NL8202638A - Cancer cell-fighting lymphocytes, process for their production and anti-cancer agents, containing these lymphocytes. - Google Patents

Description

ν '4' 5 4 -

Cancer cell-fighting lymphocytes, method of production thereof and anti-cancer agents containing these lymphocytes.

The invention relates to cancer cell-fighting lymphocytes (hereinafter referred to as "killing cells") and a method of production thereof. In particular, it relates to killing cells which specifically act on cancer cells with a glycopants antigen derived from the cancer cells (this antigen is hereinafter referred to as "GRA") and destroys the cancer cells and production of these killing cells.

In addition, the invention relates to a new anti-cancer agent, particularly containing the killing cells or GRA as an active ingredient.

Immune response effect cells, especially T lymphocytes, which play a major role in immune response and cell medium, cause rejection of grafts attributable to foreign cell antigens, but show no perceptible or very limited inhibition against cancer cells.

Thus, the cancer cells are not destroyed and multiply in vivo, finally leading to the death of the cancerous host.

However, the mechanism responsible for recognizing one's own or not one's own is not entirely clear and various studies have been conducted to gain insight into the nature of the material basis involved in such a system, for example cell-25. surface marks on mouse leukemia cells or embryonic casinoma cells using lectins, such as Dolichos biflorus agglutinin (DBA) and peanut peanut agglutinin (PNA) as described in Biochem. Biophys. Res. Comm. 8 (2) 448-455 (1979), Ibid. 96 (4) 1547-1553 (1980), J. Biochem. 89 30 473-481 (1981) and Cell 18 183-191 September 1979.

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However, no significant attempts have been made to provide new lymphocytes, which can specifically fight cancer cells and also give anti-cancer agents using lymphocyte immune response.

As a result of extensive studies of the host's immune response to cancer cells and its application to cancer treatment, it has been found that in a cancer cell there is specific antigen, GRA, that is not found in differentiated normal cells, which an immunogen acts for the host and has a very high immunogenicity causing an immune response specific for the cancer cells. In addition, it has been argued that when GRA is used to sensitize lymphocytes, killing cells can be obtained, which act specifically on cancer cells containing GRA and when the killing cells are administered to the host, they recognize GRA and are destructive on the GRA-containing cancer cells and, therefore, that they show an excellent effect in the treatment and prevention of cancer.

Therefore, an embodiment of the invention involves killing cells.

Another embodiment involves a method for the production of killing cells.

Yet another embodiment of the present invention involves an anti-cancer agent containing killer cells or GRA as an active ingredient.

From the accompanying figures, Figure 1 is a micrograph of Daudi cancer cells, Figure 2 is a micrograph showing the formation of GRA-1-KT images of the cancer cells of Figure 1, Figure 3, a micrograph of Kato-III cancer cells, Figure 4 a micrograph showing the formation of platelets by GRA-1-KT of the cancer cells of FIG. 8202638 * 4 - 3 - figure 3, FIG. 5 a micrograph of BT-I cancer cells, FIG. 6 a micrograph showing the shows platelet formation by GRA-1-KT of the cancer cells of Figure 5, Figure 7 shows a micrograph of MKN-45 cancer cells, Figure 8 shows a micrograph showing platelet formation by GRA-1-KT of the cancer cells of Figure 7, Figure 9, a micrograph of MOLT cancer cells, Figure 10, a micrograph showing platelet formation by GBA-1-KT of the cancer cells of Figure 9, Figure 11, a micrograph of BT -1, treated with a mixture of unsensitized human peripheral blood lymphocytes, Figures 12 and 13 each e and a micrograph of the cancer cell tissue of a cancerous mouse administered GBA-M-1-KT, Figure 14 a micrograph showing the state of cancer in a group of cancerous mice to which GRA-M- has been administered. 1, Figure 15 is a micrograph showing the cancer state in a cancer group of mice not administered GRA-M-1, Figure 16 is a micrograph showing the cancer cell tissue of a group of cancer mice to which GRA-M-1 has not been administered, Figure 17, a micrograph of the cancer cell tissue of the group to which GRA-M-1 has been administered, Figure 18 an SDS gel electrophoresis diagram of GRA using protein stains according to CBB method, Figures 19 to 21 each show an SDS gel 8202638 -4- * * $ electrophoresis diagram of GRA using sugar staining according to the PAS method, figure 22 a schematic of the results of an SDS gel electrophoresis diagram of GRA 5 using protein stains according to the CBB

method and Figure 23 is a graph showing tumor growth in C3H / HE mice transplanted with X5563 immune mouse spleen cells and X5563 cells.

The kills / of the invention can be prepared, for example, according to a method using GRA to sensitize lymphocytes.

GRA as used in the above method can be obtained from human or animal cancer cells, for example cultured cancer cells, transplanted cancer cells, spontaneously occurring cancer cells, chemically or virus-generated cancer cells and cancer cells derived from operated tissues, from GRA containing cancer cells. -gene.

Cell membrane components are separated from cancer cells as described above and treated with a lectin, which specifically combines with a terminal galactose or a terminal N-acetylgalactosamine, whereby GRA is combined with the lectin and can be easily separated.

Suitable examples of galactose-binding lectin are pindalectin and castorin communis lectin (see JBC, 250, 8518-8523 (1975), Biochem, Biophys. Res. Comm., 62, 144 (1975), Z. Immunitaetsforch, 138, 423-433 ( 1969), 30 Bro J Exp Pathol, 27, 228-236 (1946), Proc Nath Acad.

Sci. USA, 75, no. 5, 2215-2219 (1978), Biochemistry, 13, 196-204 (1974) and Carbohydrate Research, 51, 107-118 (1976). Suitable examples of N-acetylgalactose binding lectin are Dolichos bean (Dolichos biflorus) agglutinin, braid orange lectin, Helix pomatia lectin, lima bean (Phaseolus 8202538

A K

* 4 - 5 - limensis) lectin, soy (Glycine max) lectin and Bauhinia bean (Bauhinia purpurea) lectin.

The secretion of cancer cell membrane components can be accomplished in known ways, such as by homogenization and solubilization using a solvent. It is more advantageous to use a method in which cancer cells are homogenized in physiological saline or in a suitable buffer, a precipitated portion is removed, for example, by centrifugation and dissolved in physiological saline or a buffer using a solvent and the supernatant liquid. Solvents to be used are, for example, surfactants, which are well known to dissolve cell membrane, such as nonionic surfactants, eg Triton X-100 (manufactured by Wako Pure Chemical Industries Ltd.), NP-40 (produced by Shell Co., Ltd.), digitonin and urea and anionic surfactants, e.g. sodium dodecyl sulfonate (SDS).

From the cell membrane components thus obtained, GBA, which can combine with lectin, can be recovered using the properties of lectin in the usual physicochemical and biochemical ways.

Examples of such methods are affinity chromatography using a lectin-containing carrier column, immunoprecipitation using GBA antibody or the like, dialysis, gel filtration, electrophoresis, physical precipitation using a sugar protein precipitant, eg, polyethylene glycol and acetone and a combination thereof. Preferred is affinity chromatography using a lectin-containing support column, wherein the support column can be easily manufactured by immobilizing lectin on an insoluble support. Such immobilization of lectin to an insoluble carrier can be carried out in known ways for the immobilization of biofuels. Of these methods, activation of polysaccharide with cyanogen bromide and immobilization of the use of N-hydroxy succimide esters are preferred. The activation of polysaccharide with cyanogen bromide is a method in which an insoluble carrier is treated with cyanogen bromide and the thus obtained activated product for immobilizing lectin under soft conditions is subjected to a coupling reaction with lectin. In the treatment of the insoluble carrier with eyaam bromide, for example, basic compounds such as sodium hydroxide and sodium hydrogen carbonate are used to adjust the pH to 7.5 to 12 and the carrier is treated for 1 to 12 minutes at room temperature in a solvent as water, vinegar-15 vacuum nitrile or a buffer maintained at pH 7.5 to 12, for example, 0.1 M sodium hydrogen carbonate buffer (pH about 8.7) and 0.01 M phosphate buffer (pH about 7.7). Usually, the amount of cyanogen bromide used is preferably equal to that of the insoluble carrier. Any known insoluble carrier with low nonspecific adsorption for all biosubstances, high porosity, with a functional group capable of immobilizing biosubstances under soft conditions and chemically and is physically sufficiently stable can be used in the invention.

