WO2006006720A1 - METHOD OF CULTURING ϜδT CELLS, ϜδT CELLS AND REMEDY/PREVENTIVE - Google Patents

Abstract

It is intended to provide a method of culturing ϜδT cells whereby ϜδT cells from peripheral blood monocytes can be selectively activated and/or proliferated and cultured at a high purity in a large amount; ϜδT cells cultured by the method; and a remedy/preventive containing ϜδT cells cultured by the method which is to be used in immunotherapy targeting cancer patients or infection. By adding from 0.05 to 100 μM of bisphosphonate to peripheral blood monocytes, ϜδT cells in the peripheral blood monocytes are selectively activated and/or proliferated and further activated and/or proliferated by stimulating with from 50 to 2000 U/mL of IL-2. Thus, the ϜδT cells can be cultured at a high purity in a large amount. The ϜδT cells thus obtained contain a large amount of CD56-positive cells having a high cytotoxic activity, which makes them useful as a remedy/preventive targeting cancer patients or infection.

Description

 Description r δ τ cell culture method, δ τ cell and therapeutic / prophylactic agent TECHNICAL FIELD

 The present invention relates to a method for culturing <5 <cells. Further, the present invention relates to a cultivated cell (5Τ cells, a therapeutic-preventive agent containing a cell cultured by the culture method.

 The most common cause of death among Japanese people is malignant neoplasm (hereinafter referred to as cancer). There are three major therapies for cancer treatment: surgical treatment, chemotherapy, and radiation therapy, each of which has its own difficulties and side effects.

 In recent years, in addition to the above three major therapies, immune cell therapy has been carried out as a new treatment for cancer, and this immune cell therapy has problems such as treatment difficulties and side effects such as the above three major therapies. It is attracting attention because there are few.

 Among these immune cell therapies, there is a therapy that activates T cells called L ΑΚ (1 ymp h o kine e-a c t i v a t e d k i l l e r; lymphokine-activated killer) therapy. There are two types of T cells: α / 3-type TCR (T cellreceptor)] 3 T cells and δ-type TCR-expressing δΤ cells. This is a therapy that activates βΤ cells. Here, αΖ3Τ cells are those that are mainly responsible for acquired immunity.

 On the other hand, δδ cells are cells responsible for innate immunity. Recently, it has been found that it has cytotoxic activity (non-specific activity) against cancer cells, and research on immunotherapy using the strong antitumor activity of τδ cells has been conducted.

 Δ δ Τ cells recognize and activate non-peptidic antigens, and as a non-peptidic antigen, for example, alkylamines can be stimulated with bisphosphonates to activate and / or proliferate. There are studies on activation and / or proliferation by recognizing non-peptide antigens in r δ Τ cells isolated from peripheral blood in vitro (F um i M iyag awa eta 1. The J ou r na lof I mm uno 1 ogy 2001; 166 (9): 550 8-5514). One

However, since τδΤ cells are usually present in only 1 to 5% of peripheral blood, even if a small amount of blood is collected to activate and / or proliferate δδΤ cells, the purity and number of cells sufficient for treatment There is a problem that cannot be secured. Also treatment If the amount of blood collected from a patient is increased in order to ensure sufficient purity and cell count, there is also a problem that a great burden is placed on the patient.

 By the way, there are cells that express CD 56 (hereinafter referred to as CD 56-positive cells (5T cells) and cells that do not express them (hereinafter referred to as CD 56-negative cells δ T cells). Positive δ T cells are known to have higher cytotoxic activity than CD 56 negative δ Τ cells (Fu j imiya, Y. eta 1. Clinical Cancer Re search Vo l. 3, 6 33— 643, Ap ril 1997), and is expected to be applied to the treatment of cancer and infectious diseases.

 Here, in the above-mentioned document (Fuj im iya, Y. eta 1. Clinica 1 Cancer Research Search Vol. 3, 633-643, Apri 1 1 997), the preparation of CD 56 positive δ Τ cells Although the method is described, in order to obtain high-purity CD 56-positive 6 T cell population, it is necessary to select at least 3 times using magnetic beads, and the process is complicated. There is a problem that the cost increases.

