JPS61254527A - Cloned t cell to recognize tumor cell and t cell antigen receptor obtained from same - Google Patents

Abstract

PURPOSE:A cloned T cell to recognize specifically a tumor cell, obtained by stimulating and activating a lymphocyte with a tumor cell, lectin, etc. and cloning it. CONSTITUTION:A lymphocyte derived from a mammal, preferably a lymphocyte collected from human peripheral blood is stimulated with a tumor cell, lectin, etc., floated in a medium containing interleukins, the lymphocyte is partially added to a microwell plate in such a way that one cell is put in each well, the cell is cultivated and cloned, or a lymphocyte is stimulated and fused with a tumor cell strain or not stimulated and directly fused with it and the cell is cloned to give a cloned T cell. A cell to recognize the tumor cell, namely a cell to recognize the tumor cell and to kill it (killer activity), or a cell to recognize the tumor cell and to produce some lymphokine (helper activity) is cloned to give the aimed cloned T cell.

Description

[Detailed description of the invention] (Industrial application field) i.

The present invention relates to cloned T cells that recognize tumor cells and TIO follicle antigen receptors obtained from the cells.

(Conventional technology and problems) Conventionally, l! Identification and analysis of tumor antigens have been actively studied using antibodies, particularly monoclonal antibodies, which are antigen recognition substances of Bm cells in lymphocytes.

However, at present, a versatile monoclonal antibody that recognizes and binds to a wide range of tumor cells and does not bind to normal cells has not yet been obtained.

Recent advances in immunology have revealed that antigen recognition by T cells, which had been unknown for a long time, is carried out by the Tll8 cell antigen receptor.

(Means for Solving the Problems) In view of the above-mentioned problems with monoclonal antibodies as tumor antigen recognition substances based on the conventional technology, an object of the present invention is to treat lymphocytes, which are a different type of lymphocytes than B cells, and which are immune to immune system. The object of the present invention is to obtain an antigen receptor substance that functions as a tumor antigen recognition substance from T cells, which play the main role, and to provide a cancer diagnostic agent and a cancer therapeutic agent that have great utility and versatility.

The present inventors used human and mouse lymphocytes for the above purpose. We use various methods to activate lymphocytes using tumor cells, lectins, or interleukins, which are a type of lymphoid, and induce killer T cells that have the ability to kill tumor cells. , conducted extensive cloning experiments to establish tumoricidal killer TIH cell clones. Various types of cloned killer T cells thus obtained II! When we investigated the killer activity of tumor cells in the brain, we surprisingly discovered that there is a killer T cell clone that recognizes a wide range of F4 tumor cells and powerfully kills them, 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.

Since the tumoricidal killer T cell clones established in the present invention exhibit killer activity by distinguishing between tumor cells and normal cells, it is thought that they have antigen receptors that can recognize tumor antigens. tumorous killer T
Experiments were carried out to prepare antigen receptors in mM from m cells.

As a result, a wide range of facilities are available! It was surprisingly found that the antigen receptor obtained from the lTs killer T cell clone binds to a wide range of tumor cells and does not bind to normal I8 cells.
The present invention was completed based on the discovery that it specifically binds to the 11 cell antigen receptor monoclonal antibody and has the reported properties of a T cell antigen receptor.

In other words, the present invention provides cloned TIB cells that recognize tumor cells but not normal cells, and Ti1 cells obtained from the cells.
Pertains to cell antigen receptors.

The cloned T cells of the present invention can be obtained from lymphocytes derived from mammals such as humans, mice, rats, rabbits, and guinea pigs. However, when considering the use as a medicine, it is preferable to obtain it from human lymphocytes. Although lymphocytes can be obtained from peripheral blood, spleen, and lymph nodes, it is convenient to collect them from peripheral blood. Furthermore, the cloned rm cells of the present invention are lymphocytes without immunoglobulin on the brain surface.

The cloned ■ cell of the present invention is Il! recognizes tumor cells,
It only needs to be T1 [1 cell that does not recognize normal cells, and its function may be that of helper Tll cell or killer Tm cell. Note that tumor cell recognition by T cells in the present invention refers to the expression of various lymphocyte functions in response to tumor cells. In other words, Ta follicles react with tumor cells and express a killer effect to kill them, or react with tumor cells to express a helper effect such as the production of lymphocytes.

