JPS6232879A - Human t-cell hybridoma and production thereof - Google Patents

Abstract

PURPOSE:To keep a T-cell antigen receptor is stable state, by producing a human killer T-cell hybridoma by the fusion of a human killer T-cell clone with a 8-azaguanine-resistant human T-lymphoma. CONSTITUTION:A human killer T-cell clone is fused with a 8-azaguanine- resistant human T-lymphoma in a proper medium in the presence of a fusion accelerator. The fusion accelerator is preferably e.g. a polyethylene glycol having an average molecular weight of about 1,000-6,000, and it is added to a medium at a concentration of about 30-60W/V%. The medium is MEM medium, etc. The human killer T-cell hybridoma produced by the above process can be proliferated in a proper medium described above and is preferably selected in an HAT medium, cultured in an HT medium containing hypoxanthine, etc., for 1-2 weeks and transferred to a conventional medium.

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to a novel and stable human killer T-hybridoma, more specifically, a human killer T-hybridoma characterized by stably producing a human T cell antigen receptor; From a dark perspective, the present invention relates to a human killer T cell hybridoma characterized in that the hybridoma is a hybridoma obtained by fusing a human killer T cell clone and an 8-azaguanine resistant human T lymphoma, and a method for establishing these human killer T cell hybridomas. The inventors previously applied for a friend patent (Japanese Patent Application No. 6O-922).
As mentioned in 99), we are actively conducting research and development focusing on the importance of human T cell antigen septa in tumor antigen recognition, but it is difficult to mass culture human upper 2-T cell clones. These clones are derived from mitomycin C-treated human peripheral blood lymphocytes, which require a large amount of interleukin-2, and are very labor-intensive, such as the need to periodically add human autologous peripheral blood lymphocytes, which do not proliferate autonomously. It has become necessary to create killer T cell hybridomas that stably retain antigen receptors. That is, an object of the present invention is to create a human killer T cell hybridoma that stably retains a T cell antigen receptor. (Prior Art) No reports regarding human killer T cell hybridomas, ML/'1, have yet been found in the literature. However, a report on human helver hybridoma [M. Okada. N. Yoshimura, T. Kaieda, Y. Yamamura,
T. Kishimoto; Proceedings of the National Academy of Sciences. T-varnish T-, 78.77t'('a1): I Report on mouse killer hybridoma [Oh Kanakawa,
C. M. F. 2, 'Journal of Immunology,' 597 ('85)]. (Problems to be Solved by the Invention) The reason why a human killer T cell hybridoma was not successfully established is thought to be that a proliferative high-volume human killer T cell clone was not used for cell fusion. (Means for Solving the Problems) To achieve the above-mentioned purpose, the present inventors used human lymphocytes with @cast cells, lectin, or interleukin 2, which is a type of lymphoid protein. An experiment was conducted to activate lymphocytes using Makishin's method, induce killer T#ifU cells that have the ability to kill tumor cells, and perform cloning to establish tumoricidal killer T cell clones. Ta. When we investigated the killer activity of the cloned killer T cells obtained in this way against various tumor cells, we found that
Surprisingly, he discovered the existence of a killer T cell clone that recognizes and powerfully kills a wide range of tumor cells, but does not kill normal cells. Such broadly tumoricidal killer T cell clones could be established with good reproducibility and with various specificities if lymphocyte activation and cloning methods were kept constant. They discovered that T cell antigen receptors isolated from these killer T cell clones specifically bind to tumor cells.
We have already applied for a patent as a new tumor recognition substance (patent application filed in 1986).