 In addition, JP-A-2001-314183 describes therapeutic agents and examples using a cell group containing 50% or more of CD56-positive cells. The CD56-positive cells used here are A δΤ cells and natural killer cells (hereinafter referred to as ΝΚ cells) are mixed, and only about 10% of ƒ <5 Τ cells are included. Disclosure of the invention

 The present invention has been made in view of the above circumstances, and is capable of selectively activating and cultivating or proliferating cells from peripheral blood mononuclear cells, and capable of culturing them in high purity and in large quantities. And a therapeutic / prophylactic agent containing the δ δ cells obtained by the method in immunotherapy for cancer patients, infectious diseases, etc. With the goal.

 In the cell culture method of the present invention, the concentration of a bisphosphonate bone metabolizer (hereinafter abbreviated as bisphosphonate) in peripheral blood mononuclear cells is 0.05 to 100 / Μ and interleukin 2 (hereinafter IL— 2) (abbreviated as 2) is added so that the concentration is 50 to 20000 U / mL and cultured.

According to such a configuration of the present invention, bisphosphonate is added to peripheral blood mononuclear cells in the culture solution so as to have a concentration of 0.05 to 100 M. δ Τ cells selectively activated and expanded or proliferated and further stimulated by adding IL-12 to 50-2000 U / mL. It can be obtained in high purity and in large quantities. Also got The T 6 Τ cells contain at least 40% of cells expressing CD 56.

 The bisphosphonate is added at the start of culture.

 According to such a configuration, by adding bisphosphonate at the start of culture, unlike the conventional method of isolating 5 T cells from peripheral blood mononuclear cells, the 5 T cells are separated. Therefore, it is possible to selectively activate and / or proliferate without going through the steps, so that one step of the culture can be omitted and the culture can be efficiently performed.

 ΤδΤ cells obtained by the present invention have a non-specific cytotoxic function, a cancer antigen-specific cytotoxic function, or both cytotoxic functions. Therefore, it can be used as a therapeutic / preventive agent for cancer diseases and infectious diseases.

 In addition, the α 5 Τ cell obtained by the present invention contains a large amount of CD 56-positive δ Τ cells having a high cytotoxic function. Therefore, by selecting for CD56-positive cells, it is possible to obtain a higher therapeutic / prophylactic effect when the same number of cells are administered.

 Furthermore, the therapeutic / prophylactic agent prepared by the present invention has a non-specific cytotoxic function, a cancer antigen-specific cytotoxic function, or both cytotoxic functions. High content of r δ and high cytotoxic function among r δ Τ cells (Because it contains a lot of 5 T cells, it can identify cancer cells or infected cells without attacking normal cells. It is effective in the treatment of various cancer types and infectious diseases, especially when δ δ cells are derived from autologous lymphocytes and are administered to the patient's own immunity. It is possible to perform its function without being excluded by the system.

 Hereinafter, embodiments of the present invention will be described.

 In the method for culturing ァ 5 cells of the present invention, the concentration of the bisphosphonate bone metabolizing agent in the peripheral blood mononuclear cells is from 0.05 to ≧ 10 1/1 and the concentration of IL-1 is from 50 to It is characterized by selectively activating and / or proliferating δδΤ cells by adding and culturing at 200 U / mL to obtain a cell population containing ァ 5Τ cells with high purity. To do.

