JPS61176529A - Production of specific killer t cell - Google Patents

Abstract

PURPOSE:To obtain a large amount of killer T cell specific to sensitized cancer, by sensitizing the lymphocyte of a relative of a cancer patient with an inactivated cultured cancer cell of the same kind as the cancer of the patient, adding a T-cell proliferation factor to the sensitized T-lymphocyte obtained by the above process, and culturing the lymphocyte. CONSTITUTION:The lymphocyte of a relative of a cancer patient is sensitized with an inactivated cultured cancer cell of the same kind as the cancer of the patient, and the obtained sensitized T-lymphocyte is cultured in the presence of a T-cell proliferation factor (e.g. interleukin 2) to obtain a large amount of specific killer T cell. The T-cell proliferation factor is e.g. the lymphocyte originated from human spleen or tonsils, a factor produced by stimulating the spleen lymphocyte of crab-eating macaque or savannah monkey, etc., with mitogen, or a factor produced by genetic engineering technique. It acts specifically to the same kind of cancer as the cancer used in sensitization, and has remarkable effect also to diminish the cancer focus or to suppress the metastatis of cancer.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for mass producing killer T cells specific for specific cancer cells.

[Conventional technology and its problems]

Among head and neck cancers, nasopharyngeal cancer has a poor prognosis, with a 5-year survival rate of only about 30% even when non-specific immunotherapy is added to radiation and chemotherapy. It was pointed out that the reason for this was that the immune surveillance system was severely damaged. One possible way to improve the human body's immunity against cancer is to administer killer T cells specific to cancer cells, but killer T cells have traditionally been isolated from the patient's phospho/9 cells. Since the cancer cell tissue was produced by mixed culture, it was not possible to culture it in large quantities, making it impossible to administer a sufficient amount.

[Means to solve the problem]

The present inventors have developed a killer T protein that is specific to specific cancer cells.
While conducting various studies to obtain a large amount of cells,
Sensitized τ Rinno9 bulbs can be obtained even by contacting phosphorus and e bulbs obtained from a cancer patient or a person genetically closely related to the same type of cultured cancer cells, and the sensitized 19779 bulbs. We found that a large amount of killer T cells can be obtained by adding T cell growth factors to the cells.

Therefore, the present invention sensitizes inactivated cultured cancer cells of the same type as the patient's cancer with phosphorus/9 cells in the blood of a cancer patient or a person who is genetically closely related to the cancer patient. The present invention provides a method for producing killer T cells, which comprises obtaining sensitized Q cells and culturing them by adding T cell growth factors.

The phosphorus/Q cells used in the present invention must be obtained from the patient or someone who is genetically closely related to the patient. , refers to brothers (sisters), etc. In order to obtain phosphorus/Q cells, peripheral blood of the donor may be collected and separated by a conventional method, and a suspension of 9779 cells can be prepared in this manner.

Furthermore, the cancer cells that sensitize the phosphorus/Q spheres need to be inactivated and cultured cancer cells of the same type as the patient's cancer. In order to inactivate these cultured cancer cells, thoroughly centrifugally washed cultured cancer cells should be
RPMI-1640 or PB8 of lO・pcs/1111
This is prepared into a suspension and irradiated with cobalt at about 2000R, or treated with mitomycin-C at about 100piF/lR1 for 30 minutes at 37°C.

The inactivated cancer cells thus obtained are immediately thoroughly washed and used to sensitize lymphocytes. Sensitization of lymphocytes with inactivated cancer cells is usually carried out using l-
per l ~ 2 X l O' phosphorus/Q spheres and 1 ~ 2 X
1 O' inactivated cancer cells, preferably at a ratio of about 5:1-10:1, in a medium such as RPMI-1640 supplemented with 20% fresh human serum.
Mixed culture is carried out by a known method under conditions of 30 to 37° C. for up to 120 hours.

After the culture is completed, the sensitized phosphorus/9 cells thus obtained are separated from the inactivated cancer cells by a separation means such as density gradient centrifugation, and further! Culture is performed in the presence of cell growth factor (TCGF).

Examples of TCGF used for culture include interleukin-2 (IL-2). This TCGF is
In addition to those derived from human spleen or hentou, those derived from the spleen of monkeys such as cynomolgus monkeys, Japanese macaques, and green monkeys,
Genetically engineered cells and those derived from factor-producing cell hybridomas are used. This culture can be carried out by a conventional method, but preferably, 20-50% of CGF and 10-20% of fresh human serum are added, for example, RPM.
The reaction is carried out in a medium such as I-1640, and the concentration in the medium is preferably 2 to 4 X l O' particles.

Killer T specific for cancer cells thus obtained
The a-vesicles are separated from the culture medium by a conventional method and administered to the original cancer patient or another cancer patient related to the patient.

Administration: 3 to 40 x 107 pieces per 1 passage 9
It is preferable to inject 2 to 3 times intravenously or directly into the affected area.

[Action and effect]

Killer T cells prepared by the method of the present invention act specifically on cancers of the same type as those used for sensitization, and therefore have a great effect not only on eliminating cancer foci but also on suppressing metastatic cancer. .

Example 1 Preparation of killer T cells: (1) Preparation of sensitized 19779 cells Peripheral blood of a cancer patient was collected with heno-9 phosphate and separated by density gradient centrifugation to obtain a lymphocyte cell suspension (Takada et al. Full,
Immunology experiment operation method! .

(See p. 3045-3049 (1980)).

