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 comprises expanding and culturing γδ T cells based on the suppression of androgen signaling.

[0002] [Background technology]

[0003] T cells, a type of lymphocyte, together with B cells, are the backbone of adaptive immunity. T cells are generated through a process in which precursors produced by hematopoietic stem cells mature in the thymus. T cells consist of a majority of αβ T cells expressing the αβ T cell receptor (TCR) and a minority of γδ T cells expressing the γδ T cell receptor, which circulate in the thymus and peripheral lymphoid tissues (Non-Patent Document 1). γδ T cells present in epithelial tissues exhibit very different characteristics, and in some locations, γδ T cells may account for the majority or entire population (Non-Patent Document 2). γδ T cells present in such epithelial tissues exhibit restricted or consistent T cell receptor expression, a characteristic distinct 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 migrating to the skin, intestine, lungs, and epithelial tissues before birth (Non-Patent Document 3). γδ T cells do not have a diverse T cell receptor, but they recognize and respond to various biological insults in epithelial tissues. However, little is known about the autoantigens that induce these responses. Activated γδ T cells are known to regulate inflammation, destroy phenotypically altered cells, and improve wound healing (Non-Patent Document 3). Furthermore, recent research has shown that γδ T cells function as regulators, maintaining and restoring tissue homeostasis in response to external environmental stimuli (Non-Patent Document 1).

[0005] γδ T cells exist in a very small proportion of human peripheral blood, around 0.5-5%, and are known to be functionally involved in both adaptive and innate immune responses. 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 and Vδ2 (Non-Patent Document 4). Most γδ T cells in the blood possess the Vδ2 chain, which is paired with the 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. However, they initiate immune responses earlier than TCR αβ T cells, inducing innate-like immune responses. In particular, γδ T cells, like αβ T cells, have a more potent anti-cancer effect through cytolysis. However, unlike αβ T cells, which are the main cause of graft-versus-host disease (GVHD) against cells expressing non-self MHC, they are not MHC-restricted and do not cause 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 can be specifically proliferated by treatment with interleukin-2 (IL-2) and zoledronic acid (ZA, ZOL), their therapeutic efficacy has been demonstrated in various cancers through 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] The majority of γδ T cells in the body are present in peripheral blood at a very low rate (approximately 0.5–5%). Therefore, the therapeutic use of γδ T cells requires a large amount of activated γδ T cells. Therefore, methods for the ex vivo mass culture of γδ T cells isolated from blood have been actively researched. In addition to zoledronic acid and IL-2, which have been used for existing cell proliferation / activation methods, methods using IL-15 (Patent Documents 1 and 2) and methods using phytohaemagglutinin (PHA), TGF-β, concanavalin A, amphotericin B, etc. (Patent Documents 3–6) have been developed. However, these methods merely represent modifications and extensions of the previously used IL-2 method, and have not provided any groundbreaking or improved methods. Furthermore, 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 to massively expand 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 present inventors have made extensive efforts to develop a method for mass-producing and culturing TCR γδ T cells. As a result, they have 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 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 following steps:

[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 explanation 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] [Figure 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] [Figure 3]This shows the results of examining the proliferation and viability of gamma delta T cells following treatment with finasteride and IL-15 using peripheral blood mononuclear cells derived from male donors. High-concentration (10 nM) finasteride was continuously treated for up to 7 days, and IL-15 was continuously treated for a 14-day culture period. The results are a comparative analysis of the proliferation and viability of gamma delta T cells from male donors (A) and the increased number of gamma delta T cells after 14 days of culture compared to the control group (B).

[0040] [Figure 4] This shows the results of examining the proliferation and viability of gamma delta T cells following 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 results are a comparative analysis of the proliferation and viability of gamma delta T cells from female donors (A) and the increased number of gamma delta T cells after 14 days of culture compared to the control group (B).

[0041] [Figure 5] This shows the results of examining the proliferation and viability of γδT cells after 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 viability of γδT cells over 14 days, and B shows the results of analyzing the ratio of cultured γδT cells and αβT cells.

[0042] [Figure 6] This shows the results of examining the proliferation and viability of gamma delta 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 gamma delta T cells after 14 days of culture, and (B) is the result of a flow cytometer analysis of the proportion of gamma delta T cells compared to the control group after 14 days of culture.

