Methods for isolating and expanding cells

Abstract

To provide a method of isolating tissue-resident non-haematopoietic lymphocytes, such as from skin, that yields a greater amount of cells that are suitable for clinical use.SOLUTION: The invention relates to a method for the isolation of lymphocytes (in particular γδ T cells) from a non-haematopoietic tissue sample, comprising the steps of: culturing the non-haematopoietic tissue sample in the presence of (a) Interleukin-2 (IL-2) or Interleukin-9 (IL-9); (b) Interleukin-15 (IL-15); and (c) Interleukin-21 (IL-21); and collecting a population of lymphocytes cultured from the non-haematopoietic tissue sample.SELECTED DRAWING: Figure 1A

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to a method for the isolation and / or expansion of non-hematopoietic tissue resident lymphocytes, in particular γδ T cells. Such γδ T cells include non-Vδ2 cells, such as Vδ1, Vδ3, and Vδ5 cells. Such non-hematopoietic tissues include the skin and gastrointestinal tract. Isolated and / or expanded non-hematopoietic tissue resident lymphocytes can be used in a variety of therapeutic applications, including adoptive T cell therapy, chimeric receptor therapy, and other therapeutic approaches. It will be appreciated that the present invention is very useful in the fields of The present invention relates to both individual cells and populations of cells produced by the methods described herein. [Background technology]

[0002] BACKGROUND OF THEINVENTION The growing interest in T cell immunotherapy of cancer is driven by T cell receptors that recognize cancer cells and specifically target PD-1, CTLA-4, and Disinhibition is mediated by clinically mediated antagonism of inhibitory pathways mediated by acetylcholine and other receptors. Subpopulations of CD8+ and CD4+ αβ T cells that, when suppressed, mediate host defensive functional potential However, αβ T cells are MHC restricted and , which can lead to graft-versus-host disease.

[0003] Gamma delta T cells (γδ T cells) express a unique definitive γδ T cell receptor ( This represents a subset of T cells that express the TCR, which consists of one gamma (γ) chain and one deoxyribonucleic acid (DE) chain. The human γδ TCR chain is composed of three main Vδ1, Vδ2, and Vδ3 chains. There are six γ chains and δ chains, although not exclusively, specific γ and δ types are used. Human γδ T cells are often found in cells of more than one tissue type, and therefore have a TCR chain specific For example, most blood-resident γδ T cells are Vδ2 They express TCRs such as Vγ9Vδ2, which are less common among tissue-resident γδ T cells. Instead, these tissue-resident γδ T cells often use Vδ1 in the skin and Vγ4 in the gastrointestinal tract. do.

[0004] Most methods for isolating lymphocytes rely on isolating these cell types from blood. Non-hematopoietic tissue-resident lymphocytes, such as αβ T cells, γδ T cells, and NK cells, have specific functions. In particular, they may have properties that make them particularly suitable for targeting, for example, non-hematopoietic tumors and other targets. However, isolation of such tissue-resident lymphocytes in clinically relevant quantities remains a challenge. In particular, 10 8 It remains to be seen whether clinical doses in the range of 100–200 cells / mL or more are required for many indications. Importantly, significant cell loss during production has led to the need to produce even more starting cells. This means that cells must be generated.

[0005] Non-hematopoietic tissue-resident lymphocytes, particularly αβ T cells, γδ T cells, and NK cells, are readily recruited in large numbers. Since these cannot be fully characterized or used for therapeutic applications, Therefore, non-hematopoietic tissue-resident lymphocytes, especially γδ T cells, Sufficient for study and potential application as a therapy, e.g., as adoptive T cell therapy. There is a need in the art for methods to isolate and scale up to large quantities.

[0006] Clark et al. (2006) J. Invest. Dermatol. 126(5): 1059-70, in which normal and diseased skin A method for isolating skin-resident T cells from the skin has been described. The methods described so far have been limited in their ability to isolate cells, particularly due to the presence of animal products, and the relatively low yield of isolated cells. Relatively low, i.e., 1 cm of tissue 2 10 cells per 6 Because the number of The method described by Clark et al. uses comminuted samples, which This results in the intentional destruction of the structural integrity of the tissue sample. No. 2018 / 202808 provides that lymphocytes obtained from non-hematopoietic tissues are treated with at least interleukin-2 (IL-2) -2) and / or interleukin-15 (IL-15) in vitro. This document relates to a method for expanding non-hematopoietic tissue resident γδ T cells. WO2015189356 describes a method for expanding non-hematopoietic tissue resident γδ T cells by inducing the expression of IL-2, IL-15, and / or IL-2-associated IL-15. and IL-21. A composition for expanding lymphocytes obtained from a sample obtained by Thus, a method for isolating tissue-resident non-hematopoietic lymphocytes, for example from skin, that is suitable for clinical use is provided. There remains a need for methods that yield larger quantities of cells that are suitable for use. Summary of the Invention

[0007] (Summary of the invention) According to a first aspect of the present invention there is provided a method for isolating lymphocytes from a non-hematopoietic tissue sample comprising the steps of: (i) removing said non-hematopoietic tissue sample; (a) interleukin-2 (IL-2) or interleukin-9 (IL-9); (b) interleukin-15 (IL-15); and (c) Interleukin-21 (IL-21) Cultivating in the presence of: (ii) recovering a population of cultured lymphocytes from the non-hematopoietic tissue sample. A method is provided that includes:

[0008] According to a further aspect of the invention, there is provided a method for isolating γδ T cells from a non-hematopoietic tissue sample, comprising: , (i) removing said non-hematopoietic tissue sample; (a) IL-2 or IL-9; (b) IL-15; and (c)IL-21 Cultivating in the presence of: (ii) recovering the cultured population of γδ T cells from the non-hematopoietic tissue sample. A method is provided that includes:

[0009] According to a further aspect of the present invention, a method for isolating and expanding lymphocytes from a non-hematopoietic tissue sample is provided. There was, (i) isolating a population of lymphocytes from said non-hematopoietic tissue sample according to the methods defined herein. and (ii) further culturing the population of lymphocytes for at least 5 days to obtain an expanded population of lymphocytes; The process of producing A method is provided that includes:

[0010] According to a further aspect of the invention, a method for isolating and expanding γδ T cells from a non-hematopoietic tissue sample is provided. And, (i) isolating a population of γδ T cells from said non-hematopoietic tissue sample according to the methods defined herein. and (ii) further culturing the population of γδ T cells for at least 5 days to obtain an expanded population of γδ T cells; The process of producing the group A method is provided that includes: [Brief description of the drawings]

[0011] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] Figure 1: A: Total cell yield and percentage of γδ T cells and Vδ1 cells were determined for a two cytokine (IL-2 and IL-15) and a four cytokine (IL-2, IL-4, IL-15, and IL-21) isolation method. B: Percentage of γδ T cells and Vδ1 cells obtained using the four cytokine method compared to the two cytokine method. [Diagram 2] Figure 2: A: Total cell yields were determined for isolation methods using two cytokines "2CK" (IL-2 and IL-15), three cytokines "3CK" (IL-2, IL-15, and IL-21), and four cytokines "4CK" (IL-2, IL-4, IL-15, and IL-21) in AIM-V medium + 5% serum replacement in G-REX6. B: Percentage of γδ T cells, and C: Percentage of Vδ1 cells among γδ T cells are also shown. [Diagram 3] Figure 3: The phenotype of Vδ1 cells isolated using the 2CK and 4CK methods in AIM-V containing 5% human AB serum in 24-well plates was analyzed by measuring the percentage of TIGIT and CD27 expression. [Figure 4] Figure 4: The phenotype of V51 cells isolated using the 2CK, 3CK, and 4CK methods in (AIM-V medium + 5% serum replacement in G-REX6 was analyzed by measuring A: percentage of CD27 expression, and B: percentage of TIGIT expression. [Diagram 5] Figure 5: Initial study comparing total cell yield from 3 mm punch biopsies and standard skin comminution methods. [Figure 6] Figure 6: γδ cell yield from isolated punch biopsies of various sizes in AIM-V with 5% human AB serum in 24-well plates compared to comminuted controls sampled with a scalpel. [Figure 7]Figure 7: Total cell yield per biopsy (top graph) and total cell yield per plate (bottom graph) using various culture vessels containing AIM-V with 5% human AB serum. [Figure 8] FIG. 8: CD27 expression levels in cells isolated using the two-cytokine, three-cytokine, and four-cytokine isolation protocols. [Figure 9] Figure 9: The phenotype of Vδ1 cells isolated using the 2CK and 4CK methods in G-REX6 in medium containing 10% human AB serum (results on the left side of the graph) or 5% serum replacement (results on the right side of the graph) was analyzed by measuring A: percentage of CD27 expression and B: percentage of TIGIT expression. C: PD-1 expression was also measured in αβ T cells (CD3+, pan-γδ- cells) isolated using the 2CK and 4CK methods in medium containing 10% human AB serum. [Figure 10] FIG. 10: Graph showing a comparison of the various media used in the isolation method described in Example 6. [Figure 11] FIG. 11: Comparison of total cell yields after 2 weeks (top graph) or 3 weeks (bottom graph) of isolation in AIM-V with the indicated serum supplements in G-REX6. [Figure 12] Figure 12: Total cell yield and percentage of V51 cells isolated using AIM-V medium containing 5% or 10% of 5% serum replacement (SR) versus human AB serum (AB) in G-REX6. [Figure 13] FIG. 13: Distribution of cell types in populations isolated using A: 2CK or B: 4CK methods and subsequently expanded using the 4CK method. [Figure 14] Figure 14: Analysis of the expression of various markers of Vδ1 cells isolated using the 2CK or 4CK method and subsequently expanded using the 4CK method. [Figure 15] Figure 15: Total numbers of γδ and Vδ1 cells isolated using the 2CK or 4CK method and subsequently expanded using the 4CK method. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Detailed Description of the Invention According to a first aspect of the present invention there is provided a method for isolating lymphocytes from a non-hematopoietic tissue sample comprising the steps of: (i) removing said non-hematopoietic tissue sample; (a) interleukin-2 (IL-2) or interleukin-9 (IL-9); (b) interleukin-15 (IL-15); and (c) Interleukin-21 (IL-21) Cultivating in the presence of: (ii) recovering a population of cultured lymphocytes from the non-hematopoietic tissue sample. A method is provided that includes:

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

[0014] "Isolation" or "isolation" of cells, particularly lymphocytes and / or γδ T cells References herein to "cell culture" refers to cells that have been removed from a tissue or pool of cells or A method or process by which substances are separated, purified, concentrated, or otherwise extracted. Such references include "separated," "removed," "purified," "enriched," It will be understood that the term "reduced" includes the term "reduced" and similar terms. Isolation may be performed from intact non-hematopoietic tissue samples or from stromal cells (e.g., fibroblasts or Such isolation may alternatively or alternatively include the isolation or separation of cells from a cultured tissue (e.g., a cultured tissue or an epithelial cell). In addition, the elimination of γδ T cells from other hematopoietic cells (e.g., αβ T cells or other lymphocytes) Isolation may include the isolation or separation of a subject for a defined period of time, e.g., a tissue explant or biopsy is isolated. Starting from the time the cells are placed in isolation culture, they are then isolated, for example by centrifugation or isolation. The cell population may be harvested from the culture by other means for transfer to expansion culture or for other purposes. The culture is then either used periodically or terminated when the original tissue explant or biopsy is removed from the culture. The isolation step may be carried out for at least about 3 days to about 45 days. In an embodiment, the isolation process is for at least about 10 days to at least 28 days. In an embodiment, the isolation process lasts for at least 14 days to at least 21 days. The isolation process should last at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days. 4th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th The period may be 28, 29, 30, 31, 32, about 35, about 40, or about 45 days. During the isolation process, there may not be significant proliferation of the isolated cells, but the cells It can be seen that proliferation is not necessarily absent. For some skilled in the art, isolated cells may begin to divide and form a plurality of such cells. and / or may be produced in an isolated vessel containing the scaffold. can be.

[0015] Thus, the terms "isolated γδ T cells," "isolated γδ T cell population," and "isolated "population of isolated γδ T cells," "isolated γδ T cells," "isolated γδ T cell population," or References herein to an "isolated population of γδ T cells" include those referring to non-hematopoietic or The non-hematopoietic tissue of origin sample is then subjected to a cellular endothelial cell transplantation procedure so as to avoid substantial contact with cells contained within the intact non-hematopoietic tissue. γδ isolated, separated, removed, purified, or enriched from a sample It will be understood that the term "hematopoietic cell or population of hematopoietic cells" refers to a hematopoietic cell or population of hematopoietic cells that includes a single cell. Reference herein to an "isolated or separated population of V51 T cells" means that the cells are non- The non-hematopoietic tissue of origin is isolated from the blood cells or cells contained within the intact non-hematopoietic tissue so as to be substantially free of contact with the blood cells or cells contained within the intact non-hematopoietic tissue. Isolated, separated, removed, purified, or enriched from a blood or tissue sample Isolation or separation therefore refers to hematopoietic cells, including V51 T cells, isolated from non-hematopoietic cells (e.g. , stromal cells, fibroblasts, and / or epithelial cells) from hematopoietic cells (e.g., γδ T cells or The term refers to the isolation, separation, removal, purification, or enrichment of lymphocytes (other lymphocytes).

[0016] The method for isolating γδ T cells as defined herein comprises disruption of tissue (e.g., mincing) followed by Preferably, the method comprises isolating γδ T cells from other cell types, as defined herein. The methods for isolating γδ T cells described herein involve the use of intact non-hematopoietic tissue samples or tissue samples from explants or biopsies. This may include the "egress" of γδ T cells and other cell types from the Trkx, where Tissue-resident lymphocytes can secrete cytokines from tissue matrices without the need for tissue matrix destruction. By maintaining the integrity of the tissue matrix, the tissue Resident lymphocytes can later be easily removed at the end of the isolation, during explants or biopsies. The tissue matrix is ​​retained, with little or no escape of inhibitory cell types such as fibroblasts. It has been found that the β-amyloid ... In contrast, the use of intact non-hematopoietic tissue samples or tissue matrices allows for the formation of small numbers of fibroblasts. Such cells are released from intact non-hematopoietic tissue or tissue matrix into culture. Such a "crawling" method reduces the need for excessive processing of non-hematopoietic tissue samples or tissue matrices. maintains the structural integrity of the non-hematopoietic tissue or tissue matrix; and may provide the unexpected advantage of resulting in higher isolated cell yields.

[0017] Thus, the method for isolating non-hematopoietic tissue-derived lymphocytes defined herein comprises the steps of: The present invention also includes a method for isolating non-hematopoietic tissue-derived lymphocytes from intact biopsies or explants of such tissues. An intact biopsy or explant is one in which the structural integrity of the biopsy or explant is sufficient to render the biopsy or explant a tissue sample. Such intact living tissue is not intentionally destroyed within the perimeter of the incision to be removed from the The specimen or explant has a three-dimensional structure that is largely maintained, except for minor disruptions caused by manipulation. Therefore, the intact biopsy or explant can be dissected, for example, by grinding or mincing. have not been mechanically or chemically or enzymatically disrupted. However, disrupted tissue may also be used in the isolation methods of the present invention. In an alternative embodiment, the isolated lymphocytes are αβ T cells. In another embodiment, the isolated lymphocytes are γδ T cells. It is understood that multiple types of lymphocytes can be isolated from the same isolation step. It is possible.

[0018] Methods for isolating γδ T cells utilizing "crawling" or e.g., the methods defined herein induces the isolation or separation of γδ T cells and / or other lymphocytes as defined herein a cell and / or non-hematopoietic tissue sample in the presence of cytokines and / or chemokines sufficient to Thus, in one embodiment of the present invention, a non-hematopoietic tissue sample can be cultured. Isolation of γδ T cells from non-hematopoietic tissue samples was achieved by culturing them in the presence of IL-2, IL-15, and IL-21. In an alternative embodiment, the isolation of γδ T cells from a non-hematopoietic tissue sample comprises: The method includes culturing a non-hematopoietic tissue sample in the presence of IL-9, IL-15, and IL-21.

[0019] In one embodiment, the isolation of γδ T cells according to the first aspect of the invention comprises the step of isolating γδ T cells by interleukin-1 (IL-1) or γδ T cells by immunohistochemistry. The method further comprises culturing the non-hematopoietic tissue sample in the presence of interleukin-4 (IL-4). In certain embodiments, the non-hematopoietic tissue sample is cultured in the presence of IL-2, IL-15, IL-21, and IL-4. In an alternative further embodiment, the non-hematopoietic tissue sample is and cultured in the presence of IL-4.

