Method for expanding and culturing gamma delta T cells

Abstract

The present invention relates to a method for producing γδ T cells. Compared with the currently known general γδ T cell culture methods or culture methods using feeder cells, the present invention has the advantage that γδ T cells having high cell-killing ability and cell survival rate can be produced with high purity and high efficiency in a short period of time by a clinically friendly method, thereby increasing the productivity of allogeneic γδ T cell immunotherapeutic agents.

Description

[Technical field]

[0001] The present invention relates to a method for producing γδ T cells, and more specifically, to a method for producing γδ T cells, which is characterized by proliferating and culturing γδ T cells based on the suppression of androgen signaling.

[0002] [Background technology]

[0003] T cells, a type of lymphocyte, are the mainstay of adaptive immunity together with B cells. T cells are generated through a process in which precursors made from hematopoietic cells undergo maturation in the thymus. T cells are composed of a majority of αβT cells expressing αβT cell receptors (TCRs) that circulate in the thymus and peripheral lymphoid tissues, and a small number of γδT cells expressing γδT cell receptors (Non-Patent Document 1). Tissue-resident γδT cells present in epithelial tissues exhibit a very different pattern, and in some sites, γδT cells may account for the majority or entirety of the cells (Non-Patent Document 2). Such epithelial tissue-resident γδT cells exhibit a restricted or constant pattern of T cell receptor expression, a characteristic different from that of T cells present in lymphoid tissues.

[0004] γδ T cells are produced in the thymus and are the T cells that migrate to other sites the earliest, with most of them migrating to skin, intestines, lungs, and epithelial tissues before birth (Non-Patent Document 3). γδ T cells do not have a diversity of T cell receptors, but they recognize and respond to various biological damages inflicted on epithelial tissues. However, little is known about the self-antigens that induce such responses. Activated γδ T cells are known to regulate inflammation, destroy cells with altered cellular phenotypes, and improve wound healing (Non-Patent Document 3). In addition, recent research results have shown that γδ T cells act as regulators that maintain and restore homeostasis in tissues exposed to stimuli from the external environment (Non-Patent Document 1).

[0005] γδ T cells exist in a very small proportion of human peripheral blood, about 0.5-5%, and are known to be functionally involved in both types of immune responses: adaptive immunity and innate immunity. γδ T cells, which play an important role in immune responses, are known to react to phosphoantigens produced by cancer cells and bacteria.

[0006] Human γδ T cells have two major TCR chain subtypes, Vδ1 chain and Vδ2 chain (Non-Patent Document 4), and most γδ T cells in the blood have Vδ2 chain, which is paired with Vγ9 chain. TCR γδ T cells develop in the thymus before TCR αβ T cells and have limited TCR diversity against some antigens including phosphoantigens, but they induce an immune response earlier than TCR αβ T cells to induce an innate-like immune response. In particular, γδ T cells, like αβ T cells, have a stronger anticancer effect by cytolysis, but unlike αβ T cells, which are the main cause of graft-versus-host disease (GVHD) against cells expressing non-self MHC, they are known to have no MHC restriction and do not cause graft-versus-host disease (GVHD) (Non-Patent Document 5).

[0007] Therefore, gamma delta T cells are a promising candidate cell group for anti-cancer immune cell therapy, and since it was discovered that gamma delta T cells proliferate specifically when treated with interleukin-2 (IL-2) and zoledronic acid (ZA, ZOL), their therapeutic effects on various cancers have been demonstrated in preclinical and clinical trials. The development of cancer immunotherapy methods, including haploidentical transplantation using gamma delta T cells and the expansion of gamma delta T cells with zoledronic acid and interleukin-2, is expected to maximize the therapeutic efficacy of refractory high-risk patients.

[0008] Most of the γδ T cells present in the body are present in peripheral blood at a very low rate of about 0.5 to 5%, and in order to actually use γδ T cells for therapeutic purposes, a large amount of activated γδ T cells is required, so methods for culturing γδ T cells isolated from blood in large quantities outside the body are being actively researched. In addition to zoledronic acid and IL-2 used in existing cell proliferation / activation, methods using IL-15 (Patent Documents 1 and 2), methods using PHA (phytohaemagglutinin), TGF-β, concanavalin A, amphotericin B, etc. (Patent Documents 3 to 6) have been developed for culturing γδ T cells, but these have only found new proliferation substances in the form of modifications and developments of the previously used IL-2 use, and have not presented any groundbreaking or improved methods. In addition, cases have been reported in which γδ T cells were expanded using feeder cells. When feeder cells are used, specific cancer cell lines, rather than normal cells, are transformed or transduced and used for the mass expansion of γδ T cells (Non-Patent Document 6).

[0009] This method is not suitable for ensuring safety, which is important for clinical application, and has the limitation of producing γδ T cells with priming specificity for specific cancer cells.

[0010] Therefore, the inventors made extensive efforts to develop a method for mass-producing and culturing TCR γδ T cells, and as a result, they confirmed that when undifferentiated T cells isolated from human peripheral blood mononuclear cells are cultured in a medium containing the 5α-reductase inhibitor finasteride as an active ingredient in addition to the previously known stimulants zoledronic acid and IL-2, the growth of γδ T cells increases and the cell yield improves, thereby completing the present invention.

[0011]

[0012]

[0013] [Prior art documents] [Patent documents]

[0014] [Patent Document 1] U.S. Patent No. 11,135,245B

[0015] [Patent Document 2] European Patent Registration No. 3,154,567B

[0016] [Patent Document 3] U.S. Patent No. 10,370,452B

[0017] [Patent Document 4] U.S. Patent No. 10,557,117B2

[0018] [Patent Document 5] WO2020-172555 publication

[0019] [Patent Document 6] U.S. Patent Publication No. 2019-0175650A

[0020]

[0021] [Non-patent literature]

[0022] [Non-Patent Document 1] Jameson J.,Havran WL,Immunol.Rev.,215:114-22.,2007

[0023] [Non-Patent Document 2] Allison JP, Havran WL, Annu.Rev.Immunol.,9:679-705,1991

[0024] [Non-Patent Document 3] Komori HK, Meehan TF, Havran WL, Curr. Opin. Immunol., 18(5):534-8, 2006

[0025] [Non-Patent Document 4] Kabelitz D., Marischen, L., Oberg, H.-H., Holtmeier, W.; Wesch, D. Int. Arch. Allergy Immunol., 137, 73-81, 2005

[0026] [Non-Patent Document 5] Sharma A., Zumwalde NA, Gumperz, J., In Advanced Structural Safety Studies; Humana Press Inc.: New York, NY, USA, pp.57-72, 2019

[0027] [Non-Patent Document 6] Tan WK,Tay JCK,Zheng J.,Zheng M.,Wang S.,J.Immunol Sci.,2(3):6-12,2018

[0028]

[0029] Summary of the Invention

[0030] An object of the present invention is to provide a method for producing γδ T cells, which can increase the growth of γδ T cells and improve the cell recovery rate.