[0043] [Figure 7]This shows the results of examining the proliferation and viability of gamma delta T cells after treatment with a mixture of finasteride (0.01 nM) and cytokines using peripheral blood mononuclear cells derived from female donors. Panel A shows the results of comparing the proliferation and viability of gamma delta T cells over 14 days, and Panel B shows the results of analyzing the ratio of cultured gamma delta T cells and alpha beta T cells.

[0044] [Figure 8] This shows the results of examining the proliferation and viability of gamma delta 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 gamma delta T cells after 14 days of culture, and (B) is the result of a flow cytometer analysis of the proportion of gamma delta T cells 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 14 days after treatment with finasteride using peripheral blood mononuclear cells from a healthy donor for 7, 10, and 14 days from 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 INVENTION

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

[0049]

[0050] In the present invention, we aimed 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-containing 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 used to treat hypercalcemia associated with 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. However, unlike existing bisphosphonates (e.g., clodronate, etidronate, and tiludronate), it contains a nitrogen-containing imidazole ring, which allows it to exhibit highly potent bone resorption inhibitory effects. Zoledronic acid inhibits calcium resorption by chelating with calcium from hydroxyapaptite, a component of bone matrix. In addition, by inhibiting farnesyl diphosphate synthase, it prevents the prenylation of guanine nucleotide-binding proteins (G-proteins) in osteoclasts, inducing apoptosis of osteoclasts, which promote bone resorption, thereby reducing osteoclast numbers and regulating blood calcium levels. Zoledronic acid is rapidly absorbed from the blood into bone and stored there. Because of its long half-life in bone, a single intravenous infusion can sustain bone resorption. However, because 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, dosage adjustment is required for patients with renal impairment.

[0055] In this invention, 5α-reductase is a biocatalyst that reduces the male hormone testosterone to dihydrotestosterone (DHT), and exists in types 1 to 3. Abnormalities in type 1 result in reduced bone mass 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 inhibit the conversion of substrates to their activated forms, reducing the amount of activated substrate, resulting in a slight increase in the substrate level. The mechanism of 5α-reductase inhibition involves the binding of NADPH to the enzyme followed by the substrate. Specific substrates include testosterone, progesterone, androstene, epitestosterone, cortisol, aldosterone, and dihydroxycorticosterone.

[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] Finasteride used in the present invention is a 5α-reductase inhibitor that inhibits the conversion of testosterone to the active form dihydrotestosterone. Dihydrotestosterone, the active form of testosterone, is known to be involved in hair loss and benign prostatic hyperplasia. Therefore, 5α-reductase inhibitors that inhibit the production of dihydrotestosterone in tissues are used as therapeutic agents for benign 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, alfatradiol, 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 peripheral cell membrane proteins that appear on activated antigen-transmitting cells (APCs) and are capable of generating costimulatory signals that increase or decrease MHC-TCR signaling between APCs and T cells by interacting with T cell surface membrane proteins. The costimulatory molecule may be an antibody or a ligand, such as 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, or 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. The present invention is not limited to CD80 used in the examples, and it will be apparent to a person skilled in the art to which the present invention pertains that any costimulatory molecule that meets the objectives of the present invention can be used.

[0064] The concentration of the costimulatory molecule in the medium used for culturing 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 can 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. 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.

[0066] In the present invention, in the mass expansion culture of TCR γδ T cells, finasteride, an androgen signaling inhibitor, is used while simultaneously treating with zoledronic acid and IL-2. In addition, cytokines IL-15 and IL-18 and CD80 costimulatory molecules are used in the above method, which enables more efficient mass expansion of TCR γδ T cells. In this case, 0.4 × 10 6 ~3.0×10 6 It is necessary to maintain a cell concentration of 0.5 to 1.0 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 present invention, the method for culturing γδ T cells is as follows:

[0068] (a) stimulating a peripheral blood mononuclear cell culture containing γδ T cells and then statically culturing it;

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

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

[0071] After the step (c) above, 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, but are not limited to, 24-well plates, 12-well plates, 6-well plates, shaking flasks, T25 flasks, T75 flasks, T175 flasks, T225 flasks, and disposable cell culture bags. Any reaction vessel that can be easily adopted by a person skilled in the art to which the present invention pertains can be used.