Insoluble carriers which can be used are, for example, a carrier made of cellulose, for example aminoethyl cellulose, carboxymethyl cellulose, bromoacetyl cellulose and p-anilino cellulose, a carrier of cross-linked dextran, for example Sephadex and CM-Sephadex (manufactured by Farmacia Corp. 25). ), and an agarose carrier, for example

Sepharose 2B, Sepharose 4B and Sepharose 6B (manufactured by Farmacia Corp.).

In the coupling reaction of the cyanogen bromide-activated carrier described above with lectin, the cyanogen bromide-activated carrier is used in an 8202638

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i-7 - amount from 30 to 80 times the lectin and carry out the reaction for 10 to 20 hours at a temperature from 0 to 40 ° C, preferably from 2 to 8 ° C in a suitable solvent, such as a 0, 1 mol / l sodium hydrogen carbonate aqueous solution 5 (containing 0.5 mol / l sodium chloride, pH 8.4). In this way, a lectin-containing support for affinity chromatography can be prepared.

By chromatography using the lectin-containing carrier prepared above, the desired GRA combines with the lectin contained in the carrier and is trapped in the column. Subsequently, substances which can combine with, for example, lectin, can be passed through the column in order to effect an exchange reaction, or, optionally, an adsorption separator (eluent solution), for instance a highly concentrated salt solution, a potassium thiocyanate solution in water or a hydrate buffer, can be dissociated by the column and obtain the desired GBA.

Examples of substances to be used in the exchange which can combine with lectin when a galactose-binding lectin-containing carrier for affinity chromatography have been used are those which can combine with a terminal galactose-combining lectin, for example galactose, disaccharides with terminal ga -25 lactose and oligosaccharides with a terminal galactose and examples of substances which can combine with lectin, when an N-acetylgalactoseamine binding lectin has been used for affinity chromatography, are those which can combine with a terminal N-acetylgalactosamine combining lectin, for example N -acetylgalactosamine, disaccharides with an N-acetylgalactosamine terminal and oil saccharides with an N-acetylgalactosamine terminal.

The GRA thus obtained contains glycoprotein with a galactose and / or N-acetylgalactosamine in the final position, glycolipid and / or saccharide, 8202638 - 8 -

The GRA thus prepared can, if desired, be lyophilized and further purified using the conventional separation method. For example, the GRA preparations bound with a galactose-binding lectin can be subjected to a separation method using an N-acetylgalactosamine-combining lectin and those recovered with an N-acetylgalactosamine-combining lectin can be subjected to a separation method using a galactose binding lectin.

The lymphocytes used here are not critical and a variety of lymphocytes from normal or cancerous people or animals can be used. Examples are lymphocytes from peripheral blood, bone marrow, lymph nodes, spleen, tonsils and thymus. These lymphocytes are obtained physically or chemically or by a surface membrane method or the like.

Lymphocyte sensitization with GRA is performed by culturing the lymphocytes for 1 to 10 days, preferably 2 to 7 days, on a GRA-containing culture medium.

As a culture medium for the sensitization of lymphocytes, one can use various conventional nutrient media for such a cell culture. Preferred examples are an RPMI 1640 medium, an Eagle MEM culture, etc., to which human serum, calf fetal serum (KFS), calf serum, horse serum or the like has been added. The amount of GRA to be added to the culture is usually 1 to 1000 ng / ml, and preferably 1 to 500 ng / ml, as calculated as g an amount of sugar per 1 x 10 / ml lymphocyte.

The cultivation is carried out in the usual manner, for example at a pH of about 7.2 and a temperature of about 37 ° C.

The killing cells of the invention prepared as above are substantially normal lymphocytes and have GRA-specific cell-fighting activity. For example, 8202638 * V 4 - 9 - GRA-1-KT, which is one of the killing cells of the invention, has properties common to human peripheral blood T cells and exhibits cell-fighting activity specific for GRA containing cancer cells, as shown in the example described below.

Good examples of the novel killing cells of the invention are GRA-1-KT and GRA-M-1, which are prepared in the examples below. All killing cells of the invention are available from applicant.

The killing cells of the invention can be multiplied indefinitely on culture medium described above containing a T cell growth factor (TCGF, IL-2). In this case, selective culture of cloning of the killing cells can be performed according to the conventional ultra-dilution method. These killing cells can be stored stably for a long time, for example in liquid nitrogen.

The GRA can be used separately as an active ingredient and can additionally be used in combination with other bacterial and cancer inhibiting agents. Cancer inhibitors containing the GRA of the invention as an active ingredient can be in any form, provided they are in such a state that they contain the GRA in effective amounts. Usually, the agent is administered intravenously, subcutaneously, intradermally or intramuscularly as a solution, a suspension or an emulsion. Furthermore, it can be supplied as a drying product, which can be liquefied by adding a suitable carrier before use. This liquid agent may contain a suspending agent, for example, methyl-cellulose, an emulsifying agent, for example, lecithin, preservatives, for example, methyl p-hydroxybenzoate, a stabilizer which as such does not adversely affect the immunizing action on humans and animals, a buffer, and so on.

An aqueous carrier to be used is, for example, physiological saline and non-aqueous carriers to be used are, for example, vegetable oil, for example sesame oil, mineral oil, for example paraffin, animal oil, for example squalene and propylene glycol. In addition, suitable additives can be included to enhance the immunological activity. Additives to be used are, for example, complete additives of Freund, saponin for animals and aluminum hydroxide for humans.

The anti-cancer agent of the invention may be administered once or repeatedly for a long time to a cancer patient for treatment or may be administered to a person at risk of developing cancer for the prevention of cancer.

Since the LD, q (mouse, intraperitoneal) of GRA is at least 500 mg / kg, calculated as an amount of sugar, the anti-cancer agent of the invention has low toxicity and can therefore be administered in a wide dosage range. Although the GRA concentration in the anti-cancer agent of the invention is not critical, it is usually preferred that it be 0.001 to 100 µg / ml as calculated as an amount of sugar. Regarding the dose of the anti-cancer agent, it is usually preferred to administer the agent in an amount of 0.001 to 1000 µg / kg / day at a time or in varying portions, although this dose varies with the extent of the disease and the age and sex of the patient.

The anti-cancer agent thus prepared, containing the killing cells as an active ingredient, is preferably used as an injectable solution in combination with carriers used in the preparation of such blood medications. The carriers used are not critical, but carriers with a tone similar to that of blood are preferred. Particular preference is given to physiological saline. In the preparation of the agent, it is preferred that the killing cells become sufficient 8202638%.

- washed with physiological saline or the like, so that the culture medium described above is removed and the cells are then floated in a carrier.

The concentration of the killing cells in the agent is not specifically limited, but is preferably from 5 to 10 per milliliter. When the killing cells are administered intraperitoneally at a dose of 10 per mouse, no toxicity is observed. Although the dose of the anti-cancer agent of the invention varies with the severity of the disease and the age and sex of the patient, it is usually preferred that the agent be administered at a dose of 10 to 10 / kg / day, in a portion or in portions.

The following examples illustrate the invention.

Reference Example I Location of GRA - 1A. Preparation of FITC labeled lectin (PNA-FITC).

Ten mg of pindalectin (PNA, produced by EY Co.) was dissolved in 2 ml of 0.01 M phosphate buffer, containing 0.85%

NaCl (pH 7.2). 2 mg of FITC (manufactured by Sigma Laboratories Inc.) was dissolved in 1 ml of 0.5 M hydrogen carbonate buffer (pH 9.0) and 0.5 ml of the resulting solution was added to PNA buffer prepared above. The mixture was then stirred at room temperature for 2 hours and then separated over Sephadex G25 (10mm x 300mm, produced by Farmacia Corp.). The initial peak was absorbed. FITC / PNA ratio 1.0.

30 1B. Preparation of FITC labeled lectin (DBA-FITC).

