Method for gene transfer into gamma delta T cells

Abstract

To provide a method for producing a γδT cell population which expresses functional TCR or CAR by preparing large quantities of γδT cells with high purity and efficiently introducing a foreign gene encoding TCR or CAR into the obtained γδT cells.SOLUTION: The present invention provides a method for efficiently producing highly pure γδT cells introduced with foreign genes by culturing in the presence of IL-7 and IL-15 after stimulating the γδT cells with a bisphosphonic acid ester derivative, followed by gene introduction. When a T cell receptor (TCR) or a chimeric antigen receptor (CAR) is introduced as the foreign gene, the γδT cells in which TCR and CAR are functionally expressed can be obtained.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing highly purified γδ T cells into which an exogenous gene has been introduced with high efficiency. [Background technology]

[0002] In recent years, T cell infusion therapy, in which cancer patients are transfected with exogenous genes encoding T cell receptors (TCRs) or chimeric antigen receptors (CARs) to deliver T cells with specificity to cancer antigens, has attracted attention as a cancer treatment method. In fact, a clinical trial is underway in which TCR gene-modified T cells expressing a TCR gene that recognizes the cancer antigen MAGE-A4 are administered to esophageal cancer patients (Non-Patent Document 1). Another clinical trial is underway in which CAR gene-modified T cells expressing a CAR gene incorporating a single-chain antibody (single chain variable fragment: scFv) that binds to CD19 in the ligand-binding domain are administered to relapsed / refractory acute lymphoblastic leukemia patients (Non-Patent Document 2).

[0003] T cells with αβ TCR (αβ T cells) have generally been used as target cells for transfection of foreign genes. The reason for this is as follows: viral vectors are primarily used to transfect foreign genes, and this usually requires stimulating the proliferation of the target cells. When transfecting αβ T cells as target cells, the proliferation of the target cells can be easily stimulated using anti-CD3 and anti-CD28 antibodies.

[0004] T cells with γδ TCR (γδ T cells) have been shown to damage virus-infected cells and cancer cells. Therapy involving the infusion of antigen-specific γδ T cells into patients is expected to be more effective than the use of αβ T cells, and γδ T cell infusion therapy has been attempted (Non-Patent Document 3). However, the cytological characteristics of γδ T cells, such as their cellular function, have not been fully characterized. This is due to the fact that γδ T cells are present in small quantities, accounting for approximately 5% of peripheral blood T cells (Non-Patent Document 4), making it difficult to secure the necessary number of cells for cellular analysis. Unlike αβ T cells, γδ T cells cannot be sufficiently expanded with anti-CD3 or anti-CD28 antibodies. Therefore, attempts have been made to expand γδ T cells using zoledronate, a bisphosphonate derivative used as a bone resorption inhibitor, and interleukin-2 (IL-2) (Non-Patent Documents 5 and 6). However, even when zoledronic acid and IL-2 were used, the purity of the gamma delta T cells obtained by expansion was low, which was a problem.

[0005] A method for expanding highly purified γδ T cells has been reported, in which mononuclear cells obtained from human peripheral blood are cultured in the presence of tetrakispivaloyloxymethyl 2-(thiazol-2-ylamino)ethylidene-1,1-bisphosphonate, a bisphosphonate derivative, and IL-2 is further added (Patent Document 1 and Non-Patent Document 7). On the other hand, to effectively perform γδ T cell infusion therapy, it is desirable to prepare a large number of highly purified γδ T cells and, at the same time, to efficiently introduce an exogenous gene encoding a TCR or CAR into the obtained γδ T cells. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] WO2016 / 098904 [Non-patent literature]

[0007] [Non-Patent Document 1] Kageyama S, et al. Adoptive transfer of MAGE-A4 T-cell receptor gene-transduced lymphocytes in patients with recurrent esophageal cancer. Clin Cancer Res. 2015 May 15;21(10):2268-2277. [Non-patent document 2] Maude SL, et al. Chimeric antigen receptor T cells for sustained remissions in leukemia. N Engl J Med. 2014 Oct 16;371(16):1507-1517. [Non-patent document 3] Kobayashi H, et al. Safety profile and anti-tumor effects of adoptive immunotherapy using gamma-delta T cells against advanced renal cell carcinoma: a pilot study. Cancer Immunol Immunother. 2007 Apr;56(4):469-476. [Non-patent document 4] Carding SR and Egan PJ. Gammadelta T cells: functional plasticity and heterogeneity. Nat Rev Immunol. 2002 May;2(5):336-345. [Non-patent document 5] Nicol AJ, et al. Clinical evaluation of autologous gamma delta T cell-based immunotherapy for metastatic solid tumors. Br J Cancer. 2011 Sep 6;105(6):778-786. [Non-patent document 6] Kobayashi H, et al. Phase I / II study of adoptive transfer of γδ T cells in combination with zoledronic acid and IL-2 to patients with advanced renal cell carcinoma. Cancer Immunol Immunother. 2011 Aug;60(8):1075-1084. [Non-Patent Document 7] Tanaka Y, et al. Expansion of human γδ T cells for adoptive immunotherapy using a bisphosphonate prodrug. Cancer Sci. 2018 Mar;109(3):587-599. Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a method for preparing highly purified γδ T cells in large quantities, and then efficiently introducing an exogenous gene encoding a TCR or CAR into the obtained γδ T cells to produce a γδ T cell population expressing a functional TCR or CAR. [Means for solving the problem]