[0073] In the present invention, "appropriate 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 a "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 a concentration of 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 term "cytokine mixture" refers to an additive containing a mixture of cytokines IL-15 and IL-18, a CD80 costimulator, and additional active ingredients, which 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, 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 basal 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. Other common 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 for culturing immune cells including T cells, may also 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 to be added may vary depending on whether the peripheral blood mononuclear cells used for γδ T cell induction and culture are derived from a male or female individual.

[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, more preferably 1 to 20 nM finasteride.

[0082] In the present invention, when the peripheral blood mononuclear cells used for γδ T cell induction and culture are derived from a woman, the γδ T cell induction and culture medium preferably contains 0.001 to 10 nM finasteride, more preferably 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, alfatradiol, 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 collectively term for peripheral cell membrane proteins that appear on activated antigen-transmitting cells (APCs) and can generate costimulatory signals that increase or decrease MHC-TCR signaling between APCs and T cells by interacting with T cell surface membrane proteins. The costimulatory molecule may be an antibody or a ligand, such as 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, or 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. The present invention is not limited to CD80 used in the examples, and it will be apparent to a person skilled in the art to which the present invention pertains that any costimulatory molecule that meets the objectives of the present invention can be used.

[0090] The concentration of the costimulatory molecule in the medium used for culturing 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 can 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). However, 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 to be construed as limiting the scope of the present invention.

[0094]

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

[0096] 1-1.Peripheral blood mononuclear cell (hPBMC) separation

[0097] For each 100 cc of blood collected from a healthy donor by a medical professional and placed in a heparinized 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 carefully dispensed vertically into a new 50 mL conical tube, avoiding contact with the wall. The diluted blood was then gradually poured onto the top of the 50 mL conical tube containing the Lymphoprep solution, along the wall. The conical tubes were then 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 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 were divided into three or four 50 mL conical tubes, resuspended in 45 mL of 1X DPBS, and then pooled into one 50 mL conical tube. The pooled cells were centrifuged twice at 1,250 rpm (350 x g) for 10 minutes at room temperature. The cell concentration was 1.0 x 10 6 1.0 x 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 per 1000 cells, frozen and stored in a nitrogen tank (LN2Tank), and thawed for use when needed.

[0098]

[0099] 1-2. Proliferation and survival rates of gamma delta T cells in peripheral blood mononuclear cells as a function of 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 γδT cell culture. 3After continuous treatment with 1U / mL IL-2 and 0.01nM and 10nM finasteride as active ingredients, respectively, the cells were cultured for a total of 14 days, and the activation and proliferation rates of γδT cells were compared.

[0101] Frozen peripheral blood mononuclear cells were thawed in a 37°C water bath and then suspended in 9 mL of OpTmizer medium (Themo-Fisher Scientific, USA) containing 10% heat-inactivated fetal bovine serum (FBS, Gibco, USA). The cell count was determined 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 complete OpTmizer medium (10% fetal bovine serum + OpTmizer medium, cOpTmizer) and the cell count was determined. The cells were then resuspended in cOpTmizer medium at a concentration of 1.0 x 10 6 The cells were diluted to a concentration of 1000 cells / mL.

[0102] 1.0 x 10 total in a 24-well plate 6 0.01 nM and 10 nM finasteride, 3 μM zoledronic acid, IL-2 (10 μM), and 10 μM zoledronic acid were added to stimulate and induce differentiation of γδ T cells. 3 IU / mL) was added, and the cells were cultured at 37°C in a CO2 incubator for 3 days.

[0103] Three days after the start of culture, the aggregated cells were resuspended using a 1000P micropipette and added to the same volume of cOpTmizer culture medium and 0.01 nM, 10 nM finasteride, and 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 re-induction, 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 further added, and the cells were cultured at 37°C in a CO2 incubator.

[0105] Six days after the start of culture, half the amount of cOpTmizer culture medium was added based on the cell culture medium during culture, and 0.01 nM, 10 nM finasteride, and IL-2 (10 3 IU / mL) was further added, and the cells were cultured 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 number was measured. 6 After diluting the cells in the cOpTmizer culture medium to a cell concentration of 10 cells / mL, IL-2 (10 3 IU / mL) was added, and the cells were cultured at 37°C in a CO2 incubator for 3 days.

[0107] Ten days after the start of 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 1000 cells / ml. 3 IU / mL) was added, and the cells were cultured at 37°C in a CO2 incubator for 2 days.