[0020] As used herein, "IL-2" refers to an agonist of one or more IL-2 receptor (IL-2R) subunits. Agonists (e.g., mutants, muteins, analogs, subunits, receptor complexes, Natural or recombinant IL-1 acting as a peptide mimetic (fragments, isoforms, and peptidomimetics) Such agents refer to IL-2-dependent cell line CTLL-2 (33; American Type Culture Collection (ATC C®) TIB 214) can support the proliferation of mature human IL-2. Cell 1986.46.3:401-407, a 133 amino acid sequence (an additional 20 The IL-2 mutein is generated as follows: has specific substitutions for the interleukin-2 protein, whereas IL-2Rβ Polypeptides that retain the ability to bind to, for example, those described in US 2014 / 0046026. An IL-2 mutein is a polypeptide in which the amino acid sequence of the native IL-2 polypeptide chain is replaced by another amino acid sequence of the native IL-2 polypeptide chain. The polypeptides can be characterized by amino acid insertions, deletions, substitutions, and modifications at one or more sites. According to the present disclosure, any such insertions, deletions, substitutions, and modifications can be used to inhibit IL-2Rβ binding. IL-2 muteins that retain binding activity have been produced. Exemplary muteins include 1, 2, 3, 4, 5, 6, 7, 8 , 9, 10 or more amino acid substitutions.

[0021] Nucleic acid encoding human IL-2 can be isolated by conventional procedures such as polymerase chain reaction (PCR). The amino acid sequence of human IL-2 (Gene ID 3558) can be obtained from Genbank under the following heading: NP_00 It is found at the accession locator 0577.2 GI: 28178861. The amino acid sequence of Mus musculus IL-2 (Gene ID 16183) is located in Genbank under the accession number NP_032392. 1 GI: 7110653.

[0022] IL-2 has been reported in a variety of mammalian species, including, for example, humans, monkeys, cows, pigs, horses, and mice. Variants may also refer to IL-2 derived from a species. Variants may include sequences with conservative substitutions. It is possible to obtain a sequence in which a given amino acid residue is replaced by a residue having similar physicochemical characteristics. An example of a conservative substitution is the replacement of one aliphatic residue with another, e.g., Substitutions of Ile, Val, Leu, or Ala for one another, or substitutions of one polar residue for another, e.g. , between Lys and Arg; between Glu and Asp; or between Gln and Asn. Other such conservative substitutions, for example substitutions of entire regions having similar hydrophobicity characteristics, are well known. Naturally occurring IL-2 variants are also encompassed by the present invention. Examples of such variants are: by mRNA splicing events or by proteolytic cleavage of the IL-2 protein. The resulting protein retains the IL-2 binding properties. Protein synthesis can result in a truncated, but biologically active, IL-2 protein. Mutations resulting from proteolysis include, for example, the deletion of one or more terminal amino acids from the IL-2 protein (usually Expression in various types of host cells by proteolytic removal of a sequence of 1–10 amino acids This includes differences in the current N- or C-terminus. In some embodiments, the termini of the protein or modified internally with chemical groups, e.g., polyethylene glycol, to alter its physical properties. (Yang et al., Cancer 1995. 76: 687-694). In embodiments, the protein can be modified at its termini or internally with additional amino acids (Cla rk-Lewis et al., PNAS 1993. 90:3574-3577).

[0023] As used herein, "IL-15" refers to one or more IL-15 receptor (IL-15R) subunits. Agonists of (e.g., mutants, muteins, analogs, subunits, receptor complexes thereof) Natural or recombinant peptides acting as peptide mimetics (synthetic peptides, fragments, isoforms, and peptidomimetics) IL-15 or its variants. IL-15, like IL-2, is a cytotoxic inhibitor of CTLL-2, an IL-2-dependent cell line. IL-15 is a known T cell growth factor that can support proliferation. As the mature protein, Grabstein et al. (Grabstein et al., Science 1994. 264.516 1: 965-969). As used herein, the term "IL-15" refers to a Natural or recombinant IL-15 and its muteins, analogs, subunits, or complexes thereof ( For example, the receptor complex, e.g., the sushi peptide described in WO 2007 / 046006, is intended to be Each of these is capable of stimulating the proliferation of CTLL-2 cells. Transfected with recombinantly expressed in-frame fusions of precursor and mature IL-15. The supernatant of the injected cells is capable of inducing CTLL-2 cell proliferation.

[0024] Human IL-15 was synthesized by the method described in Grabstein et al. (Grabstein et al., Science 1994. 264.5161: 965- 969) or by conventional methods such as polymerase chain reaction (PCR). The human IL-15 cDNA was deposited with the ATCC on February 19, 1993 (Accession No. 123366). Trademark) and assigned accession number 69245.

[0025] The amino acid sequence of human IL-15 (Gene ID 3600) is located in Genbank under NP000576.1 GI: 10835 The accessions 153 (isoform 1) and NP_751915.1 GI: 26787986 (isoform 2) were Mouse (Mus musculus) IL-15 amino acid sequence The column (Gene ID 16168) corresponds to the Genbank accession NP_001241676.1 GI: 363000984. can be found at the tion locator.

[0026] IL-15 is expressed in a variety of mammalian species, including, for example, humans, monkeys, cows, pigs, horses, and mice. The term "IL-15" may also refer to IL-15 derived from a species. "Mutants" are those that are substantially homologous to the sequence of a native mammalian IL-15, but which have amino acid deletions, The polypeptides have an amino acid sequence that differs from a native mammalian IL-15 polypeptide by virtue of an insertion or substitution. A variant can include conservatively substituted sequences and is a polypeptide having the same structure as the given This means that the amino acid residues in the Examples of conservative substitutions include the replacement of one aliphatic residue with another, e.g., Ile, Val, Substitution of Leu or Ala for each other, or substitution of one polar residue for another, e.g., Lys for Arg or between Gln and Asn. Conservative substitutions such as substitutions of entire regions with similar hydrophobicity characteristics are well known. Natural IL-15 variants are also encompassed by the present invention. Examples of such variants include those that have alternate mRNA sequences. Proteins that arise from splicing events or from proteolytic cleavage of the IL-15 protein The protein is a cytoplasmic protein in which the IL-15 binding properties are retained. Alternative splicing of the mRNA results in This can result in a truncated, but biologically active, IL-15 protein. Mutations resulting from, for example, one or more terminal amino acids (usually 1-10) from the IL-15 protein Upon expression in various types of host cells, by proteolytic removal of 1 amino acid In some embodiments, the termini of the protein may be changed to For example, modifying it with chemical groups such as polyethylene glycol to change its physical properties (Yang et al., Cancer 1995. 76:687-694). In some embodiments, Alternatively, the protein can be modified at its terminus or internally with additional amino acids (Clark-Lewis et al., Literature, PNAS 1993. 90:3574-3577).

[0027] As used herein, "IL-4" refers to an agonist of one or more IL-4 receptor (IL-4R) subunits. Agonists (e.g., mutants, muteins, analogs, subunits, receptor complexes, Natural or recombinant IL-1 acting as a peptide mimetic (fragments, isoforms, and peptidomimetics) 4 or a variant thereof. Such agents are capable of converting naive helper T cells (Th0 cells) to T Mature human IL-4 is capable of supporting differentiation into human h2 cells. The IL-4 mutant is generated as a nucleotide sequence (minus the signal peptide consisting of 24 N-terminal amino acids). The interleukin-4 protein has specific substitutions, whereas the IL-4R Polypeptides that retain the ability to bind to α, e.g., those described in U.S. Pat. No. 6,313,272 IL-4 muteins are polypeptides that have been shown to have the same structure as other residues of the native IL-4 polypeptide chain. and characterized by amino acid insertions, deletions, substitutions, and modifications at one or more sites in According to the present disclosure, any such insertions, deletions, substitutions, and modifications can be used to enhance the expression of IL-2R IL-4 muteins that retain α-binding activity are generated. Exemplary muteins include 1, 2, 3, 4, 5, 6, It may contain 7, 8, 9, 10 or more amino acid substitutions.

[0028] Nucleic acid encoding human IL-4 can be isolated by conventional procedures such as polymerase chain reaction (PCR). The amino acid sequence of human IL-4 (Gene ID 3565) can be obtained in Genbank under the following heading: NG_02 It is found under the accession locator 3252. Murine (Mus musculus )) The IL-4 amino acid sequence (Gene ID 16189) is located in Genbank under the accession number NC_000077.6. can be found at the tion locator.

[0029] IL-4 has been reported in a variety of mammalian species, including, for example, humans, monkeys, cows, pigs, horses, and mice. Variants may also refer to IL-4 derived from a species. Variants may include sequences with conservative substitutions. It is possible to obtain a sequence in which a given amino acid residue is replaced by a residue having similar physicochemical characteristics. An example of a conservative substitution is the replacement of one aliphatic residue with another, e.g., Substitutions of Ile, Val, Leu, or Ala for one another, or substitutions of one polar residue for another, e.g. , between Lys and Arg; between Glu and Asp; or between Gln and Asn. Other such conservative substitutions, for example substitutions of entire regions having similar hydrophobicity characteristics, are well known. Naturally occurring IL-4 variants are also encompassed by the present invention. Examples of such variants include: This may result from an mRNA splicing event or from proteolytic cleavage of the IL-4 protein. The protein is a cytoplasmic protein in which the IL-4 binding properties are retained. can produce a truncated, but biologically active, IL-4 protein. Mutations resulting from the deletion can include, for example, deletion of one or more terminal amino acids (usually 1-1) from the IL-4 protein. Upon expression in various types of host cells, In some embodiments, the termini of the protein may be changed to For example, modifying it with chemical groups such as polyethylene glycol to change its physical properties (Yang et al., Cancer 1995. 76:687-694). In some embodiments, Alternatively, the protein can be modified at its terminus or internally with additional amino acids (Clark-Lewis et al., Literature, PNAS 1993. 90:3574-3577).

[0030] As used herein, "IL-21" refers to one or more IL-21 receptor (IL-21R) subunits. Agonists of (e.g., mutants, muteins, analogs, subunits, receptor complexes thereof) Natural or recombinant peptides acting as peptide mimetics (synthetic peptides, fragments, isoforms, and peptidomimetics) Such agents are known to be involved in the regulation of natural killer (NK) and IL-21. Injurious (CD8 +) It can support the proliferation of T cells. Mature human IL-21 consists of 133 amino acids. The sequence (minus a signal peptide consisting of an additional 22 N-terminal amino acids) is IL-21 muteins are a type of gene that contains specific substitutions in the interleukin-21 protein. On the other hand, polypeptides that retain the ability to bind to IL-21Rα, such as those described in U.S. Pat. No. 9,388, IL-21 muteins are polypeptides that are similar to the native IL-21 polypeptides described in US Pat. No. 5,993,333. The peptides are characterized by amino acid insertions, deletions, substitutions, and modifications at one or more sites of other residues in the peptide chain. In accordance with the present disclosure, any such insertions, deletions, substitutions, and modifications may be This results in IL-21 muteins that retain IL-21R binding activity. Exemplary muteins include 1, 2 , 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions. .

[0031] Nucleic acid encoding human IL-21 can be isolated by conventional procedures such as polymerase chain reaction (PCR). The amino acid sequence of human IL-21 (Gene ID 59067) can be obtained in Genbank under the ID NC_ It is found under the accession locator 000004.12. The amino acid sequence of IL-21 (Gene ID 60505) is identified in Genbank as NC_000069.6. Found at the accession locator.

[0032] IL-21 is expressed in a variety of mammalian species, including, for example, humans, monkeys, cows, pigs, horses, and mice. Variants may also refer to IL-21 derived from a species. Variants may include sequences with conservative substitutions. and a given amino acid residue is replaced by a residue with similar physicochemical characteristics. An example of a conservative substitution is the replacement of one aliphatic residue with another, e.g. For example, substitutions of Ile, Val, Leu, or Ala for each other, or substitutions of one polar residue for another, e.g. For example, substitutions between Lys and Arg; between Glu and Asp; or between Gln and Asn. Other such conservative substitutions, such as substitutions of entire regions with similar hydrophobicity characteristics, may be used. Naturally occurring IL-21 variants are also encompassed by the present invention. Examples of such variants include , either by alternative mRNA splicing events or by proteolytic cleavage of the IL-21 protein The protein is derived from the IL-21-binding domain of the mRNA, where the IL-21-binding properties are retained. Lysing can result in a truncated, but biologically active, IL-21 protein. Mutations resulting from proteolysis include, for example, mutations in one or more terminal amino acids from the IL-21 protein. Various types of host cells by proteolytic removal of acids (usually 1-10 amino acids) In some embodiments, the difference in the N- or C-terminus of the protein upon expression in The ends of the protein are modified with chemical groups, such as polyethylene glycol, to improve their physical properties. (Yang et al., Cancer 1995. 76: 687-694). In embodiments, the protein can be modified at its terminus or internally with additional amino acids (C Lark-Lewis et al., PNAS 1993. 90:3574-3577).

[0033] As used herein, "IL-9" refers to an antigen binding domain of one or more IL-9 receptor (IL-9R) subunits. Agonists (e.g., mutants, muteins, analogs, subunits, receptor complexes, Natural or recombinant IL-1 acting as a peptide mimetic (fragments, isoforms, and peptidomimetics) Mature human IL-9 occurs as a 144 amino acid sequence. IL-9 mutein In the study, specific substitutions were made to the interleukin-9 protein, whereas IL-9 An IL-9 mutein is a polypeptide that retains the ability to bind to the native IL-9 polypeptide. by amino acid insertions, deletions, substitutions, and modifications at one or more sites of other residues in the peptide chain. According to the present disclosure, any such insertions, deletions, substitutions, and and modifications result in IL-9 muteins that retain IL-9R binding activity. Exemplary muteins include: , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions. do.

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

[0035] IL-9 has been reported in a variety of mammalian species, including, for example, humans, monkeys, cows, pigs, horses, and mice. Variants may also refer to IL-9 derived from a species. Variants may include sequences with conservative substitutions. It is possible to obtain a sequence in which a given amino acid residue is replaced by a residue having similar physicochemical characteristics. An example of a conservative substitution is the replacement of one aliphatic residue with another, e.g., Substitutions of Ile, Val, Leu, or Ala for one another, or substitutions of one polar residue for another, e.g. , between Lys and Arg; between Glu and Asp; or between Gln and Asn. Other such conservative substitutions, for example substitutions of entire regions having similar hydrophobicity characteristics, are well known. Naturally occurring IL-9 variants are also encompassed by the present invention. Examples of such variants include: This may result from an mRNA splicing event or from proteolytic cleavage of the IL-9 protein. The protein is a cytoplasmic protein in which the IL-9 binding properties are retained. can produce a truncated, but biologically active, IL-9 protein. Mutations resulting from the deletion can include, for example, deletion of one or more terminal amino acids (usually 1-1) from the IL-9 protein. Upon expression in various types of host cells, In some embodiments, the termini of the protein may be changed to For example, modifying it with chemical groups such as polyethylene glycol to change its physical properties (Yang et al., Cancer 1995. 76:687-694). In some embodiments, Alternatively, the protein can be modified at its terminus or internally with additional amino acids (Clark-Lewis et al., Literature, PNAS 1993. 90:3574-3577).

[0036] In certain embodiments, the methods defined herein typically provide a dose of at least 10 IU / m L, for example, at least 100 IU / mL (for example, 10 IU / mL to 1,000 IU / mL, 20 IU / mL to 800 IU / mL, 25 IU / mL~750IU / mL, 30IU / mL~700IU / mL, 40IU / mL~600IU / mL, 50IU / mL~500IU / mL, 75IU / m L~250IU / mL, or 100IU / mL~200IU / mL, such as 10IU / mL~20IU / mL, 20IU / mL~30IU / mL , 30IU / mL~40IU / mL, 40IU / mL~50IU / mL, 50IU / mL~75IU / mL, 75IU / mL~100IU / mL, 100IU / mL~150IU / mL, 150IU / mL~200IU / mL, 200IU / mL~500IU / mL, or 500IU / mL~1,000IU / mL) In one embodiment, the method defined herein typically comprises administering to the patient a therapeutically effective amount of IL-2 at a concentration of In some embodiments, the IL-2 concentration is less than 1,000 IU / mL, e.g., less than 500 IU / mL. In one embodiment, the method includes administering IL-2 at a concentration of about 100 IU / mL.

[0037] In further embodiments, the methods defined herein typically involve at least 0. 1 ng / mL, e.g., at least 10 ng / mL (e.g., 0.1 ng / mL to 10,000 ng / mL, 1.0 ng / mL to 1,000 ng / mL, 5ng / mL~800ng / mL, 10ng / mL~750ng / mL, 20ng / mL~500ng / mL, 50ng / mL~400ng / mL , or 100ng / mL to 250ng / mL, for example, 0.1ng / mL to 1.0ng / mL, 1.0ng / mL to 5.0ng / mL, 5.0ng / mL~10ng / mL, 10ng / mL~20ng / mL, 20ng / mL~100ng / mL, 20ng / mL~50ng / mL, 40ng / mL~70n g / mL, 50ng / mL~100ng / mL, 50ng / mL~60ng / mL, 100ng / mL~200ng / mL, 200ng / mL~500ng / m In a further embodiment, the present invention comprises IL-15 at a concentration of 500 ng / mL to 1,000 ng / mL. The methods defined herein typically involve administering IL-1 at a concentration of less than 500 ng / mL, e.g., less than 100 ng / mL. In some embodiments, the method includes IL-15 at a concentration of about 50 ng / mL.