In the same manner as under 1A above, DBA-FITC was obtained using DBA produced by EY Co. FITC / DBA ratio 0.9.

35 8202638

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«% · - 12 - 1C. Soy Glutinin-FITC (FITC-SBA) is available from EY Co. FITC / SBA ratio 1.4.

2. Locality of GRA in different 5 cancer cells.

a. Cultured human cancer cells (1 xg 10) were washed three times with a 0.05 M tris hydrochloric acid buffer containing 0.85% NaCl (pH 7.2) by a centrifugal method and then 100 µl PNA-FITC was added , DBA-FITC 10 or SBA-FITC (200 µg / ml) as prepared under 1. above.

The resulting mixture was allowed to stand at room temperature for 30 minutes to react. When the reaction was complete, the reaction mixture was washed three times with 0.01 M phosphate buffer containing 0.85% NaCl (pH 7.2) and then the cells were placed on a glass plate and examined under a fluorescent microscope.

The results are shown in Table A.

The cancer cells used are alien known and obtained from the First Pathology Laboratories, Medical Department of 20 Niigata University.

TABLE A

Cancer cells positive ratio -K-a.n5.erce.fien of GRA (%)

PNA DBA SBA

25 Raji (Burkitt's Lymphoma) 98.3 1.4

Dauji (Burkitt lymphoma) 93.1 5.2 BT-1 (Burkitt lymphoma) 50.1 0 P-12 (T cell lymphoma) 44.3 6.7 MOLT (T cell leukemia) 0.6 4.8 Fujimaki (B-cell lymphoma) 19.1 5.3

Oda (IgD myeloma) 0.6 10.0 QG-56 (lung cancer, scaly) 70.4 2.0 PC-1 (lung cancer, scaly) 78.4 0.4 PC-3 (lung cancer, adenocarcinoma) 77.1 0 35 QG-90 (lung cancer, small cell) 68.0 0 8202638 * «- 13 - TABLE A (continued) ,, ,, positive ratio

GBA cell cells (%) _

PNA DBA SBA

PC-13 (lung cancer, large cell) 17.0 0 5 MK-2 (gastric cancer, poor 63.7 0.1 differentiation) KATO-III (gastric cancer, signet ring cancer 57.3 0 EQS-45 (gastric cancer, poor 1 .0 40.3 differentiation) 10 EQS-1 (gastric cancer, adenocarcinoma 4.6 0.4 scaly) EQS-28 (gastric cancer) 0.4 0.1 EQS-74 (gastric cancer) 0.5 0 MGH-U1 (urinary bladder cancer ) 37.4 0 15 KU-2 (urinary bladder cancer) 4.5 21.4 T-24 (urinary bladder cancer) 14.6 0 NBT-2 (urinary bladder cancer) 13.1 1.0 NRC-12 (kidney cancer) 23.9 0 KU-1 (kidney cancer) 3.3 0.6 20 Kuramochi (ovarian cancer) 80.0 0 NB-1 (neuroblastoma) 50.9 1.7 YT-nu (neuroblastoma) 3.6 0.5 TGW-nu-1 (neuroblastoma) 4.1 0 TGW-nu-11 (neuroblastoma) 2.0 1.0 25 GOTO (neuroblastoma) 0.5 0 ITO (embryonic carcinoma) 96.9 12.3 NEC-8 (embryonic carcinoma) 44, 6 0 SCH (choriocarcinoma, stomach) 14.6 3.1 GCH (chriocarcinoma, uterus) 5.4 0 30 YN-1 (rhabdomyosarcoma) 5.7 1.7

Mouse X5563 (plasmacytoma) 92.0 0 90.6

Mouse MH134 (hepatoma) 18.6 0 6.4 35 8202638

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14) b) The malignant tissues of cancer patients were passed through a stainless steel screen ($ 150) to obtain a single cell suspension. After washing twice with 0.01 M tris-HCl buffer (pH 7.4) containing 2 mM 5 CaCl 3, 2mM MgCl 2 and 0.85% NaCl, 5x10 4 cells were resuspended in 100 µl buffer.

One hundred µl FITC-PNA or FITC-DBA (100 µg / ml) was added to the cell suspension and the mixture incubated at room temperature for 20 minutes. After washing three times with cold PBS, the cells were examined under a fluorescent microscope.

The results are shown in Table B.

Cancer patients' malignant tissues were obtained from Kansai Medical University.

15

GSA

No. Cancer patient tissue PNA DBA - 1 gastric cancer + + 2 gastric cancer + + 20 3 gastric cancer 4 gastric cancer + 5 gastric cancer + + 6 gastric cancer + 7 breast cancer + 25 8 breast cancer + 9 breast cancer + 10 breast cancer + + 11 colon cancer + + 12 colon cancer - + 30 13 Esophageal cancer + 14 hepatoma - +

Note: Symbol "+" means GEA is expressed on the cell surface.

Symbol means GRA is not expressed on the cell-35 surface.

8202638 i + v - - 15 -

Reference example II

Preparation of GRA

IA. Preparation of Immobilized Lectin (PNA-Sepharose).

5 Three grams of CNBr-activated Sepharose 4B

(produced by Farmacia Corp.) was washed thoroughly with 1 mM HCl and suspended in 200 ml 0.1 H sodium hydrogen carbonate (pH 8.5). Then, 5 ml of 0.01 M phosphate buffer (pH 8.5) containing 20 mg of PNA was added and the picture was reacted for 2 hours at 25 ° C with occasional stirring to prepare PNA-Sepharose.

IB. In the same way as in 1A. above, except using DBA instead of PNA, DBA-Sepharose was obtained.

15 2. Preparation of GRA.

a. BT-1 (Burkitt's lymphoma) cells (1.3 x 10) were washed three times with physiological saline and 30 ml of 0.01 M tris hydrochloric acid buffer (pH 7.4) was added, containing 20 2% "TRITON X-100 (produced by Wako Pure Chemical Indus tries Ltd.), contained 0.85% NaCl, 2 mM CaCl2 and 2 mM MgCl2.

The mixture was stirred at 4 ° C for 15 minutes and then subjected to ultracentrifuge separation at 100,000 x g. Of 28 ml supernatant thus obtained, a 14 ml portion was passed through an affinity chromatography column (diameter 0.5, length 1 cm), filled with PNA agarose beads (produced by Maruzen Co., Ltd.), which equilibrated with a tris hydrochloric acid buffer (pH 7.4) containing 0.1% TRITON X-100, 0.85% NaCl, 2 mM CaCl 2 and 2 mM MgCl 2. After washing with the same buffer as used above, the column was eluted with 0.01 M tris hydrochloric acid buffer (pH 7.4) containing 0.1 M lactose, 0.85% NaCl, 2 mM CaCl 2, 2 mM MgCl 2 and 0.1% TRITON X-100. The portion thus eluted was dialyzed for 48 hours with 0.01 M tris hydrochloric acid buffer containing 0.85% NaCl, 2 mM 35 MgCl 2 and 2 mM CaCl 2 to give 17 ml of GRA 8202638% solution. With this GRA solution, the amount of protein and the amount of sugar were measured by the Folin-Lowry method and the phenol-sulfuric acid method, respectively, and were found to be 644 µg and 120 µg, respectively. This is hereinafter referred to as "GRA-1".

b. C3H / He mouse mammary tumor (MMT) cells (1 x 10 ^) were washed three times with physiological saline and 30 ml of 0.01 M tris hydrochloric acid buffer (pH 7.4), containing 2% TRITON X-100, were added. 0.85% NaCl, 2 mM CaCl 2 and 2 mM

10 MgCl2. The mixture was stirred at 4 ° C for 30 minutes.

Then the mixture was ultracentrifuged at 100,000 xg for 2 hours and the supernatant was dialyzed overnight with 0.1 M tris hydrochloric acid buffer (pH 7.4) containing 0.85% NaCl, 2 mM CaCl 2 and 2 mM MgCl ^ 15 contained. The thus dialyzed liquid was concentrated to 3 ml and a 1 ml portion was then passed through an affinity chromatography column (diameter 0.5, length 2 cm) filled with the same PNA-Sepharose used above, equilibrated with a tris hydrochloric acid buffer (pH 7.4), 20 containing 0.005% TRITON X-100, 0.85% NaCl, 2 mM CaCl2 and 2 mM MgCl2

contained. After being completely washed with the same buffer as used above, the column was eluted with a 0.01 M tris hydrochloric acid buffer (pH 7.4) containing 0.1 M lactose, 0.85% NaCl, 2 mM CaCl2, 2 mM MgCl2 and 0.005% TRITON X-100 and the thus-eluted portion was dialyzed with 0.01 M for 48 hours

tris hydrochloric acid buffer (pH 7.4) containing 0.85% NaCl, 2 mM CaCl 2 and 2 mM MgCl 2 to give 2 ml GRA solution. With the GRA solution thus obtained, the amount of protein and the amount of sugar were 156 µg and 30 µg respectively. This is referred to as "GRA-M-1".

c. About 120 g (wet weight) of KATO-III was homogenized in 100 ml PBS with a Waring blender.

