FI77157B - FOERFARANDE FOER FRAMSTAELLNING AV GLYKOBUNDEN ANTIGEN OCH FOERFARANDE FOER FRAMSTAELLNING AV FOER KANCERCELLER TOXISKA LYMFOCYTER, SOM AER SPECIFIKA MOT DENNA Antigen. - Google Patents

Description

1 77157

The present invention relates to a process for the preparation of a glycosidone antigen isolated from a component of the human or animal cancer cell membrane ascosylate, which binds to a galacto-terminal galactose component and binds to a lectin

10 The antigen is hereinafter referred to as "GRA". The invention further relates to a method for producing lymphocytes toxic to cancer cells, specific for a glycosidone antigen isolated from a human or animal cancer cell membrane component and associated with a lectin that binds to galactose at the terminal cell or to the N-acetyl lactone and they act against cancer cells that contain a glycosidone-derived antigen from the cancer cells.

It is known that cells that elicit an immune response, especially T lymphocytes, which play a major role in the cell-mediated immune response, cause graft rejection due to foreign cell antigens, but inhibit cancer cells to a very limited or negligible extent. Thus, cancer cells are not destroyed, but divide in vivo, eventually causing cancer death.

However, the mechanism that causes the separation of own cells from foreign ones is not entirely clear and various studies have been performed to elucidate the nature of the material-base of such a system. For example, surface markers for mouse Leu chemistry cells or fetal cancer cells using lectins such as Dolichos biflorus 35 agglutinin (DBA) and peanut agglutinin (PNA), 2 77157 are described in Biochem. Biophys. Res. Conan. 89 (2) (1979) 448-455, ibid. 96 (4) (1980) 1547-1553, J. Biochem. 89 (1981) 473-481 and Cell 18 (September 1979) 183-191.

However, to date, no significant attempts have been made 5 to produce new lymphocytes capable of acting specifically against cancer cells and also as anti-cancer agents that take advantage of the lymphocyte immune response.

Extensive research into the host immune response to cancer cells and its application to cancer treatment has now shown that an antigen specific to cancer cells, which is not present in differentiated normal cells, contains a GRA that acts as an immunogen to the host and has a very potent immunogenic effect. effect caused by a specific immune response against cancer cells. In addition, it has been found that when GRA is used to sensitize lymphocytes, killer cells can be obtained that act specifically against GRA-containing cancer cells, destroying them, and thus have an excellent effect in the treatment and prevention of cancer.

The method of the present invention for the preparation of GRA is characterized in that the cell membrane components of human or animal cancer cells are obtained by homogenization or dissolution, the cell membrane components are treated with lectin which specifically binds to terminal galactose or terminal N-acetyl-lactose-lactose lactose. cell membrane substance sub-complex and isolating the lectin-bound glycos-bound antigen and the glycos-bound antigen therefrom.

The method of the present invention for producing killer-30 cells is characterized in that human or animal lymphocytes are sensitized by growing them for 1-10 days in a conventional medium further containing 1-1000 ng / ml of a glycosidic antigen prepared according to the invention, calculated as 1 ^ per lymphocyte per milliliter.

3 77157

Brief explanation of the figures

Figure 1 is a photomicrograph of Daudi cancer cells;

Figure 2 is a photomicrograph showing GRA-1-K-T-induced spot formation on the cancer cells of Figure 1; Figure 3 is a photomicrograph of KATO-III cancer cells;

Figure 4 is a photomicrograph showing GRA-1-K-T-induced spot formation on the cancer cells of Figure 3;

Figure 5 is a photomicrograph of BT-1 cancer cells;

Fig. 6 is a photomicrograph showing the formation of spots on the cancer cells of Fig. 5 caused by GRA-1-K-T 10;

Figure 7 is a photomicrograph of MKN-45 cancer cells;

Figure 8 is a photomicrograph showing GRA-1-K-T-induced spot formation on cancer cells of Figure 7;

Figure 9 is a photomicrograph of MOLT cancer cells; Fig. 10 is a photomicrograph showing GRA-1-K-T-induced spot formation on the cancer cells of Fig. 9;

Figure 11 is a photomicrograph of BT-1 treated with a mixture of non-sensitized human peripheral blood lymphocytes; Figures 12 and 13 are both photomicrographs showing a cancer cell of a cancerous mouse given GRA-M-1: K;

Figure 14 is a photomicrograph showing the cancer status in a group of cancer-treated mice given GRA-M-1;

Figure 15 is a photomicrograph showing the cancer status in a group of cancer-naïve mice not given GRA-M-1;

Figure 16 is a photomicrograph showing a cancer cell of a group of 30 murine mice not administered GRA-M-1.