O-92299). However, as mentioned above, maintaining killer T cell clones obtained in this way is extremely time-consuming and requires the creation of killer T cell hybridomas that proliferate autonomously and stably retain antigen receptors. It has become. Therefore, the present inventors repeatedly attempted fusion experiments between 8-azaguanine-resistant human T-lymphoma and the highly proliferative human killer T cell clones obtained in the above-mentioned manner. Regarding T cell clones, T
The present invention was completed by obtaining a Ki7-T cell hybridoma that stably retains cell antigen receptors. That is, the present invention relates to a human killer T cell hybridoma, and a method for producing a human killer T cell hybridoma, which is characterized by fusing a human killer T cell clone with an 8-azaguanine-resistant human T lymphoma. Lymphocytes can be obtained from peripheral blood, spleen, and lymph nodes, but it is easier to collect from peripheral blood. The method for obtaining human killer T cell clones of the present invention is as follows. That is, first, lymphocytes are stimulated with tumor cells, lectin, interleukin 2, etc. In this case, using lectin or interleukin 2,
This is preferred because cloned T cells with different strain specificities can be obtained. Examples of lectins include 7 bean lectin (PHA) derived from Pha-seolus vulgaris and Concanavalia enraformis.
concanavalin A (Co
n A ) s Riceteria aoribanda (Wis
Nodakuduma lectin (WFA) derived from Lens culinalis (Le
Lentil lectin (LCH) from Phytolacca americana (Phytolacca nsculinaris)
lacca americana) is used. Among these lectins, PHA and PWM are preferred because they strongly activate tumor-recognizing T cells. Stimulated activated talymphocytes are suspended in interleukin-2-containing medium, dispensed so that one cell is in each well of a microwell plate, cultured and cloned [T.Kayeda; Journal Op Immunology]. , 129°46 ('82)) to obtain cloned T cells. Among the many cloned T cells obtained by such a method, those that recognize tumor cell lt are screened. Screening is performed by culturing a mixture of cloned T cells and tumor cells, and evaluating their ability to recognize and kill tumor cells (killer activity). The human killer T cell clone of the present invention recognizes at least solid cancer cells, including human gastric cancer cell lines MKN-1 and KATO-III, and human lung cancer cell lines PC-1 and PC-9. , PC-10, PC-9, P
C-14, human colon cancer cell line C-1, 1f17609,
Human rectal cancer cell line CaR-1°S-7512, human cholangiocarcinoma cell line H-1, human liver cancer cell line HLE, HLF, human bladder cancer cell line NET-2, KU-1% human throat cancer cell line KB, Human gastric cancer cell line W-2, NRC-12, human breast cancer cell line MBC-4, HBC-6, human uterine cancer cell line He
La, human melanoma cell line HMV-1,) (at least two types of MV-2, normal cells that do not recognize healthy human lymphocytes, healthy human red blood cells, human fetal fibroblast cell line HET%HEL) %MRHF.In addition, whether or not cloned T cells recognize tumor cells is determined by using the 31Cr labeling method and g/T ratio (mixing ratio of cloned T cells and tumor cells). 4 hours at 10, 3
Cultured with 7C and exhibiting killer activity of 10 or more
l - Suppose that it is recognized. By the lymphocyte stimulation, cloning, and screening methods described in the present invention, tumor-recognizing human killer T cell clones with various specificities can be obtained. That is, at least the Oni human gastric cancer cell lines MKN-1 and KATO-[
Kill I, healthy human lymphocytes, fetal-derived fibroblasts HEL
A human killer T cell clone thought to be specific for gastric cancer that does not kill cancer has been obtained. A cloned human killer that kills at least human lung cancer cell lines pc-1, PC-1, PC-9, and PC-14, but does not kill lymphocytes of healthy people or fetal-derived fibroblasts WET, and is thought to be lung cancer-specific. Next, T cells were obtained. Furthermore, at least human liver cancer cell lines HLE, H
A human killer T cell clone thought to be specific for liver cancer that kills LFi but not lymphocytes of healthy individuals was obtained. T-cell antigen receptors can be isolated more efficiently than T-cell hybridomas using a first-order method [Niss C. Moyer, Journal Op Experimental Medicine, 7'05 ('85)]. That is, human killer T-hyperid-7t-1% Trito obtained from human killer T cell clones by the Tomo method described below.