After centrifugation at 100,000 g for 1 hour, the pellet was dissolved in 100 ml of 2% Triton X-100 in 0.01 M tris-HCl buffer 35 (pH 7.6) containing 0.15 M NaCl. The supernatant 8202638% 4 - 17 - after centrifugation at 100,000 g for one hour was passed through a column of PNA-Sepharose 4B (0.8 x 15 cm) equilibrated with 0.015% Triton X-100 in 0, 01M tris-HCl (pH 7.6) containing 0.15 M NaCl. After washing with 50 ml of the buffer, the GRA was eluted with the buffer containing 0.1 M lactose. The eluted GRA was dialyzed at 0.85%

NaCl, concentrated with Sephadex Pharmacia Co. and stored at -20 ° C for use.

The amounts of protein and sugar determined in the same manner as under a. Above were found to be 2.0 mg and 0.8, respectively. This is referred to as "GBA-2n."

d. In the same way as under c. above, the following GRA samples were obtained.

15

TABLE C

PRA

GRA Material -r - ^^ - - protein- sugar- sample source amount (g) content (mg) content (mg) GRA-3 BT-1 33 0.5 0.09 20 GRA-4 Breast jug- 5 0, 24 0.5 ker (cut) GRA-5 QG-56 24 0.6 0.38 GRA-6 QG-90 26 1.0 0.54 GRA-7 Raji 29 0.78 0.45 25 GRA- M-2 MMT 200 11.3 28.4 GRA-M-3 LLC 14.4 0.06 0.07 GRA-M-4 MH-134 85 0.65 0.35 GRA-M-5 X-5563 25 0.56 0.23 30 e. In the same way as under c. above, except that MKN-45 (about 29 g) was used in place of Kato-III and DBA-Sepharose obtained under 1B. above was used in place of PNA-Sepharose and elution was performed with N-acetylgalactosamine, a GRA preparation having a protein content of 0.03 mg and a sugar content of 0.01 8202638-18 mg. This is hereinafter referred to as "GRA-8".

f. Five ml of GRA-3 prepared under d. was placed in a DBA Sepharose column above and eluted with tris-HCl buffer (0.015% Triton X-100, 2 mM MgCl 2 CaCl 2, 0.85% 5 NaCl) to give 4 ml fractions nos. 1-12. Fractions 1-3 are referred to as "GRA-3-A" and fractions 4-11 as "GRA-3-B". Then, the column was eluted with the same buffer, but containing 0.1 M N-acetylgalactosamine to yield fractions, designated "GKA-3-C".

10 g. SDS gel electrophoresis.

Each of the GRA preparations obtained by the above methods was subjected to electrophoresis by the method described in Fairbanks et al: Biochemistry 10, 2606, 1971.

The results obtained are shown in Figures 18 to 22.

In Figures 18-19, numbers 1 to 5 mean the following: 1: Standard, 2: GRA-M-3, 3: GRA-7, 4: GRA-1 20, and 5: GRA-5.

In Figure 20, numbers 1 to 4 mean the following: 1: Standard, 2: GRA-M-2, 3: GRA-6, and 4: GRA-5.

25 In figure 21, numbers 1 to 3 mean the following: 1: GRA-M-4, 2: GRA-M-5 and 3: standard.

In Figure 22, numbers 1 to 4 mean the following: 30 1: GRA-3, 2: GRA-3-A, 3: GRA-3-B and 4: GRA-3-C.

Figure 18 shows the state of GRA subjected to protein stain reaction according to the C.B.B. method (Fairbanks et al: Biochemistry 10, biz. 2606, 1971).

Figures 19-21 show the state of GRA, that 8202638 * - 19 - has been subjected to sugar color reaction by the PAS method (R.M. Zacharius et al: Anal. Biochem. 30, biz. 148 (1962)).

Figure 22 shows a schematic representation of the results of the stain formation according to the C.B.B. described above. method.

Of the standard fabrics listed below, the following were used from Biorad Lab. Calif. U.S.A ..

200 K dalton; myosin 116 "; ph-galactosidase 10 92.5 π; phosphorylase

66.2 "; BSA

45 "; egg albumin 21.5 i; soy trypsin inhibitor.

Reference example III

15 Preparation of TCGF.

a. Four kg spleen of Japanese monkeys (obtained from Japan Plymates Co., Ltd.) was cut and washed twice with an RPMI-1640 culture medium (produced by

Flow Laboratory Co., Ltd.). The cells were filtered using Mesh (manufactured by Millipore Inc., 150 mesh) and subjected to a specific gravity centrifuge method (specific gravity, 1.076) to obtain 9 2 liters of 2 x 10 / ml lymphocytes. The lymphocytes thus obtained were washed 3 times with an RPMI-1640 culture medium and the lymphocyte number was adjusted to 5 x 10 µ per ml using the same medium as above containing 10% FCS. The whole was then allowed to stand at 37 ° C for 1 hour in a carbon dioxide fermenter. The lymphocytes from the supernatant were collected and the lymphocyte g number was adjusted to 1 x 10 per ml using the same culture medium as above containing 1% FCS. Then 1 µg / ml indomethacin (produced by Sigma Laboratories, Inc.) and 0.2% PHA-P (produced by Difco Co.) was added and subjected to culture in a carbon dioxide gas fermenter for 48 hours at 37 ° C. Centrifuge at 3000 rpm for 10 minutes and the resulting supernatant recovered, which was sterilized by filtration through a Millipore filter (produced by Millipore Inc., 0.2 µm) to obtain 2 liters of TCGF.

5 b. The source of TCGF was the supernatant of mixed culture peripheral blood lymphocytes from ten healthy donors. Non-adherent lymphocytes prepared from C.F. (Conray. Ficol (Japan Immunoresearch Co.)) secreted lymphocytes by adsorption on plastic surface at 37 ° C for 1 hour were suspended in RPMI-1640 medium containing 1% FCS (1.5 x 10 cells / ml) and incubated with 0.2% PHA.-P, indomethacin (1 µg / ml) and human 5 B cell line (BT-1) (1.5 x 10 cells / ml) pretreated with mitomycin C (50 µg / ml). After 48 hours, the culture supernatant was collected and used as a TCGF source.

(H. Inoue et al., Scand. J. Immunol. 12, biz. 149-154 (1980)).

Reference example IV.

Preparation of lymphocytes.

1. Human peripheral blood lymphocytes.

Fifty ml of blood, obtained from a healthy adult or various cancer patients by pararinization, was centrifuged using Ficol Pack (manufactured by Farmacia Japan Co., Ltd.) to obtain 5 x 10 4 peripheral blood lymphocytes.

25 2. Mouse spleen lymphocytes.

The spleen of a C3H / He mouse (male, 6w) was excised and washed twice with an RPMI-1640 culture medium. The spleen was then detached with an injection needle and passed through a stainless steel screen (100 mesh) to remove large pieces. The cells thus filtered were washed twice with the same culture medium as above and subjected to centrifugal separation at 1200 rpm for 10 minutes to obtain 4x10 1 spleen lymphocytes.