Bisphosphonates may have any action to suppress bone resorption and are generally used as a therapeutic agent for osteoporosis. Examples thereof include pamidronic acid, a salt thereof and / or a hydrate thereof (eg, pamidronic acid Disodium pentahydrate (A redia, Novartis Pharma), alendronate, its salts and / or their hydrates (eg, sodium alendronate trihydrate (O ne 1 ast, 萬 有) Pharmaceutical)), zoledronic acid, its salts and / or their hydrates (for example, zole Sodium Dronate Hydrate (Zome ta, Novartis Pharma), Risedronic acid, its salts and Z or their hydrates (eg, risedronate sodium 'hydrate), ibandronic acid, its salts and Z or a hydrate thereof (eg, ibandronate disodium), incadronic acid, a salt thereof and Z or a hydrate thereof (eg, disodium incadronate), etidronic acid, a salt thereof and / or water thereof Among them, pamidronic acid, alendronic acid, zoledronic acid, its salt and ZX are those having nitrogen atoms such as their hydrates (amamino). Bisphosphonates) are preferred.

(1) First embodiment; r δ Τ cell culture method

 Hereinafter, the method for culturing cells <5> cells of the present invention will be described in order.

 1) Obtain peripheral blood by collecting blood. The appropriate amount is 15-25 mL. If this amount can be obtained, it can be suitably cultured. However, the range is not limited to this range as long as the peripheral blood volume is sufficient at the start of culture, and the upper limit may be further increased if the burden on the donor to collect blood is small.

2) Obtain peripheral blood mononuclear cells by, for example, density gradient centrifugation. About 10 to ⁷ peripheral blood mononuclear cells can be obtained from 15 to 25 mL of peripheral blood.

3) Suspend approximately 10 ⁷ peripheral blood mononuclear cells obtained in 2) in the culture medium AI MV (Invitrogen). Here, a suspension of peripheral blood mononuclear cells is referred to as a cell suspension. In addition to the culture medium shown here, it can be used for culturing cells such as RPMI-1640 medium (Invitrogen), Dulbecco's modified Eagle medium (Invitrogen, hereinafter referred to as DMEM), Iskov medium (Invitrogen, hereinafter referred to as IMEM), etc. You may use the commercially available culture solution used.

 If necessary, serum may be added at 0.1 to 20%. As the serum, for example, fetal calf serum (Feta1CalfSerum, hereinafter referred to as FCS), AB serum, autologous plasma, or the like may be used.

 4) Seed the cell suspension obtained in 3) in a flask, bag or plate.

5) Add bisphosphonate to peripheral blood mononuclear cells seeded in flasks, bags or plates so that the concentration is 0.05 to 100 M, preferably 0.1 to 30 AiM. Here, further, pamidronic acid, its salt and / or hydrate thereof is 1 to 30 M, alendronic acid, its salt and Z or hydrate thereof is 1 to 30 iM, zoledronic For acids, their salts and Z or their hydrates, 0.1 to 10 M is preferred.

6) Further, add IL-2 to the above culture solution at a concentration of 50 to 2000 UZmL, more preferably. Preferably, add 400 to 100 OUZmL.

7) 6) with IL- 2 34 to 38 ° C were added thereto, and more preferably at 37, 2-10%, and more preferably are cultured at 5% C_〇 ₂ presence. At this time, a culture solution is appropriately added according to the number of cells to be cultured. Furthermore, as the culture medium increases, the IL-12 concentration is appropriately added so as to be 50 to 200 OU / mL, more preferably 400 to LOO OUZmL.

 If the culture period is 7 days or longer, a cell group containing δΤ cells with high purity can be obtained. However, in order to further increase the number of rδΤ cells, it is preferable to culture for about 14 days.

 8) The δ δ cells obtained in this way contain a lot of CD 56 positive τ δ Τ cells with high cytotoxic function. CD 56 positive τ δ Τ cells can also be selected and used. Is possible. Examples of methods for sorting CD 56-positive δΤ cells include magnetic cell separation (Magnetic Cell Sorting, hereinafter referred to as MAC S) and flow cytometry. .

 By the above method, δδ T cells can be efficiently proliferated.

(2) Second embodiment: δ Treatment and prevention agent using sputum cells

 Next, the therapeutic / prophylactic agent using the activation: r δ sputum cells obtained by the culture method of the present invention will be described.