It was suspended in PBS (phosphate buffer containing 851NaC4, pH 7,2) containing n
Membrane proteins are eluted by stirring for a period of time, and affinity analysis is performed using 5epharose 4 B conjugated with a monoclonal antibody against T cell antigen receptor. When assembled T cell antigen receptor-111:sDs was subjected to polyacrylamide gel electrophoresis, one band was detected at a molecular weight of approximately 90,000 under non-reducing conditions, and one band was detected at a molecular weight of approximately 50,000 and 40,000 under reducing conditions. Two bands are detected in every 5,000. Furthermore, when a human killer T cell hybridoma obtained from the tumoricidal human killer T cell clone of the present invention by the method described below is treated with tunicamycin to obtain a T cell antigen receptor without a sugar moiety, two proteins each having a molecular weight of about 30,000 are obtained. A heterodimer with a molecular weight of about 60,000 is obtained in which the components are covalently linked to each other via disulfide bonds. That is, the T cell antigen receptors produced by the T cell hybridomas of the present invention include both sugar-bound and non-sugar-bound ones. The human T cell hybridoma of the present invention is stable. and provides T cell antigen receptors in large quantities. The fusion reaction between the 8-azaguanine resistant T-lymphoma of the present invention and the human killer T cell clone described above is carried out in an appropriate medium in the presence of a fusion promoter. As the fusion promoter, for example, a virus such as Sendai virus (HVJ) may be used, but it is preferable to use polyethylene glycol (PEG), which has been developed in recent years. The PEG has an average molecular weight of 1000 to 600.
It is preferably about 0, which means that about 30 to 60 W is applied to the medium.
Suitably, it is present at a concentration of /V%. In addition, as a medium, MEM medium such as the one used for the growth of the Friendship cell line mentioned above, its Dolbecco modified medium, RPMI 16
40 medium and various other usual media used for this type of cell culture can be used. However, the above-mentioned cell fusion medium may contain, if desired, a second adjuvant to increase fusion efficiency.
For example, dimethyl sulfoxide or the like may be added. In the above cell fusion, the ratio of the amount of human killer T cell clone of the present invention to 8-azaguanine-resistant human T-lymphoma used is not particularly limited, but in general, the number of cells of 8-azaguanine is 1JJ3 of the human killer T cell clone. The number of resistant human T-lymphoma cells can be increased by about 1 to 10 times. Preferred cell fusion is carried out, for example, as follows. Specifically, predetermined amounts of 8-azaguanine-resistant human) T lymphoma and human killer T cell clones were mixed well in an appropriate medium, centrifuged, the supernatant was removed, and an appropriate amount of a PEG solution preheated to 37C was added. Add and mix. This initiates a cell fusion reaction. Thereafter, the appearance of desired fused cells is observed by repeating the following steps: incubating the cells for an appropriate period of time, adding an appropriate medium, centrifuging, and discarding the upper groove. Isolation of the desired fused cells is carried out by culturing the cells after the cell fusion described above in a normal hybrid selection medium. This selection medium is a medium in which the parent cells cannot proliferate, and only the desired hybrid cells can proliferate (human killer cell clones cannot originally proliferate). Examples include a medium (HAT medium) containing the following. The HAT medium is, for example, 10% FCS-containing MEM medium.