Figure 17 is a photomicrograph of a cancer cell from a group given GRA-M-1;

Figure 18 shows 35 grams of GRA SDS gel electrophoresiside using protein staining according to the C.B.B method; 4 77157

Figures 19-21 show an SDS gel electrophoresis diagram of GRA stained according to the PAS method with sugar staining;

Fig. 22 is a schematic diagram showing the results of an SDS electrophoresis diagram of 5 GRAs using protein staining according to the C.B.B method;

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

10 The GRA used in the production of lymphocytes can be obtained from human or animal cancer cells containing the GRA, for example, cultured cancer cells, transplanted cancer cells, spontaneously occurring cancer cells, chemical or virus-induced cancer cells, and cut tissue derived cells.

The cell membrane components are separated from the cancer cells described above and treated with lectin specifically associated with terminal galactose 20 or N-acetylgalactosamine, whereby GRA is associated with lectin and is easily distinguished.

Suitable examples of galactose-binding lectins are peanut lectin, Ricinus communis lectin and soybean (Glycine max) lectin / les.

25 J.B.C. 250 (1975) 8518-8523; Biochem. Biophys. Res. Comm. 62 (1975) 144; Z. Immuniteatsforch 138 (1969) 423-433;

Br.J.Exp. Pathol. 27 (1946) 228-236; Proc. Nath. Acad.

Sci. USA 75 (5) (1978) 2215-2219; Biochemistry 13 (1974) 196-204; and Carbohydrate Research 51 (1976) 107-11S7.

Suitable examples of lectins that bind to N-acetylgalactose amines are Dolichos bean (Dolichos biflorus) agglutinin, braid-orange lectin. Helix pomatia lectin, mucus bean (Phaseolus limensis) lectin, soybean (Glycine max) lectin and Bauhinia bean 35 (Bauhinia purpurea) lectin.

5,771 57

Separation of the components of the cancer cell membrane can be accomplished by known methods, such as homogenization and dissolution with the aid of a solubilizing agent. It is more preferred to use a method in which cancer cells are homogenized in physiological saline or a suitable buffer, the precipitated portion is collected, for example, by separation by centrifugation and dissolved in physiological saline or buffer with the aid of a solubilizing agent, and the supernatant is separated by centrifugation. Solvents that can be used include surfactants that are generally known to be capable of dissolving the cell membrane, such as nonionic surfactants, for example, TRITON X-100 (manufactured by Wako Pure Chemical Industries Ltd., NP-40). Shell Co., Ltd.), digitonin and urea and anionic surfactants, for example sodium dodecyl sulfonate (SDS).

The GRA capable of binding to lectin can be separated from the cell membrane components thus obtained by conventional physical, chemical or biochemical methods, -; utilizing the properties of lectin. Examples of such methods include affinity chromatography utilizing a lectin-containing support on a column, immunoprecipitation using 25 GRA antibodies or the like, dialysis, gel filtration, electrophoresis, physical precipitation using sugars and proteins as precipitant, e.g. acetone and combinations thereof.

More preferred is affinity chromatography in which 1 /. 30 uses a lectin-containing support in a column which is easy to prepare by binding the lectin to the soluble. to the ground. Such attachment of lectin can be done by known methods conventionally used for binding biological agents. Of these methods, it is preferable to use the cyanogen bromide polysaccharide method and the attachment method using N-hydroxysuccinimide esters. The cyanogen bromide activation polysaccharide method is a method in which an insoluble support is treated with cyanogen bromide and the activated product thus obtained is subjected to a coupling reaction with lectin under mild conditions to fix the lectin. When treating an insoluble medium with cyanogen bromide, for example, basic compounds such as sodium hydroxide and sodium bicarbonate are used to adjust the pH to 7.5-12 and the medium is treated in a solvent such as water, acetonitrile or a buffer adjusted to pH 7.5-12. In 1 M sodium bicarbonate buffer (pH about 8.7) and 0.01 M phosphate buffer (pH about 7.7) at room temperature for about 1-12 minutes. The amount of cyanogen bromide is usually preferably equal to the amount of insoluble support.

Any known insoluble support having a low non-specific adsorption capacity for all biological agents, a high degree of porosity and containing a functional group capable of binding biological agents under mild conditions and sufficiently chemically and physically stable can be used in the invention. Useful insoluble media include media made from cellulose, for example aminoethylcellulose, carboxymethylcellulose, bromoacetylcellulose and p-aniline cellulose, cross-linked dextran from Farm, for example, from Sephadex and CM-sephadex. media made from agarose, for example Sepharose 2B, Sepharose 4B and Sepharose 6B (manufactured by Farmacia Corp.).