35 8202638 * - 21 -

Example I

GRA.-1 (amount of protein: 40 µg / ml, amount of sugar: 7.5 µg / ml) as obtained in reference example II under Ila. was diluted to 1000 times the original 5 volume with a KPMI-1640 culture medium containing 15% FCS

contained a sensitizing culture medium for preparation.

Human peripheral blood lymphocytes (5 x g 10/5 ml) as obtained in Reference Example IV under 1. were added to 5 ml of the sensitizing culture medium, prepared as above, which was placed on a laboratory dish and grown for two days at 37 ° C. Cultivation was continued for 5 days on the KPMI-1640 culture medium containing 20% JTCGF and 15% FCS as obtained in Reference Example III to obtain 20 ml of killing cell solution containing 1 x 10 killing cells per ml. This is hereinafter referred to as "GBA-1-K-T".

Example II

Mouse murine lymphocytes as obtained in Reference Example IV under 2. were adjusted to a number g of 5 x 10 / ml using a KPMI-1640 culture medium containing 15% FCS. Then GRA.-M-1 as obtained in Reference Example II under lib. so that the final amounts of protein and sugar were 1.5 µg / ml and 0.9 µg / ml, respectively. A 5 ml portion of the resulting mixture was grown on a laboratory dish (6 ml x 15 ml, manufactured by Falcon Co.) for two days at 37 ° C. Clone formation was observed. The aliquot was cultured for a further four days on a KPMI-1640 culture medium containing 15% FCS containing 25 vol% TCGF (produced by Japan Immuno 30 Research Laboratories Co., Ltd.) to obtain g of 50 ml of killing cell solution containing 1 x 10 killing cells per ml. This is hereinafter referred to as "GRA-M-1-KT".

Example III

Peripheral blood lymphocytes (5 x 10) from a healthy donor were incubated at 37 ° C in RPMI-1640 medium, 8202638 ψ-22 - containing 50 ng / ml (protein content) GRA-2 and 15% FCS.

On day 2, the human TCGF described above was added to the medium until the concentration reached 20% and incubation was continued for another 3 days to obtain killing cells. This is referred to as "GRA-2-K-T".

Example IV.

Killing cell preparations GRA.-8-KT, GM-3-AKT and GRA-3-CKT were prepared in the same manner as in example using GRA-8, GEA-10 3-A and GSA-3-C, respectively ( protein content: 50 ng / ml) and the TCGF obtained in reference example Illb.

Example V.

C3H / He mice (females, 8 weeks) were implanted with intradermal X5563 cells (10) of the same strain and after 7 days the tumor was surgically excised. After a further 7 days, X5563 cells (10 ^) from the same strain were inoculated and the vaccination resistant mice were called immune mice.

The spleen cells of the immune mice and normal C3H / He mice were prepared according to the usual method.

g

Each batch of spleen cells (5 x 10 / source) was sensitized for 5 days with 40 ng / ml (protein content) GRA-M-5 in RPMI-1640 medium containing 15% FCS.

Killing cell preparation obtained from normal spleen cells is hereinafter designated "GRA-M-5-K-T-1" and that obtained from immune spleen cells is hereinafter designated "GRA-M-5-K-T-2".

Example VI.

Human peripheral blood lymphocytes (5 x 10 ^) were incubated for two days at 37 ° C RPMI-1640 medium containing 50 ng / ml (protein content) GRA-1. On day 3, the lymphocytes were transferred to RPMI-1640 medium containing 10% lymphocyte donor serum and 0 to 100 ng / ml 8202638 - i - 23 - (protein content) GBA-1 and incubation 5 more days continued to obtain killing cell preparations as shown in Table D below.

5 TABE1 D

GRA concentration (ng / ml)

Day 3 Day 3 Killing cells.

50 0 GRA-1-KT-1 50 1.6 GBA-1 -KT-2 10 50 3.2 GBA-1-KT-3 50 6 GBA-1-KT-4 50 12.5 GBA-1-KT -5 50 25 GRA-1-KT-6 50 50 GBA-1-KT-7 15 50 100 GBA-1-KT-8

Example VII

a. Peripheral blood lymphocytes (PBL) from various cancer patients after surgery were sensitized with GRA-3 to obtain killing cell preparations. PBL

g (5 x 10) of the patients were incubated for 2 days in BPMI-1640 medium containing 50 ng / ml (protein content) of GRA-3 and then 5 more days in RPMI-1640 medium containing 20% TCGF and 15% FCS containing killing cells as shown in Table E.

TABLE E

Peripheral blood lymphocytes. , ----- days after

Ranker P-GBA D-GRA surgery killing cells 30 stomach cancer + + 14 GBA-3-KT-1 stomach cancer ++ 35 GRA-3-KT-2 breast cancer + - 21 GRA-3-KT-3 breast cancer + - 7 GRA- 3-KT-4 breast cancer + - 35 GBA-3-KT-5 35 stomach cancer + - 21 GRA-3-KT-6 8202638 - 24 -

In the table above, P-GRA and D-GRA indicate the GRA locality on the malignant tissue (excised by surgery) of the cancer patients, from which PBL was collected as determined in the same manner as in Reference Example I under 2b. P-GRA and D-GRA are results obtained using FITC-PNA and FITC-DBA, respectively. The days after surgery indicate the time in which PBL was collected after surgery.

g b. PBL (5 x 10) collected from breast cancer patients 21 days after surgery was incubated for 7 days in RPMI-1640 medium containing 50 ng / ml (protein content) GRA-3 and

Contained 10% patient's serum to sensitize PBL

and to obtain killing cellular formulation. This is hereinafter referred to as "GRA-S-K-T-y".

Example VIII

8 GRA-1-K-T (10) as obtained in Example I was dissolved in 10 ml of physiological saline to prepare an injectable solution.

Example IX

I. GRA-M-1 as obtained in Reference Example II under lib. was diluted with physiological saline so that the amounts of sugar and protein were 1.0 µg / ml and 1.6 µg / ml, respectively, to thereby prepare an anti-cancer agent no 1.

25 II. A tumor lobe of C3H / He spontaneously occurring breast cancer was sterile excised into a 5 mm cube and transplanted under the back skin of each of ten C3H / He mice of the same strain as above (7 weeks old, male nets). Fixation and tumor multiplication were confirmed seven days after transplantation. 5 of the mice were administered each subcutaneous anti-cancer agent no 1 as prepared above under I. at a dose of 3 µl per day at two day intervals. The remaining 5 mice were used as untreated control animals. Ten days after the first administration, the tumor was excised by surgery and the average weight was measured. At the same time, patho-histological examination was performed.

Tumor volume:

Infused group 22.3 mm3 (figure 14) 5 Control 162.7 mm3 (figure 15).

This means that there was an 86.3% reduction in the tumor.

Pathohistological examination:

In the control group (Figure 16), bird-nest cancer bodies were formed, the tissue type was equal to medullary canalicular cancer, and the multiplication of tumor cells was observed over the entire tissue. On the other hand, the cancer cells in the drug-treated group (Figure 17) caused liquefaction necrosis at the sites where the cancer cells were formed and calcification and firosis occurred leaving only a very limited amount of cancer cells. Thus, the anti-tumor properties of the anti-cancer agent of the invention were observed.

Test example I.

GRA-1-K-T (1 µL) as obtained in Example I was applied to a microplate (manufactured by Falcon Corp.) and left to stand at room temperature for 15 minutes. Then 4 µl FCS (produced by Falcon Corp.) was added and the mixture was allowed to stand at room temperature for 30 minutes. Sheep neuraminidase-treated red blood cells (SRBC4) were adjusted to a number of

Q

Add 1 x 10 per milliliter and 5 µl of 0.01 M phosphate buffer (pH 7.2), to which 0.85% NaCl was added, and centrifuged the plate at 600 rpm for 5 minutes.

The plate was then inverted and unresponsive

N

SRBC was removed. A dye solution (Brilliant Cresyl Blue, from Merck & Co.) was added to stain the lymphocytes and a positive rosette effect was observed. Thus, it was found that at least 98% rosette formation is positive (T cells).

8202638 - 26 -

Test example II

Specific cancer cells killing effect. a. Of the cells shown in Table A, the following five cell strains with different GRA positive ratios were used as human target cancer cells.