[0009] The object of the present invention is achieved by the following inventions. (1) A method for producing a transgene-expressing γδ T cell population, comprising: (Step 1) Gamma delta T cells were cultured using 2-(thiazol-2-ylamino)ethylidene-1,1-bisphosphonate, 2-(3-bromopyridin-2-ylamino)ethylidene-1,1-bisphosphonate, 2-(5-fluoropyridin-2-ylamino)ethylidene-1,1-bisphosphonate, 2-(pyrimidin-2-ylamino)ethylidene-1,1-bisphosphonate, or 2-(7-azaindole-1-yl)ethylidene-1,1-bisphosphonate. in the presence of one or more compounds selected from the group consisting of tetrakispivaloyloxymethyl ester derivatives of 2-(pyrimidin-4-ylamino)ethylidene-1,1-bisphosphonate, 2-(5-methylthiazol-2-ylamino)ethylidene-1,1-bisphosphonate, 2-(4-phenylthiazol-2-ylamino)ethylidene-1,1-bisphosphonate, and 2-(pyrimidin-4-ylamino)ethylidene-1,1-bisphosphonate, and pharmaceutically acceptable salts thereof; (Step 2) culturing the γδ T cells cultured in Step 1 in the presence of IL-7 and IL-15; and (Step 3) introducing a gene into the γδ T cells cultured in Step 2; A method comprising: (2) A method for producing a γδ T cell population expressing a transgene, comprising: (Step 1) culturing γδ T cells in the presence of tetrakispivaloyloxymethyl 2-(thiazol-2-ylamino)ethylidene-1,1-bisphosphonate or a pharmaceutically acceptable salt thereof; (Step 2) culturing the γδ T cells cultured in Step 1 in the presence of IL-7 and IL-15; and (Step 3) introducing a gene into the γδ T cells cultured in Step 2; A method comprising: (3) The method according to (2), wherein the concentration of tetrakispivaloyloxymethyl 2-(thiazol-2-ylamino)ethylidene-1,1-bisphosphonate or a pharmaceutically acceptable salt thereof in step 1 is 0.01 to 1 μM. (4) The method according to (1) or (2), wherein the γδ T cells are derived from a mammal. (5) The method according to (4), wherein the mammal is a cancer patient or a non-cancer patient. (6) The method according to (5), wherein the cancer is selected from the group consisting of breast cancer, lung cancer, liver cancer, oral cancer, nasopharyngeal cancer, head and neck cancer, gastric cancer, esophageal cancer, colon cancer, skin cancer, malignant melanoma, kidney cancer, pancreatic cancer, bile duct cancer, brain tumor, prostate cancer, ovarian cancer, cervical cancer, colorectal cancer, bladder cancer, synovial sarcoma, leukemia, malignant lymphoma, and multiple myeloma. (7) The method according to (1) or (2), wherein the step of introducing a gene in step 3 uses a DNA vector or an RNA vector. (8) The method according to (1) or (2), characterized in that the step of introducing a gene in step 3 uses a vector selected from the group consisting of a plasmid vector, a lentivirus vector, an adenovirus vector, an adeno-associated virus, and a retrovirus vector. (9) The method according to (1) or (2), wherein the step of introducing a gene in step 3 uses a retroviral vector. (10) The method according to (1) or (2), wherein the gene is a gene encoding a T cell receptor (TCR) or a chimeric antigen receptor (CAR). (11) The T cell receptor (TCR) is NY-ESO-1 p157-165 / HLA-A2 complex-specific TCR, HTLV-1p40Tax p11-19 / HLA-A2 complex-specific TCR, HTLV-1p40Tax p301-309 / HLA-A24 complex-specific TCR and MAGE-A4 p143-151 / HLA-A24 complex-specific TCRs. (12) The chimeric antigen receptor (CAR) is MAGE-A4 p230-239 The method according to (10), wherein the CAR is selected from the group consisting of an HLA-A2 complex-specific CAR, a CEA-specific CAR, a GD2-specific CAR, and a CD19-specific CAR. (13) A genetically modified γδ T cell population obtained by the method according to any one of (1) to (12). (14) A pharmaceutical composition comprising the genetically modified γδ T cell population according to (13) and a pharmaceutically acceptable additive. (15) The pharmaceutical composition according to (14) for treating a cancer patient by autologous or allogeneic transplantation. (16) A genetically modified γδ T cell population having the following characteristics: (i) NY-ESO-1 p157-165 / HLA-A2 complex, HTLV-1p40Tax p11-19 / HLA-A2 complex, HTLV-1p40Tax p301-309 / HLA-A24 complex or MAGE-A4 p143-151 / expresses a TCR that specifically recognizes the HLA-A24 complex, (ii) CD3 and NKG2D positive; (iii) produce cytokines and chemokines, including IFN-γ and TNFα; and (iv) It has cytotoxic activity. (17) A genetically modified γδ T cell population having the following characteristics: (i) MAGE-A4 p230-239 / expressing a CAR that specifically recognizes the HLA-A2 complex, CEA, GD2 or CD19, (ii) CD3 and NKG2D positive; (iii) produce cytokines and chemokines, including IFN-γ and TNFα; and (iv) It has cytotoxic activity. (18) The genetically modified γδ T cell population according to (16) or (17), wherein the genetically modified γδ T cell population is a γδ T cell population into which a gene has been introduced using a DNA vector or an RNA vector. (19) The genetically modified γδ T cell population according to (16) or (17), wherein the genetically modified γδ T cell population is a γδ T cell population into which a gene has been introduced using a vector selected from the group consisting of a plasmid vector, a lentivirus vector, an adenovirus vector, an adeno-associated virus, and a retrovirus vector. (20) The genetically modified γδ T cell population according to (16) or (17), wherein the genetically modified γδ T cell population is a γδ T cell population into which a gene has been introduced using a retroviral vector. (21) The genetically modified γδ T cell population according to (16) or (17), wherein the gene is a gene encoding a T cell receptor (TCR) or a chimeric antigen receptor (CAR). (22) A pharmaceutical composition comprising the genetically modified γδ T cell population according to any one of (16) to (21). (23) A cell preparation comprising the genetically modified γδ T cell population according to any one of (16) to (21). (24) The genetically modified γδ T cell population according to any one of (16) to (21) for treating a cancer patient. (25) An anticancer agent comprising the genetically modified γδ T cell population according to any one of (16) to (21). (26) A method for treating cancer, characterized by using the genetically modified γδ T cell population according to any one of (16) to (21). (27) A therapeutic agent for infectious diseases, comprising the genetically modified γδ T cell population according to any one of (16) to (21). (28) A method for treating an infectious disease, characterized by using the genetically modified γδ T cell population according to any one of (16) to (21). [Effects of the Invention]

[0010] According to the present invention, mononuclear cells obtained from peripheral blood are stimulated with a bisphosphonate derivative and then cultured in the presence of IL-7 and IL-15, thereby enabling the production of a large number of highly purified γδ T cells, compared to the conventional method of culturing in the presence of IL-2. Furthermore, foreign genes can be efficiently introduced into γδ T cells that proliferate upon stimulation with a bisphosphonate derivative, for example, by using a retroviral vector. When TCR and CAR are introduced as foreign genes, γδ T cells in which the TCR and CAR are functionally expressed can be obtained.

[0011] The present invention makes it possible to obtain a large number of γδ T cells from collected blood, even when the amount of blood collected is small, thereby reducing the burden on subjects who require blood collection. Furthermore, γδ T cells with antigen specificity can be prepared in sufficient quantities through genetic modification in a short culture period. The resulting genetically modified γδ T cells exhibit potent antitumor effects and, because their function is not impaired by freeze-thawing, can be cryopreserved. This facilitates the implementation of effective genetically modified γδ T cell infusion therapy. [Brief explanation of the drawings]