There are the following two methods for obtaining the cloned Ti1l cells of the present invention. The first method is a method using inter-Oikins. That is, first, lymphocytes are transformed into tumor cells,
Stimulate with lectins, interleukins, etc. In this case, 1' detection using lectin or interleukins is preferable because cloned Ta cells with various specificities can be obtained. Lectins include red bean lectin (PHA) derived from PhaSeolus vulgaris and Concanavalia entuformis.
Concanavalin A (CONA) derived from P. siformis
), Wisteria
aoribamba) derived from Nodakuduzu bean lectin (W
FA>, Lensculina
Lentil lectin (LCH) from ris), filtrate
``Lacquer 7 Phytolaccaame
ricana) derived from pokeweed lectin (P
WM). Among these lectins, PHA
, PWM is preferred because it strongly activates tumor-recognizing T cells.

Stimulated activated lymphocytes were suspended in an interleukins-containing medium, dispensed into each well of a microwell plate, cultured, and cloned [(T, Kaieda; J.

Immunol, 129.46 ('82)] Cloning T [lII! get. The second method is to stimulate lymphocytes with tumor cells or lectins, or to use tumor cell lines as they are without stimulation [(M, 0kada).
, N. Yoshimura.

■, aieda, Proc, Natl, Acad, S
ci 78, 7717 ('81)] to create a fused cell line, cloning was performed, and the cloned fusion T
This is a method of obtaining cells. Among the many cloned T cells obtained by this method, those that recognize tumor cells are screened. Screening involves culturing a mixture of cloned Ti1 cells and tumor cells, and either recognizing and killing the tumor cells (killer activity) or recognizing flu tumor cells and injecting some kind of lymphoid activity (interferon,
This is done by evaluating the ability to recognize tumor fjA1fA follicles based on whether they produce interferon, etc. (helper activity).

Cloning Ti1l of the present invention! ! The tumor cells that are recognized are at least solid cancers, including the human gastric cancer cell line MKN-1, KATO-Ill, and the human lung cancer cell line P.
C-1, PC-9, PC-101PC-13, PC-1
4. Human colon cancer cell line C-1, M7609, human rectal cancer cell line CaR-1, S-7512, human cholangiocarcinoma cell line H
-1, human liver cancer cell lines HLE, HL, F, human bladder cancer cell lines NBT-2, KU-1, human throat cancer cell line KB, human kidney cancer cell line W-2, NRC-12, human breast cancer cell line In
HBC-4, l-I B C-6, human uterine cancer cell line H
eLa, human melanoma cell lines HMV-1, and at least two or more of HMV-2, and normal cells that do not recognize
At least one of healthy human lymphocytes, healthy human red blood cells, and human fetal fibroblast cell lines HET, HEL, and MRHF. Whether or not the roaned TI cells recognize tumor cells is determined by evaluating the recognition ability based on killer activity.51
Cr4! Using a detection method, cells are cultured at 37°C for 4 hours at an E/T ratio (mixing ratio of cloned T cells and tumor cells) of 10, and those exhibiting killer activity of 10% or more are recognized.

Tumor-recognizing cloned TIl cells having various specificities can be obtained by the lymphocyte stimulation, cloning, and screening methods described in the present invention.

That is, at least human gastric cancer cell lines MKN-1, KA
A cloned human killer TtIA cell, which is considered to be specific for gastric cancer, was obtained which kills TO-I[I but does not kill normal human lymphocytes or fetal-derived fibroblasts HEL.

Furthermore, at least human lung cancer cell lines PC-1, PC-9,
It killed PC-10, PC-13, and PC-14, but did not kill healthy human lymphocytes or fetal-derived fibroblast HET cells, and cloned human killer T cells considered to be specific for lung cancer were obtained. Also, at least human colon cancer cell line C-1, M760
Cloned human killer T cells thought to be specific for colon cancer were obtained, which kill 9 and not lymphocytes of healthy individuals.

Also, at least human rectal cancer I cell lines CaR-1, S-75
Cloned human killer T cells that are thought to be specific for rectal cancer and kill lymphocytes of 12 but not lymphocytes of healthy individuals were obtained. Furthermore, killing at least human liver cancer cell lines HLE and HLF,
Cloned human killer T cells thought to be specific for liver cancer that do not kill lymphocytes from healthy individuals; clones believed to be specific for bladder cancer that kill at least the human bladder cancer cell line NBT=2, KU-1 but do not kill lymphocytes from healthy individuals Human Killer TI
[1r11 was obtained.