Target cancer cells:

No. 1 BT-1 (Burkitt's lymphoma)

No. 2 Daudi (Burkitt's lymphoma)

No. 3 KATO-III (gastric cancer) 10 No. 4 EQS-45 (gastric cancer)

No. 5 MOLT (T cell leukemia).

On a micro plate (produced by Falcon

Corp.), 5 x 10 4 per well of target cancer cells were laminated according to a centrifugal procedure at 800 rpm for 3 minutes. Then 4 x 10 per well of GRA-1 "K-T as obtained in Example was slowly added and incubated for 1 hour.

The killing activity was determined according to the degree of platelet formation and was evaluated as follows: ++ killing activity is clearly observed.

+ killing effect observed.

± killing effect slightly observed.

- killing effect not observed.

Unsensitized human peripheral blood lymphocytes obtained in the same manner as in Example 1 were used in a control group, except that no GRA was used. The results are shown in Table F.

Table F shows that the killing T cells produced according to the method of the invention have a strong GRA specific cytotoxic activity.

35 8202638 - 27 -

TABLE F

Target cancer- GRA positive Determination of platelet ratio ___ ratio (%) of formation _ GRA-1-KT Daudi (fig. D 93,1 ++ (fig. 2) 5 §group KATO-III (fig. 3) 57,3 + (Fig. 4) BT-1 (Fig. 5) 50.1 ++ (Fig. 6) MKN-45 (Fig. 7) 1.0 ± (Fig. 8) MOLT (Fig. 9) 0.6 - (fig. 10)

Control- BT-1 50.1 - (Fig. 11)

§ o §roeP

b. The same target cancer cells as used 6 5 under a. Above (3.2 x 10) were mixed with 8 x 10 GRA-1-KT (cell ratio: 5/1) and the resulting cell mixture 6 (total number: 4 x 10) was grown at an RFMI-1640 medium, containing 15% FCS. After one hour, the number of remaining cells were counted and the% cytotoxicity was calculated by the following equation: number of cells after% cytotoxicity = (1 - (---- 1 ...........) x 100 ).

20 cell count for culture (4 x 10 ^)

The results are given in Table G.

TABLE G

25 _Number of cells

Target- Before Na% Cytotoxi cells culture culture city_

Daudi 4 x 106 2.9 x 106 91 r £ KATO-III 4 x 10 3.7 x 10 24 30 BT-1 - 4 x 106 3.2 x 106 65 EQS-45 4 x 106 3.9 x 106 7 .2 MOLT 4 x 106 4.2 x 106 -3 c. The procedure of b. above was repeated except that the mixing ratio of GRA-1-K-T to 8202638-28 target cancer cells was changed to 5/3. The results are given in Table H.

TABLE H

5 _ Number of cells_

Target- Before Na% Cytotoxi cells culture culture city_

Daudi 4 x 106 3.6 x 105 91 KATO-III 4 x 106 3.6 x 106 24 10 BT-1 4 x 106 1.4 x 106 65 EQS-45 4 x 106 3.7 x 106 7.2 MOLT 4 x 106 4.1 x 106 -3

In the above experiment it was observed that GBA-1-K-T exerts a strong binding activity on

Daudi, KATO-III and BT-1, but only a minor binding effect on MKN-45 and MOLT.

Test Example III

C3H / He 20 spontaneous breast cancer patients received GRA-M-1-K-T subcutaneously as obtained in Example II at a dose of 3 x 10 / 0.3 ml / mouse 3 times a week every other day. After 10 days, the focus was removed and examined.

As shown in Figure 12, lymphocyte infiltration into cancer cells occurred and fracture in the tumor region was observed. It is also apparent from Figure 13 that it was observed that calcification of the tumor area occurred and thus it can be seen that the killing cells of the invention have anti-tumor activity.

Comparative Example I

In this example, cancer cells as such were used as specific antigens in the method of the invention in place of GRA.

Lymphocytes sensitized to cancer cells were obtained in the same manner as in Example 1, 8202638 * t - 29 - except that BT-1 was used instead of GEA,

Daudi, KATO-III or MKN-45 in an amount of 1x10 "* per laboratory scale.

In these lymphocytes, the cytotoxic activity was examined in the same manner as in test example II

under a. The results are given in Table I.

It can be seen from Table I that the lymphocytes prepared above do not have any cytotoxic activity.

10 TABLE I

Cell for use in lymphocyte sensitization .__

Target cells BT-1 Daudi KATO-III MKN-45 BT-1 15 Daudi ---- KATO-III - MKN-45 ----

Test example IV

In the same manner as in test example II

under b. the cancer cell killing activity of GRA-8-K-T, GRA-3-A-K-T and GRA-3-C-K-T obtained in Example 1 was determined.

The results are shown in Table J.

25 TABLE J

_Cytotoxicity_

Target cells GRA-8-K-T GRA-3-A-K-T GKA-3-C-K-T

KATO-III 8.0 5.0 5.0 BT-1 4.3 5.0 4.0 30 EQS-45 20 5.0 20 MOLT 000

Test example V

a. The cytotoxicity of each of the killing cell preparations obtained in Example VI was determined 8202638 51-30 by the Cr release assay (J. Immunol. 122, 2527-2533 (1979).

51

That is, 50 µCi radioactive Cr (Japan Isotope Asso- 7 ciation) was added to KATO-III (2x10) and the cells were incubated in RPMI-1640 medium for 1 hour at 37 ° C and obtained with 51 Cr-labeled target cells washed sufficiently by centrifugation. Killing cells (effector cells) 6 (2 x 10 °) were added to the target cells (1 x 10J) (thus E / T = 20/1) and the mixture was incubated in RPMI-1640 medium at 37 ° C for 4 hours. The supernatant was centrifuged off and its radioactivity was determined by liquid quintillation counting.

51

The specific Cr release (%) corresponding to the cytolytic activity of effector cells was calculated according to the following equation.

15 Specific ~ ^ Cr release (%) (trial release) - (spontaneous release) χ (maximum release) - (spontaneous release) (The maximum release means the radioactivity when all cells are lysed).

The results obtained are given in Table K.

TABLE K

51

Killing cells Specific Cr release (%) 25 GRA-1-KT-1 13 GRA-1-KT-2 25 GRA-1-KT-3 22 GRA-1-KT-4 20 GRA-1-KT-5 22 30 GRA-1-KT-6 25 GRA-1-KT-7 27 GRA-1-KT-8 32

The results of Table K show that 35 TCGF and FCS are not related to the generation of killing 8202638 - 31 * *> ψ cells.

b. The cytotoxicity of GRA-2-K-T, 51 gene in Example III was determined by Cr release assay in the same manner as above under a. A specific Cr. Release of 14.3% was observed on Cr-labeled KATO-III as target cells (E / T = 20/1).

Test example VI

a. Using KATO-III (E / T = 20/1) as target cells, the cytotoxicity of killing cells obtained in Example VII under a. was determined in the same manner as in Example 2 under b.

The results are given in Table L below.

15 TABLE L

Killing cells% Cytotoxicity GRA-3-KT-1 38.0 GRA-3-KT-2 18.2 GRA-3-KT-3 20.9 20 GRA-3-KT-4 23.2 GRA-3-KT -5 24.5 GRA-3-KT-6 20.2

b. The cytotoxicity of Example VII

51

25 under b. GRA-3-K-T-7 obtained was determined with Cr-KATO-III

(E / T = 20/1) as target cells in the same manner as in. Test Example V. The specific Cr release of the killing cells was found to be 25.5%.

Test example VII

The cytotoxicity of the Example V

51 killing cells obtained were determined by the Cr release assay in the same manner as in Test Example V. The 51 target cells used were Cr-labeled X5563 cells. Lymphocytes obtained in the same manner as in Example V were used as control, except that sensitization of spleen cells 8202638 - 32 - was performed with 1 x 10 4 / well mitocyne C-treated g X5563 cells (5 x 10 / ml X5563 cells were treated with 50 µg / ml mitomycin C) for 60 minutes instead of using GRA-M-5.

The results obtained are shown in Table M below.