Aminopterin 2x10-M, Hiboki Jyochin 1X
An example is a medium supplemented with 10-4M, thymidine L6 x 10-'M, and glycine 3X 10'M. The H
Cells are cultured in AT medium according to the usual limiting dilution method for a sufficient period of time, usually about several days to several weeks, to kill cells other than the desired hybridized cells (unfused cells, etc.). . This selectively proliferates only the target human killer T cell hybridoma. The hybridoma thus obtained has new characteristics that are clearly different from 8-azaguanine-resistant human T lymphoma and human killer T cell clones in terms of karyotype (number of nuclear chromosomes), cell surface phenotype, mitogen reactivity, lymphokine production ability, etc. are doing. This hybridoma can be grown and maintained in the same suitable medium as above, but it is better to culture it in HT medium containing hypoxanthin and thymidine for 1 to 2 weeks after selection in HAT medium, and then transfer it to normal medium. Preferably, human killer T cell hybridomas are hybridomas obtained by fusing human killer T cell clones and 8-azaguanine-resistant human T lymphomas, and include those with and without killer activity, and at least antigen-specific hybridomas. It is a hybridoma that can be recognized by The antigen targeted by the present invention is a tumor antigen such as a tumor-specific antigen or a tumor-related antigen, or a surface antigen of a malignant cell of a living body that has a virus antigen or the like. (Effect of the invention) The binding of the purified Td3 cell antigen receptor obtained from the human killer T cell hybridoma obtained by the method described above from the tumoricidal human killer T cells to tumor cells and normal cells was determined using an anti-Td3 antibody as a primary antibody. Surprisingly, we investigated using a cell antigen receptor monoclonal antibody and a FITC-labeled anti-mouse immunoglobulin antibody as a secondary antibody.
It has been found that the T cell antigen receptor, which has the same recognition properties as the original human killer T cell clone and is thus isolated from the cell membrane, specifically binds to tumor cells. That is, it is gastric cancer-specific, lung cancer-specific, or pan-tumor-specific, and can be used as a diagnostic and therapeutic agent for cancer as an extremely useful and novel tumor cell-binding substance that binds to tumor cells but not to normal cells. It has been found. The tumor cell-recognizing human killer cell hybridoma of the present invention and the T cell antigen receptor obtained from the cell can be obtained through basic research for tumor antigen analysis, determination of whether the cell is a cancer cell, and serum testing. It is used as a cancer diagnostic drug to determine whether a patient is a cancer patient, to detect metastatic sites by binding a labeled substance and administering it to a cancer patient, and to be used as a cancer treatment drug by binding a toxin or radioactive substance. It is something to do. However, the usefulness of the human killer T and @B cell hybridomas of the present invention is not limited to 11≠, but also includes those that have T cell antigen receptors that recognize malignant cells in living organisms that have viral antigens and the like. (Example) Hereinafter, embodiments of the present invention will be described in more detail with reference to Examples. Example 1 Human T: Cell cloning and screening for tumor 4Ji-recognizing human killer T cell clones were performed as follows. Human lymphocytes are as follows

[I got it. That is, the collected heparinized human peripheral blood was diluted 2 times with Hank's solution, layered over Ficollbac solution (manufactured by Pharmacia), and centrifuged at 2000 rpm for 20 minutes. The intermediate lymphocyte layer was collected, washed with Hank's solution, and then suspended in RPM11640 medium supplemented with 10% fetal bovine serum at a concentration of 2 x 10' cells/- cells. PHA-P (Difco fissure) was added to this at a concentration of 0.1 liter, transferred to a culture bottle, and incubated at 48 °C in 57C, 5% CO1.
I did it safely over time. Lymphocytes contain lectin (PI(A-P)
) and induced various killer T cells and helper T cells that recognize various human tumor cell lines. In order to make these polyclonal activated lymphocytes monoclonal, known methods C. Goji Kaieda; Immunology Experimental Procedures XI. P, 5689, edited by the Japanese Society of Immunology] for cloning. That is, activated lymphocytes were transferred to commercially available RPM1 containing IA p-leukin 2 (manufactured by Boehringer Mannheim).