[0012] [Figure 1] These figures show the proliferation of γδ T cells when peripheral blood mononuclear cells (PBMCs) from two healthy individuals (HD1 and HD2) were cultured for 11 days in the presence of tetrakispivaloyloxymethyl 2-(thiazol-2-ylamino)ethylidene-1,1-bisphosphonate (Compound 7) (1 μM), IL-7 (25 ng / mL), and IL-15 (25 ng / mL). (A) Flow cytometry analysis of each cell population was performed at the start of culture (Day 0) and after 11 days of culture (Day 11). The results show that γδ T cells, which accounted for only a few percent of the PBMCs before culture, proliferated to a high purity after culture. (B) Cell counts were measured over time after starting culture with 3 × 10 PBMCs. For comparison, IL-2 (100 IU / mL or 300 IU / mL) was added instead of IL-7 and IL-15 for culture. [Figure 2] Peripheral blood mononuclear cells (PBMCs) from two healthy individuals (HD1 and HD2) were stimulated with compound 7 (1 μM) on Day 0. On Days 4 and 5, NY-ESO-1-specific TCRs, which specifically recognize the complex between HLA-A2 and the NY-ESO-1 p157-165 peptide (SLLMWITQC), were introduced using a retroviral vector. The cells were cultured for 11 days in the presence of IL-7 (25 ng / mL) and IL-15 (25 ng / mL). The figure shows the expression rate of the introduced TCR in the resulting γδ T cells analyzed by flow cytometry using specific tetramers. [Figure 3]This figure shows the results of measuring cytokine (IFN-γ and TNFα) production and CD107a expression by γδ T cells into which NY-ESO-1-specific TCR was introduced. The target cells used were HLA-A2-positive T2 cell lines to which the NY-ESO-1p157-165 peptide was added. The results show that TCR-introduced γδ T cells express IFN-γ, TNFα, and CD107a in an antigen-specific manner. [Figure 4] This figure shows that γδ T cells introduced with an NY-ESO-1-specific TCR that specifically recognizes the complex of HLA-A2 and the NY-ESO-1 p157-165 peptide recognize the HLA-A2-positive, NY-ESO-1 antigen-positive cell line SK-MEL-37 and produce the cytokine IFN-γ. For comparison, the HLA-A2-positive, NY-ESO-1 antigen-negative cell line MEL72 did not produce any cytokine. [Figure 5A] These figures show that gamma delta T cells transfected with a TCR that specifically recognizes the complex of HLA-A24 and the HTLV-1 viral antigen p40Tax-derived peptide (SFHSLHLLF) (HTLV-1p40Taxp301-309 / HLA-A24 complex) do not recognize the HLA-A24-negative HTLV-1-positive cell line ILT-#37, but specifically recognize the HLA-A24-positive HTLV-1-positive cell line ILT-Hod. Figure 5A shows the results of measuring IFN-γ production. The HLA-A24-positive cell line T2A24 (T2A24-p40) supplemented with the p40Tax-derived peptide served as a positive control, and the T2A24 cell line (T2A24-ESO) supplemented with the NY-ESO-1 p157-165 peptide served as a negative control. [Figure 5B]Figure 5B shows that γδ T cells transfected with a TCR specifically recognizing the complex of HLA-A24 and a peptide derived from the HTLV-1 viral antigen p40Tax (SFHSLHLLF) (HTLV-1p40Taxp301-309 / HLA-A24 complex) specifically recognized the HLA-A24-positive HTLV-1-positive cell line ILT-Hod but not the HLA-A24-negative HTLV-1-positive cell line ILT-#37. Figure 5B shows the results of cytotoxicity assays. TCR-transfected γδ T cells were cocultured with 1 × 10 4 target ILT-Hod or ILT-#37 cells at 1 × 10 5 , 3 × 10 4 , 1 × 10 4 , or 3.3 × 10 3 cells (E / T ratios of 10:1, 3:1, 1:1, or 0.3:1, respectively). [Figure 6] Peripheral blood mononuclear cells from healthy donors were stimulated with compound 7 (1 μM) on day 0. Then, on days 4 and 5, a CAR that specifically recognizes the complex of HLA-A2 and the MAGE-A4p230-239 peptide (GVYDGREHTV) was introduced using a retroviral vector. The cells were cultured for 11 days in the presence of IL-7 (25 ng / mL) and IL-15 (25 ng / mL). The figure shows the expression rate of the introduced CAR in the resulting γδ T cells analyzed by flow cytometry using a specific tetramer. [Figure 7] Peripheral blood mononuclear cells (PBMCs) from healthy donors were stimulated with compound 7 (1 μM) on day 0, then retrovirally transduced with a CAR that specifically recognizes GD2 (disialoganglioside 2) on days 4 and 5. The cells were cultured for 11 days in the presence of IL-7 (25 ng / mL) and IL-15 (25 ng / mL). The figure shows the expression of the transduced CAR in the resulting γδ T cells analyzed by flow cytometry using a specific tetramer. The GD2-specific CAR-transduced γδ T cells were also cocultured with the GD2-positive target tumor cell line AS, and IFN-γ production was analyzed. The upper panel shows the results for control γδ T cells, and the lower panel shows the results for γδ T cells transduced with the GD2-specific CAR. [Figure 8]Peripheral blood mononuclear cells (PBMCs) from healthy donors were stimulated with compound 7 (1 μM) on day 0, then transduced with a CD19-specific CAR using a retroviral vector on days 4 and 5. The cells were cultured for 11 days in the presence of IL-7 (25 ng / mL) and IL-15 (25 ng / mL). The figure shows the expression rate of the transduced CAR in the resulting γδ T cells analyzed by flow cytometry using a specific tetramer. The CD19-specific CAR-transduced γδ T cells were also cocultured with CD19-positive tumor cells (NALM6) and analyzed for IFN-γ production. The upper panel shows the results for control γδ T cells, and the lower panel shows the results for GD2-specific CAR-transduced γδ T cells. [Figure 9] This figure shows that gamma-delta T cells transfected with a CAR that specifically recognizes the complex of HLA-A2 and the MAGE-A4p230-239 peptide (GVYDGREHTV) recognize tumor cell lines in an antigen-specific manner. This figure also shows that the cells specifically recognize HLA-A2-positive MAGE-A4-positive cell lines (SK-MEL-37 and NW-MEL-38). As a positive control, an HLA-A2-positive T2 cell line (T2-MAGE) supplemented with the MAGE-A4p230-239 peptide was used, and as a negative control, a T2 cell line (T2-ESO1) supplemented with the NY-ESO-1p157-165 peptide was used. [Figure 10A] (A) shows the tumor-suppressing effect of γδ T cells co-expressing tumor-specific TCR and CD8αβ. (B) shows the tumor-suppressing effect of γδ T cells co-expressing tumor-specific TCR and CD8αβ in tumor-injected NOG mice, measured as tumor volume, after overnight co-culture of γδ T cells with tumor cells. [Figure 10B]10B shows the tumor-suppressing effect of γδ T cells co-expressing tumor-specific TCR and CD8αβ, and Fig. 10B shows the tumor-suppressing effect of infusion of γδ T cells co-expressing tumor-specific TCR and CD8αβ in NOG mice transplanted with tumor cells, measured as tumor volume. [Figure 11A] 11A shows the tumor-suppressing effect of γδ T cells into which tumor-specific TCRs have been introduced, and FIG 11B shows the results of visualization by bioimaging of tumor formation in NOG mice. [Figure 11B] 11B is a graph showing tumor suppression by γδ T cells into which tumor-specific TCRs have been introduced. FIG. 11B is a graph showing tumor formation in NOG mice in terms of mean radiance. [Figure 12] Figure 1 shows the results of analyzing the effect of freeze-thawing on the function of genetically modified γδ T cells stimulated with compound 7. (A) shows the results for unfrozen cells, and (B) shows the results for freeze-thawed cells. [Figure 13A] 13A shows the effect of Compound 7 on the proliferation of γδ T cells compared with zoledronic acid, and FIG. 13B shows the effect on peripheral blood mononuclear cells obtained from each subject. [Figure 13B] 13A and 13B show the effect of Compound 7 on the proliferation of γδ T cells compared with zoledronic acid, and Fig. 13B shows mean values. [Figure 14] 1 shows the effect of Compound 7 on the purity of γδ T cells, compared with zoledronic acid. (A) shows the frequency of γδ T cells in CD3-positive cells (Vd2 / CD3). (B) shows the frequency of γδ T cells in the lymphocyte fraction (P1) (Vd2 / P1). DETAILED DESCRIPTION OF THE INVENTION

[0013] [Bisphosphonate Derivatives] Bisphosphonic acids are analogs of diphosphates, and are compounds (PCPs) in which the O (oxygen atom) in the diphosphate backbone POP is replaced with a C (carbon atom). Nitrogen-containing bisphosphonic acids are compounds having an N (nitrogen atom) in the bisphosphonic acid molecule. The bisphosphonate ester derivatives used in the present invention are pivaloyloxymethyl (POM) esters of the nitrogen-containing bisphosphonic acids listed in Table 1, or pharmaceutically acceptable salts, hydrates, or solvates thereof. These compounds are described in Patent Document 1 (WO2016 / 098904) and can be produced based on the description therein. Alternatively, they can be synthesized using synthetic methods well known to those skilled in the art.

[0014] [Table 1]

[0015] Gamma-delta T cells can be cultured in the presence of one or more of the nitrogen-containing bisphosphonic acid pivaloyloxymethyl esters listed in Table 1, but it is preferable to use tetrakispivaloyloxymethyl 2-(thiazol-2-ylamino)ethylidene-1,1-bisphosphonate (Compound 7) or a pharmaceutically acceptable salt thereof. A pharmaceutically acceptable salt is a common salt that is pharmacologically and pharmaceutically acceptable and is not toxic to cells even when added to a cell culture medium.