TABLE M 51

Specific Gr release 10 E / T ratio

Killing cells 40: 1 20: 1 10: 1 GRA-M-5-K-T-1 12.7 6.3 5.7 GRA-M-5-K-T-2 18.4 20.6 13.7

Control 0.0 0.0 0.0 15

Test Example VIII (H-2 Test) GRA-M-1, GRA-M-3 and GRA-M-4 obtained in Reference Example 2 above were serially diluted with PBS (0.85% NaCl) to prepare samples.

20 Anti-H-2 serum from National Institute of

Genetic Research and the above sample were mixed and incubated for 2 hours at 4 ° C adding target cells corresponding to the anti-H-2 serum used. Spleen cells or lymph node cells obtained in the usual manner from B10 (H-2) and B10.Br (H-21) mice were used as target cells. After the cells were washed with PBS, complement (rabbit) was added to the cells and the cells were incubated at 37 ° C for 1 hour and stained with 0.2% tryptane blue-PBS to determine% cytotoxicity.

The anti-H-2 serum was used in such a maximum dilution that it showed at least 95% cytotoxicity in the absence of GRA.

The blocking effect by GRA was determined for systems shown in Table N below.

35 8202638

F

% I

- 33 -

TABLE N

GRA Anti-H ~ 2 serum, target cell concentration GRA-M-1 D-23 (anti-H-2Kk), X80 B10BR (H-2k) D-32 (anti-H-2Dk), X300 spleen cells 5 GRA- M-3 D-33 (anti-H-2Kb), X600 B10 (H-2b) T. or D- "(anti-H-2D), X80 spleen cells GRA-M-4 D-23, X80 B10.BR (h-2k) or D-32, X300 lymph node cells. The results obtained are given in Table 0.

TABLE 0% Cytotoxicity 15 Anti-H-2 _ Dilution of sample _ GRA serum X2 X4 X8 X16 X32 X64 X128 X256 X512 0 * I - - 13 14 12 13 14 13 14 13 14 13 D-23 - 97 99 96 97 97 96 97 96 96 14 D-32 - 95 95 95 95 94 95 95 96 95 13 20 Π 19 15 14 12 15 12 11 12 13 14 D-33 - 98 98 99 99 98 99 99 99 99 15 D “2 - 95 96 95 95 97 95 97 95 95 17 III D-23 100 100 100 100 --- - - 100 10 D32 99 99 99 99 - - - - 99 10 25 Remarks: GRA I: GRA-M-1 GRA II: GRA -M-3 GRA III: GRA-M-4 * means% cytotoxicity when only complement was used.

30

From the results of Table 0 above, it can be seen that GRA-M-1, GRA-M-3 and GRA-M-4 lack H-2.

Test Example IX

C57BL / 6 mice received subcutaneous transplantation of LLC (2 x 10) from the same strain and after 6 days 1 mg (protein) GRA-M-3, obtained in Reference Example II under d. administered. Then the administration was repeated once a day at the same dose for 4 days.

Tumor cells were excised and weighed the day after the last administration. Animals to which physiological saline had been administered were used as a control. Each test group included 5 animals.

As a result, an average tumor-10 weight of the control group was found to be approximately 500 mg. In the group administered GRA-M-3, three animals had tumor disappearance and two had an average tumor weight of 100 mg.

Test example X

C57BL / 6 mice were immunized subcutaneously with 1 ng (protein) GRA-M-3, obtained in Reference Example II under d, once daily for three days. and spleen cells were harvested from the animal on day 5 to obtain effector cells. A mixture of effector cells and Lewis lung carcinoma (LLC) as target cells at a ratio of E / T = 50: 1 was prepared and a 5 (1 x 10) portion thereof was transplanted into mice of the same strain and fed Winn's experiment (J. Immunol.

86, biz. 228-239 (1961)).

The results obtained are shown in Table P below.

TABLE P

Effector Tumor growth (%) Day 20 mortality / 30 cell_ Day 15 Day 17 Day 18 Day 19 Day 20 group A 5.7 5.5 5.7 3.1 1.4 0/10 B 21.4 39. 5 37.1 55.4 76.8 4/10 C 39.3 65.1 61.7 98.1 96.4 4/10

Comments: Group A are spleen cells of GRA-M-3 immune 8202638

• I

- 35 - mice.

Group B are spleen cells from normal mice.

Group C are mouse spleen cells 10 days g after transplantation of LLC (1 x 10).

Tumor growth was calculated according to the following equation:

T - T

Tumor growth (%) = ——— x 100

T

N

where T. represents the thickness of the sole of the foot on the side where A

10 the tumor was transplanted and T ^. represents the thickness of the normal sole of the foot.

Test example XI

C3H / He mice were immunized with 4.5 µg (protein) of GRA-M-4 obtained in Reference Example 15 II under d. and 0.1 ml complete Freund's additive at the sacrococcygeal portion. After two weeks, lymph nodes were collected in the usual manner and used as response cells to determine proliferative response in GRA-M-4.

For this purpose, response cells (5 x 10 ^) were incubated in RPMI-1640 medium containing 15% FCS for 5 days in the presence of GRA-M-4. During the last 18 hours of the incubation period, 1 uCi H-thymidine (H-TdR) was added to the medium and its uptake counted in the cells.

The results obtained are given in Table Q below.

TABLE Q

Concentration 3H-TdR. incorporation GRA-M-4 (ng / ml) (mean cpm + S.E.) S.I.

30 Control 0 5,302 + 1,761 - - 0.5 7,903 ± 1,290 1.5 1 10,076 ± 936 1.9

Experiment 5 10,686 ± 429 2.0 20 10,615 ± 1,270 2.0 35 40 8,565 + 1,419 1.6 8202638 «- 36 -

Note: "S.I." means stimulation index in terms of experiment / control.

Test example XII

Delayed type (DTH) hypersensitivity response (DTH) of normal C3H / He mice, X5563 immune mice and MH134 immune mice was obtained in the same manner as in Example V and GRA-M-4 immune mice and GRA-M-5 immune mice, obtained in the same manner as in Test Example XI by footpad reaction (FPR). That is, tumor cells treated with GRA or MMC were seeded on the footpad skin of the hind leg of the animal and the swelling of the footpad was determined 24 hours after inoculation. The DTH response was calculated by subtracting the swelling before the inoculation -2 from that after the inoculation (10 mm).

The results obtained are shown in Tables R-V.

TABLE R

-2 _ Average footpad swelling (10 mm)

Inoculation Normal mice ME134 Immune mice 20 1 2.8 13.6 2 12.4 32.0 3 3.6 3.6 4 3.2 22.0 5 6.8 27.0 25 Remarks: g

Group 1: syngeneic normal spleen 1 x 10/20 ul medium (Hanks solution).

/ g

Group 2: MH134 cell 1 x 10/20 µl medium. Group 3: medium 20 µl.

Group 4: GRA-M-4, 0.8 µg (protein) / 20 µl medium.

Group 5: „0.4 yUg„ !! 35 8202638 - 37 -

TABLE S

_2 _ Average footpad swelling (10 mm) _

Eating Normal mice X5563 immune mice MH134 immune mice I 5.4 4.3 1.1 5 2 0.9 - 24.3 3 2.0 16.9

Comments:

Group 1: medium (Hanks solution) 20 yUl.

Group 2: GRA-M-4, 4 yUg (protein) 20 yul 10 medium.

Group 3: GRA-M-5, „„ „

II

TABLE T

15 -2

Average footpad swelling (10 mm)

Inoculation Normal mice GRA-M-4, immune mice 1 1.7 -0.3 2 5.1 24.6

Comments: 20

Group 1: medium (Hanks solution) 20 yul.

Group 2: GRA-M-4, 4 yUg (protein) / 20 yul medium.

TABLE U

25. .-2

Average footpad swelling (10 mm)

Inoculation Normal mice GRA-M-4, immune mice 1 -1.8 0.6 2 6.3 20.0 3 6.8 6.3 30

Comments:

Group 1: medium (Hanks solution) 20 yul.

Group 2: GRA-M-4, 4 yUg (protein) / 20 yul medium.

Group 3: 4 µg (protein / 20 µl medium fraction, 35 '8202638 - - 38 - passed through PNA column at the time of GRA-M-4 (hereinafter "C.P.").