 1) The cells obtained by the culture method of the present invention are collected by centrifugation or the like.

2) Wash the collected cells with a washing solution. The washing solution is preferably an isotonic solution having an osmotic pressure equal to that of cells, and more preferably a liquid that can be used as a pharmaceutical product. Here, in consideration of administration to a patient, it is preferable to use, for example, physiological saline, PBS (ρhosphatebuferfelineseline; phosphate buffered saline), or the like.

 3) The r 3 τ cells obtained after washing are collected using a centrifugal method, etc., and suspended in a liquid that can be used as a medicine, such as physiological saline, to prepare the therapeutic / preventive agent of the present invention. can do. At this time, the amount of the suspension liquid used is appropriately adjusted according to the number of cells to be administered and the administration method.

The number of r (5 τ cells used in the therapeutic / prophylactic agent of the present invention is appropriately selected according to the administration method, the type of disease, the symptom of the patient, etc., but is usually 10 ⁸ to 10 ¹² Z. More preferably, it is 10 ⁹ or more.

 4) Suspend in physiological saline to obtain a therapeutic / preventive agent using δΤ cells.

In addition, when used as a cancer treatment / prevention agent, it is also possible to combine cytokines such as 1 to 2 and 1 to 1 2 with the prevention and treatment agent of the present invention. The

 Furthermore, when used as a therapeutic / preventive agent for viral infections, it is possible to combine the interferon (IFN-r) and the like with the prophylactic / therapeutic agent of the present invention.

 5) As a method of administration, for example, it may be injected intravenously, intradermally, subcutaneously, etc., may be directly injected into the affected area, or may be administered systemically as an instillation. Furthermore, it may be injected from an artery near the lesion.

 The therapeutic / prophylactic agent of the present invention has a high content of T <5 T cells having non-specific cytotoxic function, cancer antigen-specific cytotoxic function or both cytotoxic functions, In particular, it contains many CD 56 positive rδ Τ cells with high cytotoxic function, so it has the ability to specifically attack cancer cells or infected cells without attacking normal cells. It is an effective treatment and prevention agent for the treatment of cancer types and infectious diseases. In particular, if the <5 cells are derived from autologous lymphocytes, they can function without being excluded by the patient's own immune system when administered to the patient. Example

 Hereinafter, the present invention will be described in detail with reference to examples. However, the present invention is not limited to this. Example 1

 <Study of suitable concentration by various bisphosphonates in the culture method of τ δ τ cells

>

 1) First, 18 OmL of peripheral blood was collected from a healthy donor, and peripheral blood mononuclear cells were separated using a specific gravity solution for blood cell separation.

 2) The obtained peripheral blood mononuclear cells were suspended in a culture solution AIM-V (hereinafter referred to as AIM-V (10 FCS)) to which FCS was added at 10%.

To 3) 24we 1 1 plate (SUM I LON), were seeded healthy human peripheral blood mononuclear cells of l X l 0 ⁶ ZlmL.

4) Ar edia, Onc last, and Zome ta were added as bisphosphonates to activate and Z or proliferate _r δτ cells, respectively, and the culture was started under conditions of 37 ° C and 5% CO ₂ concentration.

 5) Next, 0, 5, 7, 9, 11 days after the start of culture, I L-2 was added at a concentration of 400,

1000 and 200 OU / mL were added. Furthermore, according to the growth of the cells, the culture solution AIMV was added and cultured for 14 days.

6) As described above, the percentage of cells expressing <5 T cells in the cell group obtained after 14 days of culture was determined as the fluorescence—Ac tivated C It was measured by ell Sorter (hereinafter referred to as FAC S, Epics XL-MCL ADC, Beckman Cole Yuichi). The values obtained by measurement are shown in Tables 1 to 3 below.