[0031] Another object of the present invention is to provide a medium composition for inducing and culturing γδ T cells, which can improve the growth of γδ T cells and the cell yield.

[0032] To achieve the above object, the present invention provides a method for producing γδ T cells, comprising the steps of:

[0033] (a) culturing peripheral blood mononuclear cells (PBMCs) in a medium containing (i) zoledronic acid, (ii) IL-2, and (iii) a 5α-reductase inhibitor or an androgen receptor antagonist to induce and culture γδ T cells; and

[0034] (b) obtaining the cultured γδ T cells.

[0035] The present invention also provides a medium composition for inducing and culturing γδ T cells, comprising (i) zoledronic acid, (ii) IL-2, and (iii) a 5α-reductase inhibitor or an androgen receptor antagonist.

[0036] [Brief description of the drawings]

[0037] [Figure 1] When culturing gamma delta T cells using peripheral blood mononuclear cells derived from male donors, basic culture components and finasteride were continuously treated at different concentrations, and the proliferation rate and survival rate of gamma delta T cells over a 14-day culture period were compared and analyzed.

[0038] [Diagram 2] When culturing gamma delta T cells using peripheral blood mononuclear cells derived from female donors, basic culture components and finasteride were continuously treated at different concentrations, and the proliferation rate and survival rate of gamma delta T cells over a 14-day culture period were compared and analyzed.

[0039] [Diagram 3]This shows the results of confirming the proliferation and survival rate of γδ T cells by treatment with finasteride and IL-15 using peripheral blood mononuclear cells derived from male donors. Finasteride was administered at a high concentration (10 nM) for up to 7 days, and IL-15 was administered for a 14-day culture period. The proliferation and survival rate of γδ T cells from male donors (A) and the increased number of γδ T cells after 14 days of culture compared to the control group (B) were compared.

[0040] [Figure 4] This shows the results of confirming the proliferation and survival rate of γδ T cells by treatment with finasteride and IL-15 using peripheral blood mononuclear cells derived from female donors. Finasteride was administered at a low concentration (0.01 nM) for up to 7 days, and IL-15 was administered for a 14-day culture period. The proliferation and survival rate of γδ T cells from female donors (A) and the increased number of γδ T cells after 14 days of culture compared to the control group (B) were compared.

[0041] [Diagram 5] This shows the results of confirming the proliferation and survival rate of γδT cells by treatment with a mixture of finasteride (10 nM) and cytokines using peripheral blood mononuclear cells derived from male donors. (A) shows the results of comparing the cell proliferation and survival rate of γδT cells over a 14-day period, and (B) shows the results of analyzing the ratio of cultured γδT cells and αβT cells.

[0042] [Figure 6] This shows the results of investigating the proliferation and survival rate of γδ T cells after treatment with a mixture of finasteride (10 nM) and cytokines using peripheral blood mononuclear cells derived from male donors. (A) is the result of a comparative analysis of the number of γδ T cells after 14 days of culture, and (B) is the result of an analysis of the proportion of γδ T cells using a flow cytometer compared to the control group after 14 days of culture.

[0043] [Figure 7]This shows the results of investigating the proliferation and survival rate of γδT cells by treatment with a mixture of finasteride (0.01 nM) and cytokines using peripheral blood mononuclear cells derived from female donors. (A) shows the results of comparing the proliferation and survival rate of γδT cells over a 14-day period, and (B) shows the results of analyzing the ratio of cultured γδT cells and αβT cells.

[0044] [Figure 8] This shows the results of investigating the proliferation and survival rate of γδ T cells after treatment with a mixture of finasteride (0.01 nM) and cytokines using peripheral blood mononuclear cells derived from female donors. (A) is the result of a comparative analysis of the number of γδ T cells after 14 days of culture, and (B) is the result of an analysis of the proportion of γδ T cells using a flow cytometer compared to the control group after 14 days of culture.

[0045] [Figure 9] A shows the results of a comparative analysis of the number of γδ T cells over a 14-day period after peripheral blood mononuclear cells from a healthy donor were treated with finasteride for 7, 10, and 14 days following the start of culture, and B shows the results of an analysis of the ratio of γδ T cells and αβ T cells cultured for up to 14 days after treatment using the above culture method.

[0046]

[0047] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein is one that is well known and commonly used in the art.

[0049]

[0050] In the present invention, we attempted to develop a method for explosively proliferating γδ T cells from peripheral blood mononuclear cells in a short period of time, and confirmed that the proliferation rate and survival rate of γδ T cells can be increased when peripheral blood mononuclear cells are cultured in a serum medium containing an androgen inhibitor such as a 5α-reductase inhibitor or an androgen receptor antagonist.

[0051] Thus, in one aspect, the present invention relates to a method for producing γδ T cells, comprising the steps of:

[0052] (a) culturing peripheral blood mononuclear cells (PBMCs) in a medium containing (i) zoledronic acid, (ii) IL-2, and (iii) a 5α-reductase inhibitor or an androgen receptor antagonist to induce and culture γδ T cells; and

[0053] (b) Obtaining cultured γδ T cells.

[0054] Zoledronic acid used in the present invention is a bisphosphonate drug and is used to treat hypercalcemia caused by cancers such as Paget's disease, metastatic breast cancer, parathyroid gland tumors, and multiple myeloma. Zoledronic acid has a similar structure to endogenous pyrophosphate, which regulates blood calcium levels, but unlike existing bisphosphonates (clodronate, etidronate, tiludronate, etc.), it has a nitrogen-containing imidazole ring, and is therefore characterized by its strong effect in inhibiting bone resorption. Zoledronic acid inhibits calcium resorption into the blood by chelating with calcium from hydroxyapaptite, a component of the bone matrix. It also inhibits farnesyl diphosphate synthase, which prevents prenylation of guanine nucleotide-binding proteins (G-proteins) in osteoclasts, and induces apoptosis of osteoclasts that promote bone resorption, thereby reducing the number of osteoclasts and regulating blood calcium levels. Zoledronic acid is rapidly absorbed from the blood into bones and stored in the bones, and has a long half-life in bones, so it can sustain bone resorption with a single intravenous injection. However, bisphosphonates in the blood have a very short half-life of 0.5 to 2 hours and are excreted through the kidneys without being metabolized, so dosage adjustment is required for patients with kidney damage.