[0038] In some embodiments, the isolation of γδ T cells from a non-hematopoietic tissue sample comprises any of the methods described above. The present invention also includes culturing in the presence of both IL-2 and IL-15 at any concentration. The concentration is approximately 100 IU / mL and the concentration of IL-15 is 55 ng / mL.

[0039] In further embodiments, the methods defined herein typically involve at least 0. 1 ng / mL, e.g., at least 1.0 ng / mL (e.g., 0.1 ng / mL to 1,000 ng / mL, 1.0 ng / mL to 100 ng / mL, 1.0ng / mL~50ng / mL, 2ng / mL~50ng / mL, 3ng / mL~10ng / mL, 4ng / mL~8ng / mL, 5ng / mL ~10ng / mL, 6ng / mL~8ng / mL, e.g. 0.1ng / mL~10ng / mL, 1.0ng / mL~5ng / mL, 1.0ng / mL In a further embodiment, the present invention comprises a concentration of IL-21 in the range of 1.0 ng / mL to 10 ng / mL, 1.0 ng / mL to 20 ng / mL. The methods defined herein typically involve administering IL-1 at a concentration of less than 100 ng / mL, for example less than 50 ng / mL. In some embodiments, the method includes administering a 100% CI 0.01 to 1.5 mg / mL of 100% CO2, including about 6 ng / mL, e.g., about 6.25 ng / mL. Contains IL-21 at a concentration of 100 mM.

[0040] In further embodiments, the methods defined herein typically involve at least 0. 1 ng / mL, e.g., at least 10 ng / mL (e.g., 0.1 ng / mL to 1,000 ng / mL, 1.0 ng / mL to 100 ng / mL) mL, 1.0ng / mL~50ng / mL, 2ng / mL~50ng / mL, 3ng / mL~40ng / mL, 4ng / mL~30ng / mL, 5ng / mL ~20ng / mL, 10ng / mL~20ng / mL, e.g. 0.1ng / mL~50ng / mL, 1.0ng / mL~25ng / mL, 5ng / m In a further embodiment, the method as defined herein comprises administering to the patient an IL-4 concentration of 100 ng / mL to 25 ng / mL. The method typically involves administering IL-1 at a concentration of less than 100 ng / mL, for example less than 50 ng / mL, in particular less than 20 ng / mL. In some embodiments, the method includes IL-4 at a concentration of about 15 ng / mL.

[0041] References herein to "non-hematopoietic tissue" or "non-hematopoietic tissue sample" include those made from skin (e.g., Non-hematopoietic tissues include blood, bone marrow, or thymus tissue. In one embodiment, the non-hematopoietic tissue sample is a tissue other than skin (e.g., human skin). In further embodiments, the non-hematopoietic tissue sample is from the gastrointestinal or digestive tract (e.g., human In some embodiments, lymphocytes and / or γδ T cells are The cells are not obtained from certain types of samples of biological fluids, such as blood or synovial fluid. In an embodiment, lymphocytes and / or γδ T cells are isolated according to the methods defined herein. The non-hematopoietic tissue sample from which the hematopoietic tissue is derived is skin (e.g., human skin), which has been described in detail in the art. Alternatively, the lymphocytes and / or Methods for isolating γδ T cells include those derived from the digestive tract (e.g., colon or gastrointestinal), mammary gland, lung, prostate, liver, The present invention can be applied to the spleen, pancreas, uterus, vagina, and other skin membranes, mucous membranes, or serosal membranes. Lymphocytes and / or γδ T cells are resident in human cancer tissue samples, e.g., breast or prostate tumors. In some embodiments, the lymphocytes and / or γδ T cells are derived from human cancer tissue samples. In another embodiment, the cells may be derived from lymphocytes and and / or γδ T cells are expressed in non-human cancer tissue (e.g., tissue that does not contain significant numbers of tumor cells). For example, lymphocytes and / or γδ T cells may be derived from a hematopoietic tissue sample. or may be from an area of ​​skin (e.g., healthy skin) away from adjacent cancerous tissue. Thus, in some embodiments, the γδ T cells are not obtained from human cancer tissue. In a further embodiment, the lymphocytes are not obtained from human cancer tissue.

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

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

[0044] In some embodiments of the invention, the non-hematopoietic tissue sample is an intact biopsy. References herein to a "biopsy" or "explant" refer to a tissue that is substantially intact or The structural integrity of the biopsy or explant is not destroyed, thereby Including tissue and tissue samples that are not intentionally destroyed within the perimeter of the resection to be removed from the tissue sample. Such intact biopsies or explants are largely intact except for minor disruptions caused by manipulation. The intact biopsy or explant has a maintained three-dimensional structure. It may be mechanically disrupted by crushing or shredding, or it may be chemically or enzymatically disrupted, for example. An intact biopsy or intact tissue sample is defined as a whole tissue, intact tissue, tissue fragment, or tissue fragment. For example, in one embodiment, an intact biopsy may include a portion of, or all of the elements of, the tissue. In a further embodiment, the biopsy comprises the epidermal and dermal layers of the skin. In those embodiments in which the biopsy is intact, separation and differentiation of such layers is It will be understood that the term "intact" is used herein to refer to a substance that is in a state where it is intact. Reference further includes full thickness biopsies of non-hematopoietic tissue samples.

[0045] Thus, in one particular embodiment of the invention, the non-hematopoietic tissue sample is not comminuted. In a further embodiment, the intact biopsy is a punch biopsy. In such cases, intact biopsies are obtained by punch biopsy. The embodiments presented herein are directed to non-comminuted and / or intact non-hematopoietic tissue. It provides the surprising advantage of obtaining large numbers of isolated or separated cells from a sample. As indicated herein, uncomminuted according to the methods defined herein and / or cells obtained from intact non-hematopoietic tissue samples may be subjected to subsequent expansion as known in the art. and / or may possess a phenotype useful in engineering methods.

[0046] In further embodiments, the intact biopsy is skin (e.g., human skin) or intact. The biopsy is gastrointestinal (e.g., human gastrointestinal). In one embodiment, the non-hematopoietic tissue sample is at least The "minimum cross section" is measured through the center of gravity of the tissue sample. It will be understood that the term "maximum cross-section" refers to the smallest or shortest length of a tissue sample. It will be further understood that the term refers to the maximum or greatest length measured through the center of gravity. As used herein, the term "centroid" refers to the average or mean of all the points in a tissue sample. In a further embodiment, the non-hematopoietic tissue sample is at least 2 mm , at least 3mm, at least 4mm, at least 5mm, at least 6mm, at least It will be appreciated that the minimum cross section of at least 7 mm, or at least 8 mm, may be used. In some embodiments, the non-hematopoietic tissue sample is 8 mm or less, 7 mm or less, 6 mm or less, 5 mm or less, 4 mm or less, 3 mm or less. In one embodiment, the non-hematopoietic tissue sample has a minimum cross-section of 1 mm or less, or 2 mm or less. In one particular embodiment, the membrane has a minimum cross section of from 2 mm to 8 mm inclusive, for example from 2 mm to 4 mm. In one particular embodiment, the non-hematopoietic tissue sample has a minimum cross-section of about 3 mm. The hematopoietic tissue sample has a cross-section of about 3 mm. In a further embodiment, the non-hematopoietic tissue sample At least 2 mm, at least 3 mm, at least 4 mm, at least 5 mm, at least It is understood that each of the 1000 .mu.m and 1000 .mu.m diameters has a maximum cross section of at least 6 mm, at least 7 mm, or at least 8 mm. In further embodiments, the non-hematopoietic tissue sample may be 8 mm or less, 7 mm or less, 6 mm or less, 5 mm or less, In one embodiment, the fluororesin has a maximum cross-section of no more than 1 mm, no more than 4 mm, no more than 3 mm, or no more than 2 mm. The non-hematopoietic tissue sample has a maximum cross-section of 1 mm to 8 mm (inclusive), for example, 2 mm to 4 mm. In certain embodiments, the non-hematopoietic tissue sample has a maximum cross-section of about 3 mm.

[0047] According to a further embodiment, the non-hematopoietic tissue sample is at least 1 mm 2 have a minimum cross-sectional area of "Minimum cross-sectional area" refers to the area of ​​the smallest cross section measured around the center of gravity of the tissue sample. It will be understood that the "maximum cross-sectional area" is the maximum area measured around the center of gravity of the tissue sample. It will be further understood that "centre of gravity" refers to the area of ​​the major cross section. The term is the average or mean position of all the points in the tissue sample. In one embodiment, the non-hematopoietic tissue sample comprises at least 2 mm 2 , at least 3 mm 2 At least Also 4mm 2 , at least 5 mm 2 , at least 6mm 2 , at least 7mm 2 , at least 8mm 2 At least Also 9mm 2 , or at least 10 mm 2 In a further embodiment, the cross-sectional area is Non-hematopoietic tissue samples were 50 mm 2 Below, 40mm 2 Below, 30mm 2 Below, 25mm 2 Below, 20mm 2 Below, 15mm 2 Below Bottom, 10mm 2 Less than or equal to 8mm 2 In one embodiment, the non-hematopoietic The tissue samples were 1 mm 2 ~50mm 2 , for example, 3 mm 2 ~12mm2 A minimum cross-sectional area of ​​1. In an embodiment, the non-hematopoietic tissue sample is about 7 mm 2 Further embodiments have a minimum cross-sectional area of In such cases, the non-hematopoietic tissue sample is at least 2 mm 2 , at least 3 mm 2 , at least 4 mm 2 , small At least 5mm 2 , at least 6mm 2 , at least 7mm 2 , at least 8mm 2 , at least 9mm 2 ,also is at least 10 mm 2 In a further embodiment, the non-hematopoietic tissue has a maximum cross-sectional area of The sample is 50 mm 2 Below, 40mm 2 Below, 30mm 2 Below, 25mm 2 Below, 20mm 2 Below, 15mm 2 Below, 10mm 2 Less than or equal to 8mm 2 In one embodiment, the non-hematopoietic tissue sample has a maximum cross-sectional area of , 1mm 2 ~50mm 2 , for example, 3 mm 2 ~12mm 2 In one particular embodiment, the cross-sectional area is The non-hematopoietic tissue samples were approximately 7 mm 2 has a maximum cross-sectional area of

[0048] According to a further embodiment, the non-hematopoietic tissue sample is at least 5 mm 3 It has a volume of . In one embodiment, the non-hematopoietic tissue sample is at least 8 mm 3 , at least 10 mm 3 , less Both are 15mm 3 , at least 20 mm 3, at least 25mm 3 , at least 30 mm 3 , at least 35mm 3 , At least 40mm 3 , at least 50 mm 3 , or at least 60 mm 3 Further experiments In an embodiment, the non-hematopoietic tissue sample is 250 mm 3 Below, 200mm 3 For example, 180 mm 3 Below, 16 00mm 3 Below, 140mm 3 Below, 120mm 3 Below, 100mm 3 Below, 80mm 3 Below, 60mm 3 Below, 50mm 3 below, Or 40mm 3 In one embodiment, the non-hematopoietic tissue sample has a volume of 5 mm 3 ~250mm 3 , for example, 15mm 3 ~65mm 3 In one particular embodiment, the non-hematopoietic tissue sample has a volume of , about 35mm 3 has a volume of

[0049] In one embodiment, the non-hematopoietic tissue sample is a punch biopsy. A punch biopsy can be any form of It may be of round shape, but is conveniently of circular cross section, preferably at least 1.5 mm in diameter. In yet a further embodiment, the non-hematopoietic tissue sample is at least 2 mm in diameter. , for example, at least 3 mm in diameter, at least 4 mm in diameter, at least 5 mm in diameter, Further examples include punch biopsies of at least 6 mm in diameter, at least 7 mm in diameter, or at least 8 mm in diameter. In embodiments, the non-hematopoietic tissue sample is 8 mm or less in diameter, e.g., 7 mm or less in diameter, 6 mm or less in diameter, In one embodiment, non-hematopoietic tissue samples include punch biopsies of 5 mm or less in diameter, or 3 mm or less in diameter. The sample includes a punch biopsy having a diameter of 1 mm to 8 mm, for example, a diameter of 2 mm to 4 mm. In this regard, non-hematopoietic tissue samples include 3 mm diameter punch biopsies.

[0050] In certain embodiments, the non-hematopoietic tissue sample has a size, area, volume, and / or diameter biopsy (e.g. punch biopsy, in particular a circular cross-sectional punch biopsy), The maximum depth is determined by the site from which the biopsy is obtained (although that depth may be reduced). In one embodiment, the biopsy is a skin biopsy and includes the epidermal and dermal layers. In an embodiment, the biopsy is substantially free of subcutaneous fat. In some embodiments, the biopsy includes the epidermal and dermal layers and is substantially free of the subcutaneous fat layer. In some cases, the biopsy does not include subcutaneous fat. Alternatively, the subcutaneous fat is not removed and therefore the biopsy Thus, in yet a further embodiment, In one embodiment, the biopsy consists of the epithelial and dermal layers. In one embodiment, the biopsy is a non-hematopoietic tissue sample. Includes all layers of the

[0051] The method of the present invention comprises culturing a non-hematopoietic tissue sample as defined herein. References herein to "isolating or separating" from a non-hematopoietic tissue sample include those obtained by Cells and / or non-hematopoietic tissues, including cells that have been isolated, removed, purified, or enriched. A culture medium containing growth factors and / or essential nutrients required and / or preferred by the tissue sample. Such culture conditions include the addition of the cells and / or non-hematopoietic tissue sample to the culture medium according to the present invention. According to the present invention, the method is adapted for isolating a cell or cell population from a non-hematopoietic tissue sample. or cells to be isolated and expanded from a non-hematopoietic tissue sample. It will be appreciated that this can be adapted according to the population.

[0052] In certain embodiments, the culturing of the non-hematopoietic tissue sample comprises culturing γδ T cells from the non-hematopoietic tissue sample. In an alternative embodiment, the culture of a non-hematopoietic tissue sample is for a period of time sufficient for isolation. The culture may be performed using lymphocytes other than γδ T cells (e.g., αβ T cells and / or NK cells) derived from a non-hematopoietic tissue sample. The period is sufficient for the isolation of (natural killer) cells. Thus, the culture period according to the methods defined herein is at least 14 days. In the method defined herein, the culture period is less than 45 days, for example less than 30 days, For example, less than 25 days. In a further embodiment, The culture period is 14 to 35 days, for example, 14 to 21 days. The culture period according to the method defined herein is approximately 21 days.

[0053] In a particular embodiment of the present invention, phosphorus is isolated according to the method defined herein. The lymphocytes and / or gamma delta T cells are isolated from the culture of the non-hematopoietic tissue sample after the culture of the non-hematopoietic tissue sample. The recovery of lymphocytes and / or γδ T cells as defined herein is carried out by harvesting the lymphocytes and / or γδ T cells from the culture. the physical collection of lymphocytes and / or γδ T cells, the physical collection of other lymphocytes (e.g., αβ T cells, Isolation of lymphocytes and / or γδ T cells from the host (γδ T cells, and / or NK cells), or The present invention relates to the isolation and / or proteolytic isolation of lymphocytes and / or γδ T cells from stromal cells (e.g., fibroblasts). In one embodiment, lymphocytes and / or γδ T cells may be isolated by mechanical In a further embodiment, the lymphocytes are collected by means of a suitable means (e.g., pipetting). The globulins and / or γδ T cells are then collected by magnetic separation and / or labeling. In an embodiment, lymphocytes and / or γδ T cells are identified using flow cytometry techniques, e.g. For example, they are collected by FACS. Thus, in one embodiment, the γδ T cells are In a further embodiment, lymphocytes are recovered by specific labeling of T cells. The lymphocytes are recovered by specific labeling to distinguish them from other cells in the culture. The recovery of such lymphocytes and / or γδ T cells requires physical removal from the culture of a non-hematopoietic tissue sample. This may include removing the cells from the culture medium, transferring them to a separate culture vessel, or transferring them to separate or different culture conditions. It will be understood that.

[0054] The recovery of such lymphocytes and / or γδ T cells may be achieved by the use of lymphocytes isolated from non-hematopoietic tissue samples. It is understood that the assay is performed after a period of time sufficient to obtain a population of lymphocytes and / or γδ T cells. In certain embodiments, lymphocytes and / or γδ T cells will be isolated from a non-hematopoietic tissue sample. At least 1 week, at least 10 days, at least 11 days, at least 12 days, at least Preferably, lymphocytes and / or γδ T cells are harvested after at least 13 days, or at least 14 days. The cells may be cultured for up to 40 days, e.g., up to 38 days, up to 36 days, up to 34 days, up to 32 days, up to 30 days, up to 28 days, In one embodiment, the lymphocytes and / or γδ T cells are harvested after at least 14 days of culture of the non-hematopoietic tissue sample. In an embodiment, lymphocytes and / or γδ T cells are isolated from cultures of non-hematopoietic tissue samples from 14 to 21 days of culture. It will be collected after a few days.