In the coupling reaction between the cyanogen bromide-activated medium obtained in the above-mentioned manner and the lectin, the cyanogen bromide-activated medium is used in an amount of 30 to 80 times that of lectin and reacted in a suitable solvent such as 0.1 M aqueous sodium bicarbonate solution (containing , 5 mol / l sodium chloride: pH 8.4) at a temperature of 0-40 ° C, preferably 2-8 ° C, for about 10-20 hours. In this way, a lectin-containing support for affinity chromatography can be prepared.

In chromatography using a lectin-containing support prepared as described above, the desired GRA binds to the lectin in the support and remains on the column. Substances capable of binding to, for example, lectin may then be passed through the column to effect an exchange reaction, or alternatively a desorbent (eluting solution), for example concentrated brine, aqueous potassium thiocyanate or nitrate buffer, may be passed through the column to give the desired GRA. and recovered.

In the exchange reaction, examples of substances which V "20 are capable of binding to lectin when a carrier containing galactose-binding lectin is used in affinity chromatography are substances which may bind to a galactose-bound lectin at the terminal position, for example galactose and di- and oligosaccharides. . . galaktoosiryhmän; Examples of substances that can bind to lectin when a carrier containing N-acetyl-galactosamine-binding lectin is used in affinity chromatography are substances that are able to: bind to a terminal N-acetyl-galactosamine-bound lectin, e.g. N-acetyl di- and oligosaccharides containing the terminal N-acetylgalactosamine.

The GRA thus obtained contains a glycoprotein which contains 8 77157 galactose and / or N-acetyl ligactose amine as well as a glycolipid and / or saccharide.

The GRA thus prepared can, if desired, be freeze-dried and further purified using conventional separation methods. For example, GRA preparations isolated by galactose-binding lectin can be separated by a method using N-acetylgalactosamine-binding lectin and those isolated by N-galactose amine-binding lectin can be treated by a separation method using galactose-binding lectin.

The lymphocytes used in this context are not critical and any lymphocytes obtained from cancerous humans or animals can be used. Examples are lymphocytes from the peripheral bloodstream, bone marrow, lymph nodes, spleen, tonsils, and thymus. These lymphocytes are isolated by physical or chemical methods, surface membrane method or the like.

Sensitization of lymphocytes to GRA is performed by growing lymphocytes in a culture medium containing GRA for 1-10 days, preferably 2-7 days.

A variety of common nutrient media conventionally used in such cell culture can be used as the culture medium for lymphocyte sensitization. Preferred examples are RPMI-1640 medium, Eagle MEM medium, etc. supplemented with human serum, fetal bovine serum (FCS), calf serum, horse serum or the like. The amount of GRA to be added to the culture is usually 1 to 1000 ng / ml and preferably 1 to 500 ng / ml, calculated as the amount of sugar and the number of lymphocytes is 1 x 10 6 / ml.

The culture is carried out by a conventional method, for example at pH 7.2 and a temperature of about 37 ° C.

The killer cells prepared according to the invention are approximately 77,7157 normal lymphocytes and have a GRA-specific anti-cellular activity. For example, GRA-1-KT, one of the killer cells of the invention, has properties common to human peripheral blood T cells and has an anti-cellular activity specific for GRA-containing cancer cells, as shown in in the example described below.

Typical examples of novel killer cells selected according to the invention are GRA-1-KT and GRA-M-1, which are prepared in the examples described below. All killer cells prepared according to the invention are available from the patent applicant.

The killer cells 15 prepared according to the invention can be amplified indefinitely on the medium described above containing T cell growth factor (TCGF, IL-2). In this case, selective growth of the killer cell clone can be performed by a conventional ultra-dilution method. These killer cells can be stored for long periods of time, for example in liquid nitrogen.

GRA can be used alone as the active ingredient and in addition in combination with other antibacterial and anti-cancer agents. Anti-cancer agents containing GRA as an active ingredient may be in any form, as long as they are in such preparations that there is an effective amount of V- and GRA. The substance is usually administered intravenously, subcutaneously, intradermally or intramuscularly as a solution, suspension or emulsion. In addition, it may be in the form of a dry product which can be made into a liquid-30 by the addition of a suitable carrier before use. These liquid substances may contain a suspending agent, for example methylcellulose, an emulsifier, for example lecithin, preservatives, for example methyl p-hydroxybenzoate, a stabilizer which as such does not have adverse effects on the human and animal immune buffer, .