Furthermore, at least human gastric cancer cell line W-2, NPC-
Cloned human killer T cells thought to be specific for gastric cancer that kill 12 but not lymphocytes of healthy individuals, and at least breast cancer-specific killer T cells that kill human breast cancer cell lines HBC-4 and HBG-6 but do not kill lymphocytes of healthy individuals. Possible cloned human killer Tel cells were obtained. Furthermore, at least
Human lung cancer cell lines PC-10, PC-14, human gastric cancer cell lines MKN-1, KATO-1, human bladder cancer cell line NBT
Cloned human killer T cells that are thought to be pan-tumor-specific and kill lymphocytes of -2 but not lymphocytes of healthy individuals were obtained.

These tumoricidal killer T cell clones can distinguish between tumor cells and normal cells and exhibit killer activity.
Since it was thought that these tumoricidal killer T cells have antigen receptors that can recognize is antigens, we energetically worked to separate, purify, and identify antigen receptors from these tumoricidal killer T cells. As a result, it was found that this antigen receptor corresponds to a reported T cell antigen receptor. That is, by producing an anti-TII cell antigen receptor monoclonal antibody that can specifically bind to the reported Tel cell antigen receptor, and using this antibody, membrane proteins obtained from tumoricidal killer T cell clones can be obtained. It has been found that an antigen receptor-like substance that specifically binds to tumor cells and is recognized by the anti-T cell antigen or septa monoclonal antibody is present in the fraction. Furthermore, when this antigen receptor-like substance was immunoprecipitated with an anti-T cell antigen receptor monoclonal antibody and the precipitate was subjected to SDS polyacrylamide electrophoresis, approximately 90,000 bands were detected under non-reducing conditions. Under the conditions, the molecular weight is approximately 50,000 and 45,000.
Two bands were detected. That is, the antigen receptor of the tumoricidal killer T cell clone of the present invention is Tel, which is a reported heterodimer in which two protein components with molecular weights of approximately 50,000 and 45,000 are covalently bonded via disulfide bonds. It turned out to be a cell antigen receptor.

Furthermore, when the tumoricidal killer T cell clone of the present invention is treated with tinicamycin to obtain a T cell antigen receptor without a sugar moiety, two protein components each having a molecular weight of about 30,000 are covalently bonded to each other via disulfide bonds. A heterodimer with a molecular weight of about 60,000 is obtained. That is, the T cell antigen receptor of the present invention includes both sugar-bound and non-sugar-bound T cell antigen receptors.

T cell antigen receptors can be detected using known methods [(s, c.

Heuer; J, ExD, Hed, 157.705 ('
[83)] can be used to isolate cells more efficiently than T cells. That is, phosphorylated T cells were treated with 1% Trit.
on PBS (0,85% NaCl
Contains phosphoric acid I! The membrane protein is eluted by suspending it in a buffer solution, pH 7.2) and stirring in water for 1 hour,
This is subjected to affinity purification using 5epharose 4 B coupled with a monoclonal antibody against pigs that list Tm cell antigens. When the purified Tl0III antigen richpter was subjected to SDS 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 2 at a molecular weight of approximately 45,000 under reducing conditions. The book band is detected.

(Effect of the invention) The binding properties of the purified cell antigen receptor obtained from the tumoricidal human killer T cells to tumor cells and normal cells were determined using anti-T cell antigen receptor monoclonal antibody as the primary antibody and FITC as the secondary antibody. When examined using a labeled anti-mouse immunoglobulin antibody, it was surprisingly found that the T111 antigen receptor isolated from the cell membrane had the same recognition properties as the original cloned T cell.
It was found that it specifically binds to tumor cells.

In other words, 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. There was found. That is, the present invention is based on the discovery for the first time that T cell antigen receptors isolated from useful T cell clones are extremely useful as antigen recognition substances.

The cloned TI cells recognizing tumor IllI cells and the Tm cell antigen receptor obtained from the cells of the present invention are useful for basic research for tumor antigen analysis, determination of whether cells are cancer cells, and serum tests. It can be used as a cancer diagnostic agent 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 as a cancer treatment agent by binding a toxin or radioactive substance. This is what we are trying to do.

(Example) The embodiments of the present invention will be described in more detail with reference to Examples below.

Example 1 Cloning of human Tel cells and f! Tumor-recognizing chestnuts
Screening for converted killer Tl cells was carried out as follows.