[0055] In one embodiment of the invention, the non-hematopoietic tissue sample is cultured in a medium that is substantially free of serum (e.g. For example, they are cultured in serum-free medium or medium containing serum replacement (SR). In an embodiment, the non-hematopoietic tissue sample is cultured in serum-free medium. The serum substitute is a chemically defined compound that avoids the use of serum of human or animal origin. In an alternative embodiment, the non-hematopoietic tissue sample may also include a serum replacement medium based on are cultured in medium containing serum (e.g., human AB serum or fetal bovine serum (FBS)). In one embodiment, the non-hematopoietic tissue sample is cultured in a medium containing serum replacement. In an embodiment, the non-hematopoietic tissue sample is cultured in a medium that does not contain animal-derived products.

[0056] Embodiments according to the invention in which non-hematopoietic tissue samples are cultured in serum-free medium may be used without the need for serum filtration, precipitation, or precipitation. It will be appreciated that this has the advantage of avoiding problems with contamination and supply. Furthermore, animal-derived products are not preferred for use in the manufacture of clinical grade human therapeutics. As seen herein, the inventors have surprisingly found that cells, in particular V51 γδ cells The use of serum-free medium for the isolation of non-hematopoietic tissues compared to the use of medium containing AB serum. It has also been found to substantially increase the number of cells obtained from a sample, particularly in serum-free medium. Isolation of γδ T cells from cultured non-hematopoietic tissue samples increases the yield of Vδ1 cells.

[0057] In one embodiment, the methods defined herein are carried out in an isolated vessel. Reference to a "container" includes lymphocytes and / or Refers to a container that contains a non-hematopoietic tissue sample for isolation of γδ T cells. The isolation container may vary depending on the isolation method. It was noted that the ELISA kit can be used for the sole purpose of amplification and cannot be used for further amplification steps. stomach.

[0058] In one embodiment, the method defined herein comprises the step of: Such materials are typically used in an isolation vessel (e.g., an isolation vessel). It is permeable to all gases, allowing gas exchange between the contents of the container and the surrounding environment. References herein to "container" include culture dishes, culture plates, single-well dishes, and the like. Flasks, multi-well dishes, multi-well plates, flasks, multi-layer flasks, bowls These may include tanks (e.g., roller bottles), bioreactors, bags, tubes, etc. It will be appreciated that such vessels are suitable for use in methods involving the expansion of non-adherent cells and other lymphocytes. However, as shown herein, Surprisingly, the container containing the gas permeable material is capable of preventing adhesion of the γδ T cells, which are normally considered to be adhesive. The use of such vessels for the culture of non-hematopoietic tissue samples also finds utility in isolating hematopoietic cells. It has been found that such a vessel significantly increases the yield of isolated γδ T cells from the have shown that γδ T cells are more abundant than other stromal cells (e.g., epithelial cells), including fibroblasts and adherent cell types. It has also been found that the IL-16 / ... The container comprising the gas permeable material as defined herein is capable of retaining γδ T cells as well as other lymphocytes ( In a further embodiment, the IL-16 / ... Fibroblasts and / or other stromal cells (e.g., epithelial cells) are cultured in a container comprising a gas permeable material. It is not present in cultures performed at .

[0059] Such containers that include a gas permeable material may further include a gas permeable material that is non-porous. Thus, in one embodiment, the gas permeable material is non-porous. In an embodiment, the gas permeable material is silicone, fluoroethylene polypropylene, polypropylene. and membrane films such as polyolefins or ethylene vinyl acetate copolymers. Such containers are made of gas permeable materials, gas permeable membrane films, or non-porous gas permeable materials. Thus, according to yet a further embodiment, the container may include a lid, a base, and and at least one sidewall, wherein at least a portion of the bottom of the container is in contact with the lid. In one embodiment, the gas permeable material is located on a substantially horizontal surface. The container includes a lid, a bottom, and at least one sidewall, wherein at least a portion of the bottom includes a gas permeable material that is in a horizontal plane when the lid is above the bottom. In an embodiment, the container comprises a lid, a bottom, and at least one sidewall, wherein the at least At least one side wall may be a vertical surface when the lid is above the bottom, or If the lid is not above the base, it includes a gas permeable material that may be on a horizontal surface. It is understood that in embodiments, only a portion of the bottom or sidewall may include a gas permeable material. Alternatively, the entire bottom or the entire sidewall may comprise a gas permeable material. In further embodiments, the lid of the container comprises a gas permeable material, e.g., an O-ring. Depending on the application, the container may be sealed. Such an embodiment may prevent leakage of the contents of the container. It will be appreciated that the present invention may be advantageous in that it may reduce or eliminate the evaporation of the polymer. In this case, the container comprises a liquid-tight container that includes a gas permeable material to allow gas exchange. In an alternative embodiment, the lid of the container, which comprises a gas permeable material, is on a horizontal surface or The lid is located above the bottom and is not sealed. Thus, in one embodiment, the lid is The container is configured to allow gas exchange through the lid. The bottom of the gas permeable container is configured to allow gas exchange from the bottom of the container. In yet a further embodiment, the container comprising the gas permeable material is a liquid-tight container. and may further include an inlet and an outlet or drain. wherein the container comprising the gas permeable material comprises a lid, a bottom, and optionally at least one sidewall; wherein at least a portion of the lid and the base comprise a gas permeable material, and when present, at least At least a portion of at least one sidewall comprises a gas permeable material. These are described in WO2005035728 and US9255243, which are incorporated herein by reference. Containers are also commercially available, for example the GR 1.111 provided by Wilson Wolf Manufacturing. EX® cell culture devices, such as G-REX 6-well plates, G-REX 24-well plates, and G-REX10 containers.

[0060] In one embodiment, the non-hematopoietic tissue sample is placed on a synthetic scaffold. As used in the Notes, "synthetic scaffold," "scaffold," and "glycolic acid" are used interchangeably. The terms "adiploid" and "adiploids" are used interchangeably to describe non-native three-dimensional structures suitable for supporting cell growth. A non-hematopoietic tissue sample is placed on or attached to a synthetic scaffold. This allows lymphocytes to attach to the scaffold and promote lymphocyte egress from the explant onto the scaffold. Synthetic scaffolds can be made of polymers (e.g., natural or synthetic polymers, e.g., poly(ethylene glycol)). Vinylpyrrolidone, polymethyl methacrylate, methylcellulose, polystyrene, polypropylene, polyurethane), ceramics (e.g., tricalcium phosphate, calcium aluminate calcium, calcium hydroxyapatite), or metals (e.g., tantalum, titanium, Platinum and metals of the same element group as platinum, niobium, hafnium, tungsten, and The material may be constructed from natural and / or synthetic materials, such as alloys of the above. In an embodiment, the synthetic scaffold is tantalum coated. (e.g., collagen (e.g., collagen I or collagen II), fibronectin, laminin Nin, integrin, angiogenic factors, anti-inflammatory factors, glycosaminoglycans, vitrogens, Antibodies and fragments thereof, cytokines (e.g., IL-2, IL-15, IL-4, IL-21, IL9, and the like) (a combination of) is coated on the scaffold surface according to a method known in the art. They can be embedded in scaffold materials or added to culture media to promote cell attachment, migration, Using this and other methods, several other It has been possible to isolate lymphocytes from various non-hematopoietic tissue types, such as skin, gastrointestinal, prostate, and breast. can.

[0061] In one embodiment, the non-hematopoietic tissue sample is subjected to a step of isolating lymphocytes from the non-hematopoietic tissue sample. In a further embodiment, the composite scaffold is placed inside a container used for the In this study, the synthetic scaffold was designed to transport lymphocytes and / or gamma-erythrocytes from a non-hematopoietic tissue sample to the bottom of the vessel. δ T cell egress. Such embodiments are adapted to enhance the expression of hematopoietic T cells in non-hematopoietic tissue samples. and / or lymphocytes (e.g., lymphocytes from stromal cells (e.g., fibroblasts and / or epithelial cells). For example, the method allows for the isolation and / or separation of γδ T cells, αβ T cells, and / or NK cells. Moreover, such an embodiment has the advantage that it is possible to reduce the amount of hematopoietic tissue from the non-hematopoietic tissue sample to the bottom of the culture vessel. This allows for the recovery of lymphocytes (e.g., γδ T cells, αβ T cells, and / or NK cells). In certain embodiments, the synthetic scaffold is capable of stimulating the depletion of γδ T cells from a non-hematopoietic tissue sample. In a further embodiment, the synthetic scaffold is configured to promote extrusion. promotes the egress of lymphocytes, e.g., αβ T cells and / or NK cells, from non-hematopoietic tissue samples. It is configured to:

[0062] Thus, in one embodiment of the methods defined herein, the synthetic scaffold comprises: The present invention is configured to promote lymphocyte egress from a non-hematopoietic tissue sample to the bottom of a culture vessel. In a further embodiment of the method defined herein, the synthetic scaffold is It is configured to promote egress of γδ T cells from the sample to the bottom of the culture vessel.

[0063] The methods of the present invention provide a much greater total cell yield than previously described. In one embodiment, the total isolated cell count is at least 10 of the tissue sample. 6 cells / cm2 , at least 2 ×10 6 cells / cm 2 , at least 5 × 10 6 cells / cm 2 , at least 10 x 10 6 cells / cm 2 , at least 20 ×10 6 cells / cm 2 , at least 30 x 10 6 cells / cm 2 , at least 40 x 10 6 cells / cm 2 , at least 5 0×10 6 cells / cm 2 , at least 60×10 6 cells / cm 2 , at least 70×10 6 cells / cm 2 ,at least 80×10 6 cells / cm 2 , at least 90×10 6 cells / cm 2 , at least 100 x 10 6 cells / cm 2 At least Also 150×10 6 cells / cm 2 , at least 200×10 6 cells / cm 2 In a specific embodiment, The total number of isolated cells should be at least 50 x 10 6 cells / cm 2 In another embodiment, the total isolated cell The number of cells should be at least 100 × 10 6 cells / cm 2 It is.

[0064] The predominant γδ T cells in blood are primarily Vδ2 T cells, whereas the predominant γδ T cells in non-hematopoietic tissues are Vδ2 T cells. The predominant γδ T cells are predominantly Vδ1 T cells, and as a result, Vδ1 T cells are predominantly found in non-hematopoietic tissues. They comprise approximately 70-80% of the resident γδ T cell population. However, a portion of Vδ2 T cells resides in non-hematopoietic tissues. They are also found in tissues such as the gastrointestinal tract, where Vδ2 T cells comprise approximately 10-20% of γδ T cells. A subset of γδ T cells resident in non-hematopoietic tissues expresses neither Vδ1 nor Vδ2 TCRs. In the literature, these are called double negative (DN) γδ T cells. These DN γδ T cells A large proportion of T cells are likely to express Vδ3, with Vδ5 expressing T cells being in the minority. γδ T cells routinely resident in blood tissues and isolated by the methods of the present invention , preferably non-V52 T cells, e.g., V51 T cells, and less abundant DN γδ T cells. include.

[0065] Thus, in one preferred embodiment, a single The isolated γδ T cells comprise a population of Vδ1 T cells. The γδ T cells isolated by the method described herein comprise a population of DN γδ T cells. In the present invention, the γδ T cells isolated by the method defined herein are a population of Vδ3 T cells. In one embodiment, the γδ T cells isolated by the method defined herein include The cells contain a population of V55 T cells.

[0066] γδ T cells can also be defined by the type of γ chain they express. In an embodiment, the γδ T cells isolated by the methods defined herein are Vγ4 T In most cases, Vγ4 T cells are obtained from a gastrointestinal tissue sample.

[0067] The isolation method should be such that the number of isolates is greater than the number of isolates in the reference population (e.g., at least two-fold greater, at least three-fold greater). number, at least four times as many, at least five times as many, at least six times as many, at least seven times as many, At least 8 times as many, at least 9 times as many, at least 10 times as many, at least 15 times as many, At least 20 times as many, at least 25 times as many, at least 30 times as many, at least 35 times as many, At least 40 times as many, at least 50 times as many, at least 60 times as many, at least 70 times as many, At least 80 times as many, at least 90 times as many, at least 100 times as many, at least 200 times as many, At least 300 times as many, at least 400 times as many, at least 500 times as many, at least 600 times as many number, at least 700 times the number, at least 800 times the number, at least 900 times the number, at least 1,0 00-fold, at least 5,000-fold, at least 10,000-fold) of isolated γδ T cells Provide a group.

[0068] In some embodiments, the population of γδ T cells isolated according to the methods of the invention comprises: For example, a population of isolated γδ T cells has a low percentage of cells expressing TIGIT. Less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or Alternatively, the isolated population of γδ T cells may have a frequency of TIGIT+ cells of less than 10%. 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, or about 10% of the time In one embodiment, the isolated population of γδ T cells may comprise less than 80% TIGIT+ cells. Thus, in one embodiment, isolated γδ T cells have a frequency of TIGIT+ cells of at least 100%. In a further embodiment, the isolated population of cells has a frequency of about 70% TIGIT+ cells. The population of γδ T cells has a frequency of TIGIT+ cells of less than 60%. In this study, the isolated population of γδ T cells had a frequency of TIGIT+ cells of approximately 30%. Thus, in one embodiment, the isolated γδ T cells do not substantially express TIGIT.

[0069] In some embodiments, the isolated population of V51 T cells comprises a low frequency of TIGIT+ cells. For example, the isolated population of V51 T cells may be less than 90%, less than 80%, less than 70%, less than 60% population of Vδ1 T cells at a frequency of less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% Alternatively, the isolated population of V51 T cells may be about 90%, about 80%, or more than about 10%. %, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, or about 10% frequency of TIGIT+ cells In one embodiment, the isolated population of V51 T cells may have a frequency of less than 80%. Thus, in one embodiment, the isolated population of V51 T cells comprises In a further embodiment, the isolated V51 T cells have a frequency of about 70%. In yet a further embodiment, the population of cells has a frequency of TIGIT+ cells of less than 60%. The isolated V51 T cell population has a frequency of TIGIT+ cells of about 30%. In an embodiment, the isolated V51 T cells do not substantially express TIGIT.

[0070] In some embodiments, the population of γδ T cells isolated according to the methods of the invention comprises: For example, the isolated population of γδ T cells may be greater than 10%, greater than 20%, greater than 30%, greater than 40%, or greater than 50%. %, greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90% frequency of CD27+ cells. Alternatively, the isolated population of γδ T cells may be about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 70%. In certain embodiments, the subject may have a frequency of CD27+ cells of about 50%, about 70%, about 80%, or about 90%. Thus, the isolated population of γδ T cells has a frequency of CD27+ cells of more than 10%. In an embodiment, the isolated population of γδ T cells has a frequency of about 20% CD27+ cells. In a further embodiment, the isolated population of γδ T cells comprises CD27+ cells with a frequency of greater than 20%. In one embodiment, the isolated population of γδ T cells has a frequency of about 20% CD27 + cells.

[0071] In some embodiments, the isolated population of V51 T cells expresses CD27. In some embodiments, the isolated γδ T cells express CD27. In the isolated Vδ1 T cell population, the proportion of Vδ1 T cells was greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, or having a frequency of CD27+ cells of greater than 60%, greater than 70%, greater than 80%, or greater than 90%. The population of γδ T cells is approximately 10%, approximately 20%, approximately 30%, approximately 40%, approximately 50%, approximately 60%, approximately 70%, approximately 80%, approximately 90%, approximately 100%, approximately 110%, approximately 120%, approximately 130%, approximately 140%, approximately 150%, approximately 160%, approximately 170%, approximately 180%, approximately 190%, approximately 200%, approximately 210%, approximately In one embodiment, the isolated V51 may have a frequency of CD27+ cells of about 90% or about 90%. The population of T cells has a frequency of CD27+ cells of greater than 10%. Thus, in one embodiment, The isolated population of V51 T cells has a frequency of CD27+ cells of about 20%. In one embodiment, the isolated population of V51 T cells has a frequency of CD27+ cells of more than 20%. In an embodiment, the population of isolated V51 T cells has a frequency of CD27+ cells of about 20%.