5 Cells in 1640 medium (containing 20% tallow serum)
The cells were suspended at a cell concentration of 100a? /ml
, 37C, 45 minutes) were incubated with IX 10'
Sickle added at a cell concentration of 200 μif cells/- 96 wells 2
00μ microwell plate C Falcon black 30
72) K was dispensed and cultured in 37C, 5% CO7. The cloned T cells proliferated in about 2 weeks. Whether or not these cloned T cells had tumor cell recognition ability was evaluated by determining whether they exhibited killer activity against tumor cells as follows. That is, various tumor cells were labeled with 51Cr using a known method [R.M. 'cloned T cells were cultured in 200 μt of RPMI medium (5
Mixed culture for 4 hours in C57C, 5%
After Co, )l, the radioactivity of 51Cr released in the supernatant was counted to evaluate whether it had killer activity. Killer activity was calculated using the following formula. Killer activity-((B-C)/(A-C))X100A=
Radioactivity of labeled tumor cells I x 10' B: labeled tumor cells I x 10' and cloned T, penile cells I x 1
Radioactivity in the supernatant when 0' cells were mixed and cultured C: Radioactivity in the supernatant when only 104 labeled tumor cells were cultured 10,000 activated lymphocytes were cloned under the above conditions. As a result, approximately 500 T cell clones were obtained. Among these, there were approximately 100 human killer T cell clones that had killer activity against at least one of various human tumor cell lines. Among the human cloned killer T cells obtained by repeating the cloning experiments in this manner, the killer activity against various typical tumor cells and fetal-derived fibroblasts is shown in Table 1. These clones require interleukin 2 for culture, and in order to maintain them in culture for a long time, RPM11 containing interleukin 2 is required.
Culture is performed in 640 medium (containing 20% tallow baby serum). Further, on days 7 to 10, autologous human peripheral blood lymphocytes from which PHA-i, PHA-P, and these clones are derived are treated with mitomycin and then added to the culture. By culturing under these conditions, it was possible to maintain the culture for a long period of 6 months or more. In addition, such tumor cell-recognizing human killer T cell clones could be obtained with good reproducibility by lectin stimulation, cloning, and screening of human peripheral blood lymphocytes under the same moxibustion conditions as described above. Table 1 Seeds of Human Killer T Cell Clones Killer T-hybridoma with tumor recognition for target cells was obtained as follows. 8-Azaguanine resistance)
OEM-AC was used as the T-shirt. The OEM-A GR was created using a known method (Japanese Patent Application Laid-open No. 5
7-206383). Parental cells CEM-AGR were grown in culture medium (RPM11640+20
% FC8 + 100 μM 8-azaguanine).
The cells were kept in a state of active proliferation and suspended in a medium in which 100 μM-Azaguanine had been removed from the above medium one day before. 6 x 107 cells of the above CEM-AGR and 8 x 10' cells of the broadly tumor-introducing human killer T cell clone 51 shown in Table 1 above were used for cell fusion. That is, each of the above cells was heated and maintained at 37C in advance with Fe2.
Washed 3 times with MEM medium without water, transferred to a 50-inch conical tube, mixed well, and incubated at room temperature at 800 rpm.
The mixture was centrifuged at m for 10 minutes. The fine salt pellet obtained by removing the supernatant was lightly shaken, and PE heated to 57C was placed on top of it.
0.5 d of G-6000 (Cotsholite Co., Ltd.) dissolved in MEM to a volume of 45 was poured into the solution. After shaking well for 30 seconds, the mixture was left standing in a carbon dioxide incubator containing 5% carbon dioxide and 95% air at 37C and Vc for 6 minutes. This includes MEM that does not contain Fe2 (37CI in advance)
C. warming) was added at a rate of 2 minutes per minute for a total of 12 hours while rotating the conical tube. Furthermore, MEM25
After the quick addition of WLt, the
The mixture was centrifuged for 0 minutes and the supernatant was removed. Loosen the pellet well and gently add 120- of RPMI 1640 medium containing FC3 (pre-warmed to 57C) to OEM-A.