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

[0109] After 14 days from the start of 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 αβ T cells and γδ T cells, were analyzed using the antibodies listed in Table 1 on the start of culture and on days 5, 7, 10, and 14 using a BD Bioscience Fortessa-X20 flow cytometry instrument.

[0110] 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. Compared to the control group treated with IL-2 and ZA alone, the male donors showed an increased proliferation rate of γδ T cells at high concentrations of finasteride (Figure 1). Conversely, the female donors showed an increased proliferation rate of γδ T cells at low concentrations of finasteride (Figure 2).

[0111] [Table 1]

[0112]

[0113] Example 2. Proliferation and survival rates 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 PBS. 3 In addition to 1.1U / mL IL-2, the active ingredient finasteride was administered at a concentration of 10nM in male donors or 0.01nM in female donors for up to 7 days, and 0.8nM IL-15 was also administered continuously. The activation and proliferation rates of γδT 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 determined 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, and the cell count was determined. A 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 γδT cells (final volume 1 mL) were seeded into one well. To stimulate and induce differentiation, γδT cells were cultured in a 10 nM and 0.01 nM finasteride culture medium for males and females, respectively, along with 3 μM zoledronic acid (ZA), IL-2 (10 3 IL-15 (0.8 nM, 0.1U / 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 and 0.01 nM finasteride, and 0.8 nM IL-15 were further added to male and female mice, respectively, and the mice were cultured in a 37°C CO2 incubator for 2 days.

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

[0119] Six days after the start of culture, half the amount of cOpTmizer culture medium was added to the culture medium of the cells in culture, and 10 nM and 0.01 nM finasteride, IL-2 (10 3 1.1U / 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 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 with cOpTmizer culture medium to 10 cells / mL, IL-2 (10 3 IL-15 (0.111 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 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 concentration of 10 cells / mL. The added cOpTmizer culture medium contained IL-2 (10 3 IL-15 (0.111 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 IL-15 (0.111 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 in the culture. 3 IL-15 (0.111 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 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 αβ T cells and γδ T cells were analyzed using the antibodies listed in Table 1 on the start of culture and on days 5, 7, 10, and 14 using a BD Bioscience Fortessa-X20 flow cytometry instrument.

[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 (Control) treated with only IL-2 and ZA (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 base components (3 μM zoledronic acid and 10 μM zoledronic acid). 3 In addition to 100 IU / mL IL-2, males and females were treated with 10nM and 0.01nM finasteride as the active ingredient once on the day of culture initiation, respectively. An additional ingredient, 6nM IL-18, was also treated once on the day of culture initiation, and 100nM CD80 and 0.8nM IL-15 were continuously treated for a total of 14 days of culture, and the activation and proliferation rates of γδT cells were compared.

[0129] Frozen peripheral blood mononuclear cells were thawed in a 37°C water bath and suspended in 9 mL of OpTmizer medium (cOpTmizer, Themo-Fisher Scientific, USA) containing 10% fetal bovine serum (FBS). The cell count was determined 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) and the cell count was determined. The cells were then diluted to 1.0 x 10 in cOpTmizer medium. 6 The cells were diluted to a cell concentration of 1000 / mL.

[0130] 1.0 x 10 total cells in a 24-well plate 6 1 mL of cells was dispensed per well. To induce γδT cell stimulation and differentiation, the active ingredient, finasteride, was administered at a concentration of 10 nM for male donors and 0.01 nM for female donors. 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 in a CO2 incubator at 37°C 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 in a CO2 incubator at 37°C.

[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 in a CO2 incubator at 37°C.

[0133] Six days after the start of 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 in a CO2 incubator at 37°C.

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

[0137] After 14 days from the start of 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 αβ T cells and γδ T cells, were analyzed using the antibodies listed in Table 1 on the start of culture and on days 3, 4, 5, 6, 7, 10, and 14 using a BD Bioscience Fortessa-X20 flow cytometry analyzer.

[0138] As shown in Figures 5 and 6, the proliferation rate of γδ T cells in the male donor experimental group, which was further supplemented with finasteride (10 nM), IL-15, IL-18, and CD80, 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 over 95% of the cells were γδ T cells, and approximately 2-3% were αβ T cells.