[0072] In some embodiments of any of the aforementioned aspects, the isolated population of γδ T cells comprises , relative to a reference population (e.g., relative to a population of γδ T cells isolated using alternative methods). (larger) CD124, CD215, CD360, CTLA4, CD1b, BTLA, CD39, CD45RA, Fas ligand, A marker selected from the group consisting of CD25, ICAM-1, CD31, KLRG1, CD30, and CD2 Additionally or alternatively, an isolated population of γδ T cells, Greater frequencies of CD124, CD215, CD360, CTLA4, CD1b, BTLA, CD39, and CD 45RA, Fas ligand, CD25, ICAM-1, CD31, KLRG1, CD30, and CD2. In particular, the markers may be CD45R, CD46R, CD47R, CD48R, CD49R, CD50R, or CD51R. In some embodiments, the isolated population of γδ T cells is selected from CD25, CD26, CD27, and CD28. had lower NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, and LAG3 than the reference population , CCR4, CD69, PD-1, and CD64. Additionally or alternatively, the isolated population of γδ T cells has surface expression of Lower frequencies of NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, expressing one or more of the markers selected from the group consisting of CD69, PD-1, and CD64. The cell may have

[0073] In some embodiments, the isolated population of V51 T cells is more highly expressed compared to a reference population. Abundant CD124, CD215, CD360, CTLA4, CD1b, BTLA, CD39, CD45RA, Fas ligand, CD25, ICA one or more of the markers selected from the group consisting of M-1, CD31, KLRG1, CD30, and CD2 In some embodiments, the isolated population of γδ T cells has surface expression of: Greater frequency of CD124, CD215, CD360, CTLA4, CD1b, BTLA, CD39, CD45RA compared to reference , Fas ligand, CD25, ICAM-1, CD31, KLRG1, CD30, and CD2. In some embodiments, the cells express one or more of the markers. The isolated γδ T cell population exhibited lower NKp44, NKp46, ICAM-2, and CD70 expression levels compared to the reference population. , CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1, and CD64. In another embodiment, the isolated γ The delta T cell population had lower frequencies of NKp44, NKp46, ICAM-2, CD70, and CD28 compared to the reference population. , CD103, NKp30, LAG3, CCR4, CD69, PD-1, and CD64. The present invention relates to a method for the preparation of ... cell that expresses one or more of the following:

[0074] When isolated from non-hematopoietic tissues (e.g., skin), γδ T cells typically outgrow, e.g., αβ T cells. Part of a larger population of lymphocytes that includes lymphocytes, B cells, and natural killer (NK) cells In some embodiments, between 1% and 10% of the isolated lymphocyte population is γδ T cells (e.g., 1–10% of the isolated skin-derived lymphocyte population are γδ T cells). In most cases, γδ T cell populations (e.g., skin-derived γδ T cell populations) are characterized by large Vδ T cell populations. In some embodiments, the isolated lymphocytes (e.g., skin 1-10% of the population of isolated lymphocytes are Vδ1 T cells (e.g., Vδ1 T cells are isolated The population of γδ T cells may represent more than 50%, more than 60%, more than 70%, more than 80%, or more than 90% of the population of γδ T cells. In some cases, less than 10% of the isolated population of γδ T cells are Vδ2 T cells (e.g., Less than 10% of the isolated skin-derived γδ T cell population are Vδ2 T cells).

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

[0076] Isolated γδ T cells (e.g., γδ T cells isolated from the skin, e.g., isolated from the skin isolated Vδ1 T cells) can be expressed in response to corresponding hematopoietic tissue-derived cells (e.g., blood-derived γδ T cells and / or or blood-derived Vδ2 T cells). For example, a population of isolated γδ T cells The panel may be a reference population, e.g., a population of TCR-activated non-hematopoietic tissue-resident γδ T cells or a corresponding Hematopoietic tissue-derived cells (e.g., blood-derived γδ T cells and / or blood-derived Vδ2 T cells) Some populations may express higher levels of CCR3, CCR4, CCR7, CCR8, or CD103 than other populations. In one embodiment, the isolated population of γδ T cells is at least 5%, 10%, 15%, 20%, %, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of CCR3 + cell; At least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% , or more CCR4 + Cells; at least 5%, 10%, 15%, 20%, 25%, 30%, 40% , 50%, 60%, 70%, 80%, 90% or more of CCR7 + Cells; at least 5%, 10% , 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more CCR8 + cells; and / or at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60% , 70%, 80%, 90% or more CD103 + The isolated γδ T cell population The group consists of one or more, two or more, three or more, four or more, five or more of CCR3, CCR4, CCR7, CCR8, or CD103. , or all six may be expressed.

[0077] In some embodiments, an isolated population of γδ T cells (e.g., skin-derived γδ T cells) is The reference population, e.g., TCR-activated non-hematopoietic T cells, may be a reference population, e.g., a TCR-activated non-hematopoietic T cell. A population of tissue-resident γδ T cells and / or the corresponding hematopoietic tissue-derived cells (e.g., blood-derived γδ T T cells and / or blood-derived Vδ2 T cells) and / or express TIM3. In some embodiments, the isolated population of γδ T cells is at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 9 0% or more NKGD2 + Cells; at least 5%, 10%, 15%, 20%, 25%, 30%, 4 0%, 50%, 60%, 70%, 80%, 90% or more CD56 + Cells; at least 5%, 10 %, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more CD69 + cells; and / or at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60 %, 70%, 80%, 90% or more of TIM3 + The isolated γδ T cells The population may include one or more, two or more, three or more, four or more of NKGD2, CD56, CD69, and / or TIM3. All five may be expressed.

[0078] Isolated non-hematopoietic tissue-derived γδ T cells (e.g., skin-derived γδ T cells and / or skin-derived A population of V51 T cells can also be characterized by function. Functions known in the art Assays can be performed to detect and / or elucidate the expression of any non-hematopoietic tissue-derived cell of the invention (e.g., isolated γδ T cells). a population of skin-derived Vδ1 T cells, or expanded γδ T cells and / or skin-derived Vδ1 T cell population) and a reference cell (e.g., a population of TCR-activated non-hematopoietic tissue-resident γδ T cells or are associated with corresponding hematopoietic tissue-derived cells, e.g., blood-derived γδ T cells and / or blood-derived Vδ2 Such assays can determine functional differences between T cell populations. , proliferation assays, cytotoxicity assays, binding assays, persistence and / or location Assays and the like can be mentioned.

[0079] Thus, in one aspect of the invention, lymphocytes and / or γδ cells as defined herein are Methods for isolating T cell populations include isolating non-depleted lymphocyte and / or γδ T cell populations The resulting population contains a surface phenotype consistent with

[0080] According to one aspect of the present invention, an isolated A population of isolated lymphocytes (eg, skin-derived αβ T cells and / or NK cells) is provided.

[0081] According to one aspect of the invention, a composition comprising a compound obtained by any of the processes defined herein Provided are isolated populations of lymphocytes (e.g., skin-derived αβ T cells and / or NK cells) that can be used to infect, treat, and / or immunize patients with, or against, the following conditions: can be.

[0082] According to a further aspect of the present invention, there is provided a method for treating a pulmonary circulation disorder, comprising the steps of: An isolated population of γδ T cells is provided.

[0083] According to a further aspect of the present invention, a method for producing a composition comprising the steps of: The present invention provides an isolated population of γδ T cells capable of

[0084] In one embodiment, the isolated population comprises more than 5% γδ T cells, e.g., between 7% and 12% γ In one embodiment, the isolated population comprises V51 cells, wherein the V51 T cells Less than 50%, such as less than 40%, of the δ1 cells express TIGIT. The selected population comprises V51 cells, wherein more than 50%, e.g., more than 60%, of the V51 cells express CD27. do.

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

[0086] In one embodiment, the present invention relates to a method for the detection of non-hematopoietic tissue resident lymphocytes and / or γδ T cells (e.g. For example, skin-derived αβ T cells, NK cells, γδ T cells, and / or non-Vδ2 T cells, e.g., Vδ The present invention features methods for expanding in vitro induced T cells and / or DN T cells. These methods include In some embodiments, γδ T cells can be cultured in vitro. Expanded from a population of γδ T cells isolated from non-hematopoietic tissue samples according to a defined method In general, non-hematopoietic tissue-resident γδ T cells associate physically with stromal cells (e.g., skin fibroblasts). When contact is removed, the cells can expand spontaneously. Inducing such a separation, leading to disinhibition of γδ T cells and inducing their expansion. In one embodiment, lymphocytes (e.g., skin-derived αβ T cells and / or NK cells) can be used. The non-hematopoietic cells (cells, gastrointestinal derived αβ T cells and / or NK cells) are treated according to the methods defined herein. It is expanded from a population of lymphocytes isolated from a tissue sample.

[0087] As used herein, "expanded" or "expanded lymphocytes and / or References to a "population of γδ T cells" refer to a population that is larger or expanded than the unexpanded population. Such a population includes a population of cells that contains a greater number of It may be a mixed population with a small number of cells or an expansion of a certain cell type or cell types within the population. The term "expansion step" refers to the process that results in an expansion or expanded population. It will be understood that an expansion or expanded population therefore refers to the number of times that an expansion step is performed. The number may be greater or be greater than that of the population before any expansion step or before Any number given herein to indicate magnification (e.g., For example, fold increase or expansion) refers to an increase in the number or size of a population of cells or the number of cells. It will be further understood that the above is indicative and is indicative of the amount of magnification.

[0088] Thus, in one embodiment, the γδ T cells isolated according to the methods of the invention are expanded Such expansion is achieved by γ-activation in the presence of IL-2, IL-15, and IL-21, optionally with IL-4. Alternatively, the expansion may include culturing the δ T cells in the presence of IL-9, IL-15, optionally including IL-4. The optional expansion step may include culturing the γδ T cells in the presence of IL-21 and IL-21. The method is performed for a period of time effective to generate a population of expanded lymphocytes and / or γδ T cells. It will be appreciated that in one embodiment, expanded lymphocytes and / or γδ T cells The effective period for producing a population of is at least 5 days. In this study, expansion produced an expanded population of γδ T cells in the presence of IL-2, IL-15, and IL-21. The method further comprises culturing the γδ T cells for at least 5 days in an amount effective to induce proliferation of the γδ T cells. In an embodiment, the expansion is achieved by the administration of expanded γδ T Culturing the γδ T cells for at least 5 days in an amount effective to produce a population of cells. In yet a further embodiment, the expansion comprises expansion in the presence of IL-9, IL-15, and IL-21. and culturing the γδ T cells for at least 5 days in an amount effective to produce a population of γδ T cells that is In one embodiment, the expansion includes increasing the presence of IL-9, IL-15, IL-21, and IL-4. Below, the γδ T cells are administered for at least 5 days in an amount effective to generate an expanded population of γδ T cells. This involves culturing the cells.

[0089] In a further embodiment, the expansion is effective to generate an expanded population of γδ T cells. in a sufficient amount for a period of time (e.g., at least 5 days, at least 6 days, at least 7 days, At least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days at least 13 days, at least 14 days, at least 21 days, at least 28 days, or For a longer period, for example, 5 to 40 days, 7 to 35 days, 14 to 28 days, or about 21 days ), and culturing lymphocytes and / or γδ T cells. In some embodiments, The lymphocytes and / or γδ T cells may be cultured for several hours (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 18, or 21 hours) to about 35 days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 In one embodiment, lymphocytes and / or γδ T cells are expanded for 10 days (for 30 days, 31 days, 32 days, 33 days, 34 days, or 35 days). is expanded for 14 to 21 days. Therefore, (e.g., 1 to 40 days, e.g., 14 to 21 days) The isolation and expansion process, including the isolation culture period, is, in some embodiments, for 28 to 56 days. Or it can last for about 41 days.

[0090] In further embodiments, the expansion comprises incubating the γδ T cells for at least 5 days, at least 6 days, At least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 1 1 day, at least 12 days, at least 13 days, at least 14 days, at least 21 days, At least 28 days or longer, for example, 5 to 40 days, 7 to 35 days, 14 to In one embodiment, the expansion step comprises culturing γδ T cells for about 28 days, or for about 21 days. The cells are cultured for at least 10, 15, or 20 days to produce an expanded population. In one embodiment, the expansion step comprises culturing the γδ T cells for 5 to 25 days, for example, 14 to 21 days. In a further embodiment, the expansion step comprises culturing the γδ T cells for about 20 days. This includes culturing the cells for a period of time.

[0091] In some embodiments, IL-2 effective to generate an expanded population of γδ T cells. Typical doses range from 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, for example, 5 IU / mL to 10 IU / mL , 10IU / mL~20IU / mL, 20IU / mL~30IU / mL, 30IU / mL~40IU / mL, 40IU / mL~50IU / mL, 50IU / m L~60IU / mL, 60IU / mL~70IU / mL, 70IU / mL~80IU / mL, 80IU / mL~90IU / mL, 90IU / mL~100IU / mL, 100IU / mL~120IU / mL, 120IU / mL~140IU / mL, 140IU / mL~150IU / mL, 150IU / mL~175IU / mL, 175IU / mL~200IU / mL, 200IU / mL~300IU / mL, 300IU / mL~400IU / mL, 400IU / mL~500IU / mL, 500IU / mL~1,000IU / mL, 1,000IU / mL~1,500IU / mL, 1,500IU / mL~2,000IU / mL, or In some embodiments, the expanded population of γδ T cells is produced. The effective amount of IL-2 to stimulate proliferation is approximately 100 IU / mL.

[0092] In some embodiments, expanded γδ T cells (e.g., skin-derived γδ T cells and and / or non-V52 T cells, e.g., V51 T cells and / or DN T cells). A typical amount of effective IL-15 is at least 0.1 ng / mL (e.g., 0.1 ng / mL to 10,000 ng / mL, 1.0 ng / mL to 20,000 ng / mL). / mL~1,000ng / mL, 5ng / mL~800ng / mL, 10ng / mL~750ng / mL, 20ng / mL~500ng / mL, 50ng / mL ~400ng / mL, or 100ng / mL to 250ng / mL, for example, 0.1ng / mL to 1.0ng / mL, 1.0ng / mL to 5.0ng / mL mL, 5.0ng / mL~10ng / mL, 10ng / mL~20ng / mL, 20ng / mL~50ng / mL, 50ng / mL~100ng / mL, 10 0ng / mL to 200ng / mL, 200ng / mL to 500ng / mL, or 500ng / mL to 1,000ng / mL). In one embodiment, the amount of IL-15 effective to generate an expanded population of γδ T cells is It is about 10ng / mL.

[0093] In some embodiments, γδ T cells (e.g., skin-derived γδ T cells and / or non-V and the administration of IL-21 effective to generate a population of Vδ1 T cells and / or DN T cells. A typical amount is at least 0.1 ng / mL, for example at least 1.0 ng / mL (e.g., 0.1 ng / mL to 1 ,000ng / mL, 1.0ng / mL~100ng / mL, 1.0ng / mL~50ng / mL, 2ng / mL~50ng / mL, 3ng / mL~10ng / mL, 4ng / mL~8ng / mL, 5ng / mL~10ng / mL, 6ng / mL~8ng / mL, e.g. 0.1ng / mL~10ng / 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). Further embodiments In the present invention, the amount of IL-21 is typically at a concentration of less than 100 ng / mL, e.g., less than 50 ng / mL. In some embodiments, the method involves administering IL-1 at a concentration of about 6 ng / mL, e.g., about 6.25 ng / mL. Includes 21.

[0094] In further embodiments, the methods defined herein typically involve at least 0. 1 ng / mL, e.g., at least 10 ng / mL (e.g., 0.1 ng / mL to 1,000 ng / mL, 1.0 ng / mL to 100 ng / mL) mL, 1.0ng / mL~50ng / mL, 2ng / mL~50ng / mL, 3ng / mL~40ng / mL, 4ng / mL~30ng / mL, 5ng / mL ~20ng / mL, 10ng / mL~20ng / mL, e.g. 0.1ng / mL~50ng / mL, 1.0ng / mL~25ng / mL, 5ng / m In a further embodiment, the method as defined herein comprises administering to the patient an IL-4 concentration of 100 ng / mL to 25 ng / mL. The method typically involves administering IL-1 at a concentration of less than 100 ng / mL, for example less than 50 ng / mL, in particular less than 20 ng / mL. In some embodiments, the method includes IL-4 at a concentration of about 15 ng / mL.

[0095] Substitute for other factors in the expansion of non-hematopoietic tissue-resident lymphocytes and / or γδ T cells Additional antibodies are also provided herein. For example, in some embodiments, 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 and stromal cell-derived factor-1 (SDF-1), In addition to or instead of any one of IL-2 and IL-15. Such additional or alternative factors for the expansion of lymphocytes, such as NK cells, are disclosed in the art. In one embodiment, such factors selectively stimulate the expansion of γδ T cells. In a further embodiment, such factors are used in promoting the expansion of αβ For use in selectively enhancing the expansion of lymphocytes such as T cells and / or NK cells do.

[0096] Each of the above cytokines required to generate an expanded population of γδ T cells It will be understood that the amount 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 decreases accordingly, respectively. As noted above, an amount effective to produce an expanded population is defined herein as In , it refers to the combined effect of all factors on cell expansion.