G 9 degrees is 5 x 10' pieces/rnt, and this is 2
4-hole culture plate (Co5tar genus 3524)
One dose of each was dispensed into each of 120 wells. 24 hours 1, HAT medium [hyboxanthin (Sigma)
I X 10-'M, aminopterin (Sigma) 2
x 10-'M, and thymidine (Sigma) 1.6
RPMI 1640-1-20%F containing X10-5M
C8 medium]1- was added to each well. Discard half of the supernatant every 2 days and add HAT medium 11I! The operation of adding / to each well was repeated, and the cells were cultured at 37C in a 5% carbon dioxide gas incubator for 2 to 4 weeks. The growing cell line was then grown in HT medium without aminopterin (A) (HAT
Remove A from the medium) Transfer K and culture for another - week i
, RPMI 1640-1-20%F without HAT
Transferred to C8 medium (normal medium). As a result of repeating the same cell fusion twice, a total of 6 hybridoma clones were obtained. In order to check whether the obtained hybridoma produced T cell antigen receptor, we tested the T cell surface antigen T, which is thought to bind to the T cell antigen receptor.
3 antibody 0KT3 was used as the primary antibody, and FITC-labeled mouse Ig antibody (Tabosha!
Among the hybridoma clones, ring-shaped cells were observed in only one clone. This clone is H51
I decided to call it -4. In addition, the parent cell line CEM-AGR
There was no staining at all using this staining method. Among H51-4 cells, while there were mtQ cells that clearly expressed T3, there were also many cells that could barely excrete T3. Therefore, in order to increase T cell antigen receptor production, cloning was carried out in accordance with conventional methods in order to obtain subclones clearly expressing 9T3. That is, 1 x 10' CEM-AG were washed with normal medium, suspended in AT medium for 60 days, diluted with !M-4 to 2.5 cells/go, and added to the above HAT medium. 0.2rrIt/
The supernatant was dispensed into wells, half of the supernatant was added every two weeks, and HAT medium preheated to 37C was added to continue the proliferation of the cells. A single clone of the 1tIl cell line was thus obtained by cross-crossing. As a result of staining the obtained single clone by the above-mentioned indirect method, almost all cells were stained palely, and two clones were obtained in which a considerable number of ring-shaped cells were stained. This is H5
1-4-A2, called H51-4-DI. The production of T, i (B cell antigen receptor) of these T hybridomas was very stable.
The T cell antigen receptor isolated according to the method of Example 4 of 299 was isolated from the human gastric cancer cell line MKN-1, K A
T O-■, human lung cancer cell lines PC-10, PC-9, P
C-14, human bladder cancer cell line NBT-2, human throat cancer cell line KB, human uterine cancer cell line HeLa, human melanoma cell line HMV-1, binds to HMV-2, clone 5
It did not bind to cells that 1 did not recognize. The T cell antigen receptor purified from the cell membrane was derived from the original clone 51.
showed the same recognition ability. Example 2 Example 1 from human killer T cell clone 8 shown in Table 1
When hybridomas were created using the human killer using the method described in 1., they stably produced T5. Next, T
When we prepared a cell antigen receptor and examined its tumor cell binding properties, we found that it bound to MKN-1 and KATO-117, but did not bind to clone 8 non-recognizing cells. Example 3 A human killer T hybridoma was created using the method shown in Example 1 from the human killer T cell clone 19 shown in Table 1, and it stably produced T3, followed by L0.
When T cell antigen resegregators were prepared and their tumor cell binding properties were investigated, they were found to be PC-1, PC-1, PC-9, and PC-14.
and did not bind to clone 19 non-recognized Fa cells. Example 4 Human key y-T shown in Table 1, ll! cell clone 24
When a human killer T hybridoma was created using the method shown in Example 1, it stably produced T5. Subsequently, the T cell antigen receptor was purified and its binding to tumor cells was examined, and it was found that it was HLE. It bound to HLF and did not bind to clone 24 non-recognizing cells. Example 5 When a human killer T cell clone 75 shown in Table 1 was used to create a human Ki 7-T hybridoma using the method shown in Example 1, it stably produced T3. continue,
When we produced T cell antigen receptors and examined their tumor cell binding properties, we found that MKN-1, KATOrII, PC-9
, PC-10, PC-9, PC-14, C-1, H-1
, NBT-2, KBX-bV-2, II e La
-, bound to HMV 1 and did not bind to clone 75 non-recognizing cells. Although the present invention has been described above with reference to representative examples, the present invention is not limited to these only.