[0055] In the present invention, 5α-reductase is a biocatalyst that reduces the male hormone testosterone to dihydrotestosterone (DHT), and there are three types: Type 1, Type 2, and Type 3. Abnormalities in Type 1 result in reduced bone weight and muscle mass, while abnormalities in Type 2 cause typical intersex symptoms. Abnormalities in Type 3 result in abnormalities in cell growth and vision, and reduced production of testosterone, androstenedione, and progesterone.

[0056] 5α-reductase inhibitors are known to inhibit the conversion of substrates to their activated forms, decreasing the amount of activated substrates, which in turn increases the substrate levels somewhat. The mechanism of inhibition of 5α-reductase involves NADPH binding to the enzyme followed by the substrate. Specific substrates include testosterone, progesterone, androstene, epitestosterone, cortisol, aldosterone, and dioxycorticosterone.

[0057] Substrate + NADPH + H+ → 5α-Substrate + NADP+

[0058] 5α-reductase isoforms Type I and II reduce testosterone to dihydrotestosterone (DHT), progesterone to 5α-dihydroprogesterone (5α-DHP), and dioxycorticosterone to dihydrodioxycorticosterone (DHDOC).

[0059] The "finasteride" used in the present invention is a 5α-reductase inhibitor that inhibits the conversion of testosterone into the activated form of dihydrotestosterone. Dihydrotestosterone, the activated form of testosterone, is known to be involved in hair loss and prostatic hyperplasia. Therefore, 5α-reductase inhibitors that inhibit the production of dihydrotestosterone in tissues are used as therapeutic agents for prostatic hyperplasia, and are also used to prevent and treat hair loss.

[0060] In the present invention, the 5α-reductase inhibitor may be, but is not limited to, finasteride, dutasteride, epristeride, alphatradiol, saw palmetto extract, etc.

[0061] In the present invention, the androgen receptor antagonist may be, but is not limited to, bicalutamide, flutamide, nilutamide, etc.

[0062] The "5α-reductase inhibitor" used in the present invention is preferably finasteride.

[0063] In the present invention, the medium may further contain a costimulatory molecule. In the present invention, the costimulatory molecule that may be contained in the culture medium is a collective term for a peripheral cell membrane protein that appears on activated antigen-transmitting cells (APCs) and is capable of generating a costimulatory signal that increases or decreases MHC-TCR signaling between APCs and T cells by interacting with a surface membrane protein of a T cell. An antibody or a ligand may be used as the costimulatory molecule, and examples of the costimulatory molecule include 5A6.E9, Bl, TS8.2, 15D, B6, B3, TS-1, γ3.20, 7A5, IMMU510, R9.12, 11F2, or a combination thereof, phorbol 12-myristate-13-acetate (TPA), mezerein, staphylococcal enterotoxin A (SEA), streptococcal protein A, amphotericin B, and the like. B) or a combination thereof, αTCR, βTCR, γTCR, δTCR, CD277, CD28, CD46, CTLA4, ICOS, PD-1, CD30, NKG2D, NKG2A, HVEM, 4-1BB (CD137), OX40 (CD134), CD70, CD80, CD86, DAP, CD122, GITR, FceRIg, CD1, CD16, CD161, DNAX, accessory molecule-1 (DNAM-1), SLAM, coxsackievirus and adenovirus receptors or a combination thereof, and preferably, CD80, 4-1BB, CD86 or CD276 can be used, and the present invention is not limited to CD80 used in the examples of the present invention, and it is obvious to a person skilled in the art to which the present invention belongs that any costimulatory molecule that meets the purpose of the present invention can be used.

[0064] The concentration of the costimulatory molecule in the medium used for culturing the γδ T cells of the present invention is 1 to 2,000 nM, specifically 5 to 1,000 nM, more specifically 10 to 500 nM, and the concentration of the cytokine in the medium is 1 to 2,000 IU, specifically 10 to 1,000 IU, more specifically about 20 to 400 IU.

[0065] In the present invention, the cytokine that may be contained in the culture medium may be one or more selected from interleukins. Interleukin is a general term for proteinaceous biologically active substances produced by immunocompetent cells. The interleukins that can be used in the present invention may be one or more selected from the group consisting of interleukin-2 (IL-2), interleukin-15 (IL-15), and interleukin-18 (IL-18). The interleukins used in the present invention are not limited to IL-2, IL-15, and IL-18. It will be apparent to a person having ordinary skill in the art to which the present invention pertains that any cytokine that meets the objectives of the present invention may be used.

[0066] In the present invention, in the mass proliferation culture of TCR γδ T cells, finasteride, an androgen signal transduction inhibitor, is used while simultaneously treating with solredox acid and IL-2, and further, by using cytokines IL-15 and IL-18 and CD80 costimulatory molecule in the above method, mass proliferation of TCR γδ T cells can be achieved with higher efficiency. 6 ~3.0×10 6 It is necessary to maintain a cell concentration of 1000 cells / mL. By statically culturing γδ T cells while maintaining the above conditions, γδ T cells can be expanded and cultured in large quantities.

[0067] In one embodiment of the invention, a method for culturing γδ T cells is as follows:

[0068] (a) stimulating a peripheral blood mononuclear cell culture containing γδ T cells and then subjecting the culture to static culture;

[0069] (b) diluting the statically cultured cell culture solution to a certain cell ratio after a certain period of time and statically culturing the cell culture solution; and

[0070] (c) When the number of γδ T cells in the cell culture medium reaches a certain level, the culture medium containing the statically cultured cells is re-induced, and then a culture medium containing additional components is added and the cells are statically cultured.

[0071] After the above step (c), a step of collecting the obtained γδ T cells in large quantities may be further included.