Human lymphocytes were obtained as follows. That is,
The collected heparinized human peripheral blood was diluted 2 times with Hank's solution, layered over Ficoll-Birk solution (manufactured by Fufurmacia), and centrifuged at 2000 rpm for 20 minutes. After collecting the intermediate lymphocyte layer and washing it with Hank's solution, RPM was added with 10% fetal bovine serum.
11640 medium) at a concentration of 2 x 106 cells/d. Add 0.0% PHA-P (manufactured by Difco) to this.
Add at a concentration of 1%, transfer to a culture bottle, and incubate at 37°C with 5%
Culture was performed in CO2 for 48 hours. Lymphocytes were activated by lectin (PHA-P) and various killer T cells, helper Tel cells, recognizing various human tumor cell lines were induced. A known method (Goji Kaieda;
Immunology Experimental Procedures XI, p. 3689, Japanese Society of Immunology g)
Cloning was performed at. That is, activated lymphocytes were suspended in a commercially available RPM I 1640 medium (containing 20% tallow serum) containing Interleukins (manufactured by Boehringer Mannheim) at a cell concentration of 5 cells/me, and mitomycin C was added to the cell suspension medium. (Made by Kyowa M Hakko)
After adding treated (100μ (1/d, 37°C, 45 minutes) autologous lymphocytes at a cell concentration of 1 x 105 cells/d, 20
Dispense into 96-hole 200 μm microwell plate (Fashion Nα3072) at 37°C, 5% C.
Culture was performed in O2. The cloned engineered cells proliferated in about two weeks. Whether the cloned T cells had tumor cell recognition ability was evaluated by determining whether they had killer activity against tumor cells as follows. In other words, 5 types of tumor cells
1c [R, H, Thorn et al.
, J. Immunol Methods 4,301 (
' Labeled with 74 months, l x 10 ' labeled tumors WAIll
Cells and I x 10” cloned TIt [200 cells
After 4 hours of mixed culture (37°C, 5% CO2) in μm RPMI medium (containing 5% tallow serum), the radioactivity of 51Cr released in the supernatant was counted to determine whether it has killer activity. evaluated.

Killer activity was determined using the following formula.

Killer activity = [(B-C)/(A-C)IX 10
0A: Radioactivity of 1 x 104 labeled tumor cells B: Radioactivity in the supernatant when 1 x 104 labeled tumor cells and 1 x 105 cloned engineered cells are mixed and cultured C: Only 1 x 10' labeled tumor cells are cultured As a result of cloning 10,000 activated lymphocytes with radioactivity in the supernatant under the above conditions, approximately 500 cloned T cells were obtained. Of these, approximately 100 human cloned killer T cells have killer activity against at least one type of human tumor cell line.
There were several. Tables 1 and 2 show the killer activity of typical human cloned killer T cells obtained through repeated cloning experiments against various tumor cells and fetal-derived fibroblasts. These clones require interleukins for culture, and in order to maintain them in culture for a long period of time, R P containing interleukins is required.
Culture is performed in MI 1640 medium (containing 20% tallow serum). Furthermore, once every 7 to 10 days), human autologous peripheral blood and lymphocytes from which PHA-P and these clones are derived are added to the culture after treatment with mitomycin. By culturing K under these conditions, 6
It was possible to maintain the culture for a long period of time for more than several months. Moreover, 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 conditions as above.

Table 1 Killer that targets the clones of "Kento V no Goro" Table 2. Killer activity of human killer Tln cells against various target cells: -(10%
(below), -(10% or more) Example 2 Tumor-recognizing human cloned helper cells were obtained as follows. Human lymphocyte collection and lectin stimulation;
Cloning was carried out in the same manner as in Example 1, and 100 glue-O-ionized T cells were obtained.

It was investigated in the following manner whether or not there were tumor fs-recognizing helper cell clones among these cloned T cells that recognized tumor alB cells and exerted a helper effect. That is, 1×106 each cloned Tl cell was 1alt RPM.
Autologous lymphocytes suspended in I medium (containing 10% tallow serum) and treated with mitomycin were cultured at I x 10''.
5 x 1 tumor cells added and further treated with mitomycin
05 cells were added and cultured for 40 hours at 37°C in 5% CO2.

The activity of interleukins in the supernatant after culture is known 1...
The method of W [(S, G11lis et al; J, 111
11un01.120.202γ('78)] and the helper activity was calculated using the following formula.

Helper activity = (B/A)-1 A: Intermolar Ikins activity in the supernatant when mitomycin-treated autologous lymphocytes and tumor cells were cultured.

B: Interleukins activity in the supernatant when cloned Til cells were cultured with mitomycin-treated autologous lymphocytes and hepatoma cells.

Using the above method, 5 clones having helper activity against at least two types of tumor 1a cells were found among 100 cloned TIB cells.