Claims (13)

[Claims] (1) Human killer T cell hybridoma. (2) The human killer T cell hybridoma according to claim 1, which produces a T cell antigen receptor. (3) T cell antigen receptors are antigen recognition molecules on the surface of T cell membranes, and two polypeptides, one with a molecular weight of about 50 kilodaltons and the other with a molecular weight of about 45 kilodaltons, form a disulfide bond. 3. The human killer T-cell hybridoma according to claim 2, which is a heterodimer covalently bonded to each other via a cell line. (4) Hybridoma is human killer T cell clone 8
- The human killer T cell hybridoma according to claim 1, which is a hybridoma obtained by fusing an azaguanine-resistant human T lymphoma. (5) Human killer T cell clone is interleukin 2
The human killer T cell hybridoma according to claim 4, which is a cell obtained by cloning using the human killer T cell hybridoma. (6) 8-azaguanine-resistant human T lymphoma is CE
M, JurKat, HPB-ALL, HPB-MLT,
The human killer T cell hybridoma according to claim 4, which is an 8-azaguanine resistant strain derived from Molt3 and Molt4. (7) Human killer T cell clone is human gastric cancer cell line MK
N-1, KATO-III, human lung cancer cell line PC-1, P
C-9, PC-10, PC-13, PC-14, human colon cancer cell line C-1, M7609, human rectal cancer cell line Ca
R-1, S-7512, human cholangiocarcinoma cell line H-1, human liver cancer cell line HLE, HLF, human bladder cancer cell line NBT-
2. KU-1, human throat cancer cell line KB, human kidney cancer cell line W-2, NRC-12, human breast cancer cell line HBC-4, H
Claim 4, which is a killer T cell having killer activity against at least two of BC-6, human uterine cancer cell line HeLa, human melanoma cell line HMV-1, and HMV-2. Human killer T-hybridoma. (8) The human killer T cell hybridoma according to claim 4, wherein the human killer T cell clone is a killer T cell having killer activity against at least human gastric cancer cell lines MKN-1 and KATO-III. (9) Human killer T cell clones are at least human lung cancer cell lines PC-1, PC-10, PC-13, and PC-14.
The human killer T cell hybridoma according to claim 4, which is a killer T cell having killer activity against. (10) The human killer T cell hybridoma according to claim 4, wherein the human killer T cell clone is a killer T cell having killer activity against at least human liver cancer cell lines HLE and HLF. (11) Human killer T cell clones include at least human gastric cancer cell lines MKN-1, KATO-III, human lung cancer cell lines PC-10, PC-13, PC-14, human bladder cancer cell line NBT-2, and human throat cancer cells. strain KB, human uterine cancer cell line HeLa, human melanoma cell line HMV-1, HMV-2
The human killer T cell hybridoma according to claim 4, which is a killer T cell having killer activity against. (12) Human killer T cell clones include at least gastric cancer cell line MKN-1, KATO-III, and lung cancer cell line PC-9.
, PC-10, PC-13, PC-14, colon cancer cell line C-1, cholangiocarcinoma cell line H-1, bladder cancer cell line NBT-2
, throat cancer cell line KB, kidney cancer cell line W-2, uterine cancer cell line HeLa, and melanoma cell line HMV-1, which are killer T cells having killer T cells according to claim 4. T cell hybridoma. (13) A method for producing a human killer T cell hybridoma, which comprises fusing a human killer T cell clone with an 8-azaguanine-resistant human T lymphoma.