 Based on these results, in order to obtain high-purity δ δ T cells, the concentration of bisphosphonate bone metabolism-improving drug should be 0 · 1 to 30 χΜ, and IL-1 should be added to 400 to 200 OUZmL. It was confirmed that it was preferable. Further, it was confirmed that Ar ed i a is preferably added at a concentration of 1 to 30 ζΜ, On c l a st at a concentration of 1 to 30 / iM, and Zome t a at a concentration of 0.1 to 10 M. Table 1:% of T cells contained in mononuclear cells in which the concentration of IL-12 was changed when Ar e dia was used as the bisphosphonate (%)

表 2 ： ビスホスホネートとして On c l a s tを使用した場合に I L— 2 の濃度を変化させた単核球中に含まれるァ δΤ細胞の割合 （％)

Table 2: Percentage of δδ cells contained in mononuclear cells with varying IL-2 concentrations when On clast was used as the bisphosphonate

 IL-2 concentration (U / mL)

 O n c 1 a s t

 400 1000 2000

 (^ M)

 0 10. 1 9. 7 10. 4

 1 68. 5 57. 1 69. 3

 5 82. 8 82. 1 82. 3

 10 89. 7 86. 5 84. 1

 30 90. 2 84. 6 85. 7

 50 88. 5 81. 2 77. 0

100 74. 4 62. 9 70. 9 Table 3: Percentage of δ δ cells contained in mononuclear cells with varying IL-2 concentration when using Zome ta as bisphosphonate

実施例 2

Example 2

 <T <5 Examination of optimal concentration of IL-2 for efficient proliferation of T cells>

 1) Peripheral blood was collected from healthy individuals, and peripheral blood mononuclear cells were separated using a specific gravity solution for blood cell separation.

 2) The peripheral blood mononuclear cells obtained in 1) were suspended in the culture medium AIMV (10% FCS).

3) A 1 × 10 ⁶ Zl mL peripheral blood mononuclear cell suspension was seeded on a 24we 11 plate.

 4) Ardia was added to the peripheral blood mononuclear cell suspension to a concentration of 10 M.

 5) After 0, 3, 7, 9, 11 and 14 days from the start of culture, IL 1 was added so that the concentration of IL-1 was 20 00, 40 0, 700, 1 000 and 1 50 OUZmL.

 6) Culture medium AIMV (10% FCS) was added according to cell growth.

7) Cultured for 14 days.

 8) 1 The number of viable cells obtained after 4 days of culture was examined using trypan blue.

 9) Further, the surface antigens of the cells obtained after 14 days of culture were examined by FACS, and the ratio of ΤδΤ cells was measured. As a result, it was confirmed that a large amount of δ δ cells can be obtained by adding IL 1-2 at 400 0 to 100 U / mL.

Table 4 and Table 5 show the percentage (%) of 5 Τ cells when the amount of IL-2 is changed. Table 4 shows the number of cells on days 7, 9, 1 and 14 and Table 5 shows the number of cells on days 0, 3, 7, 9, 1 and 14 (X 1 0 ⁶ ) respectively.

The peripheral blood mononuclear cells in Table 4 a) and Table 5 a) are derived from the same donor. Thus, the peripheral blood mononuclear cells in Tables 4b) and 5b) are from the same donor. From these results, it was confirmed that the amount of IL-12 is preferably 400-1500 UZmL and the number of culture days is 11 days or more in order to obtain high purity and large amount of δδ cells. Table 4: A ratio of <5 T cells when the amount of IL-12 is changed ()

 a)

Table 5 ： Number of δΤ cells when the amount of IL-1 2 is changed (X 10 ⁶ ) a)

 IL-2 concentration

 0 曰 3 曰 7 曰 9 曰 11 曰 14 曰 (U / mL)

 200 1. 00 1. 11 3. 42 4. 88 8. 48 13. 6

400 1. 00 1. 12 3. 81 10. 6 30. 6 57. 6

700 1. 00 1. 29 4. 65 19. 5 42. 9 92. 0

1000 1. 00 1. 40 5. 43 20. 6 44. 2 97. 6

1500 1. 00 1. 35 5. 55 24. 2 48. 8 129 IL-1 concentration

 0 曰 3 曰 7 曰 9 曰 11 曰 14 曰 (U / mL)