Useful aqueous carriers include physiological saline and useful anhydrous carriers include vegetable oils, for example sesame oil, mineral oils, for example paraffin oil, animal oils, for example squalene and propylene glycol. In addition, suitable excipients may be added to provide immunological enhancement. Excipients that can be used include e.g. Freund's complete adjuvants, saponin for animals and aluminum hydroxide for humans.

The anti-cancer agent can be administered once or repeatedly over a long period of time to a cancer patient to treat the disease or to prevent cancer in a person at risk for cancer.

Since the LD50 value of the GRA (mouse, intraperitoneal) is at least 500 mg / kg in terms of sugar, the toxicity of the anticancer agent is low and the dosage can therefore vary widely. Although the GRA concentration in the anti-cancer agent is not critical, it is usually preferable to use a concentration of 0.001 to 100 pg / ml calculated as the amount of sugar. With respect to the dosage of the anti-cancer agent, it is usually preferable to administer the agent from 0.001 to 100 pg / kg / day, in a single dose or in several doses, although the dosage will vary depending on the extent of the disease and the age and sex of the patient.

An anti-cancer agent prepared by containing killer cells as an active ingredient is preferably used as a solution for injection together with carriers for the manufacture of such drugs. The carriers used in this connection are not critical, but isotonic carriers are preferred. Physiological saline is particularly preferred. In preparing the agent, it is preferable to wash the killer cells 35 sufficiently with physiological saline or the like to remove the culture medium described above and then slurry them in the carrier.

The concentration of killer cells in the substance is not strictly limited, but is preferably in the range of 10 to 10 milliliters. When the killer cells are administered intraperitoneally at a dose of 10 / mouse, no toxic effect is observed. Although the dosage of the anti-cancer agent according to the invention varies depending on the degree of the disease and the age and sex of the patient, it is usually preferable to administer the agent at a dose of 10-10 / kg / day in one or more doses.

The following examples illustrate the invention in more detail.

Reference Example 1 Location of GRA (1) -A Preparation of FITC-labeled lectin (PNA-FITC) 10 mg of peanut lectin (PNA, manufactured by EC Co.) was dissolved in 0.01 M phosphate buffer (2 ml) containing 0.85% NaCl (pH = 7.2). 0.5 mg of FITC (manufactured by Sigma Laboratories Inc.) was dissolved in 0.5 M bicarbonate buffer (1 ml) (pH = 9.0), and 0.5 ml of the resulting solution was added to the PNA buffer prepared above. The mixture was stirred at room temperature for two ;; 25 hours and then separated on a Sephadex G25 column · '*' (10 x 300 mm, gel manufactured by Farmacia Corp.). The initial peak was collected. FITC / PNA ratio = 1.0.

(1) -B Preparation of FITC-labeled lectin (DBA-FITC) DBA-FITC was prepared as described in (1) -A using DBA manufactured by EC Corp. FITC / DBA ratio = 0.9.

(1) -C Soybean agglutinin FITC (FITC-SBA) is sold by EY Co. FITC / SBA ratio = 1.4.

12 771 57 (2) Location of GRA in different cancer cells g (a) Human cultured cancer cells (1 x 10) were washed three times with 0.05 M Tris-hydrochloric acid buffer containing 0.85% NaCl (pH = 7.2) by centrifugation method and then 100 μl of PNA-FITC, DBA-FITC or SBA-FITC (200 μg / ml) prepared according to (1) were added. 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 plated and examined by fluorescence microscopy.

Mice X5563 and MH134 were treated and examined in the same manner as above.

15 The results are shown in Table 1. The cancer cells used are all known and were obtained from First Pathology Laboratories, Medical Department of Niigata University.

table 1

Cancer cells GRA-positive 20 (%)

PNA DBA SBA

Raji (Burkitt's lymphoma) 98.3 1.4

Dauji - "- 93.1 5.2 BT-1 -" - 50.1 0 25 P-12 (T-cell lymphoma) 44.3 6.7 MOLT (T-cell leukemia) 0.6 4, 8

Fujimaki (B-cell lymphoid tumor) 19.1 5.3

Oda (IgD bone marrow tumor) 0.6 10.0 QG-56 (lung cancer, squamous) 70.4 2.0 30 PC-1 - "- 78.4 0.4 PC-3 (lung cancer, glandular cancer) 77.1 0 QG-90 (lung cancer, small cell) 68.0 0 PC-13 (lung cancer, large cell) 17.0 0 MK-2 (stomach cancer, poor differentiation) 63.7 0.1 KATO-III (stomach cancer) , Signet ring cancer) 57.3 0 13 771 57