[0016] Nitrogen-containing bisphosphonates, such as zoledronic acid, which is used as a bone resorption inhibitor, inhibit farnesyl diphosphate synthase (FDPS) in cells (van Beek E, et al. Biochem Biophys Res Commun. 1999 Oct 14;264(1):108-111). Inhibition of FDPS by nitrogen-containing bisphosphonates increases the level of isopentenyl diphosphate, an upstream metabolite of FDPS in the biosynthetic pathway, resulting in stimulation of gamma-delta T cells (Wang H, J Immunol. 2011 Nov 15;187(10):5099-5113 and Tanaka Y, Sci Rep. 2017 Jul 20;7(1):5987). When the compounds listed in Table 1 herein are taken up into cells, the pivaloyloxymethyl group is hydrolyzed by intracellular esterases to form the free acid. This free acid inhibits FDPS in gamma delta T cells, thereby stimulating gamma delta T cells (Patent Document 1, Non-Patent Document 7, and Tanaka Y, Sci Rep. 2017 Jul 20;7(1):5987).

[0017] Nitrogen-containing bisphosphonic acids have low permeability into cells in the free acid state. The compounds listed in Table 1 of this specification are pivaloyloxymethyl (POM) esters, which confer permeability to cells. The compounds listed in Table 1 are preferably substituted with four POM groups per molecule, but may have one to three POM groups per nitrogen-containing bisphosphonic acid molecule as long as permeability into cells is maintained. Furthermore, other esters may be used as long as permeability into cells is maintained and they are hydrolyzed by intracellular esterases. Examples include n-butyloxymethyl ester derivatives and n-heptyloxymethyl ester derivatives. These nitrogen-containing bisphosphonic acid ester derivatives may also have one to four ester groups per molecule, but preferably four ester groups.

[0018] [Gamma-delta T cell culture] The γδ T cells used in the present invention are derived from mononuclear cells isolated from peripheral blood, umbilical cord blood, or cancer tissue biopsy tissue of mammals, particularly cancer or non-cancer patients. The γδ T cells can be preferably obtained from whole blood by density centrifugation, a method well known to those skilled in the art. Density centrifugation methods include, but are not limited to, methods using lymphocyte separation solutions such as Ficoll and Lymphoprep (registered trademark). Furthermore, cells may be separated by fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS).

[0019] Cancer types from which mononuclear cells can be collected include, but are not limited to, breast cancer, lung cancer, liver cancer, oral cancer, nasopharyngeal cancer, head and neck cancer, gastric cancer, esophageal cancer, colon cancer, skin cancer, malignant melanoma, kidney cancer, pancreatic cancer, brain tumor, prostate cancer, ovarian cancer, cervical cancer, colorectal cancer, bladder cancer, synovial sarcoma, liposarcoma, leukemia, malignant lymphoma, and multiple myeloma.

[0020] Gamma-delta T cells can be expanded from peripheral blood mononuclear cells, umbilical cord blood mononuclear cells, or tissue-derived mononuclear cells by culturing them in a cell culture medium containing a nitrogen-containing bisphosphonic acid POM ester compound listed in Table 1 herein, as well as IL-7 and IL-15. The scope of the present invention includes IL-7 and IL-15 mutants or fragments that bind to their respective receptors and exert biological activity. Examples of cell culture medium include, but are not limited to, RPMI 1640 medium and Yssel's medium. For example, fetal bovine serum, human AB serum, autologous plasma, human albumin, or serum substitutes can be added to the cell culture medium at a concentration of 0.1 to 20% (v / v). The nitrogen-containing bisphosphonic acid POM ester can be added to the cell culture medium at a concentration of 0.01 to 1 μM. The concentrations of IL-7 and IL-15 added to the cell culture medium can be varied depending on the specific activity of the lot used, but both cytokines can be added at a concentration of 5 to 100 ng / mL.

[0021] Gamma-delta T cells are stimulated by culturing them in the presence of the nitrogen-containing bisphosphonic acid POM ester in an environment of 5% CO at 37°C for 1 to 7 days. IL-7 and IL-15 may be added to the culture medium simultaneously with the nitrogen-containing bisphosphonic acid POM ester, or may be added 1 to 5 days after the addition of the bisphosphonic acid POM ester.

[0022] [Gene transfer into gamma-delta T cells] Mononuclear cells derived from peripheral blood or tissues are cultured in the presence of the nitrogen-containing bisphosphonic acid POM ester, IL-7, and IL-15, and the resulting γδ T cells can be transduced with a gene using a gene transfer method well known to those skilled in the art. Gene transfer can be performed at any time during the γδ T cell culture period, but is preferably performed between the day after the start of culture and 14 days after the start of culture. Methods for transducing genes into γδ T cells include lipofection, calcium phosphate coprecipitation, DEAE-dextran, electroporation, microinjection, plasmid vectors, and viral vectors. DNA or RNA vectors are used as vectors. Viral vectors include lentiviral vectors, adenoviral vectors, adeno-associated virus, and retroviral vectors, with retroviral vectors being preferred.

[0023] When using a viral vector to transfer genes into γδ T cells, functional substances that improve viral infection efficiency can be used. Examples include functional substances that have the activity of binding to viral vectors, such as fibronectin, fibronectin fragments, and polypeptides. In particular, in the case of retroviral vectors, it is preferable to use RetroNectin (registered trademark) or Vecofusin-1 (registered trademark), which are fibronectin fragments with a heparin-binding site. These functional substances can be used in a state where they are immobilized on an appropriate solid phase (e.g., plate, Petri dish, conical tube, microtube, etc.) or carrier (e.g., microbeads).

[0024] Genes to be introduced into γδ T cells include, but are not limited to, NY-ESO-1-specific TCR, HTLV-1 Tax-specific TCR, MAGE-A4-specific TCR, MAGE-A4-specific CAR, CEA-specific CAR, GD2-specific CAR, CD19-specific CAR, etc. The retroviral vector structure for TCR expression is a structure in which the α-chain gene and β-chain gene of each specific TCR are incorporated between LTRs (Long Terminal Repeats) at both ends containing an enhancer and promoter. The retroviral vector structure for CAR expression is a structure in which the α-chain gene and β-chain gene of each specific TCR are incorporated between the LTRs at both ends containing an enhancer and promoter, followed by a leader sequence, followed by HLA-A2 and MAGE-A4. p230-239 The VH and VL are components of an antibody that specifically recognize a complex with a peptide, followed by CL, CD28TM, and an intracellular domain. To express and introduce individual TCRs and CARs, other means, such as lentiviral vectors, may be used to express and introduce the corresponding TCRs and CARs.

[0025] After introducing a gene into γδ T cells using a vector, gene expression in the cells can be confirmed by methods known to those skilled in the art, such as flow cytometry, RT-PCR, Northern blotting, Western blotting, ELISA, and fluorescent immunostaining.

[0026] Gamma delta T cells transduced with TCR or CAR genes express the corresponding TCR or CAR on the cell membrane. When the specific antigens recognized by these TCRs or CARs bind to the TCR or CAR, respectively, the genetically modified gamma delta T cells are activated, producing cytokines such as interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNFα), and expressing cytotoxic molecules such as CD107a.

[0027] The genetically modified γδ T cells obtained as described above exhibit specific cytotoxicity against cells expressing an antigen specifically recognized by the TCR or CAR expressed by the cells. Therefore, the genetically modified γδ T cell population can be administered to patients for the treatment or prevention of diseases involving cells expressing the antigen, such as viral infections, bacterial infections, fungal infections, protozoan infections, or cancer. Administration to patients is preferably by injection or infusion. Although intravenous administration is preferred, direct injection into living tissues is also acceptable. Furthermore, because they do not induce graft-versus-host disease (GVHD), they are suitable for both autologous and allogeneic transplantation.