[0072] In the present invention, reaction vessels that can be used for static culture include 24-well plates, 12-well plates, and 6-well plates, shaking flasks, T25 flasks, T75 flasks, T175 flasks, T225 flasks, and disposable cell culture bags, but are not limited to these. Any reaction vessel that can be easily adopted by a person having ordinary skill in the technical field to which the present invention belongs can be used.

[0073] In the present invention, "suitable culture conditions" means static culture of cells in an incubator at 37°C and 5% CO2.

[0074] In the present invention, the term "certain culture period" refers to a period of about 2 to 5 days during which γδ T cells proliferate to reach a certain ratio or number of cells.

[0075] In the present invention, a "certain ratio" or "certain number of cells" refers to a cell number that grows in a certain culture period and is 0.4 × 10 6 ~3.0×10 6 Mean number of γδ T cells with concentration in cells / mL.

[0076] In the present invention, "re-induction" refers to re-suspending aggregated cells in the culture medium containing the cells after a certain culture period using a micropipette or serological pipette, and further adding a mixture of cell culture medium and cytokines (cytokine mix) to re-induce γδ T cell proliferation.

[0077] In the present invention, the "cytokine mixture" refers to an additive that is a mixture of cytokines IL-15 and IL-18, CD80 costimulatory factor, and additional active ingredients that are further added after re-induction during the culture period to induce re-induction and cell proliferation.

[0078] In the present invention, the active ingredient is preferably finasteride, which is a 5α-reductase inhibitor, but is not limited thereto. Other active ingredients include dutasteride, epristeride, alpha-tradiol, saw palmetto extract, and androgen receptor antagonists (ARA), such as bicalutamide, flutamide, and nilutamide.

[0079] The basic culture medium used in the present invention may be Gibco CTS OpTmizer T cell Expansion Serum Free Media (Thermo-Fisher Scientific, Cat# A1048501), but is not limited thereto. Conventional T cell culture media such as Gibco CTS AIM-V medium (Thermo-Fisher Scientific), LymphoONE T cell expansion XFM (Takara), X-VIVO 10, -15 and -20 Hematopoietic Cell medium (Lonza), PRIME-SV T cell expansion XSFM (Fujifilm-Irvine Scientific), and RPMI-1640 medium, which are used in the culture of immune cells including T cells, may be used.

[0080] Finasteride used in the present invention may be contained in the γδ T cell induction and culture medium at a concentration of 0.001 to 100 nM, preferably 0.001 to 50 nM. The optimal concentration of finasteride added may differ depending on whether the peripheral blood mononuclear cells used for γδ T cell induction and culture are derived from a male or female.

[0081] In the present invention, when the peripheral blood mononuclear cells used for γδ T cell induction and culture are derived from a male, the γδ T cell induction and culture medium preferably contains 1 to 50 nM finasteride, and more preferably contains 1 to 20 nM finasteride.

[0082] In the present invention, when the peripheral blood mononuclear cells used for the induction and culture of γδ T cells are derived from a woman, the γδ T cell induction and culture medium preferably contains 0.001 to 10 nM finasteride, and more preferably contains 0.001 to 1 nM finasteride.

[0083] In the present invention, γδT is preferably cultured for a total of 3 to 21 days, more preferably 7 to 14 days.

[0084] According to the method for inducing and culturing γδT of the present invention, it is possible to obtain cells in which the ratio of γδT cells to the total cultured cells is 90% or more on the 14th day of culture.

[0085] In another aspect, the present invention relates to a medium composition for inducing and culturing γδ T cells, comprising (i) zoledronic acid, (ii) IL-2, and (iii) a 5α-reductase inhibitor or an androgen receptor antagonist.

[0086] In the present invention, the 5α-reductase inhibitor may be, but is not limited to, finasteride, dutasteride, epristeride, alphatradiol, saw palmetto extract, etc.

[0087] In the present invention, the androgen receptor antagonist may be, but is not limited to, bicalutamide, flutamide, nilutamide, etc.

[0088] The "5α-reductase inhibitor" used in the present invention is preferably finasteride.

[0089] In the present invention, the medium may further contain a costimulatory molecule. The costimulatory molecule that may be contained in the culture medium in the present invention is a collective term for a peripheral cell membrane protein that appears on activated antigen-transmitting cells (APCs) and can generate a costimulatory signal that increases or decreases MHC-TCR signaling between APCs and T cells by interacting with a surface membrane protein of a T cell. An antibody or a ligand can be used as the costimulatory molecule, and examples of the costimulatory molecule include 5A6.E9, Bl, TS8.2, 15D, B6, B3, TS-1, γ3.20, 7A5, IMMU510, R9.12, 11F2, or a combination thereof, phorbol 12-myristate-13-acetate (TPA), mezerein, staphylococcal enterotoxin A (SEA), streptococcal protein A, amphotericin B, and the like. B) or a combination thereof, αTCR, βTCR, γTCR, δTCR, CD277, CD28, CD46, CTLA4, ICOS, PD-1, CD30, NKG2D, NKG2A, HVEM, 4-1BB (CD137), OX40 (CD134), CD70, CD80, CD86, DAP, CD122, GITR, FceRIg, CD1, CD16, CD161, DNAX, accessory molecule-1 (DNAM-1), SLAM, coxsackievirus and adenovirus receptors or a combination thereof, and preferably, CD80, 4-1BB, CD86 or CD276 can be used, and the present invention is not limited to CD80 used in the examples of the present invention, and it is obvious to a person skilled in the art to which the present invention belongs that any costimulatory molecule that meets the purpose of the present invention can be used.

[0090] The concentration of the costimulatory molecule in the medium used for culturing the γδ T cells of the present invention is 1 to 2,000 nM, specifically 5 to 1,000 nM, more specifically 10 to 500 nM, and the concentration of the cytokine in the medium is 1 to 2,000 IU, specifically 10 to 1,000 IU, more specifically about 20 to 400 IU.

[0091] In the present invention, the cytokines that may be contained in the culture medium are one or more selected from interleukins. Interleukin is a general term for proteinaceous biologically active substances produced by immunocompetent cells. Interleukins that can be used in the present invention may be one or more selected from the group consisting of interleukin-2 (IL-2), interleukin-15 (IL-15), and interleukin-18 (IL-18). The interleukins used in the present invention are not limited to IL-2, IL-15, and IL-18. It will be apparent to those skilled in the art to which the present invention pertains that any cytokine that meets the objectives of the present invention can be used.