[0097] The expansion method provides a population of expanded γδ T cells that are greater in number than the reference population. In some embodiments, the expanded population of γδ T cells comprises isolated γδ T cells prior to the expansion step. are greater in number than the population of cells (e.g., compared to the population of isolated γδ T cells prior to the expansion step) , at least twice as many, at least three times as many, at least four times as many, at least five times as many, At least six times as many, at least seven times as many, at least eight times as many, at least nine times as many, at least At least 10 times as many, at least 15 times as many, at least 20 times as many, at least 25 times as many, at least At least 30 times as many, at least 35 times as many, at least 40 times as many, at least 50 times as many, at least At least 60 times as many, at least 70 times as many, at least 80 times as many, at least 90 times as many, at least At least 100 times as many, at least 200 times as many, at least 300 times as many, at least 400 times as many, At least 500 times as many, at least 600 times as many, at least 700 times as many, at least 800 times as many , at least 900 times as many, at least 1,000 times as many, at least 5,000 times as many, at least 1 0,000 times more).

[0098] In one embodiment, the expansion step comprises exposing the isolated γδ T cells to substantial stromal cell contact. In a further embodiment, the expansion step comprises culturing the isolated γδ The method includes culturing the T cells in the absence of substantial fibroblast contact.

[0099] In a further embodiment, the expansion step comprises culturing the isolated γδ T cells in the presence of IL-4. Thus, in one embodiment, the expansion further comprises: In one embodiment, the expansion is performed in the presence of IL-2, IL-15, IL-4, and IL-21. The method may include culturing the isolated γδ T cells in the presence of IL-9, IL-15, IL-4, and IL-21. do.

[0100] The expansion methods provided herein can be used to expand other lymphocytes (e.g., αβ T cells and / or NK cells). It will be understood that the present invention also applies to the enlargement of The process comprises providing the isolated lymphocytes with relevant growth factors and / or nutrients (e.g., cytokines, αβ T The method includes producing a population of immune cells (cells and / or NK cells).

[0101] In one embodiment, the method for expanding a population of γδ T cells defined herein comprises: Culturing the T cells or other lymphocytes in serum-free medium. Thus, the method for expanding a population of γδ T cells defined herein comprises: This involves culturing the cells in a medium containing a serum-free or serum-replacement-containing medium. It is understood that the expansion of such γδ T cells in the subsurface may confer similar benefits to those described above. It will be.

[0102] In some embodiments, substantial TCR pathway activation is absent during the expansion process ( (e.g., no exogenous TCR pathway activators are included in the culture). A major step involves the absence of exogenous TCR pathway agonists. is a method for expanding γδ T cells isolated according to the method defined herein, The expansion method involves contact with feeder cells, tumor cells, and / or antigen-presenting cells. Thus, in a further embodiment of the method defined herein, Cell expansion involves culturing γδ T cells in the absence of substantial stromal cell contact.

[0103] Also provided are large non-hematopoietic tissue-derived γδ T cells (e.g., skin-derived γδ T cells). and / or non-V52 T cells, e.g., V51 T cells and / or DN T cells) (e.g., stromal By removing cell contact and / or TCR stimulation or by culturing in the presence of an effective amount of a factor. In some embodiments, the present invention provides a method for producing the β-glucosidase inhibitor (β-glucosidase inhibitor) at high rates (by culturing the β-glucosidase inhibitor). The expansion process described in expands γδ T cells with a low population doubling time given by the following formula: Larger:

number

[0104] Given the information provided herein, the skilled artisan will appreciate that the present invention is directed to the treatment of non-hematopoietic tissue-derived γδ T cells (e.g., skin-derived γδ T cells and / or non-Vδ2 T cells, e.g., Vδ1 T cells and / or DNA T cells) for less than 5 days (e.g., less than 4.5 days, less than 4.0 days, less than 3.9 days, less than 3.8 days, less than 3.7 days). , Less than 3.6 days, Less than 3.5 days, Less than 3.4 days, Less than 3.3 days, Less than 3.2 days, Less than 3.1 days, Less than 3.0 days, 2 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 Less than days, Less than 2.1 days, Less than 2.0 days, Less than 46 hours, Less than 42 hours, Less than 38 hours, Less than 35 hours, 32 One would recognize that this method would expand the population with a population doubling time of less than 1 hour.

[0105] In some embodiments, within 7 days of culture, an expanded population of γδ T cells (e.g., For example, a population of expanded Vδ1 T cells and / or DN T cells may be derived from isolated γδ T cells prior to expansion. At least 10-fold more γδ T cells (e.g., isolated γδ T cells prior to expansion) than the population of At least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, fold, at least 60 times, at least 70 times, at least 80 times, at least 90 times, at least 10 times 0x, at least 150x, at least 200x, at least 300x, at least 400x, at least 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 at least 5,000 times, at least 6,000 times, at least 7,000 times, or at least 8,000 times as many In some embodiments, the expanded γδ T cells are A population of Vδ1 T cells (e.g., an expanded population of Vδ1 T cells and / or DN T cells) may be obtained by dividing the Vδ1 T cells by the number of T cells prior to expansion. At least 20-fold more γδ T cells are obtained (e.g., expanded) than the isolated γδ T cell population. at least 30-fold, at least 40-fold, at least At least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, at least 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 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 In some embodiments, the number of γδ T cells is at least 9,000, or at least 10,000 times higher. Within 21 days of culture, expanded populations of γδ T cells (e.g., expanded Vδ1 The population of isolated γδ T cells and / or DN T cells was smaller than the population of isolated γδ T cells before expansion. at least 50-fold greater number of γδ T cells (e.g., compared to the population of isolated γδ T cells prior to expansion) , at least 60 times, at least 70 times, at least 80 times, at least 90 times, at least 100 times times, at least 150 times, at least 200 times, at least 300 times, at least 400 times, at least 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 At least 5,000 times, at least 6,000 times, at least 7,000 times, at least 8,000 times, at least In some embodiments, the number of γδ T cells is at least 9,000, or at least 10,000 times higher. Within 28 days of culture, expanded populations of γδ T cells (e.g., expanded Vδ1 T cells) and / or DN T cell populations) are at least At least 100-fold more γδ T cells (e.g., compared to the population of isolated γδ T cells prior to expansion) were obtained. 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 At least 6,000 times, at least 7,000 times, at least 8,000 times, at least 9,000 times, at least 10,000-fold, at least 12,000-fold, or at least 15,000-fold greater numbers of γδ T cells).

[0106] Non-hematopoietic tissue-derived γδ T cells expanded by the methods provided herein (e.g., cutaneous Skin-derived γδ T cells and / or non-Vδ2 T cells, e.g., Vδ1 T cells and / or DN T cells, In some embodiments, the cells may have a phenotype well suited for anti-tumor efficacy. The expanded population of γδ T cells (e.g., skin-derived Vδ1 T cells) can be used as a reference population (e.g., expansion process have a greater mean expression of CD27 than previously isolated populations of γδ T cells. In one embodiment, the expanded population of γδ T cells is compared to the isolated population of γδ T cells. at least twice as many T cells as isolated γδ T cell populations (e.g., at least 3x, at least 4x, at least 5x, at least 6x, at least 7x, at least 8x, At least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 25 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 times, at least 300 times, at least 400 times, at least 500 times, at least 600 times, at least At least 700 times, at least 800 times, at least 900 times, at least 1,000 times, at least 5,000 times , at least 10,000-fold, at least 20,000-fold, or more) do.

[0107] Expanded populations of γδ T cells (e.g., skin-derived γδ T cells and / or non-Vδ2 T cells, e.g. For example, a unique portion of Vδ1 T cells and / or DN T cells can upregulate CD27. Meanwhile, another part is CD27 low or CD27 negative In this case, isolated γδ T cells CD27 in the expanded population compared to the control population positive The frequency of cells is greater For example, the expanded population of γδ T cells may be a population of isolated γδ T cells prior to expansion. A frequency of CD27 that is at least 5% greater than the frequency of positive Cells (e.g., isolated cells before expansion) at least 10%, at least 15%, at least 20% compared to the frequency of the γδ T cell population obtained %, at least 25%, at least 30%, at least 35%, at least 40%, at least 45 %, at least 50%, at least 60%, at least 70%, at least 80%, at least 90 %, or up to 100% greater frequency of CD27 positive In some embodiments, the cells may include In the expanded population, CD27 compared with the isolated γδ T cell population positive cell For example, the expanded population of γδ T cells may be increased in number by increasing the number of isolated γδ T cells prior to expansion. At least twice as many CD27 positive It may have cells. The population of γδ T cells was >10%, >20%, >30%, >40%, >50%, >60%, >70%, >80% Alternatively, the expanded γδ T cell population may have a frequency of CD27+ cells of greater than 90%, or greater than 90%. , about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% of the time In one embodiment, the expanded population of γδ T cells may have a concentration of 50 % frequency of CD27+ cells.

[0108] The expansion methods provided herein, in some embodiments, include a reference population (e.g., The expanded γδ T cell population has low expression of TIGIT compared to the isolated γδ T cell population before the expansion step. non-hematopoietic tissue-derived γδ T cells (e.g., skin-derived γδ T cells and / or non-Vδ2 T cells, e.g., In some embodiments, the expanded T cells produce a population of T cells (e.g., V51 T cells and / or DN T cells). The expanded population of γδ T cells can be compared to a reference population (e.g., isolated γδ T cells prior to the expansion step). In some embodiments, the expanded population has a lower average expression of TIGIT than the expanded population. The population of γδ T cells is at least 10% smaller than the population of isolated γδ T cells (e.g. , at least 20% less than the isolated population of γδ T cells, at least 30% less than the isolated population of γδ T cells, At least 40% less, at least 50% less, at least 60% less, at least 70% less None, at least 80% less, at least 90% less, or up to 100% less) The expanded γδ T cell population has an even expression of <90%, <80%, <70%, <60% have a frequency of TIGIT+ cells of less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% Alternatively, the expanded population of γδ T cells may be about 90%, about 80%, about 70%, about 60%, about In some embodiments, the TIGIT+ cells may have a frequency of about 50%, about 40%, about 30%, about 20%, or about 10%. In some cases, the isolated population of γδ T cells has a frequency of TIGIT+ cells below 80%.

[0109] In some embodiments, expanded γδ T cells (e.g., skin-derived γδ T cells or A population of non-V52 T cells, e.g., V51 T cells and / or DN T cells, may be associated with a high or frequent CD4 T cell population. 27 + Cells and low frequency of TIGIT + In some embodiments, the expanded γ The population of γδ T cells is compared to a reference population (e.g., the population of isolated γδ T cells prior to expansion). (compared to) high frequency of CD27 + TIGIT - For example, an expanded population of γδ T cells may include A frequency of CD27 that is at least 5% greater than the frequency of the isolated γδ T cell population before expansion + TIGIT - cells (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or up to 100% greater frequency of CD27 + TIGIT - cell) In some embodiments, the expansion of the γδ T cells relative to the isolated population of γδ T cells may be achieved. CD27 in selected populations + TIGIT - The number of cells can be increased, for example by expanding γδ T cells. The cell population contained at least twice as many CD27 T cells as the isolated γδ T cell population before expansion. + TIGIT - cells (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or up to 100% greater frequency of CD27 + TIGIT - cell) may have:

[0110] Optionally, expanded γδ T cells (e.g., skin-derived γδ T cells and / or non-Vδ2 T cells) are CD27 in populations of T cells (e.g., Vδ1 T cells and / or DN T cells) + In the γδ T cell population The average expression of TIGIT is low compared to the reference population. D27 + The population of γδ T cells can be determined by comparing it with a reference population (e.g., isolated CD27 T cells prior to the expansion step). + γδ T cells In some embodiments, the expanded population has a lower average expression of TIGIT than the expanded population. CD27 + A population of γδ T cells was isolated using CD27 + At least 10% smaller than the γδ T cell population (e.g., isolated CD27 + At least 20% less than the population of γδ T cells At least 30% less, at least 40% less, at least 50% less, at least 60% less , at least 70% less, at least 80% less, at least 90% less, or up to 100 % less) on average expression of TIGIT.

[0111] Additionally or alternatively, expanded γδ T cells (e.g., skin-derived γδ T cells and / or non- TIGIT in a population of V52 T cells, e.g., V51 T cells and / or DN T cells - Enrichment of γδ T cells The median expression of CD27 in the expanded TIGIT - γδ The T cell population was isolated from TIGIT - At least, compared with the frequency of the γδ T cell population 5% greater frequency of CD27 + Cells (e.g., isolated TIGIT cells before expansion) - Frequency of γδ T cell population At least 10%, at least 15%, at least 20%, at least 25% less than the at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least at least 60%, at least 70%, at least 80%, at least 90%, or up to 100% greater Frequency of CD27 + In some embodiments, the isolated TIGIT - γδ T CD27 in the expanded population compared to the cell population + The number of cells can be increased. For example, Expanded TIGIT - The γδ T cell population was isolated from TIGIT - γδ T cell population and At least twice as many CD27 + Cells (e.g., isolated TIGIT cells before expansion) - γδ T cells at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or up to 10 0% greater frequency of CD27 + The present invention may include a method for producing a cellular endothelial cell.

[0112] 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 In addition, increased or decreased expression of other markers, including CCR4, CD69, PD-1, and CD64, may also be detected. Alternatively, one or more expanded non-hematopoietic tissue-derived γδ T cells (e.g., skin-derived γδ T cells) may be used. and characterizing a population of V51 T cells and / or non-V52 T cells, e.g., V51 T cells and / or DN T cells. Optionally, expanded γδ T cells (e.g., skin-derived γδ T cells and A population of V51 T cells and / or non-V52 T cells, e.g., V51 T cells and / or DN T cells, can be, for example, expanded. Greater CD124, CD215, CD360, CTLA4, and CD1b expression compared to previously isolated γδ T cell populations , BTLA, CD39, CD45RA, Fas ligand, CD25, ICAM-1, CD31, KLRG1, CD30, and CD2 In addition, or alternatively, the present invention has an average expression of one or more of the markers selected from the group consisting of: Instead, the expanded γδ T cell population was larger than the isolated γδ T cell population. CD124, CD215, CD360, CTLA4, CD1b, BTLA, CD39, CD45RA, Fas ligand, CD25, ICAM- 1, one or more of the markers selected from the group consisting of CD31, KLRG1, CD30, and CD2 In some embodiments, the expanded γδ T cells may have a frequency of cells expressing The population of γδ T cells expresses lower levels of NKp44, NKp46, ICAM-2, CD70, and NKp47 compared to isolated γδ T cell populations. A marker selected from the group consisting of CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1, and CD64. The expanded population has average expression of one or more of the following markers: Lower NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, and CC compared to the cytoplasmic population R4, CD69, PD-1, and CD64. The frequency of cells expressing the marker may also be measured.

[0113] Thus, the non-hematopoietic tissue resident γδ T cells produced by the methods of the present invention have the following characteristics: (i) CD69 high , TIM3 high , and CD28 low / absent Representation of (ii) upregulates one or more of CCR3, CD39, CD11b, and CD9; i) produce IFN-γ in response to NKG2D ligands in the absence of a TCR agonist; (iv) produce IFN-γ in response to a TCR agonist (v) in response to TCR activation, they produce IFN-γ, TNF-α, and GM (vi) produce one or more of the following in response to TCR activation: IL-17 or IL-20. (vii) grow in culture medium containing IL-2 without additional growth factors; (viii) exhibits a cytotoxic T cell response in the absence of a TCR agonist; and / or (ix) is normal It exhibits selective cytotoxicity against tumor cells rather than normal cells.

[0114] Optionally, the non-hematopoietic tissue resident γδ T cells produced by the methods of the invention are TCR antagonists. and / or produces IL-13 in the absence of a TCR agonist and / or produces NKG2D ligands in the absence of a TCR agonist. In response to this, they produce IFN-γ.

[0115] A number of basal culture media are suitable for use in the expansion of γδ T cells, in particular AIM-V, Iscoves medium, Media such as RPMI-1640 (Life Technologies) and RPMI-1640 (Life Technologies) are available. The medium may be supplemented with other media factors as defined in, for example, serum, serum proteins, and selective agents, antibiotics. For example, in some embodiments, 2 mM glutamine, 10% FBS, 10 mM H EPES, pH 7.2, 1% penicillin-streptomycin, sodium pyruvate (1 mM; Life Technologies echnologies), non-essential amino acids (e.g., 100 μM Gly, Ala, Asn, Asp, Glu, Pro, and Se 1×MEM non-essential amino acids (Life Technologies), and 10 μl / L β-mercaptoethanol In an alternative embodiment, the AIM-V medium contains RPMI-1640 medium containing 100% erythrocytes from CTS Immune. It may be supplemented with serum replacement and amphotericin B. Certain embodiments as defined herein Advantageously, the culture medium may be further supplemented with IL-2 and IL-15. During isolation and / or expansion, the cells were cultured in suitable culture medium in a humidified atmosphere containing 5% CO2 for 37 min. It is cultured at ℃.