[0028] When the antigen is a cancer-specific antigen, a cell preparation or pharmaceutical composition containing the genetically modified γδ T cell population of the present invention can be used as an anticancer agent. Examples of cancer-specific antigens include NY-ESO-1, MAGE-A4, and CEA (carcinoembryonic antigen). Examples of cancers expressing these cancer-specific antigens include breast cancer, lung cancer, gastric cancer, esophageal cancer, bile duct cancer, malignant melanoma, prostate cancer, ovarian cancer, synovial sarcoma, liposarcoma, and multiple myeloma. CD19, expressed on B cells, can also be used as an antigen to formulate an anticancer agent for B-cell tumors. Examples of cancer-specific antigens include GD2 (disialoganglioside 2). GD2, expressed in neuroblastoma, is expressed not only in neuroblastoma but also in a wide range of other malignant tumors of neuroectodermal origin, such as brain tumors, retinoblastoma, small cell lung cancer, and malignant melanoma. The pharmaceutical composition of the present invention can be used for the treatment or prevention of these cancers. When the antigen is an infectious disease-specific antigen, the cell preparation or pharmaceutical composition containing the genetically modified γδ T cell population of the present invention can be used as a therapeutic agent for neoplastic diseases caused by infectious diseases or infectious diseases. Examples of infectious disease-specific antigens include the HTLV-1 (human T-cell leukemia virus type 1)-derived antigen Tax. Examples of infectious diseases or neoplastic diseases expressing these infectious disease-specific antigens include HTLV-1-associated myelopathy, HTLV-1 uveitis, and ATL (adult T-cell leukemia-lymphoma). The pharmaceutical composition of the present invention can be used for the treatment or prevention of these infectious diseases.

[0029] The cell preparation or pharmaceutical composition of the present invention may contain pharmaceutically acceptable additives, such as cell culture medium and phosphate-buffered saline. [Example]

[0030] The present invention will now be described in detail with reference to examples, but the present invention is not limited to these examples. [Example]

[0031] [Proliferation of gamma-delta T cells] Mononuclear cells were prepared from the peripheral blood of two healthy individuals using Ficoll. In accordance with the research ethics regulations of the Mie University School of Medicine, consent was obtained from the individuals before blood collection. Mononuclear cells were cultured at 1.5 × 10 in Yssel's medium containing 10% human AB serum. 6 Mononuclear cells adjusted to 1000 cells / mL were cultured in the presence of tetrakispivaloyloxymethyl 2-(thiazol-2-ylamino)ethylidene-1,1-bisphosphonate (compound 7, 1 μM) for 1 day, then 25 ng / mL IL-7 (Miltenyi Biotec, catalog number 170-076-111) and 25 ng / mL IL-15 (Miltenyi Biotec, catalog number 170-076-114) were added and cultured for an additional 10 days. The frequency of γδ T cells in the mononuclear cell population was analyzed by flow cytometry (FACSCANTO II, Becton Dickinson). As a result, the percentage of γδ T cells at the start of culture (Day 0) was 2.7% and 1.7% for donor 1 (HD1) and donor 2 (HD2), respectively. However, by culturing in the presence of compound 7, IL-7, and IL-15, the percentage increased to 97.1% and 94.5%, respectively, by Day 11 (Day 11), with the majority of cells proliferating into γδ T cells with high purity (Figure 1A). Normally, peripheral blood from healthy individuals contains 1-3% γδ T cells, but 1 × 10 γδ T cells were isolated from 1.5 mL of peripheral blood. 8 It was possible to prepare a highly purified γδ T cell population of approximately 100 cells.

[0032] 3 × 10 mononuclear cells collected from 3 mL of peripheral blood 6 The cells were cultured for 13 days in the presence of compound 7 (1 μM), IL-7 (25 ng / mL), and IL-15 (25 ng / mL). The culture medium used was Yssel's medium containing 10% human AB serum. As a result, rapid cell proliferation was observed from around day 8 of culture, and by day 13 of culture, the cell count was 3 × 10 8 The number of cells increased by approximately 5,000 times (Figure 1B). The results demonstrate that γδ T cells can be stimulated and expanded with compound 7, followed by culturing them in a culture medium containing IL-7 and IL-15, resulting in the efficient expansion of a highly purified γδ T cell population.

[0033] For comparison, mononuclear cells were cultured in a culture medium containing IL-2 (PROLEUKIN, Chiron Therapeutics) instead of IL-7 and IL-15. The cell proliferation-promoting effects of IL-7 and IL-15 were superior to those of IL-2 (100 IU / mL and 300 IU / mL) alone (Figure 1B). Furthermore, the cell proliferation-promoting effects of IL-7 and IL-15 were not further enhanced by the addition of IL-2. [Example]

[0034] [Production of a γδ T cell population transfected with a foreign gene (TCR)] On Day 0, 1.5 × 10 mononuclear cells were collected from 1.5 mL of peripheral blood of a healthy donor using Ficoll. 6 Stimulation culture was initiated in the presence of Compound 7 (1 μM). On Day 1, IL-7 and IL-15 were added to the culture medium to a final concentration of 25 ng / mL. On Days 4 and 5, HLA-A2 and NY-ESO-1 were stimulated in the presence of RetroNectin (registered trademark, Takara Bio). p157-165 The cells were infected with a retroviral vector expressing NY-ESO-1-specific TCR, which specifically recognizes a complex with the peptide (SLLMWITQC). Culture was continued in a medium containing IL-7 and IL-15, and the expression efficiency of the transduced TCR was examined on Day 11. The results are shown in Figure 2. Figure 2 demonstrates that gamma delta T cells transduced with the TCR gene were obtained with high efficiency. The resulting gamma delta T cells were positive for CD3, which forms a complex with the TCR, and for the receptor NKG2D (natural killer group 2, member D).

[0035] γδ T cells into which TCR was introduced by a NY-ESO-1-specific TCR-expressing retroviral vector expressed NY-ESO-1 p157-165Co-culture (4 hours) of the (SLLMWITQC) peptide with the HLA-A2-positive cell line T2 confirmed the production of cytokines IFN-γ and TNF-α and the expression of CD107a, an indicator of functional cytotoxic activity (Figure 3). Amino acids in the amino acid sequence are indicated by single-letter abbreviations. [Example]

[0036] [Antigen-specific recognition of tumor cell lines by TCR gene-transduced γδ T cells] HLA-A*02:01 Binding to NY-ESO-1 p157-165 The tumor recognition ability of γδ T cells transfected with a TCR gene that specifically recognizes a peptide was examined. The results are shown in Figure 4. When the γδ T cells were co-cultured (4 hours) with the SK-MEL-37 cell line, an NY-ESO-1-positive, HLA-A*02:01-positive cell line, IFN-γ production was confirmed by intracellular cytokine staining using the intracellular IFN-γ staining method. However, when co-cultured with Mel72, an NY-ESO-1-negative, HLA-A*02:01-positive cell line, no IFN-γ production was observed. As a positive control, NY-ESO-1 p157-165 The peptide-added HLA-A2-positive T2 cell line (T2-ESO1) and MAGE-A4 were used as negative controls. p230-239 Using a peptide-loaded T2 cell line (T2-MAGE), the generated TCR-transduced gamma delta T cells were shown to be functional and to recognize tumor cells in an antigen-specific manner. [Example]