[0139] Furthermore, as shown in Figures 7 and 8, the proliferation rate of γδT 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 depending on the duration of finasteride treatment

[0142] Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors and cultured with γδ T cell culture base components (3 μM zoledronic acid and 10 μM zoledronic acid). 3 The active ingredient, 10 nM finasteride, was added to the IL-2 (1.1U / mL IL-2) and cultured for a period of 14 days. 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 determined 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). The cell count was determined and the cells were then diluted to 1.0 x 10 in cOpTmizer medium. 6 The cells were diluted to a concentration of 1000 cells / mL.

[0144] 1.0 x 10 total 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 cells were 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 the cells were added to the same amount of cOpTmizer culture medium as the cell culture medium during the culture, along with 10 nM finasteride and IL-2 (10 3 IU / mL) was further added, and the cells were cultured at 37°C in a CO2 incubator.

[0146] Five days after the start of 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 further added, and the cells were cultured at 37°C in a CO2 incubator.

[0147] The next day, add 10 nM finasteride and IL-2 (10 nM IL-2) to half the cOpTmizer culture medium based on the cell culture medium in the culture. 3 IU / mL) was further added, and the cells were cultured at 37°C in a CO2 incubator.

[0148] 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 number was measured. 6 After diluting the cells in the cOpTmizer culture medium to a cell concentration of 100 cells / mL, the cOpTmizer culture medium was supplemented with 10 nM finasteride and IL-2 (10 3 IU / mL) was added, and the cells were cultured at 37°C in a CO2 incubator for 3 days.

[0149] Ten days after the start of 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 100 cells / mL, the cOpTmizer culture medium was supplemented with 10 nM finasteride and IL-2 (10 3 IU / mL) was added and the cells were cultured at 37°C in a CO2 incubator.

[0150] After 12 days of culture, the cells were cultured in the same amount of cOpTmizer culture medium as the cell culture medium during culture, and 10 nM finasteride, IL-2 (10 3 IU / mL) was added and the cells were cultured 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 treated with high concentrations of finasteride in the γδ T cell culture medium for 7, 10, and 14 days, respectively, and the activation and proliferation rates of γδ T cells were compared over the 14-day period.

[0153] As a result, as shown in Figure 9, the group treated with finasteride for up to 7 days showed a 1.5-fold increase in γδ T cell proliferation rate compared to the control group, and a 1.1-fold increase compared to the groups treated with finasteride for up to 10 or 14 days. Viability was not significantly different from the control group, and sustained treatment with high concentrations of finasteride did not affect cell viability. The γδ T cell ratio was approximately 70% after 7 days of culture, and over 90% of the cells were confirmed to be γδ T cells after 10 days of culture.

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

[0155] As shown in Table 2, the proliferation rate of γδT cells increased when finasteride was added compared to the control group, and the proliferation rate of γδT 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] The present invention has the advantage that, compared to conventional γδ T cell culture methods or culture methods using feeder cells, γδ T cells with high cell-killing ability and cell viability can be produced with high purity and efficiency in a short period of time using a clinically friendly method, thereby increasing the productivity of allogeneic γδ T cell immunotherapeutic agents.

[0159]

[0160] Although certain parts of the present invention have been described in detail above, it will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the true scope of the present invention is to be 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) finasteride to induce and culture γδ T cells; and (b) obtaining the cultured γδ T cells; Here, when the peripheral blood mononuclear cells are derived from a male, the medium is characterized by containing 1 to 50 nM of finasteride, and when the peripheral blood mononuclear cells are derived from a female, the medium is characterized by containing 0.001 to 10 nM of finasteride.

2. The method of claim 1, wherein the culture medium further contains CD80 as a costimulatory molecule.

3. The method according to claim 1, wherein the medium further contains IL-15 or IL-18.

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

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

6. A medium composition for inducing and culturing γδT cells, comprising (i) zoledronic acid, (ii) IL-2, and (iii) finasteride; Here, when the peripheral blood mononuclear cells are derived from a male, the composition is characterized by containing 1 to 50 nM of finasteride, and when the peripheral blood mononuclear cells are derived from a female, the composition is characterized by containing 0.001 to 10 nM of finasteride.

7. The medium composition according to claim 6, further comprising CD80 as a costimulatory molecule.

8. The medium composition according to claim 6, further comprising IL-15 or IL-18.

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