[0092]

[0093] The present invention will be described in more detail below with reference to examples. It will be apparent to those skilled in the art that these examples are merely for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention.

[0094]

[0095] Example 1. Confirmation of proliferation rate and survival rate of γδ T cells in healthy peripheral blood depending on finasteride concentration

[0096] 1-1.Human Peripheral Blood Mononuclear Cell (hPBMC) separation

[0097] For each 100 cc of healthy donor blood collected by a medical professional and placed in a heparin tube, four 50 mL conical tubes were filled with 25 mL of 1X DPBS and 25 mL of blood. 15 mL of Lymphoprep (Axis Shield, UK) was vertically dispensed into a new 50 mL conical tube, with care taken to avoid contact with the wall as much as possible, and the diluted blood was gradually placed on top of the 50 mL conical tube containing the Lymphoprep solution along the wall. The conical tube was placed in a centrifuge and centrifuged at 800 x g (1,890 rpm) for 20 minutes at room temperature. The white buffy coat layer between the Lymphoprep and plasma layers in the blood layers separated into four layers was collected and transferred to three or four new 50 mL conical tubes and centrifuged at 1,500 rpm (500 x g) for 10 minutes at room temperature. The cells separated into three or four 50 mL conical tubes were resuspended in 45 mL of 1X DPBS and then collected into one 50 mL conical tube. The collected cells in one tube were centrifuged twice at 1,250 rpm (350 x g), 10 min, and room temperature. The cell concentration was 1.0 x 10 6 To obtain a concentration of 1.0×10 cells / mL 7 Culture medium was added to the cell stock to a final volume of 10 mL, and after pipetting 2-3 times, 1.0 mL of the cells was dispensed into the Day 0 sample and cultured. The remaining cells were 1.0 × 10 7 The cells were dispensed into cryovials at a rate of 100 cells / mL and stored frozen in a nitrogen tank (LN2Tank), and were thawed and used when necessary.

[0098]

[0099] 1-2. Proliferation and survival rate of gamma delta T cells in peripheral blood mononuclear cells according to finasteride concentration

[0100] Peripheral blood mononuclear cells (PBMCs) isolated in 1-1 were cultured in 10 mL of 100 mL of 3 μM zoledronic acid, a basic component of γδ T cell culture. 3After continuous treatment with 1.1 IU / mL IL-2 and 0.01 nM and 10 nM finasteride as the active ingredient, the cells were cultured for a total of 14 days, and the activation and proliferation rates of γδ T cells were compared.

[0101] Frozen peripheral blood mononuclear cells were thawed in a 37°C water bath and then suspended in 9 mL OpTmizer medium (Themo-Fisher Scientific, USA) containing 10% heat inactivation fetal bovine serum (Heat inactivation Fetal Bovine Serum (FBS), Gibco, USA). The cell number was measured and centrifuged at 400x g for 4 minutes at 25°C. The cell pellet obtained by centrifugation was suspended in 5 mL of complete OpTmizer medium (10% fetal bovine serum + OpTmizer medium, cOpTmizer) to measure the cell number, and then the cells were resuspended at a concentration of 1.0 × 10 in cOpTmizer medium. 6 The cells were diluted to a concentration of 1000 cells / mL.

[0102] Total of 1.0 x 10 in a 24-well plate 6 10 cells (final volume 1 mL) were seeded in one well. To stimulate and induce differentiation of γδT cells, 0.01 nM and 10 nM finasteride, 3 μM zoledronic acid, IL-2 (10 3 IU / mL) was added and the mixture was cultured at 37°C in a CO2 incubator for 3 days.

[0103] Three days after the start of the culture, the aggregated cells were resuspended using a 1000P micropipette and resuspended in the same amount of cOpTmizer culture medium and 0.01 nM or 10 nM finasteride or IL-2 (10 3 IU / mL) was further added, and the cells were cultured at 37°C in a CO2 incubator for 2 days.

[0104] Two days after reinduction, the aggregated cells were resuspended using a 1000P micropipette and the cell number was measured. The same amount of cOpTmizer culture medium was used based on the cell culture medium during the culture, and 0.01 nM and 10 nM finasteride and IL-2 (103 IU / mL) was added and the cells were incubated at 37°C in a CO2 incubator.

[0105] Six days after the start of culture, the cells were cultured in half the amount of cOpTmizer culture medium based on the cell culture medium during culture, and 0.01 nM, 10 nM finasteride, and IL-2 (10 3 IU / mL) was added and the cells were incubated at 37°C in a CO2 incubator.

[0106] Seven days after the start of culture, the aggregated cells were resuspended using a 1000P micropipette or a 10 mL serological pipette, and the cell count was measured. 6 After diluting the cells in the cOpTmizer culture medium to a cell concentration of 10 3 IU / mL) was added and the mixture was cultured at 37°C in a CO2 incubator for 3 days.

[0107] Ten days after the start of the culture, the aggregated cells were resuspended using a 1000P micropipette or a 10 mL serological pipette, and the cell number was measured. 6 The cells were diluted with cOpTmizer culture medium to a cell concentration of 10 3 IU / mL) was added and the mixture was cultured at 37°C in a CO2 incubator for 2 days.

[0108] After 12 days from the start of culture, the same amount of cOpTmizer culture medium was added based on the cell culture medium in culture, and IL-2 (10 3 IU / mL) was added and the cells were incubated at 37°C in a CO2 incubator.

[0109] After 14 days from the start of the culture, the aggregated cells were resuspended using a 1000P micropipette or a 10 mL serological pipette, and the cell number was measured. In Example 1, the composition and ratio of cells including αβT cells and γδT cells were analyzed using the antibodies in Table 1 on the start day of the culture, and on the 5th, 7th, 10th, and 14th days using a Fortessa-X20 flow cytometry machine (BD Bioscience).

[0110] As a result, as shown in Figures 1 and 2, peripheral blood mononuclear cells isolated from male and female donors were treated with finasteride at concentrations of 0 nM, 0.01 nM, and 10 nM and cultured for 14 days. The male donors showed an increased proliferation rate of γδ T cells at high concentrations of finasteride compared to the control group treated with IL-2 and ZA only (Figure 1), while the female donors showed an increased proliferation rate of γδ T cells at low concentrations of finasteride, in contrast to the male donors (Figure 2).