TABLE V

-2 5 Average footpad swelling (10 mm) _

Inoculation Normal mice GRA-M-4, immune mice 1 2.4 -1.0 2 2.6 17.7 3 2.4 1.8 10 4 5.5 18.9

Comments:

Group 1: medium (Hanks solution) 20 yul. Group 2: GRA-M-4, 3.8 yUg (protein) / 20 yul medium.

Group 3: 3.8 yUg (protein) / 20 yul medium "C.P." upstairs.

Group 4: 3.8 yUg (protein) GRA-M-4 and 3.8 yUg (protein) / 20 yul medium "C.P.".

Reference Test ♦ X5563 Immune mice were obtained in the same manner as in Example V. Spleen cells from these immune mice were used as effector cells and the effector cells (10 ^) and target cells (X5563, 10) were transplanted together on mice of the same strain. The test of

Winn was performed in the same manner as in Test Example X.

The results obtained are shown in Figure 23, in which the X axis indicates the days and the Y axis the mean tumor size (cm2) ± S.E. and the different characters mean the following: # - ->: Group where no effector cells were added.

0-0: Group, in which untreated effector cells were added.

8202638 ο - 39 - Δ-Δ: Group, in which effector cells treated with rabbit complement were added.

x-x: Group, in which effector cells treated with anti-Thy 1 (New England Nuclear Co., U.S.A.) and rabbit complement were added.

0- □: Group, in which effector cells treated with anti-Lyt (New England Nuclear Co., II.S.A.) and rabbit complement were added.

-1: Group, in which effector cells treated with anti-Lyt 2 (New England Nuclear Co., U.S.A.) and rabbit complement were added.

From the results shown in Figure 23, it can be seen that Lyt 1 type T cells play an important role in the mechanism of in vivo effector in tumor immunity. Furthermore, it is known that DTH response is mediated by Lyt-1 T cells (J. Exp. Med. 143, biz. 1534-39 (1976)).

20 8202638

Claims (35)

1. For cancer cells cytotoxic lymphocyte, which is specific for a cancer cell derived glyco-related antigen. Lymphocyte according to claim 1, 5, characterized in that it is obtained by sensitizing the lymphocyte with the glycoplastic antigen derived from cancer cells. Lymphocyte according to claim 1 or 2, characterized in that the glycoprotective antigen is a cancer cell membrane component which combines with lectin, which specifically combines with a terminal galactose or N-acetyl-galactosamine. Lymphocyte according to claim 3, characterized in that the glycopant antigen is a cancer cell membrane component which combines with lectin, which specifically combines with a terminal galactose. Lymphocyte according to claim 3, characterized in that the glycoprotective antigen is a cancer cell membrane component, which combines with lectin, which specifically combines with N-acetylgalactosamine terminal. 6. A method for preparing lymphocytes cytotoxic to cancer cells, characterized in that lymphocytes are sensitized with a glycopant antigen derived from cancer cells. The method according to claim 6, characterized in that the glycopant antigen is a cancer cell membrane component, which combines with lectin, which specifically combines with a terminal galactose or N-acetylgalactosamine. The method according to claim 6, characterized in that the glycopant antigen is a cancer cell membrane component, which combines with lectin, which specifically combines with a terminal galactose. 9. A method according to claim 6, characterized in that the glycopant antigen is a cancer cell membrane component, which combines with lectin, which specifically combines with an N-acetylgalactosamine terminal. Anti-cancer agent, characterized in that it contains as active ingredient for cancer cells cytotoxic lymphocytes specific for a cancer cell-derived glycopants antigen. Anti-cancer agent according to claim 10, characterized in that the lymphocytes cytotoxic to cancer cells are prepared by sensitizing the lymphocytes with a glycopants antigen derived from cancer cells. Anti-cancer agent according to claim 10 or 15, characterized in that the glycopant antigen is a cancer cell membrane component, which combines with lectin, which specifically combines with a terminal galactose or N-acetylgalactosamine. Anti-cancer agent according to claim 12, 20, characterized in that the glycopant antigen is a cancer cell membrane component, which combines with lectin, which specifically combines with a terminal galactose. Anti-cancer agent according to claim 12, characterized in that the glycopant antigen is a cancer cell membrane component, which combines with lectin, which specifically combines with an N-acetylgalactosamine terminal. Anti-cancer agent, characterized in that it contains a glycopant antigen from cancer cells as an active ingredient. Anti-cancer agent according to claim 15, characterized in that the glycopant antigen is a cancer cell membrane component which combines with lectin, which specifically combines with a terminal galactose or N-acetylgalactosamine. Anti-cancer agent according to claim 16, 8202638 t τ - 42 - characterized in that the glycopant antigen is a cancer-cell membrane component which combines with lectin, which specifically combines with a terminal galactose. The anti-cancer agent according to claim 16, 5, characterized in that the glycopant antigen is a carcin cell membrane component which combines with lectin, which specifically combines with an N-acetylgalactosamine terminal. 19. A method of treating cancer, characterized in that cytotoxic lymphocytes, which are specific for a cancer cell-derived glycoplane antigen or a cancer-derived glycoplane antigen, are administered to a cancer patient for cancer cells. 20. Glycup glove antigen, characterized in that it is derived from human cancer cell membrane component 15 and can combine with a lectin, which can combine with a terminal galactose or a terminal N-acetylgalactosamine. 21. The glycopant antigen according to claim 20, characterized in that the glycopant antigen is prepared by recovering a glycopant antigen from human cancer cell membrane component with a lectin which can specifically combine with a terminal galactose or a terminal N-acetylgalactosamine. Glycup mitt antigen according to claim 21, characterized in that the lectin is a lectin which specifically combines with a terminal galactose. Glycan mitt antigen according to claim 22, characterized in that the lectin is pindalectin, Ricinus communis lectin or soy lectin. Glycup glove antigen according to claim 23, characterized in that the lectin is pindalectin. Glycopant antigen according to claim 21, characterized in that the lectin is a lectin which specifically combines with an N-acetylgalactosamine terminal. Glycup mite antigen according to claim 8202638 c - 43 - 25, characterized in that the lectin is selected from the group consisting of Dolichos bean agglutinin, orange lectin, Helix pomatia lectin, lima bean lectin, soy lectin and Bauhinia bean lectin, Glycup glove antigen according to claim 26, characterized in that the lectin Dolichos bean is agglutinin. 28. Glycup antigen, characterized in that it is prepared by recovering a glycopant antigen from human cancer cell membrane component, first with a first lectin, which specifically combines with a terminal galactose and then with a second lectin, which specifically combines with a N-acetylgalactosamine terminal. Glycup glove antigen according to claim 15 28, characterized in that the first lectin is pindalectin and the second lectin Dolichos bean agglutinin. 30. A method of preparing a glycopant antigen, characterized in that a glycopant antigen is obtained, which can be combined with a lectin, which can specifically combine with a terminal galactose or a terminal N-acetylgalactosamine. 31. A method according to claim 30, characterized in that a glycopant antigen from a human cancer cell membrane component is recovered with a lectin, which specifically combines with a terminal galactose or a terminal N-acetylgalactosamine. The method according to claim 31, characterized in that the lectin specifically combines with a terminal galactose. The method according to claim 31, characterized in that the lectin specifically combines with a N-acetylgalactosamine terminal. 34. A method of preparing a glycoplaid antigen, characterized in that a glyco-35 related antigen is recovered from human cancer cell membrane components, first with a first lectin, which specifically combines with a galactose terminal and then with a second lecetin, which specifically combines with a N-acetyl-galactosamine terminal. A method according to claim 32, characterized in that the first lectin is pindalectin and the second lectin Dolichos bean agglutinin. 10 8202638 Insert in the description pertaining to patent application No. 8202638 Ned. proposed by applicant dated September 30, 1982. Insert between lines 9 and 10 of page 9 of the description submitted on June 30, 1982: "On September 27, 1982, a sample of the GRA-1-KT cell line was deposited at ATCC 12301 Parklawn Drive Rockville, Maryland USA and received ATCC No. CRL 8175. " 8202638