On the other hand, after centrifugation at 1000 rpm for 10 minutes, RPMI-
Cultured cancer cells washed 3 times with 1640 or PR8 were
A suspension of 2×10 cells/− of RPMI-1640 or PB8 was prepared and inactivated by irradiating with 200 OR cobalt at 37°C for 30 minutes (instead of cobalt irradiation, mitomycin-〇 (100 μf/ld) was also used). Mix the lymphocyte cell suspension and the inactivated cancer cell suspension to a cell number ratio of 10:1, and mix with RPMI-1640 medium supplemented with 20% human fresh disintegration. ℃ for 96 hours. The mixed culture cells were separated into cancer cells and sensitized 779 cells by density gradient centrifugation. Separately, the killer activity of the sensitized lymphocytes was determined using Garo.
uoy et al. (Manual of CIinicalIC
l1nicalI+ p 290-296 (1980
).

(2) Preparation of interleukin-2 (IL-2) Human spleen and tonsil-derived lymphocytes (1-2 x 10
After culturing on RPMI-1640 supplemented with 1% human hemagglutinin M and 2% human fresh serum, the supernatant was incubated at 37°C for 48 hours (flotation culture), and the supernatant was added to IL-2.
And so. In addition, monkey spleen-derived lymphocytes (1-2 x 10
10μf/-) in RPMI-1840 culture medium supplemented with 2% fresh human serum) in the same manner as lymphocytes, and the supernatant may be used as IL-2.

(3) Preparation of Killer T Cells The 9 sensitized cells obtained in (1) were further incubated with 50% IL-2 stock solution and 20% human freshly disrupted RPMI-1640 culture medium for 2 to 4 x 10' cells. The cells were cultured for 1 week at a cell concentration of 1/-. After that, 2-4 x 10' pieces/- every 2-3 days
Cultivation was continued in the presence of IL-2 at a cell concentration of , and the cells were cultured and propagated using killer.

Example 2 Killer T cells prepared by the method of the present invention were administered to 15 patients with nasopharyngeal cancer (NPC), and their effects were investigated.

The administration methods were all intravenous injections except for local injection in Example -. The dosage was 3 x 10' to 4 x 10'' 779 balls for sensitization, administered 2 to 3 times every week, for a total of 4 to 34 times.The contents of the phosphorus/9 balls used are shown in Table 1. Of the 15 cases, 15 cases were the patient's own phosphorus and E bulbs, 4 cases were the patient and his older brother (sisters), and 6 cases were the patient's older brother (sisters).
It was phosphorus/9 pitches.

Below is a blank jar F: fresh case, R: recurrent cancer, inside) is the metastatic site Case 4: Tumor in the nasal cavity disappears with local injection 5: Tumor that invaded into the cranium from the nasopharynx disappears, preventing distant metastasis lO: Top It was possible to control the 9719 node tumor in the pharynx and at the same time prevent distant metastasis for 1 year and 7 months (to date) 11. 12: Prevent distant metastasis 13: Prevent the expansion of lung distant metastasis 14: Distant organ metastasis As is clear from the results in Table 1, by administering killer T cells prepared by the method of the present invention, there were two cases in which tumors that had invaded locally and within the skull disappeared. In addition, an excellent suppressive effect on distant cancer metastasis was observed in many patients (case 5, 10, 11, 12, 13).
14th grade). From this result, it is expected that the killer T cells prepared by the method of the present invention will have a significant effect on early and intermediate stage cancer patients.

Example 3 Cytotoxicity enhancing effect (killer activity) by IL-2: Lin/Q in nasopharyngeal cancer (NPC) patients or brothers (sisters)
Cells and mitomycin (MMC)-treated NPC-derived cultured cells (NPC-204) (established by Mitsuru Takada et al.* GAN
N Monograph FJnl OI? 149-1
61 (1971)), 9729 balls (l ~ 2 x 1
MMC processing for 0'/-)10 4゜NPC-2
04 cells (1 to 2 x 105 cells/-) mixed at a ratio of 3
Sensitized lymphocytes were produced by culturing at 7°C for 96 hours. After partial sensitization, the Q spheres were further cultured for one week in the presence of IL-2, and then used as killer T cells, and their killer activity against NPC-204 cells was measured. Killer activity was measured using 51(r
Free $1 depending on the degree of damage of labeled NPC-2-04 cells
(This was determined by measuring r.) Killer activity was induced in the lymphocytes in each case by mixed culture (sensitization) with NPC-204 cells, but the sensitized lymphocytes were further incubated with IL-204 cells.
It was found that the killer activity was further enhanced by culturing in the presence of The results are shown in Figure 1.

vinegar.

[Brief explanation of drawings]

Figure 1 is a drawing showing the killer activity of lymphocytes and killer T cells after each treatment. that's all

Claims (1)

[Claims] 1. Lymphocytes in the blood of a cancer patient or a person genetically closely related to the cancer patient and inactivated cultured cancer cells of the same type as the patient's cancer are sensitized. Obtain produced T lymphocytes,
A method for producing killer T cells, which further comprises adding a T cell growth factor and culturing them. 2. T-cell growth factors produced by stimulating human spleen- or tonsil-derived lymphocytes or splenic lymphocytes of cynomolgus monkeys, green monkeys, and Japanese monkeys with mitogens, or factors produced by genetic manipulation. The method for producing killer T cells according to claim 1, wherein the killer T cell is a factor produced by a hybridoma of a factor-producing cell or a factor produced by a hybridoma of a factor-producing cell. 3. The method for producing killer T cells according to claim 1, wherein the T cell growth factor is interleukin-2.