[0111] [Table 1]

[0112]

[0113] Example 2. Proliferation and survival rate of γδ T cells in peripheral blood mononuclear cells by treatment with finasteride and IL-15

[0114] Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors and cultured in a γδ T cell culture medium containing 3 μM zoledronic acid (ZA) and 10 μM ethanol. 3 In addition to 100 IU / mL IL-2, the active ingredient finasteride was administered at a concentration of 10 nM in male donors or 0.01 nM in female donors for up to 7 days, and 0.8 nM IL-15 was administered continuously, and the activation and proliferation rates of γδ T cells were compared after a total of 14 days of culture.

[0115] Frozen peripheral blood mononuclear cells were thawed in a 37°C water bath and then suspended in 9 mL of OpTmizer medium (cOpTmizer, Thermo-Fisher Scientific, USA) containing 10% fetal bovine serum (FBS). The cell count was measured and the cells were centrifuged at 400x g for 4 minutes at 25°C. The cell pellet obtained by centrifugation was suspended in 5 mL of cOpTmizer medium, the cell count was measured, and the cells were then diluted to 1.0 x 10 6 The cells were diluted to a concentration of 1000 cells / mL.

[0116] 1.0 x 10 total cells in a 24-well plate 6 10 cells (final volume 1 mL) were seeded in one well. To stimulate and induce differentiation, γδT cells were cultured in the presence of 10 nM and 0.01 nM finasteride, 3 μM zoledronic acid (ZA), IL-2 (10 3 IL-15 (0.8 nM, 1.1 IU / mL) and 0.8 nM IL-15 were added, and the cells were cultured at 37°C in a CO2 incubator for 3 days.

[0117] Three days after the start of the culture, the aggregated cells were resuspended using a 1000P micropipette and the cell number was measured. 3 IU / mL), and 10 nM, 0.01 nM finasteride, and 0.8 nM IL-15 were further added to the male and female mice, respectively, and the mice were cultured at 37°C in a CO2 incubator for 2 days.

[0118] Two days after resuspension, the aggregated cells were resuspended using a 1000P micropipette and the cell count was measured. 3 IU / mL), and 10 nM, 0.01 nM finasteride, and 0.8 nM IL-15 were further added to the male and female mice, respectively, and the mice were cultured at 37°C in a CO2 incubator.

[0119] Six days after the start of culture, half the cOpTmizer culture medium was added based on the cell culture medium during culture, and 10 nM or 0.01 nM finasteride, IL-2 (10 3 IU / mL) and 0.8 nM IL-15 were further added, and the cells were cultured at 37°C in a CO2 incubator.

[0120] Seven days after the start of the culture, the aggregated cells were resuspended using a 1000P micropipette or a 10 mL serological pipette, and the cell number was measured. 6 After diluting the cells in the cOpTmizer culture medium to 10 cells / mL, IL-2 (10 3 IU / mL) and 0.8 nM IL-15 were further added and the cells were cultured at 37°C in a CO2 incubator for 3 days.

[0121] Ten days after the start of the culture, the aggregated cells were resuspended using a 1000P micropipette or a 10 mL serological pipette, and the cell number was then measured. 6 The cells were diluted with cOpTmizer culture medium to obtain a total of 10 cells / mL. The supplemented cOpTmizer culture medium contained IL-2 (10 3 IU / mL) and 0.8 nM IL-15 were further added and the cells were cultured at 37°C in a CO2 incubator.

[0122] After 12 days of culture, IL-2 (10 3 IU / mL) and 0.8 nM IL-15 were further added and the cells were cultured at 37°C in a CO2 incubator.

[0123] The next day, add IL-2 (10 mL) to half the cOpTmizer culture medium based on the cell culture medium during incubation. 3 IU / mL) and 0.8 nM IL-15 were further added and the cells were cultured at 37°C in a CO2 incubator.

[0124] After 14 days from the start of the culture, the aggregated cells were resuspended using a 1000P micropipette, and the cell number was measured. In Example 2, the composition and ratio of cells including αβT cells and γδT cells were analyzed using the antibodies in Table 1 with a Fortessa-X20 flow cytometry (BD Bioscience) on the start day of the culture, and on days 5, 7, 10, and 14.

[0125] As a result, as shown in Figures 3 and 4, the male donors showed a 1.26-fold (126%) increase in the proliferation rate of γδ T cells compared to the control group treated with IL-2 and ZA only (Figure 3), whereas the female donors, unlike the male donors, did not show much difference in the proliferation rate of γδ T cells (Figure 4).

[0126]

[0127] Example 3. Proliferation and survival of γδ T cells in healthy peripheral blood by treatment with finasteride, IL-15, IL-18, and CD80

[0128] Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors and cultured with γδ T cell culture basic components (3 μM zoledronic acid and 10 3 In addition to 100 IU / mL IL-2, men and women were treated once with 10 nM and 0.01 nM finasteride as the active ingredient on the initiation of culture, respectively, and the additional ingredient 6 nM IL-18 was treated once on the initiation of culture. 100 nM CD80 and 0.8 nM IL-15 were continuously treated for a total of 14 days of culture, and the activation and proliferation rates of γδ T cells were compared.

[0129] Frozen peripheral blood mononuclear cells were thawed in a 37°C water bath and then suspended in 9 mL of OpTmizer medium (cOpTmizer, Themo-Fisher Scientific, USA) containing 10% fetal bovine serum (FBS). The number of cells was measured and the cells were centrifuged at 400 x g for 4 minutes at 25°C. The cell pellet obtained by centrifugation was suspended in 5 mL of cOpTmizer medium (Themo-Fisher Scientific, USA) to measure the number of cells, and then diluted to 1.0 x 10 using cOpTmizer medium. 6 The cells were diluted to a cell concentration of 100 / mL.

[0130] Total 1.0 x 10 cells in a 24-well plate 6 1 mL of cells were dispensed per well. To induce γδT cell stimulation and differentiation, the active ingredient finasteride was treated at a concentration of 10 nM for male donors and 0.01 nM for female donors, and 3 μM zoledronic acid (ZA), IL-2 (10 3 IU / mL), 100 nM CD80, 0.8 nM IL-15, and 6 nM IL-18 were added, and the cells were cultured at 37°C in a CO2 incubator for 3 days.