Tumor II cells of these cloned helper TIE cells! !
Table 3 shows the intelligence.

These human cloned helper TI cells can be obtained with good reproducibility by stimulating human lymphocytes with lectin, cloning, and screening in the same manner as described above. Further, if culture is performed under the same conditions as in Example 1, long-term culture of 6 months or more is possible.

Recognition helper activity for normal cells: - (1 or less), + (1 or more) Example 3 Mouse tumor cell-recognizing cloned killer T cells were obtained as follows. Pile cells of BALB/c mice were collected by the following method. That is, dissecting BALB/c mice,
The viscera was taken out, loosened in a petri dish containing 10 d of Banks' solution, and the cell suspension was transferred to a test tube, left to stand for 3 minutes to allow the tissue pieces to settle, and then the cell suspension was subjected to another test. The mixture was transferred to a tube, centrifuged, the supernatant was discarded, 0.85% ammonium chloride aqueous solution 2IIN was added, and the mixture was kept at 37°C for 2 minutes to hemolyze the red blood cells. After centrifugation and discarding the supernatant, RPM I 1640 medium containing 10% live fetal serum was added to give a cell concentration of 2×10 6 /d.

Transfer 10 m of this mouse tile cell suspension medium to a culture bottle,
0μ9 of ConA (manufactured by E, Y Laboratories) was added and cultured for 40 hours. Lymphocytes activated by lectin were cloned in the same manner as in Example 1. However, interleukins use rat-derived interleukins (manufactured by Collaborative),
As autologous lymphocytes, BA treated with mitomycin
LB/ctl cells were used.

The killer activity of the obtained mouse cloned T cells against tumor cells was evaluated in the same manner as in Example 1.
Recognizing cloned killer T cells were obtained.

Typical examples are shown in Table 4.

Killer activity helper activity: - (1 or less), + (1 or more) Example 4 (Culture of human killer Ti1l cell clone, I labeling of membrane protein and adjustment of solubilized membrane protein fraction) Shown in Example 1 Human killer T cell clone 51 with broad tumor recognition was cultured with interleukins, and 1×109
cells were obtained. The engineered cell antigen, septa, was isolated from these cells as follows.

Cells were suspended in 5Id PBS and labeled with cell membrane protein I using the lactoperoxidase method (Immunology Experimental Procedures X1. P3491 edited by the Japanese Society of Immunology), and the cells were used three times in Hank's balanced salt solution containing 1% TritonX-100. PBS (11IIM phenylmeg rusulfonyl fluoride, 1mM EDTA, 10mM
The membrane protein was solubilized by suspending it in NaF-containing pH 7.2>2ae and stirring in water for 1 hour.

The lysate was centrifuged at 1000 CI for 20 minutes to remove cell debris and to obtain a fraction of solubilized membrane protein.

(Preparation of anti-T cell antigen receptor monoclonal antibody) Anti-T cell antigen receptor monoclonal antibody was obtained as follows. That is, 1 x 10 8 human peripheral blood lymphocytes were mixed with 100 IRIl of RPM I containing 10% beef tallow serum.
1640 medium (containing 0.1% PHA-P),
After culturing for 2 days, the cells were grown in interleukins medium to obtain 1 x 109 activated T cells. The cells were treated by the above method to solubilize membrane proteins, placed on Sephacryl S-200 (manufactured by Pharmacia), and
Gel chromatography was performed with BS, and the molecular weight was 7 to 10.
Ten thousand fractions were collected and ultraconcentrated (0.5 d). Mix the concentrate with Freund's Complete Adjuvant in equal volume and add BA
LB/c mice were immunized. After 10 and 20 days, 100 μg of the concentrated solution was given intraperitoneally, and 100 μg of the reconstituted solution was intravenously injected on the 10th day. After 3 days, the spleen of the immunized mice was taken out and subjected to conventional methods [(G, Ohler & C. , Hils
tein; Nature 256, 49. ('75)
] was fused with mouse myeloma P3U1 to create /X hybridoma. Hybridoma supernatant is human↑
We screened for the ability to bind to cells and inhibit the human killer TI cell killer activity, and further confirmed that the antibody does not recognize other currently known T cell membrane antigens, and finally determined that T111 cell membrane antigens were not antibodies that recognize other T cell membrane antigens. We performed immunoprecipitation with the exposure fraction and performed SDS polyacrylamide electrophoresis, confirmed that it recognized and bound to the reported T cell antigen receptor, and obtained an anti-T cell antigen receptor monoclonal antibody-producing hybridoma (116-23). Ta.