[0116] According to a further aspect of the present invention, a method for isolating and expanding lymphocytes from a non-hematopoietic tissue sample is provided. There is: (i) isolating a population of lymphocytes from said non-hematopoietic tissue sample according to the methods defined herein. and (ii) further culturing the population of lymphocytes (e.g., for at least 5 days) to produce expanded lymphocytes; Producing a population of spheres A method is provided that includes:

[0117] In one embodiment, the lymphocytes comprise αβ T cells. According to the present invention, there is provided a method for isolating and expanding αβ T cells from a non-hematopoietic tissue sample, comprising: (i) isolating a population of αβ T cells from said non-hematopoietic tissue sample according to the methods defined herein. and (ii) further culturing the population of αβ T cells (e.g., for at least 5 days) to obtain expanded αβ T cells. Producing a population of T cells A method is provided that includes:

[0118] The culturing in step (ii) may, for example, allow the αβ T cells to differentiate from other cells present in the isolated population of step (i). This is due to selective growth, achieved by choosing culture conditions that favor the expansion of the cytoplasmic form over the cytoplasmic form. Alternatively, the growth conditions may be non-selective and may be selective for the growth of non-target cells following the culture of step (ii). Alternatively, the expansion conditions may be selective for the removal of cells (e.g., cells other than αβ T cells). However, removal of non-target cells (e.g., cells other than αβ T cells) is performed prior to the culture in step (ii). The goal of these embodiments is to expand the total number of αβ T cells while also increasing the number of subpopulations. Note that the aim of the present invention is to increase the proportion of

[0119] In one embodiment, the lymphocytes comprise NK cells. According to the present invention, there is provided a method for isolating and expanding NK cells from a non-hematopoietic tissue sample, comprising: (i) isolating a population of NK cells from a non-hematopoietic tissue sample according to the methods defined herein. Process; and (ii) further culturing the population of NK cells (e.g., for at least 5 days) to obtain an expanded population of NK cells. Producing a population A method is provided that includes:

[0120] The culturing of step (ii) may, for example, be performed to differentiate NK cells from other cell types present in the isolated population of step (i). This was due to selective expansion by selecting culture conditions that favored expansion over Alternatively, the expansion conditions may be non-selective and may allow the non-target cells ( For example, cells other than NK cells may be removed. Alternatively, the expansion conditions may be non-selective. In addition, removal of non-target cells (e.g., cells other than NK cells) is performed prior to the culture in step (ii). The objective of these embodiments is to expand the total number of NK cells while also decreasing their proportion in the population. Note that the goal of this study is to increase the number of

[0121] In one embodiment, the lymphocytes comprise γδ T cells. According to the present invention, there is provided a method for isolating and expanding γδ T cells from a non-hematopoietic tissue sample, comprising: (i) isolating a population of γδ T cells from a non-hematopoietic tissue sample according to the methods defined herein; and (ii) further culturing the population of γδ T cells (e.g., for at least 5 days) to obtain expanded γδ T cells. Producing a population of T cells A method is provided that includes:

[0122] The culturing in step (ii) may, for example, allow the γδ T cells to differentiate from other cells present in the isolated population of step (i). This is due to selective expansion by selecting culture conditions that favor the expansion of the cytoplasmic form over the cytoplasmic form. Alternatively, the expansion conditions may be non-selective and may be selective to allow the non-target cells to grow after the culture in step (ii). Alternatively, the expansion conditions may be selective for the removal of non-γδ T cells (e.g., cells other than γδ T cells). However, removal of non-target cells (e.g., cells other than γδ T cells) is performed prior to the culture in step (ii). The goal of these embodiments is to expand the total number of γδ T cells while also increasing the number of subpopulations. Note that the aim of the present invention is to increase the proportion of

[0123] Thus, according to a further aspect of the invention, isolation of γδ T cells from a non-hematopoietic tissue sample is provided. and a magnification method, (i) isolating a population of γδ T cells from a non-hematopoietic tissue sample according to the methods defined herein; and (ii) detecting the population of γδ T cells; (a) IL-2 or IL-9; (b) IL15; and (c)IL-21 In a culture medium containing 100% γδ T cells, the 100% γδ T cells are cultured for at least 5 days in the presence of: Cultivation process A method is provided that includes:

[0124] In one embodiment of this aspect of the invention, the culture of the population of γδ T cells is in the presence of IL-4. Thus, in a further aspect of the invention, the γδ 1. A method for isolating and expanding T cells, comprising: (i) isolating a population of γδ T cells from a non-hematopoietic tissue sample according to the methods defined herein; and (ii) detecting the population of γδ T cells; (a) IL-2 or IL-9; (b) IL15; and (c) IL-21; and (d) IL-4 In a culture medium containing 100% γδ T cells, the 100% γδ T cells are cultured for at least 5 days in the presence of: Cultivation process A method is provided that includes:

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

[0126] According to a further aspect of the present invention, a method for producing a composition comprising the steps of: An expanded isolated population of lymphocyte cells capable of being isolated from the host is provided.

[0127] According to yet a further aspect of the present invention, a method for treating a pulmonary circulation comprising administering to the patient a therapeutically effective amount of ... pulmonary circulation inhibitor obtained by any of the methods defined herein. An expanded isolated population of γδ T cells is provided that is capable of being administered to a subject.

[0128] According to yet a further aspect of the present invention, a method for treating a pulmonary circulation comprising administering to the patient a therapeutically effective amount of ... pulmonary circulation inhibitor obtained by any of the methods defined herein. In one embodiment, an isolated expanded population of γδ T cells is provided that is capable of reproducing the γδ T cells.

[0129] In one embodiment, the isolated population is greater than 50% γδ T cells, for example greater than 75% γδ In one embodiment, the isolated population comprises V δ1 cells, where less than 50%, e.g., less than 25%, of the Vδ1 cells express TIGIT. In an embodiment, the isolated population comprises V51 cells, wherein more than 50% of the V51 cells, e.g. For example, over 60% express CD27.

[0130] The lymphocytes and / or γδ T cells obtained by the method of the present invention can be used, for example, in adoptive T cell therapy. The recombinant human leukocytes obtained by the method of the present invention can be used as a medicine for the treatment of leukemia. The therapy involves the transfer of lymphocytes and / or γδ T cells into the patient. i.e., the γδ T cells may be transplanted back into the same patient from which they were obtained, or The therapy may be allogeneic, i.e., using γδ T cells from a given individual. They may be transplanted into a different patient. In cases involving allogeneic transplants, γδ T cells may act as αβ T cells. For example, αβ T cells may be, for example, after expansion, Using any suitable means known in the art (e.g., using magnetic beads, e.g., negative selection, The therapeutic method may involve removing γδ T cells from the γδ T cell population; Providing a sample of non-hematopoietic tissue; culturing and expanding γδ T cells from said sample. and administering the expanded population of γδ T cells to a recipient individual. and

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

[0132] Tissue-resident V51 T and DN γδ T cells normally reside in non-hematopoietic tissues; Its systemic blood-resident counterparts could also home to and be retained within the tumor mass. The adoptive transfer of these cells has been shown to be more effective than conventional immunotherapy for solid tumors and potentially other non-hematopoietic tissue-associated diseases. It may be more effective to target associated immunopathology.

[0133] Because γδ T cells are not MHC restricted, they do not recognize the host into which they are transplanted as a foreign body. This means that γδ T cells are less likely to cause graft-versus-host disease. In some cases, γδ T cells can be used “off the shelf” to, for example, provide allogeneic adoptive T cells. This means that the cells can be transplanted into any recipient for therapy.

[0134] The non-hematopoietic tissue-resident γδ T cells obtained by the method of the present invention express NKG2D and inhibit malignant tumors. Responds to NKG2D ligands (e.g., MICA) that are strongly associated with tumors. Non-hematopoietic tissue-resident γδ T cells also express a cytotoxic profile in the absence of any activation, and therefore inhibit tumor cells. For example, the method may be effective in killing the . Non-hematopoietic tissue-resident γδ T cells respond to IFN-γ, TNF-α, GM-CSF, and IL-1 in the absence of any activation. , CCL4, IL-13, granulysin, granzymes A and B, and perforin One or more, preferably all, of the IL-17A and IL-20 may be expressed.

[0135] The findings reported herein therefore support the present invention as a novel method for the preparation of non-hematopoietic tissue normal cells. On the feasibility and suitability of clinical application of resident γδ T cells as "off-the-shelf" immunotherapy reagents We provide strong evidence that these cells possess innate-like killing and are not MHC restricted. and exhibit improved homing to and / or retention within tumors compared to other T cells.

[0136] In some embodiments, the method for treating an individual having a tumor in a non-hematopoietic tissue comprises: providing a sample of said non-hematopoietic tissue obtained from an individual; culturing γδ T cells from said sample; and culturing the expanded population of γδ T cells to produce an expanded population of γδ T cells. The method may include administering to an individual

[0137] A pharmaceutical composition may comprise one or more pharma- ceutical or physiologically acceptable carriers, diluents, or excipients. may comprise the expanded non-hematopoietic tissue resident γδ T cells described herein in combination with Such compositions may include buffers, such as neutral buffered saline, phosphate buffered saline, and the like; Carbohydrates, such as glucose, mannose, sucrose, or dextran, mannitol proteins; polypeptides or amino acids, e.g., glycine; antioxidants; chelating agents, For example, EDTA or glutathione; adjuvants (for example, aluminum hydroxide); and preservatives. Examples of cryoprotective solutions that may be used in the pharmaceutical composition of the present invention include DMSO. The composition can be formulated, for example, for intravenous administration.

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

[0139] Optionally, the therapeutic utility of expanded γδ T cells obtained by any of the above methods. A therapeutically effective amount is administered to a subject (e.g., for the treatment of cancer, e.g., for the treatment of solid tumors). Optionally, expanded γδ T cells (e.g., skin-derived γδ T cells) can be administered in a single dose. A therapeutically effective amount of V51 T cells and / or non-V52 T cells, e.g., V51 T cells and / or DN T cells) is 10 x 10 per dose 12 Fewer than 10 cells (e.g., 9 x 10 per dose) 12 < 1 cell per dose 8×10 12 Less than 7 x 10 cells 12Less than 6 x 10 cells per dose 12 Less than 1 cell, dose equivalent 5×10 12 Less than 4 x 10 cells per dose 12 Less than 3 x 10 cells per dose 12 Less than one cells, 2 x 10 per dose 12 Less than 1 x 10 cells per dose 12 < 1 cell per dose 9×10 11 Less than 8 x 10 cells per dose 11 Less than 7 x 10 cells per dose 11 Less than one piece Cells, 6 x 10 per dose 11 Less than 5 x 10 cells per dose 11 < 4 x 1 cells per dose 0 11 Less than 3 x 10 cells per dose 11 Less than 2 x 10 cells per dose 11 Less than one cell, 1 x 10 per dose 11 Less than 9 x 10 cells per dose 10 Less than 7.5 x 10 cells per dose 1 0 Less than 5 x 10 cells per dose 10 Less than 2.5 x 10 cells per dose 10 Less than one cell, 1 x 10 per dose 10 Less than 7.5 x 10 cells per dose 9 Less than 5 x 10 cells per dose 9 Less than 2.5 x 10 cells per dose 9 Less than 1 x 10 cells per dose 9 Less than one cell, dose Winnings 7.5 x 10 8 Less than 5 x 10 cells per dose 8 Less than 2.5 x 10 cells per dose 8 Not yet available Fully 1 x 10 cells per dose 8 Less than 7.5 x 10 cells per dose7 < 1 cell per dose 5×10 7 Less than 2.5 x 10 cells per dose 7 Less than 1 x 10 cells per dose 7 Less than one piece Cells, 7.5 x 10 per dose 6 Less than 5 x 10 cells per dose 6 Less than 2.5 cells per dose ×10 6 Less than 1 x 10 cells per dose 6 Less than 7.5 x 10 cells per dose 5 Less than one cell , 5 x 10 per dose 5 Less than 2.5 x 10 cells per dose 5 Less than 1 cell or per dose ×10 5 (less than 100 cells).

[0140] In some embodiments, expanded γδ T cells (e.g., skin-derived γδ T cells and A therapeutically effective amount of V5 T cells, e.g., V51 T cells and / or non-V52 T cells, e.g., V51 T cells and / or DN T cells, is administered over a course of treatment. in progress, 10×10 12 Fewer than 9 × 10 cells (e.g., during the course of treatment) 12 Less than 8 x 10 cells 12 Not yet available Full Cell, 7 x 10 12 Less than 6 x 10 cells 12 Less than 5 x 10 cells 12 Less than 4 x 10 cells 12 Less than 3 x 10 cells 12 Less than 2 x 10 cells 12 Less than 1 x 10 cells 12 Less than 9x cells 10 11 Less than 8 x 10 cells 11 Less than 7 x 10 cells 11 Less than 6 x 10 cells 11 Less than one cell , 5×10 11Less than 4 x 10 cells 11 Less than 3 x 10 cells 11 Less than 2 x 10 cells 11 Less than one cells, 1 x 10 11 Less than 9 x 10 cells 10 Less than 7.5 x 10 cells 10 Less than 5 x 10 cells 10 pieces Less than 2.5 x 10 cells 10 Less than 1 x 10 cells 10 Less than 7.5 x 10 cells 9 Less than 5 cells ×10 9 Less than 2.5 x 10 cells 9 Less than 1 x 10 cells 9 Less than 7.5 x 10 cells 8 Less than one piece cells, 5×10 8 Less than 2.5 x 10 cells 8 Less than 1 x 10 cells 8 Less than 7.5 x 10 cells 7 Less than one cells, 5 × 10 7 Less than 2.5 x 10 cells 7 Less than 1 x 10 cells 7 Less than 7.5 x 10 cells 6 pieces Less than 5 × 10 cells 6 Less than 2.5 x 10 cells 6 Less than 1 x 10 cells 6 Less than 1 cell, 7.5 x 1 0 5 Less than 5 x 10 cells 5 Less than 2.5 x 10 cells 5 Less than 1 x 10 cells or 1 x 10 5 Less than one cell ).

[0141] In some embodiments, the expanded non-hematopoietic tissue resident γδ T The cell dose was approximately 1 x 10 6 , 1.1×10 6 , 2×10 6 , 3.6×106 , 5×10 6 , 1×10 7 , 1.8×10 7 , 2 ×10 7 , 5×10 7 , 1×10 8 , 2×10 8 , or 5 × 10 8 In some embodiments, and expanding non-hematopoietic tissue-resident γδ T cells (e.g., skin-derived γδ T cells and / or non-Vδ2 The dose of T cells, e.g., V51 T cells and / or DN T cells, is at least about 1×10 6 , 1.1×1 0 6 , 2×10 6 , 3.6×10 6 , 5×10 6 , 1×10 7 , 1.8×10 7 , 2×10 7 , 5×10 7 , 1×10 8 , 2×10 8 , Or 5 x 10 8 In some embodiments, the expanded non-hematopoietic tissue resident gamma δ T cells (e.g., skin-derived γδ T cells and / or non-V52 T cells, e.g., V51 T cells and and / or DN T cells) is administered at a dose of up to about 1 × 10 6 , 1.1×10 6 , 2×10 6 , 3.6×10 6 , 5×10 6 , 1×10 7 , 1.8×10 7 , 2×10 7 , 5×10 7 , 1×10 8 , 2×10 8 , or 5 × 10 8 Contains cells / kg. In an embodiment, expanded non-hematopoietic tissue resident γδ T cells (e.g., skin-derived γδ T cells and / or non-V52 T cells, e.g., V51 T cells and / or DN T cells), is about 1.1×10 6 ~1.8×10 7 In some embodiments, the expanded non-hematopoietic tissue resident gamma δ T cells (e.g., skin-derived γδ T cells and / or non-V52 T cells, e.g., V51 T cells and and / or DN T cells) is about 1×10 7 , 2×10 7 , 5×10 7 , 1×10 8 , 2×10 8 , 5×10 8 , 1×10 9 , 2×10 9 , or 5 × 10 9 In some embodiments, the expanded non-hematopoietic tissue comprises Resident γδ T cells (e.g., skin-derived γδ T cells and / or non-Vδ2 T cells, e.g., Vδ1 T cells) The dose of the immunization target cells (immunoglobulins and / or DN T cells) is at least about 1×10 7 , 2×10 7 , 5×10 7 , 1×10 8 , 2×10 8 , 5×10 8 , 1×10 9 , 2×10 9 , or 5 × 10 9 In some embodiments, the cells are expanded. non-hematopoietic tissue-resident γδ T cells (e.g., skin-derived γδ T cells and / or non-Vδ2 T cells, For example, the dose of Vδ1 T cells and / or DN T cells) may be up to about 1×10 7 , 2×10 7 , 5×10 7 , 1×1 0 8 , 2×10 8 , 5×10 8 , 1×10 9 , 2×10 9 , or 5 × 10 9Contains cells.