[0131] Three days after the start of the culture, the aggregated cells were resuspended using a 1000P micropipette and the cell number was measured. 3 IU / mL), 100 nM CD80, and 0.8 nM IL-15 were further added, and the cells were cultured at 37°C in a CO2 incubator.

[0132] Four days after the start of culture, the aggregated cells were resuspended using a 1000P micropipette and the cell count was measured. Five days after the start of culture, the aggregated cells were resuspended using a 1000P micropipette and the cell count was measured. The same amount of cOpTmizer culture medium and IL-2 (10 3 IU / mL), 100 nM CD80, and 0.8 nM IL-15 were further added, and the cells were cultured at 37°C in a CO2 incubator.

[0133] Six days after the start of the culture, the aggregated cells were resuspended using a 1000P micropipette and the cell number was measured. 3 IU / mL), 100 nM CD80, and 0.8 nM IL-15 were further added, and the cells were cultured at 37°C in a CO2 incubator.

[0134] Seven days after the start of the culture, the aggregated cells were resuspended using a 1000P micropipette or a 10 mL serological pipette, and the cell number was measured. 6 After diluting the cells in the cOpTmizer culture medium to 10 cells / mL, IL-2 (10 3 IU / mL), 100 nM CD80, and 0.8 nM IL-15 were further added, and the cells were cultured at 37°C in a CO2 incubator for 3 days.

[0135] Ten days after the start of the culture, the aggregated cells were resuspended using a 1000P micropipette or a 10mL serological pipette, and the cell number was measured. The cell culture medium was diluted with the same amount of cOpTmizer culture medium based on the cell culture medium being cultured, and IL-2 (10 3 IU / mL), 100 nM CD80, and 0.8 nM IL-15 were further added, and the cells were cultured at 37°C in a CO2 incubator for 2 days.

[0136] The same amount of cOpTmizer culture medium and supplemented cOpTmizer culture medium were added with IL-2 (10 3 IU / mL), 100 nM CD80, and 0.8 nM IL-15 were further added, and the cells were cultured at 37°C in a CO2 incubator.

[0137] After 14 days from the start of the culture, the aggregated cells were resuspended using a 1000P micropipette and the cell number was measured. In Example 3, the composition and ratio of cells including αβT cells and γδT cells were analyzed using the antibodies in Table 1 with a Fortessa-X20 flow cytometry (BD Bioscience) on the start day of the culture, and on the 3rd, 4th, 5th, 6th, 7th, 10th, and 14th days.

[0138] As a result, as shown in Figures 5 and 6, the proliferation rate of γδ T cells in the male donors in the experimental group to which finasteride (10 nM), IL-15, IL-18, and CD80 were further added increased by approximately 1.45-fold compared to the control group treated with only IL-2 and ZA. On day 14 of culture, it was confirmed that the proportion of cells was 95% or more γδ T cells and approximately 2-3% αβ T cells.

[0139] Furthermore, as shown in Figures 7 and 8, the proliferation rate of γδ T cells in the female donors in the experimental group to which finasteride (0.01 nM), IL-15, IL-18, and CD80 were further added increased by approximately 3.8 times (380%) compared to the control group treated with only IL-2 and ZA.

[0140]

[0141] Example 4. Proliferation and survival rates of γδ T cells in healthy peripheral blood according to the duration of finasteride treatment

[0142] Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors and cultured with γδ T cell culture basic components (3 μM zoledronic acid and 10 3 The active ingredient, 10 nM finasteride, was continuously administered to the cells at 100 ng / mL IL-2 (1.1U / mL interleukin-2) for 7, 10, and 14 days after the start of culture. The activation and proliferation rates of γδ T cells were compared after culturing for a total of 14 days.

[0143] Frozen peripheral blood mononuclear cells were thawed in a 37°C water bath and then suspended in 9 mL of OpTmizer medium (cOpTmizer, Themo-Fisher Scientific, USA) containing 10% fetal bovine serum (FBS). The cell count was measured and the cells were centrifuged at 400x g for 4 minutes at 25°C. The cell pellet obtained by centrifugation was suspended in 5 mL of cOpTmizer medium (Themo-Fisher Scientific, USA), the cell count was measured, and the cells were then diluted to 1.0 × 10 in cOpTmizer medium. 6 The cells were diluted to a concentration of 1000 cells / mL.

[0144] Total 1.0 x 10 cells in a 24-well plate 6 1 mL of cells was dispensed per well. To induce γδT cell stimulation and differentiation, 10 nM finasteride, 3 μM zoledronic acid (ZA), 100 μL IL-2 (10 3 IU / mL) was added and the mixture was cultured at 37°C in a CO2 incubator for 4 days.

[0145] Four days after the start of the culture, the aggregated cells were resuspended using a 1000P micropipette and added with the same amount of cOpTmizer culture medium based on the cell culture medium during the culture, as well as 10 nM finasteride and IL-2 (10 3 IU / mL) was added and the cells were incubated at 37°C in a CO2 incubator.

[0146] Five days after the start of the culture, the aggregated cells were resuspended using a 1000P micropipette and the cell number was measured. The same amount of cOpTmizer culture medium was added with 10 nM finasteride and IL-2 (10 3 IU / mL) was added and the cells were incubated at 37°C in a CO2 incubator.

[0147] The next day, add 10 nM finasteride and IL-2 (10 3 IU / mL) was added and the cells were incubated at 37°C in a CO2 incubator.

[0148] Seven days after the start of the culture, the aggregated cells were resuspended using a 1000P micropipette or a 10 mL serological pipette, and the cell number was measured. 6 After diluting the cells in the cOpTmizer culture medium to a cell concentration of 10 cells / mL, 10 nM finasteride and IL-2 (10 3 IU / mL) was added and the mixture was cultured at 37°C in a CO2 incubator for 3 days.

[0149] Ten days after the start of the culture, the aggregated cells were resuspended using a 1000P micropipette or a 10 mL serological pipette, and the cell number was measured. 6 After diluting the cells in the cOpTmizer culture medium to a cell concentration of 10 cells / mL, 10 nM finasteride and IL-2 (10 3 IU / mL) was added and the mixture was incubated at 37°C in a CO2 incubator.

[0150] After 12 days from the start of culture, the same amount of cOpTmizer culture medium was added based on the cell culture medium in culture, along with 10 nM finasteride, IL-2 (10 3 IU / mL) was added and the mixture was incubated at 37°C in a CO2 incubator.