[0037] [Recognition of HLA-A*24:02 and p40Tax-positive cell lines by γδT cells transfected with HTLV-1 viral antigen p40Tax-specific TCR] We investigated the antigen recognition of γδ T cells transfected with a retroviral vector that specifically recognizes the HTLV-1 viral antigen p40Tax-derived peptide (SFHSLHLLF) in an HLA-A*24:02-restricted manner. The results are shown in Figure 5. When the γδ T cells were cocultured (18 hours) with the ILT-Hod cell line, an HLA-A*24:02-positive HTLV-1-positive cell line, IFN-γ production was confirmed in the culture supernatant by ELISA. However, when the cells were cocultured with the ILT-#37 cell line, an HLA-A*24:02-negative HTLV-1-positive cell line, no IFN-γ production was observed (Figure 5A). As a positive control, the HLA-A24-positive T2A24 cell line (T2A24-p40) supplemented with the p40Tax-derived peptide was used, and as a negative control, NY-ESO-1 p157-165 We used the peptide-treated T2A24 cell line (T2A24-ESO). Furthermore, when the γδ T cells were cocultured with either the ILT-Hod or ILT-#37 cell lines at varying cell ratios, strong cytotoxicity against the ILT-Hod cell line was observed, depending on the E / T ratio (Figure 5B). Thus, TCR-transduced γδ T cells generated from γδ T cells stimulated with compound 7 were shown to be functional and capable of antigen-specific tumor cell recognition and cytotoxicity. [Example]

[0038] [Production of a γδ T cell population transfected with a foreign gene (CAR)] In recent years, CAR therapy using CAR genes has been clinically applied to hematological malignancies. Therefore, we investigated whether CARs could be introduced into gamma-delta T cells. On day 0, 1.5 × 10 mononuclear cells were collected from 1.5 mL of peripheral blood of healthy individuals using Ficoll. 6 Stimulation culture was initiated in the presence of Compound 7 (1 μM) (Day 0). On Day 1, IL-7 and IL-15 were added to the culture medium to a final concentration of 25 ng / mL. On Days 4 and 5, HLA-A*02:01 and MAGE-A4 were stimulated in the presence of retronectin. p230-239The cells were infected with a retroviral vector expressing a CAR that specifically recognizes a complex with the peptide (GVYDGREHTV). After this, the cells were cultured in a medium containing IL-7 and IL-15, and the expression efficiency of the transduced CAR was examined on Day 11.

[0039] The results are shown in Figure 6. Figure 6 demonstrates that γδ T cells into which the CAR gene was introduced were obtained with high efficiency. The obtained γδ T cells were positive for CD3 and NKG2D.

[0040] Similarly, on days 4 and 5, cells were infected with a retroviral vector for expressing a CAR that specifically recognizes GD2 (disialoganglioside 2) or CD19 in the presence of retronectin. Culture was then continued in a medium containing IL-7 and IL-15, and the expression efficiency of the introduced CAR was examined on day 11.

[0041] The results are shown in Figure 7 for the expression of GD2-specific CARs when a CAR gene specifically recognizing GD2 was introduced, and in Figure 8 for the expression of CD19-specific CARs when a CAR gene specifically recognizing CD19 was introduced. Figures 7 and 8 demonstrate that γδ T cells transfected with CAR genes targeting GD2 or CD19 were obtained with higher efficiency than controls. When γδ T cells transfected with GD2-specific CAR genes were cocultured with the GD2-positive target tumor cell line AS, enhanced cytokine IFN-γ production was observed in γδ T cells transfected with GD2-specific CARs compared to controls. Similarly, when γδ T cells transfected with CD19-specific CAR genes were cocultured with CD19-positive tumor cells (NALM6), enhanced cytokine IFN-γ production was observed in γδ T cells transfected with CD19-specific CARs compared to controls. [Example]

[0042] [Antigen-specific tumor cell line recognition by CAR gene-transduced gamma-delta T cells] HLA-A*02:01 restricted to MAGE-A4 p230-239 The tumor recognition ability of gamma delta T cells transfected with a CAR gene that recognizes (GVYDGREHTV) was examined. The results are shown in Figure 9. When SK-MEL-37 and NW-MEL-38 cell lines, which are MAGE-A4-positive and HLA-A*02:01-positive cell lines, were co-cultured (4 hours), flow cytometry analysis confirmed the expression of CD107a, an indicator of functional cytotoxic activity. However, when Mel72 and HCT116 cell lines, which are MAGE-A4-negative and HLA-A*02:01-positive cell lines, were co-cultured, CD107a expression was low. As a positive control, MAGE-A4 was used. p230-239 The peptide-treated HLA-A2-positive T2 cell line (T2-MAGE) and NY-ESO-1 (NY-ESO-1) were used as negative controls. p157-165 The peptide-added T2 cell line (T2-ESO1) was used. [Example]

[0043] [Tumor-suppressive effect of γδ T cells expressing CD8αβ and tumor-specific TCR] HLA-A*02:01 Binding to NY-ESO-1 p157-165 We infected γδ T cells with a retroviral vector carrying a peptide-specific TCR (G50 TCR) gene and a retroviral vector co-expressing human CD8α and CD8β chains to generate γδ T cells expressing the TCR (G50 TCR) and CD8αβ, a costimulatory molecule that enhances TCR stimulation. We then examined the tumor-suppressive activity of these cells. For comparison, we generated control cells (NGMC, non-gene modified T cells), cells expressing only the G50 TCR, and cells expressing only CD8αβ. We compared IFN-γ and CD107a production in these γδ T cells when co-cultured with T2-ESO1, T2-MAGE, NW-MEL-38, and HCT116 cell lines. The results showed that IFN-γ and CD107a production were significantly higher than those of NY-ESO-1. p157-165The activity was enhanced when co-cultured with the peptide-added T2 cell line T2-ESO1 or the HLA-A2-positive MAGE-A4-positive cell line NW-MEL-38, and the enhancement effect was stronger in γδ T cells transfected with G50 TCR and expressing CD8αβ than in γδ T cells transfected with G50 TCR alone (Figure 10A).

[0044] On Day 0, 4 × 10 tumor cells (HCT116 and NW-MEL-38) were subcutaneously injected into the left and right dorsal regions of NOG mice. 6 On day 7, 1 × 10 γδ T cells of each of the four types were transplanted into the mice to form tumors. 7 The tumor size was measured. In mice transplanted with HCT116 cells, which did not induce IFN-γ or CD107a production in coculture with γδ T cells, no tumor-suppressing effect was observed, regardless of the γδ T cell infusion (Fig. 10B, left panel). However, in mice transplanted with NW-MEL-38 cells, which induced IFN-γ and CD107a production, significant tumor-suppressing effect was observed in mice transplanted with γδ T cells expressing G50 TCR and CD8αβ, indicating that coexpression of a tumor-specific TCR and CD8αβ is effective in tumor suppression (Fig. 10B, right panel). [Example]

[0045] [Creation of a pathological model using NOG mice and the tumor-suppressive effect of γδ T cells transduced with tumor-specific TCRs in this model] An HLA-A*24:02-positive, HTLV-1-positive cell line (TL-Su) transfected with a luciferase gene was generated and inoculated into NOG mice. Tumor formation was visualized by bioimaging. Gamma delta T cells transfected with tumor-specific TCRs were produced as described in Example 2, except that the retroviral vector used expressed a TCR that specifically recognized the HTLV-1 viral antigen p40Tax-derived peptide (SFHSLHLLF) in an HLA-A*24:02-restricted manner. NGM-g / dT cells were used as control cells.