 200 1. 00 1. 49 5. 07 22. 5 35. 0 43. 6

400 1. 00 1. 59 5. 91 25. 4 35. 3 54. 8

700 1. 00 1. 64 6. 93 24. 3 48. 3 58. 8

1000 1. 00 1. 85 5. 82 27. 0 37. 5 67. 2

1500 1. 00 1. 86 5. 82 25. 5 37. 5 48. 8 Example 3

CD 56 positive, CD 56 negative δ Selection of Τ cells and comparison of their cytotoxic activity>

 1) 40 ml of peripheral blood was collected from a healthy donor, and peripheral blood mononuclear cells were separated using a specific gravity solution for blood cell separation.

 2) The obtained peripheral blood mononuclear cells were suspended in AIM-V (10 FCS).

3) Peripheral blood mononuclear cells of l X l 0 ⁶ ZlmL were seeded on 24we 1 1 plate (SUM I LON). IL-2 was added at 700 U / mL, and Aredia was added at 10 M, and cultivation was started under the conditions of 37 ° C, 5% CO ₂ concentration.

 4) 0, 5, 7, 9, and 11 days after the start of the culture, IL-2 was added to a concentration of 700 U / mL. Furthermore, according to the proliferation of the cells, the culture solution A I M-V was added and cultured for 14 days.

5) After culturing for 14 days, the surface antigen of the obtained cell group was measured by FACS. As a result, it was clarified that the αδ cells activated and / or expanded by adding A redia to peripheral blood mononuclear cells express NKG2D. Ν KG 2 D is an active NK cell receptor that is expressed on NK cells, τ 6 T cells and CD 8 positive T cells and expresses its ligand, MI CAZB molecule. Cells are known to be injured through NKG 2DZM ICA interaction. In addition, it was confirmed that about 50% of cells expressing CD56 exist. PT / JP2005 / 013218

 11

Table 6: A <5 T cell surface antigen frequency after 14 days of culture (%)

 6) Label the resulting cells with CD 56 microbeads (M i 1 t ny i Biotec) and collect the positive fraction (CD56 positive cells) and negative fraction (CD 56 negative cells) by MACS. did. The obtained cells were used as reaction cells. Table 7: Frequency of surface antigens in each fraction separated by MACS (%)

a) Positive fraction (CD 56 positive cells)

7) Use U266 (multiple myeloma cell line) as the target cell. In order to distinguish target cells from reactive cells, the target cells were identified as PKH-26 (PKH26. Red Fluorescent Cell Cell Lines) It, SI GMA). 8) Seed in 24we 1 1 late so that the ratio of reaction cells to target cells (hereinafter referred to as EZT ratio) is 0: 1 (control group of target cells only), 1: 1, 10: 1, 37, for 4 hours co-culture with 5% C_〇 ₂ concentration conditions.

 9) Next, collect the co-cultured cells and identify the early stage of apoptosis, and identify the late stage of apoptosis and necrosis 7-AAD

(ANNEX INV—FITCZ7—AAD KIT, Beckman Coulter) was stained, and cytotoxic activity against U266 was measured by FACS. In FACS analysis, only target cells can be analyzed by gating ΡΚΗ—26 positive cells.

 In Table 8, An n + indicates the number of cells that have developed the fluorescence of Anne x i n V, and An n− indicates the number of cells that have not developed color. 7 AAD + indicates the number of cells that developed 7-AAD fluorescence, and 7 AAD- indicates the number of cells that did not. That is, An n-7 AAD + is a necrotic cell, An n + 7 AAD + is a late apoptotic cell, An n + 7 AAD- is an early apoptotic cell, Ann-7 AAD- Indicates that the cell is alive.