[0151] After 14 days from the start of the culture, the aggregated cells were resuspended using a 1000P micropipette or a 10 mL serological pipette, and the cell number was measured.

[0152] Peripheral blood mononuclear cells were isolated from healthy donors, and the γδ T cell culture medium was treated with high concentrations of finasteride for 7, 10, and 14 days, respectively, and the activation and proliferation rates of γδ T cells were compared over the 14 days.

[0153] As a result, as shown in Figure 9, the γδT cell proliferation rate in the group treated with finasteride for up to 7 days was increased by about 1.5 times compared to the control group, and the proliferation rate was increased by more than 1.1 times compared to the groups treated with finasteride for up to 10 or 14 days. There was no significant difference in viability compared to the control group, and the cell viability was not affected by continuous treatment with high concentrations of finasteride. The ratio of γδT cells was about 70% after 7 days of culture, and it was confirmed that more than 90% of the cells were γδT cells after 10 days of culture.

[0154] The IL-2 and ZA-containing medium was used as a control group, and the proliferation rate of γδ T cells due to the components added in each Example is summarized in Table 2.

[0155] As shown in Table 2, the proliferation rate of γδ T cells increased when finasteride was added compared to the control group, and the proliferation rate of γδ T cells was further increased when IL-15 and a cytokine mixture (IL-15, IL-18, and CD80) were added.

[0156] [Table 2]

[0157] [Industrial Applicability]

[0158] According to the present invention, compared to the conventional γδ T cell culture methods or culture methods using feeder cells, it is possible to produce γδ T cells with high cell-killing ability and cell survival rate with high purity and high efficiency in a short period of time using a clinically friendly method, and has the advantage of being able to increase the productivity of allogeneic γδ T cell immunotherapeutic agents.

[0159]

[0160] Although the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for producing γδ T cells, comprising the steps of: (a) culturing peripheral blood mononuclear cells (PBMCs) in a medium containing (i) zoledronic acid, (ii) IL-2, and (iii) a 5α-reductase inhibitor or an androgen receptor antagonist to induce and culture γδ T cells; and (b) obtaining the cultured γδ T-cells.

2. 2. The method according to claim 1, wherein the 5α-reductase inhibitor is selected from the group consisting of finasteride, dutasteride, epristeride, alfatradiol, and saw palmetto extract.

3. 2. The method of claim 1, wherein the androgen receptor antagonist is selected from the group consisting of bicalutamide, flutamide, and nilutamide.

4. The method according to claim 1, wherein the 5α-reductase inhibitor is finasteride.

5. The method of claim 1 , wherein the medium further contains a costimulatory molecule.

6. The costimulatory molecule may be 5A6.E9, Bl, TS8.2, 15D, B6, B3, TS-1, gamma 3.20, 7A5, IMMU510, R9.12, 11F2 or a combination thereof, phorbol 12-myristate-13-acetate (TPA), mezerein, staphylococcal enterotoxin A (SEA), streptococcal protein A, amphotericin B, B) or a combination thereof, αTCR, βTCR, γTCR, δTCR, CD277, CD28, CD46, CTLA4, ICOS, PD-1, CD30, NKG2D, NKG2A, HVEM, 4-1BB (CD137), OX40 (CD134), CD70, CD80, CD86, DAP, CD122, GITR, FceRIg, CD1, CD16, CD161, DNAX, accessory molecule-1 (DNAM-1), SLAM, coxsackievirus and adenovirus receptors or a combination thereof.

7. The method of claim 5, wherein the costimulatory molecule is selected from the group consisting of CD80, 4-1BB, CD86, and CD276.

8. 2. The method of claim 1, wherein the medium further contains a cytokine selected from the group consisting of IL-4, IL-7, IL-12, IL-15, IL-18, IL-21, and IL-23.

9. The method according to claim 8, wherein the medium further contains a cytokine selected from the group consisting of IL-12, IL-15, and IL-18.

10. The method according to claim 4, characterized in that when the peripheral blood mononuclear cells are derived from a male, the medium contains 1 to 50 nM finasteride.

11. The method according to claim 4, characterized in that when the peripheral blood mononuclear cells are derived from a woman, the culture medium contains 0.001 to 10 nM finasteride.

12. The method according to claim 1, wherein the culture is carried out for 3 to 21 days.

13. The method according to claim 1, wherein the ratio of γδ T cells to total cells is 90% or more on day 14 of culture.

14. A medium composition for inducing and culturing γδT cells, comprising (i) zoledronic acid, (ii) IL-2, and (iii) a 5α-reductase inhibitor or an androgen receptor antagonist.

15. The medium composition according to claim 14, wherein the 5α-reductase inhibitor is selected from the group consisting of finasteride, dutasteride, epristeride, alfatradiol, and saw palmetto extract.

16. 15. The medium composition according to claim 14, wherein the androgen receptor antagonist is selected from the group consisting of bicalutamide, flutamide, and nilutamide.

17. The medium composition according to claim 14, wherein the 5α-reductase inhibitor is finasteride.

18. The medium composition according to claim 14, further comprising a costimulatory molecule.

19. The costimulatory molecule may be 5A6.E9, Bl, TS8.2, 15D, B6, B3, TS-1, gamma 3.20, 7A5, IMMU510, R9.12, 11F2 or a combination thereof, phorbol 12-myristate-13-acetate (TPA), mezerein, staphylococcal enterotoxin A (SEA), streptococcal protein A, amphotericin B, B) or a combination thereof, αTCR, βTCR, γTCR, δTCR, CD277, CD28, CD46, CTLA4, ICOS, PD-1, CD30, NKG2D, NKG2A, HVEM, 4-1BB (CD137), OX40 (CD134), CD70, CD80, CD86, DAP, CD122, GITR, FceRIg, CD1, CD16, CD161, DNAX, accessory molecule-1 (DNAM-1), SLAM, coxsackievirus and adenovirus receptors or a combination thereof.

20. The medium composition according to claim 18, wherein the costimulatory molecule is selected from the group consisting of CD80, 4-1BB, CD86 and CD276.

21. The medium composition according to claim 14, further comprising a cytokine selected from the group consisting of IL-4, IL-7, IL-12, IL-15, IL-18, IL-21, and IL-23.

22. The medium composition according to claim 21, further comprising a cytokine selected from the group consisting of IL-12, IL-15, and IL-18.

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