[0046] 5 × 10 5 TL-Su cell lines were subcutaneously transplanted (Day -7). After 7 days, 5 × 10 6 The above genetically modified γδ T cells or NGM-g / dT cells were intravenously injected into mice (Day 0). Tumors were visualized by bioimaging 1 day (Day 1), 1 week (1w), 2 weeks (2w), 3 weeks (3w), 4 weeks (4w), and 6 weeks (6w). The results are shown in Figure 11(A). Tumors in mice administered phosphate-buffered saline (PBS) instead of cells showed growth over time. Even when NGM-g / dT cells were administered, tumor growth was observed and no tumor-suppressing effect was observed. However, in mice administered the above genetically modified γδ T cells, tumor growth was barely observed, and no tumors were observed after 3 weeks. The above genetically modified γδ T cells suppressed tumor growth and demonstrated therapeutic effects in NOG mouse models, resulting in tumor regression. The mean radiance in the mice is shown in Figure 11(B). It can be seen that the mean radiance increased over time in the PBS-treated group and the NGM-g / dT cell-treated group, but this was hardly observed in the genetically modified γδ T cell-treated group (FIG. 11B). [Example]

[0047] Effect of freeze-thawing on the function of genetically modified gamma-delta T cells stimulated with compound 7. To facilitate disease treatment using transfusion of genetically engineered γδ T cells, freezing is required for the transport and storage of the cells. Furthermore, to make transfusion therapy practical, the loss of cell function due to freezing must be avoided. Therefore, we investigated whether freezing affected the function of transgenic γδ T cells after cell proliferation in the presence of compound 7 (see Table 1).

[0048] On Day 0, mononuclear cells collected from peripheral blood of healthy donors using Ficoll were stimulated and cultured in the presence of Compound 7 (1 μM). On Day 1, IL-7 and IL-15 were added to the culture medium to a final concentration of 25 ng / mL. On Days 4 and 5, HLA-A2 and NY-ESO-1 were stimulated using a retroviral vector in the presence of RetroNectin. p157-165 The NY-ESO-1-specific TCR (G50 TCR), which specifically recognizes a complex with the peptide (SLLMWITQC), was transfected. Culture continued in a medium containing IL-7 and IL-15. On Day 12, the cells were frozen and thawed the following day, Day 13, for analysis of cell function. For comparison, cells were analyzed on Day 13 without freezing. The results are shown in Figure 12. Flow cytometry analysis revealed almost no difference in the expression level of the transfected NY-ESO-1-specific TCR gene between frozen-thawed cells (Figure 12B, upper panel) and unfrozen cells (Figure 12A, upper panel). We also co-cultured genetically modified γδ T cells with T2-ESO1, T2-MAGE, SK-MEL-37, NW-MEL-38, MEL72, and HCT116 cell lines to analyze the effect of freezing genetically modified γδ T cells on CD107a production. The results showed that there was little difference in antigen specificity for the complex of HLA-A2 and NY-ESO-1p157-165 peptide (SLLMWITQC) between frozen-thawed cells (Figure 12B, bottom panel) and unfrozen cells (Figure 12A, bottom panel). These results indicate that the function of genetically modified γδ T cells stimulated and cultured with compound 7 is not affected by freezing. [Example]

[0049] [Comparison of the effects of compound 7 and zoledronic acid on the proliferation and purity of gamma-delta T cells] Zoledronic acid (Zometa) is a compound with the following structure, and is a nitrogen-containing bisphosphonic acid that is common to compound 7 (see Table 1). Therefore, the usefulness of compound 7 relative to zoledronic acid was investigated using the proliferation of gamma-delta T cells and the purity of the gamma-delta T cells obtained after proliferation as indicators. JPEG0007820774000002.jpg4453 Peripheral blood mononuclear cells (PBMCs) were collected from healthy volunteers (n = 8, HD1 to HD8) in two separate experiments (n = 4 per experiment). The collected PBMCs were placed in each well of a 24-well plate at 1.5 × 10 6 After adding 100 μM of compound 7 or zoledronic acid (5 μM), stimulation culture was initiated with modified Cyssel's medium (1.5 mL) containing 10% human AB serum and compound 7 (1 μM) or zoledronic acid (5 μM) (Day 0). Next, on Day 1, IL-2 (300 U / mL) was added. IL-2 is a cytokine previously reported for the proliferation of γδ T cells (Non-Patent Documents 5 and 6). On Day 6, cells from one well were diluted 2-fold to two wells, and the cell number was counted. On Day 7, cells from two wells were diluted 2-fold to four wells, and IL-2 (300 U / mL) was added. On Day 8, half the amount of cells (approximately 3 mL) was diluted 6-fold to 18 mL and cultured in a T75 flask. On Days 8 and 11, cell number was counted and cell purity was measured by flow cytometry.

[0050] Figure 13A shows the time course of the effects of compound 7 (PTA) and zoledronic acid (Zometa) on cell proliferation in peripheral blood mononuclear cells obtained from each subject. On Day 8, there was little difference in the effects of compound 7 and zoledronic acid on cell proliferation. However, on Day 11, compound 7 (PTA) exhibited a stronger stimulatory effect on cell proliferation than zoledronic acid (Zometa) in all cells from subjects HD1 to HD4 in Experiment 1. In Experiment 2, compound 7 (PTA) exhibited a weaker stimulatory effect on cell proliferation than zoledronic acid (Zometa) in cells from subject HD8, but was comparable in cells from subject HD5. Compound 7 (PTA) exhibited a stronger effect than zoledronic acid (Zometa) in cells from subjects HD6 and HD7. Figure 13B summarizes the results of Experiments 1 and 2 on Day 11, showing the mean ± standard deviation (n = 8). It can be seen that compound 7 (PTA) is significantly superior to zoledronic acid (Zometa) in the proliferation of γδ T cells (p=0.008, t-test).

[0051] Next, on Day 8 and Day 11, cell counting and cell purity measurement by flow cytometry analysis were performed. The frequency of γδ T cells (Vd2 / CD3) among CD3-positive cells was shown to be significantly higher in the compound 7 (PTA) treatment group than in the zoledronic acid (Zometa) treatment group on both Day 8 and Day 11 (Figure 14A). Furthermore, the frequency of γδ T cells (Vd2 / P1) in the lymphocyte fraction (P1) in flow cytometry analysis was also shown to be significantly higher in the compound 7 (PTA) treatment group than in the zoledronic acid (Zometa) treatment group on Day 8 and Day 11 (Figure 14B). These results demonstrate that compound 7 (PTA) is superior to zoledronic acid (Zometa) in obtaining highly pure γδ T cells. Note that the significance test in Figure 14 was performed using a t-test. Test Example