Note that the value of the cytotoxic activity was calculated by Equation 1. Formula 1

Cytotoxic activity (%)

 [Ann + cell count (test)] ― [Ann + cell count (control)]

 ~ Number of malformed cells [Ann + number of cells (control)]

As a result, it was confirmed that CD 56-positive δ Τ cells have higher cytotoxic activity against U 2 66 than CD 56-negative δ Τ cells. In addition, the expression of NKG 2 D was also confirmed in CD 56 negative δ δ cells, confirming that δ δ cells expressing both NKG2D and CD 56 have more efficient cytotoxic activity. It was.

Cytotoxic activity against U 266 of CD 56 positive and CD 56 negative cells

 Control CD 56 positive CD 56 negative

EZT ratio 0: 1 1: 1 10: 1 1: 1 10: 1 Total number of cells (pieces) 30000 30000 30000 30000 30000

Ann- 7 AAD + 142 37 36 63 47

Ann + 7 AAD + 1958 2111 7014 1892 2694

Ann + 7 AAD— 2420 5539 13102 3298 8761

Ann— 7 AAD— 25480 22313 9848 24747 18497 Cytotoxic activity (%) 0 12. 8 61. 4 3. 2 27. 6

Claims

The scope of the claims 1. It is characterized by adding bisphosphonate bone metabolism-improving drug to peripheral blood mononuclear cells at 0.05 ~ 100 M and IL-12 concentration of 50 ~ 2000 UZmL. <5 τ cell culture method.  2. The method for culturing τδΤ cells according to claim 1, wherein the bisphosphonate bone metabolism-improving drug is added so as to have a concentration of 0.1 to 30 / χΜ. 3. The method for culturing rδΤ cells according to claim 1 or 2, wherein the bisphosphonate-based bone metabolism improving agent is added at the start of the culture.  4. The bisphosphonate bone metabolism-improving drug is pamidoramic acid, alendronic acid, zoledronic acid, risedronic acid, ibandronic acid, incadronic acid, etidobutanoic acid, their salts and sputum or hydrates thereof 4. The method of culturing rδΤ cells according to any one of claims 1 to 3, wherein  5. The method for culturing τδΤ cells according to claim 4, wherein the concentration of pamidronic acid, a salt thereof or a hydrate thereof is 1 to 30Μ.  6. The method for culturing τδΤ cells according to claim 4, wherein the concentration of the alendronic acid, a salt thereof or a hydrate thereof is 1 to 30 0.  7. The method for culturing τ δ Τ cells according to claim 4, wherein the concentration of the zoledobutanoic acid, a salt thereof, or a hydrate thereof is 0.1 to 1 ^^ Μ.  8. The method for cultivating δΤ cells according to any one of claims 1 to 7, wherein the IL-2 is added so as to have a concentration of 400 to 100 OUZmL.  9. It is obtained by adding bisphosphonate bone metabolism-improving drug and IL-12 to peripheral blood mononuclear cells at concentrations of 0.05 to 100 M and 50 to 2000 UZmL, respectively. End δ Τ cells.  10. The δδ cells according to claim 9, wherein the δδ cells contain at least 25% of CD56 positive γ6 cells.  11. The δ δ cell according to claim 9, wherein the τ δ Τ cell contains at least 40% or more of CD56-positive << 5 Τ cells.  12. Bisphosphonate bone metabolism-improving drug and IL-12 were added to peripheral blood mononuclear cells at concentrations of 0.05-: L 00 AiM and 50-200 OUZmL. A therapeutic / preventive agent for cancer and / or infection characterized by containing T cells. 13. The therapeutic / preventive agent for cancer and Z or infectious disease according to claim 12, wherein the τ δ cells contain at least 25% of CD 56 positive cells <5 cells. 14. The therapeutic and / or preventive agent for cancer and / or infectious disease according to claim 12, wherein the δδ cells contain at least 40% of CD56 positive δδ cells.  15. The therapeutic / preventive agent for cancer and epilepsy or infectious disease according to any one of claims 12 to 14, wherein the δδ cells are derived from autologous lymphocytes.