[0142] In one embodiment, the subject is administered 10 4 ~10 6 Expanded non-hematopoietic tissue Resident γδ T cells (e.g., skin-derived γδ T cells and / or non-Vδ2 T cells, e.g., Vδ1 T cells) In one embodiment, the subject is administered non-hematopoietic tissue resident γδ T cells. An initial administration of a population of T cells (e.g., 10 per kg body weight of the subject) 4 ~10 6 Priming of γδ T cells For example, 10 per kg of subject's body weight 4 ~10 5 γδ T cells), and expanded non-hematopoietic tissue One or more (e.g., 2, 3, 4, or 5) subsequent administrations of resident γδ T cells (e.g., per kg body weight of the subject) 10 per 4 ~10 6 expanded non-hematopoietic tissue resident γδ T cells, e.g., per kg body weight of the subject 10 4 ~10 5 and one or more subsequent administrations of expanded non-hematopoietic tissue-resident γδ T cells. In an embodiment, the one or more subsequent administrations are administered less than 15 days after the previous administration, e.g., 14, 13, 12 , 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days, e.g., 4, 3, or 2 days after the previous administration In one embodiment, the subject is administered at least three doses of the population of γδ T cells. During the course of treatment, a total of approximately 10 6 γδ T cells, e.g., the subject , 1×10 5 Initial administration of γδ T cells, 3 × 10 5 A second administration of 6 x 10 γδ T cells, and 5 pieces and a third administration of γδ T cells, e.g., each administration being 4, 3, or 2 days after the previous administration. It is administered in less than a day.

[0143] The non-hematopoietic tissue resident γδ T cells obtained by the methods of the invention have enhanced therapeutic properties, e.g. For example, they can be genetically modified for CAR-T therapy, which allows for new specificities, e.g. Altered T cell receptors to reprogram T cells with monoclonal antibody specificity The modified TCR is then specific to malignant cells and is therefore useful for cancer immunotherapy. Useful T cells can be produced. For example, T cells can be generated by normal somatic cells derived from a target tissue. The present invention is capable of recognizing tumor antigens that are not expressed by the human tumor, such as cancer cells that express tumor-associated antigens. Therefore, CAR-modified T cells can be used, for example, in adoptive T cell therapy for cancer patients. can.

[0144] The use of blood-resident γδ T cells for CAR has been described. The resulting non-hematopoietic tissue-resident γδ T cells are transduced with chimeric antigen-specific TCRs. On the other hand, it can retain its native-like ability to recognize transformed cells and can inhibit blood-resident γδ have better tumor penetration and retention capabilities than either T cells or conventional systemic αβ T cells This may be a particularly good vehicle for a CAR-T approach, as Furthermore, their lack of MHC-dependent antigen presentation reduces the potential for graft-versus-host disease. This allows for targeting of tumors that express low levels of MHC. For example, its independence from conventional costimulation through CD28 engagement allows for low levels of The targeting of tumors expressing ligands for the co-stimulatory receptors of the IL-1 receptor is enhanced.

[0145] In some embodiments, one or more additional therapeutic agents can be administered to the subject. The additional therapeutic agent may be an immunotherapeutic agent, a cytotoxic agent, a growth inhibitory agent, a radiation therapeutic agent, an anti-angiogenic agent, or a combination of two or more of these agents. The antibody may be administered simultaneously with, prior to, or after administration of the γδ T cells. The therapeutic agent is targeted to the subject's body (e.g., the subject's own immune system) and / or to the transplanted γδ It may be an immunotherapeutic agent capable of acting on T cells.

[0146] Administration of the compositions can be performed in any convenient manner. The composition can be administered by intraarterial, subcutaneous, intradermal, intratumoral, intranodal, intramedullary, intramuscular, intravenous injection, or It can be administered to a patient intraperitoneally, for example, by intradermal or subcutaneous injection. Compositions of hematopoietic tissue-resident γδ T cells can be injected directly into tumors, lymph nodes, or sites of infection. can be done.

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

[0148] As used herein, the term "about" means any number of units within a specified range. Including values ​​10% greater than the specified value and values ​​10% less than the specified value. Values ​​up to and including the specified value, preferably values ​​up to and including 5% greater than the specified value "Between" includes values ​​5% less than the specified value and above, specifically including the specified value. The term " " is inclusive of the specified range of values.

[0149] Specific aspects and embodiments of the present invention will now be described, by way of example and with reference to the above-mentioned drawings, in which: , I will explain. EXAMPLES

[0150] (Example) (Example 1. Analysis method) Unless otherwise stated, the following methods were used to produce the results in the following examples.

[0151] (Flow Cytometry) The following antibody-fluorophore conjugates: Ki-67-BV421, CD3-BV510, Vδ1-PeVio770, TIM-3- Flow cytometry was performed using PE, CD9-PE, CCR3-BV421, and CD39-BV421. Samples were also stained for viability using eFluor770NIR. Commercially available antibodies were used from Biolegend or Milte. Viability dye (near IR) was from eBioscience. Ki-67 staining Colors are from cells fixed and permeabilized with Foxp3 staining buffer set (eBioscience). After each experiment, the cell population was washed in PBS and split in half. To determine viability, the cells were stained with eFluor770 NIR, washed, and then stained with TrueStain (Biolegend). Half of the samples were stained for the indicated surface markers to avoid non-specific binding of the staining antibodies. One half was stained for lineage markers only (CD3, Vδ1) and the other half for equivalent surface markers. Isotype controls were stained using matched mouse IgG conjugated to the same fluorophore. Isotype controls were used at the same concentrations as previously described. Therefore, it indicates non-specific binding or false positives. The histograms are When compared to type control or presented, this is shown. Data summaries are presented in comparative Positive for the displayed markers compared, and therefore at higher levels than the isotype The percentage of cells stained with IgG is shown. Flow cytometry data analysis ,This was carried out using FLOWJO (version 10.1).

[0152] The initial and final phenotype of each cell population, including expression of CD27 and TIGIT, was also determined by the mean fluorescence intensity. The median light intensity (MFI) was used to determine the intensity of the light.

[0153] (Population analysis) Using the methods described herein, skin-resident lymphocytes were isolated. Among CD45+ cells, Anti-CD3 was used to stain T cells, and anti-CD56 was used to stain NK cells, CD3- CD56+. Within CD3+ cells, we identified skin-resident γδ T cells using an antibody against the pan-γδ T cell receptor. Anti-CD8α was used to identify and differentiate conventional CD4 and CD8 positive αβ TCR-gated CD3+, pan-γδ TCR-gated The proportion of T cells was identified.

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

[0155] Example 2. Isolation of lymphocytes from human skin samples A three-dimensional skin explant protocol was established, which is described herein. Tantalum-coated reticulated vitreous carbon scaffolds (GlcNAC) with dimensions of 1.5 mm were Autoclave and clean the lid (Ultramet, California, USA) or equivalent. Then, they were completely immersed in PBS before use.

[0156] 1L of AIM-V medium (Gibco, Life Technologies), 50mL of serum replacement from CTS Immune (Life Technologies) echnologies), human recombinant IL-2 (Miltenyi Biotech, Cat no 130-097-746), and human recombinant Contains IL-15 (Miltenyi Biotech, Cat no 130-095-766) for measuring 3 cytokines (3CK) Human recombinant IL-21 (Miltenyi Biotech, Cat no 130-095-784) and 4 cytokine (4CK) assay The complete kit also contained the following concentrations of human recombinant IL-4 (Miltenyi Biotech, Cat no 130-093-922) for determination: All isolation media was prepared. For the first 7 days of culture, 10 mL of Amphotericin B (250 μg / mL, Life Sciences) was added. A complete isolation medium containing AMP was used ("+AMP"). The target final concentrations of caine are as follows: Table 1: Final concentrations of cytokines in complete isolation medium [Table 1]

[0157] Adult skin samples were obtained, shipped and processed within 48 hours of collection. The hair was removed from the sample with a scalpel and tweezers. The skin sample was placed with the epidermis side facing up. Using an appropriate sized punch biopsy, hold the skin around the biopsy with sterile forceps. , cut the skin.

[0158] Three biopsies were placed epidermis side up, evenly spaced, and one tantalum-coated charcoal Using sterile tweezers, the grid was placed in a 30 mL complete isolation flask. Wells of a G-REX 6-well plate (Wilson Wolf Manufacturing) containing medium (+AMP) In a tissue culture vessel with a gas-permeable membrane of 100 mL or 300 mL of GR containing complete isolation medium (+AMP), One grid was transferred into a G-REX EX100 bioreactor (Wilson Wolf Manufacturing). Place three grids in each well of a 6-well plate or place three grids in a G-REX10 bioreactor. or place 10 grids in a G-REX 100 bioreactor. The cultures were incubated at 37°C in a 5% CO2 incubator. Incubated.

[0159] Unless otherwise noted, gently aspirate the top medium and replace with 2x complete isolation medium (no AMPs). and avoid disturbing the cells on the bottom of the plate or bioreactor. It was changed every 7 days.

[0160] To isolate lymphocytes, the grid containing the skin was placed in a G-REX 6-well plate or a G-REX 10 or removed from the G-REX100 bioreactor and discarded for disposal. The cells at the bottom of the bioreactor were resuspended and transferred to a 500 mL centrifuge tube. followed by centrifugation (eg, 300 g for 10 min).

[0161] When cell counts are required, lymphocytes are counted as described in Example 1. The results of an exemplary study are shown in Table 2: Table 2. Yield of isolated lymphocytes per donor [Table 2]

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

[0163] Total cell yield and percentage of γδ T cells and Vδ1 cells were determined as described in Example 1. The results are shown in Figure 1. The use of the four cytokines in the isolation significantly increased cell yield. The results showed that the number of isolated γδ T cells and Vδ1 cells was increased by 1.0 μg / mL of γδ T cells and 1.0 μg / mL of Vδ1 cells. The results shown are based on the cell yield and the isolated γδ T cells and They also showed that it can increase the numbers of Vδ1 cells and Vδ2 cells.

[0164] Isolation was performed by measuring TIGIT and CD27 expression using the method described in Example 1. The phenotype of the isolated Vδ1 cells was analyzed. Vδ1 cells with low TIGIT expression and high CD27 expression It is believed that the phenotype is desirable. The results are shown in Figures 3 and 4. Overall, , compared to cells isolated with two cytokines, four cytokines and three cytokines. Cells isolated with cytokines had low TIGIT expression and high CD27 expression.

[0165] Example 4. Optimization of punch biopsy size Early studies have shown that 3 mm punch biopsy is superior to standard skin comminution. (Figure 5).

[0166] Punch biopsy sizes of 1 mm, 2 mm, 3 mm, 4 mm, and 8 mm were tested, and 2 The optimal punch biopsy size was further investigated by using 1 mm explants as a control. Skin samples were prepared as described in Example 2. One biopsy of each size was included in the table. Test by attaching the skin, skin facing up, to the surface of a carbon grid and a 24-well plate. (Corning) wells. Each well contained AIM-V 10% human AB serum + IL-1 at the above concentration. 2 and IL-15 with standard concentrations of β-mercaptoethanol (2ME) and penicillin / streptomycin It contained syn (P / S).

[0167] The medium was changed three times a week (half medium change) and the biopsies were incubated at 37 °C in a 5% CO2 incubator. The cells were incubated at 4°C for 21 days before cell harvesting and cell yield analysis.

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

[0169] The percentage of γδ T cells present in the cell yield was determined as described in Example 1. The results are presented in Figure 6. These results indicate that biopsies with a diameter of 3 mm provided the greatest yield. These results suggest that γδ T cells are more likely to be involved in the development of inflammatory bowel disease.

[0170] Example 5. Optimization of isolation vessel Isolation in 24-well plates was performed using G-REX 6-well plates (Wilson Wolf Manufacturing). The skin samples were compared to those using a container containing any gas-permeable material as described in Example 2. Biopsies were attached to the surface of a carbon grid with the epidermis facing up, as described in The carbon grid is then placed into a well of a 24-well plate or a G-REX 6-well plate. A 9 mm grid was used for the 24-well plate and a 20 mm grid for the G-REX 6-well plate. All samples were in AIM-V 10% AB serum + P / S + 2ME + IL-2 and IL-15. For 4-well plates, the medium was changed three times a week. For G-REX 6-well plates, The biopsies were incubated at 37°C in a 5% CO2 incubator until the cells were fully grown. The cells were incubated for 21 days before cell yield analysis.

[0171] Total cell yields per plate and per biopsy were determined as described in Example 1. Experiments have shown that the G-REX 6-well plate can hold up to 1000 samples per biopsy when compared to the 24-well plate. It was shown that the G-REX6 expression level increased the cell yield per plate and the number of cells increased (Figure 7 and Table 4). Although well plates allowed for an increased amount of tissue to be cultured (compared to 24-well plates), By contrast, the plates yielded a remarkable 25-fold increase in cell numbers (2.5-fold more tissue per plate). Table 4. Total cell yields obtained with 24-well plates vs. G-REX 6-well plates [Table 4]

[0172] The use of G-REX vessels was demonstrated in the 2-cytokine, 3-cytokine, and 4-cytokine isolation protocols. TIGIT and CD27 expression was measured using the method described in Example 1. We analyzed the phenotype of Vδ1 cells by comparing PD-1 expression with that of isolated αβ T cells (CD3+, The results are shown in Figures 8 and 9. These results suggest that G-REX Vδ1 cells isolated using four cytokines in the G-REX vessel were isolated using two G-REX vessels. Compared to Vδ1 cells isolated with other cytokines, they showed lower TIGIT expression and higher While αβ T cells isolated using four cytokines had low CD27 expression, two subtypes Compared with αβ T cells isolated using cytokines, these cells had lower PD-1 expression. It is supported.

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

[0174] A comparison of different media was tested. Biopsies were placed on the grid: Contains 5% human AB serum and IL-2 / IL-15 (2CK) or IL-2 / IL-15 / IL-21 / IL-4 (4CK) AIM-V; or 10% fetal calf serum (FCS) and IL-2 / IL-15 (2CK) or IL-2 / IL-15 / IL-21 / IL-4 (4CK) Contains SKIN-T The cells were cultured in 24-well plates containing either

[0175] The biopsies were incubated at 37°C in a 5% CO2 incubator for 14 days (AIM-V) or 21 days (AIM-V) prior to cell yield analysis. The total cell yield per grid was determined as described in Example 1. The results are shown in Figure 10. Isolation in AIM-V for a shorter period of time Even so, it resulted in better cell yields and higher overall V51 cell numbers.

[0176] The duration of cell isolation was also tested. 3 mm punch biopsies were placed on grids and cultured as described in Example 2. The cultures were placed in G-REX 6-well plates or G-REX 10 bioreactors as described in the literature. The assay was performed using AIM-V (containing 5% serum replacement (SR), 5% human AB serum, or a 5% SR / 5% AB "blend"). The cells were cultured in a 5% CO2 incubator at 37°C, and the cell yield was analyzed. The total cell yield per grid was determined as described in Example 1. The results are shown in Figure 11. For all media types, Isolation after 3 weeks improved cell yield when compared to isolation after 2 weeks.

[0177] The use of serum replacements compared to human AB serum (5% or 10%) was also tested. Biopsies were incubated in 5% CO2 incubator. The cells were incubated at 37°C in an incubator for 21 days before cell analysis. Cell yield and % of V51 cells were measured as described in Example 1. The results are shown in Figure 12. Improved cell viability was observed using medium supplemented with 5% serum replacement compared to human AB serum. Improved cell yields and a higher proportion of Vδ1 cells were obtained.

[0178] Example 7. Cell expansion Once cells have been isolated using the above protocol, they can be cultured using methods known in the art. For example, selective expansion of γδ T cells can be achieved using the method described in WO2017072367. This can be achieved using the enlargement methods described in.

[0179] The expansion of γδ T cells with additional cytokines was also tested. Skin tissues isolated using either two cytokines (2CK) or four cytokines (4CK) were Lymphocytes were harvested after 21 days of culture. The harvested cells were cultured in 5% serum replacement and human serum albumin. Cultured in TexMACs (Miltenyi Biotech) medium containing recombinant IL-2, IL-4, IL-15, and IL-21. The cell types were analyzed using FACS as described in Example 1. The results were: The use of four cytokines at the time of isolation is shown in Figure 13. The use of two cytokines at the time of isolation This resulted in a larger population of γδ T cells after expansion compared to the use of

[0180] By measuring the expression of various markers using the methods described in Example 1. The phenotype of Vδ1 cells was then analyzed. The results are shown in Figure 14. Use of IgA and subsequent expansion compared to cells isolated using two cytokines , giving rise to cells with greater CD27 expression.

[0181] The total number of γδ cells and Vδ1 cells per grid was measured as described in Example 1. The results are shown in Figure 15. The use of four cytokines at isolation significantly increased the number of Vδ 1 cell yield.

Claims

【Claim 1】 A novel article, method, and manufacturing method substantially described in the present specification.

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