(Preparation of anti-T cell antigen receptor monoclonal antibody-bound Sephas 4B) 111. Hybridoma (116-23) was cultured in RPM I 1640 medium containing 20% tallow serum of 1jI, and 10 g of anti-T A cell antigen setter monoclonal antibody was obtained. This antibody 111TI+ was used to activate 20 CNBr?
It was bonded to Sefa 4B (manufactured by Pharmacia) by a pasting method.

(Purification of T cell antigen receptor by affinity chromatography) The solubilized membrane protein fraction of killer T cell clone 51 was transferred to a column packed with anti-T cell antigen receptor monoclonal antibody-conjugated Sephas 483d. Then, 888200m, which had absorbed TIO cell antigen resetter, was run to elute impurities. Then 0.1M glycine-salt 1I (P
I13. O) 10111i! The adsorbed Tt
IA follicle antigen receptors were eluted. After neutralizing the eluate, it was concentrated to obtain a concentrated solution of 0.5-. This concentrated liquid is SO
When subjected to S-polyacrylamide electrophoresis, approximately 90,000 intermolecular bands were detected under non-reducing conditions, and two bands with molecular m of 50,000 and 45,000 were detected under reducing conditions. It was found that it has an antigen resetter.

(Specific binding of T cell antigen receptor isolated from clone 51 to tumor cells) To investigate whether the Ta cell antigen receptor isolated and purified from clone 51 has the same recognition ability as the original clone. The following experiment was conducted.

Various cells were suspended in RPM I 1640 medium containing 10% live fetal serum at a cell concentration of 1×10 7 cells/Id, dropped in 50 μρ portions onto glass slides, and cultured overnight to allow them to adhere. 5 μft of Clone 51 T cell antigen receptor solution was added thereto, and after being kept in water for 30 minutes, it was immersed in a medium and washed. Next, the slide glass was immersed in a medium supplemented with 1 μg/d of anti-TI8 cell antigen or Sebutar monoclonal antibody, and kept in water for 30 minutes. After washing the slide glass with the medium, it is immersed in a medium containing FITCIK Mouse 10 antibody (manufactured by Tabo) in water for 30 minutes. After washing with a medium, it was determined whether the TIO follicular antigen receptor bound to the cells using a fluorescence microscope. By using such a method, the T cell antigen receptor isolated from clone 51 was isolated from human gastric cancer cell lines MKN-1 and KAT.
O-III, human lung cancer 18 cell lines PC-10, PC-13
, PC-14, human bladder cancer cell line NBT-2, human throat carcinoma cell line KB, human uterine cancer cell line HeLa, human melanoma cell line HMV-1,1-IMV-2, and clone 51. The T cell antigen receptor separated and purified from the cell membrane showed the same recognition ability as the original clone 51, without binding to cells that it did not recognize.

Example 5 TRI cell antigen receptor was purified from the human killer TIDla clone 8 shown in Example 1 using the method shown in Example 4, and its tumor cell binding property was examined.
1. It bound to KATO-111 but did not bind to clone 8 non-recognizing cells.

Example 6 Human killer TI shown in Example 1 [IJ1 clone 19
Using the method shown in Example 4, ① cell antigen receptor was purified and its tumor cell binding property was examined.
, PC-10, PC-13, and PC-14, but did not bind to clone 19 non-recognizing cells'.

Example 7 Using the method shown in Example 4 from the human killer Tit cell clone 24 shown in Example 1, cell antigen receptors were purified and tumor cell binding properties were examined.
It bound to LF and did not bind to clone 24 non-recognizing cells.

Example 8 Tl cell antigen receptor was purified from the human killer cell clone 36 shown in Example 1 using the method shown in Example 4, and its binding to tumor cells was examined.
1 (bound to BC-6 and did not bind to clone 24 non-recognizing cells).

Example 9 Ti1llll antigen receptor was purified from human helper Tm cell clone 55 shown in Example 2 using the method shown in Example 4, and tumor cell binding was examined.
c-io, PC-13, PC-14, MKN-1, KA
Combines with TO-111, C-1, H-1, clone 55
It did not bind to non-recognizing cells.

Although the present invention has been described above with reference to representative examples, the present invention is not limited to these.

Claims (2)

[Claims] (1) Cloned T cells that recognize tumor cells but not normal cells. (2) T cell antigen receptor obtained from cloned T cells that recognize tumor cells but not normal cells.