointestinal tract.

[0318] 19. The method according to any of the preceding claims, wherein the non-hematopoietic tissue sample has been obtained from a human body.

[0319] 20. The method according to any of the preceding claims, wherein the method is carried out in a container comprising a breathable material.

[0320] 21. The method according to any of the preceding claims, wherein the isolated lymphocyte population or γδT cell population includes a Vδ1T cell population.

[0321] 22. The method according to item 21, wherein less than 80% of the isolated Vδ1T cell population expresses CD45RA, such as less than 30% of the isolated Vδ1T cell population expresses CD45RA.

[0322] 23. The method according to item 21 or 22, wherein less than 10% of the isolated Vδ1T cell population expresses NKG2A.

[0323] 24. The method according to any of the preceding claims further includes amplifying the isolated lymphocyte population or γδT cell population.

[0324] 25. A method for isolating lymphocytes from a non-hematopoietic tissue sample, the method comprising the steps of:

[0325] (i) The non-hematopoietic tissue sample is cultured in a medium containing serum substitutes, human plasma, and / or human AB serum; and

[0326] (ii) Collect cultured lymphocyte populations from the non-hematopoietic tissue sample.

[0327] 26. The method of claim 25, comprising culturing the non-hematopoietic tissue sample in a culture medium containing a serum substitute.

[0328] 27. The method according to item 25 or 26, wherein the isolated lymphocyte population includes a TCR-negative cell population.

[0329] 28. The method according to any one of items 25 to 27, wherein step (i) further comprises culturing the non-hematopoietic tissue sample in the presence of IL-2 and IL-15.

[0330] 29. The method according to any one of items 25 to 28, wherein step (i) further comprises culturing the non-hematopoietic tissue sample in the presence of IL-4 and / or IL-1β.

[0331] 30. The method according to any one of items 25 to 29, wherein the culture is carried out in the absence of IFN-γ.

[0332] 31. The method according to any one of items 25 to 30, wherein step (i) further comprises culturing the non-hematopoietic tissue sample in the presence of IL-21 at a concentration between 15 ng / mL and 25 ng / mL.

[0333] 32. The method according to any one of items 25 to 31, wherein the lymphocytes are collected after 19 days of culture.

[0334] 33. The method according to any one of items 25 to 31, wherein the lymphocytes are collected after 21 days of culture.

[0335] 34. A method for isolating and expanding lymphocytes from a non-hematopoietic tissue sample, the method comprising the following steps:

[0336] (i) Isolating a lymphocyte population from the non-hematopoietic tissue sample according to the method described in any of the preceding claims; and

[0337] (ii) Further culture the lymphocyte population for at least 5 days to generate an expanded lymphocyte population.

[0338] 35. A method for isolating and expanding γδT cells from a non-hematopoietic tissue sample, the method comprising the following steps:

[0339] (i) Isolating a population of γδT cells from the non-hematopoietic tissue sample according to the method described in any of the preceding claims; and

[0340] (ii) Further culture the γδT cell population for at least 5 days to generate an expanded γδT cell population.

[0341] 36. The method according to item 34 or item 35, wherein the isolated lymphocyte or γδT cell population is frozen and then thawed prior to step (ii).

[0342] 37. The method according to item 35 or 36, wherein the amplification step comprises culturing the γδT cells in the presence of:

[0343] (a) IL-2 or IL-9;

[0344] (b) IL-15; and optional

[0345] (c)IL-21,

[0346] Culture for at least 5 days to generate an effective population of expanded γδT cells.

[0347] 38. The method according to item 37, further comprising culturing the γδT cells in the presence of IL-4.

[0348] 39. The method according to any one of claims 35 to 38, wherein the amplification step comprises culturing the γδT cells in the absence of a large number of stromal cell contacts.

[0349] 40. The method according to any one of claims 35 to 39, wherein the amplification step includes the absence of an exogenous TCR pathway agonist.

[0350] 41. A method for isolating γδT cells from a non-hematopoietic tissue sample, the method comprising the following steps:

[0351] (i) Isolating a population of γδT cells from the non-hematopoietic tissue sample; and

[0352] (ii) Freeze the isolated γδT cell population.

[0353] 42. The method according to item 41, wherein γδT cells are isolated by culturing the non-hematopoietic tissue sample in the presence of IL-2 and IL-15, optionally in combination with IL-1β, IL-4 and / or IL-21.

[0354] 43. The method according to item 42, wherein γδT cells are isolated by culturing the non-hematopoietic tissue sample in the presence of IL-21 at a concentration between 15 ng / mL and 25 ng / mL, such as 18 to 20 ng / mL.

[0355] 44. The method according to items 41 to 43, wherein the γδT cells are frozen 19 days after separation.

[0356] 45. The method according to any one of items 41 to 43, wherein the γδT cells are frozen 21 days after separation.

[0357] 46. ​​The method according to any one of items 41 to 44, wherein the isolated γδT cell population is frozen in a freezing solution and stored in liquid nitrogen (gas phase).

[0358] 47. The method according to any one of items 41 to 45, further comprising: (iii) thawing the frozen γδT cell population; and (iv) culturing the thawed γδT cell population for at least 5 days to produce an expanded γδT cell population.

[0359] 48. An isolated population of γδT cells, which can be obtained by any one of items 1 to 24.

[0360] 49. An isolated population of γδT cells obtained by any one of items 1 to 24.

[0361] 50. A frozen, isolated population of γδT cells, which can be obtained by any one of items 41 to 47.

[0362] 51. A frozen, isolated population of γδT cells obtained by any one of items 41 to 47.

[0363] 52. An isolated and expanded population of γδT cells, which can be obtained by any one of items 35 to 40.

[0364] 52. An isolated and expanded population of γδT cells obtained by any one of items 35 to 40.

Claims

1. A method for isolating lymphocytes from a non-hematopoietic tissue sample, the method comprising the following steps: (i) The non-hematopoietic tissue samples were cultured in the presence of interleukin-1β (IL-1β); and (ii) Collect cultured lymphocyte populations from the non-hematopoietic tissue sample.

2. A method for isolating γδT cells from a non-hematopoietic tissue sample, the method comprising the following steps: (i) The non-hematopoietic tissue samples were cultured in the presence of IL-1β; and (ii) Collect cultured γδT cell populations from the non-hematopoietic tissue samples.

3. The method according to any of the preceding claims, wherein step (i) further comprises culturing the non-hematopoietic tissue sample in the presence of interleukin 2 (IL-2) and interleukin 15 (IL-15).

4. The method according to any of the preceding claims, wherein step (i) further comprises culturing the non-hematopoietic tissue sample in the presence of interleukin-4 (IL-4) and / or interferon-γ (IFN-γ).

5. The method according to any of the preceding claims, wherein the culture is performed in the absence of interleukin-21 (IL-21).

6. The method according to any one of claims 1 to 4, wherein the culture is carried out in the presence of interleukin-21 (IL-21) at a concentration between 15 ng / mL and 25 ng / mL, such as 18 to 20 ng / mL.

7. The method according to any of the preceding claims, wherein the non-hematopoietic tissue sample is cultured in a culture medium containing serum or plasma.

8. The method of claim 7, wherein the non-hematopoietic tissue is cultured in a culture medium containing 2.5% human plasma.

9. The method of claim 7, wherein the non-hematopoietic tissue is cultured in a culture medium containing 10% human AB serum.

10. The method according to any of the preceding claims, wherein the lymphocytes or γδT cells are collected after culturing for at least 7 days.

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