[0052] Materials and Methods 1. Lymphocyte Preparation Human lymphocytes were obtained by isolating peripheral blood mononuclear cells (PBMCs) from blood donated by healthy donors using Ficoll-Paque® PLUS (GE Healthcare, catalog number 17-1440-03). The collection and analysis of human peripheral blood and other samples used in this study were conducted in accordance with the Declaration of Helsinki. All studies were conducted according to protocols approved by the Mie University School of Medicine Research Ethics Committee and with written consent from the subjects. Collected samples were encrypted to prevent personal identification and stored in an anti-theft refrigerator and liquid nitrogen tank. Subject personal information was anonymized, and strict precautions and measures were taken to prevent the disclosure of personal privacy and genetic analysis results. 2. Gene transfer into cells A retroviral vector was used to introduce the TCR / CAR gene into γδ T cells. RetroNectin (registered trademark) (Takara Bio) dissolved at 20 μg / mL in Biological Product Standard Blood Preservation Solution A (ACD-A Solution, Terumo) was used to coat a multi-dish for suspension cells at 500 mL / well for 16 hours at 4°C or 2 hours at 25°C. A retroviral vector solution was added to the multi-dish at 1 mL / well, and preloading was performed by centrifugation (2000 × g, 2 hours, 32°C). Each well was then washed twice with 1 mL of phosphate-buffered saline (PBS) containing 1.5% human serum albumin (HSA), and 3.8 × 10 lymphocytes were added. 5Cells were seeded at 0.95 mL / well and pelleted by centrifugation (1000 × g, 32°C, 10 min). After microscopic examination, the cells were cultured for 1 day in the presence of the indicated cytokines (IL-2 100 IU / mL or 300 IU / mL, or IL-7 25 ng / mL and IL-15 25 ng / mL) at 37°C in a 5% CO2 incubator. After 24 hours, the cells were diluted 4:3, and the entire volume was used for a second transfection using the same method as the first transfection. The culture was continued for 4 hours, and then diluted with 6.8 mL of culture medium and cultured again at 37°C in a 5% CO2 environment. Non-gene-modified T cells (NGMCs) were prepared by culturing the cells in the presence of the same cytokines for 6 days or more using the same method as for the transfected cells. The retroviral transduction experiments of tumor antigen-specific TCRs into human peripheral blood mononuclear cells were approved by the Mie University Recombinant DNA Experimental Review Board and the Mie University Faculty of Medicine Research Ethics Committee. These experiments were performed in an approved P2-level laboratory at Mie University. 3. Flow Cytometry The stained cells were analyzed using a BD FACS Canto® II flow cytometer (Becton Dickinson). Gamma delta T cells stimulated in the presence of compound 7 were infected with retrovirus to express the target TCR or CAR gene. After 8-11 days of culture, the cells were washed twice with 2% fetal calf serum (FCS)-PBS. Tetramers specific to each TCR / CAR were then diluted 50-fold with 2% FCS-PBS and added to the cells. The cells were incubated at 37°C for 15 minutes in the dark. Next, the cells were stained with FITC- or V500-labeled anti-human CD8 antibody (Becton Dickinson) or FITC-labeled anti-human Vδ2 antibody (Biolegend) for 15 minutes at 4°C in the dark. After washing twice with 2% FCS-PBS, the cells were analyzed using a flow cytometer. 4.ELISA ELISA was performed using a kit from eBioscience. The coating buffer was prepared by diluting 10x coating buffer 10 times with distilled water. 48 μL of primary antibody was diluted by mixing with 12 mL of coating buffer. 100 μL of this diluted solution was added to each well of a 96-well flat-bottom plate and incubated overnight at 4°C. The wells were then washed five times with 0.05% PBS-T (phosphate-buffered saline containing Tween 20). The assay diluent was prepared by diluting 5x assay diluent 5 times with distilled water. 200 μL of the assay diluent was added to each well, followed by blocking at room temperature for 1 hour and then washing five times with 0.05% PBS-T. The IFN-γ standard solution was prepared by diluting 7 times with a common ratio of 2, with the highest concentration of IFN-γ at 1000 pg / mL. The measurement samples and standard solution were added to each well of the plate and incubated at room temperature for 2 hours. After incubation, the wells were washed five times with 0.05% PBS-T. 48 μL of secondary antibody was diluted with 12 mL of assay diluent, and 100 μL of this solution was added to each well. The incubation was continued for 1 hour at room temperature. After incubation, the wells were washed five times with 0.05% PBS-T. 48 μL of streptavidin-peroxidase (horseradish peroxidase) was diluted with 12 mL of assay diluent, and 100 μL of this solution was added to each well. The incubation was continued for 30 minutes at room temperature in the dark. Next, the wells were washed seven times with 0.05% PBS-T, and 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB) substrate solution was added to each well. The incubation was continued for 15 minutes at room temperature in the dark, and the reaction was stopped by adding 50 μL of 0.18 M sulfuric acid to each well. The absorbance was immediately measured at a wavelength of 450 nm using a microplate reader (Model 680, Bio-Rad). 5. Intracellular Cytokine Staining 1 x 10 target cells 5 cells / mL, and 1 × 10 effector cells 5The cells were prepared to a concentration of 0.5 μL / mL. APC anti-human CD107a antibody was added to each sample at a target cell:effector cell ratio of 1:1, and the cells were co-cultured in a 96-well plate (U-bottom) at 37°C for 1 hour. Then, 0.7 μL of Golgistop® (Protein Transport Inhibitor, Becton Dickinson) was added to each well, and the cells were cultured at 37°C for 4 hours. The cells in each well were transferred to a 96-well V-shaped plate, centrifuged at 1200 rpm at 4°C for 5 minutes, and then washed twice with 0.5% BSA / PBS. PE-anti-human CD6 antibody (0.5 μL) was added to each well, and the wells were incubated on ice for 20 minutes in the dark, followed by washing twice with 0.5% BSA / PBS. 100 μL of cell fixative (Cytofix / Cytoperm®, Becton Dickinson) was added to each well and the plate was left on ice for 20 minutes in the dark. Then, 100 μL of Perm / Wash® (Becton Dickinson) buffer was added to each well and centrifuged. Then, the plate was washed twice more with Perm / Wash buffer. 0.5 μL of V450 IFN-γ or PE-Cy7 TNFα was added to each well. After leaving the plate on ice for 30 minutes in the dark, the wells were washed twice with 0.5% BSA / PBS. Measurements were then performed using a FACSCanto II flow cytometer (Becton Dickinson) and analyzed using FACS Diva software (Becton Dickinson).

Claims

1. 1. A method for producing a transgene-expressing γδ T cell population, comprising: (Step 1) culturing peripheral blood mononuclear cells containing γδ T cells in the presence of tetrakispivaloyloxymethyl 2-(thiazol-2-ylamino)ethylidene-1,1-bisphosphonate or a pharmaceutically acceptable salt thereof; (Step 2) culturing the cell population containing γδ T cells cultured in Step 1 in the presence of IL-7 and IL-15; and (Step 3) introducing a gene into the cell population containing γδ T cells cultured in Step 2, the gene is a gene encoding a T cell receptor (TCR) or a chimeric antigen receptor (CAR), the T cell receptor (TCR) is selected from the group consisting of NY-ESO-1p157-165 / HLA-A2 complex-specific TCR, HTLV-1p40Taxp11-19 / HLA-A2 complex-specific TCR, HTLV-1p40Taxp301-309 / HLA-A24 complex-specific TCR, and MAGE-A4p143-151 / HLA-A24 complex-specific TCR; The method, wherein the chimeric antigen receptor (CAR) is selected from the group consisting of a MAGE-A4p230-239 / HLA-A2 complex-specific CAR, a CEA-specific CAR, a GD2-specific CAR, and a CD19-specific CAR.

2. 2. The method according to claim 1, wherein the concentration of tetrakispivaloyloxymethyl 2-(thiazol-2-ylamino)ethylidene-1,1-bisphosphonate or a pharmaceutically acceptable salt thereof in step 1 is 0.01 to 1 μM.

3. The method of claim 1, wherein the γδ T cells are derived from a mammal.

4. The method of claim 3 , wherein the mammal is a cancer patient or a non-cancer patient.

5. 5. The method of claim 4, wherein the cancer is selected from the group consisting of breast cancer, lung cancer, liver cancer, oral cancer, nasopharyngeal cancer, head and neck cancer, gastric cancer, esophageal cancer, colon cancer, skin cancer, malignant melanoma, kidney cancer, pancreatic cancer, brain tumor, prostate cancer, ovarian cancer, cervical cancer, colorectal cancer, bladder cancer, synovial sarcoma, liposarcoma, leukemia, malignant lymphoma, and multiple myeloma.

6. 2. The method according to claim 1, wherein the step of introducing a gene in step 3 uses a DNA vector or an RNA vector.

7. 2. The method according to claim 1, wherein the step of introducing a gene in step 3 uses a vector selected from the group consisting of a plasmid vector, a lentiviral vector, an adenoviral vector, an adeno-associated virus, and a retroviral vector.

8. 2. The method according to claim 1, wherein the step of introducing a gene in step 3 uses a retroviral vector.

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