Table 1 (continued)

Cancer cells GRA-positive (%)

PNA DBA SBA

5 MKN-45 (abdominal cancer, poor differentiation) 1,040.3 MKN-1 (abdominal cancer, squamous cell carcinoma) 4.6 0.4 MKN-28 (abdominal cancer) 0.4 0.1 10 MKN-74 "0.5 0 MGH-Ul (bladder cancer) 37.4 0 KU-2 "4.5 21.4 T-24" 14.6 0 NBT-2 "13.1 1.0 15 NRC-12 (kidney cancer) 23.9 0 KU-1 "3.3 0.6

Kuramochi (ovarian cancer) 80.0 0 NB-1 (early nerve cell tumor) 50.9 1.7 *: YT-nu "3.6 0.5" 20 TGW-nu-l "4.10 TGW-nu-ll" 2 .0 1.0 GOTO "0.5 0 ITO (fetal cancer) 96.9 12.3 NEC-8 -" - 44.6 0 25 SCH (choroidal cancer, abdomen) 14.6 3.1 GCH (choroidal cancer, uterus ) 5.4 0 YN-1 (striated muscle tumor) 5.7 1.7

Mouse X5563 (plasma cell tumor plasmacytoma) 92.0 0 90.6: 30 Mouse MH134 (liver tumor, hepatoma) 18.6 0 6.4 .... (b) Malignant tissues from cancer patients were passed through a stainless steel screen (# 150) to provide a single cell suspension. After washing the cells twice with 0.01 M Tris-HCl buffer (pH = 7.4) in 5 mM CaCl 2 containing 0.85% NaCl, 5 x 10 cells were suspended in buffer (100 μl). ). 100 μl of FOTC-PNA or FITC-DBA (200 μg / ml) was added to the cell suspension and 14,771 5 7 mixtures were incubated at room temperature for 20 minutes. After washing the cells three times with cold PBS, they were examined under a fluorescence microscope.

The results are shown in Table 2. Malignant-5 grade tissues from cancer patients were obtained from Kansai Medical University. Table 2

No. Cancer patient tissue GRA

PNA DBA

1 Stomach cancer + + 10 2 + + 3 - 4 "+ 5" + + 6 + 15 7 Breast cancer + 8 "+ - 9" + 10 + + 11 Colon cancer + + 20 12 "+ 13 Esophageal cancer + 14 Liver tumor - +

Note: A “+” indicates that GRA is present on the cell surface.

25 The mark indicates that GRA is not present on the cell surface.

Reference Example 2 Preparation of GRA (1) -A Preparation of Attached Lectin (PNA-Sepharose) 30 Three g of CNBr-activated Sepharose 4B (manufactured by

Farmacia Corp.) was washed well with 1 mM HCl and suspended in 0.1 M sodium bicarbonate (pH 8.5, 200 mL). Then, 5 ml of 0.01 M phosphate buffer (pH = 8.5) containing 20 mg of PNA was added and allowed to react at 25 ° C for two hours with stirring to prepare PNA-Sepharose.

(1) -B In the same manner as in (1) -A, but using DBA instead of PNA, DBA-Sepharose was prepared.

15 771 57 (2) Preparation of GRA (a) BT-1 (Burkitt's lymphoma) cells (1.3 xg 10) were washed three times with physiological saline and 30 ml of 0.01 M Tris-HCl buffer (pH = 7.4), containing 2% TRITON X-100 (manufactured by Wako Pure Chemical Industries Ltd.), 0.85% NaCl and 2 mM CaCl 2 and 2 mM MgCl 2. The mixture was stirred at 4 ° C for 15 minutes and then subjected to ultracentrifugation (100,000 x g). A 10 ml aliquot of the supernatant thus obtained (28 ml) was passed through an affinity chromatography column (diameter 0.5 cm length 1 cm) packed with PNA-agarose beads (manufactured by Maruzen Co., Ltd.) equilibrated with tris -HCl buffer (pH 7.4) containing 0.1% TRITON X-100, 0.85% NaCl 2 mM CaCl 2 and 2 mM MgCl 2 · After washing the beads with the same buffer as used above, 0 .01 M in Tris-HCl buffer (pH = 7.4) containing 0.85% NaCl and 0.1 M lactose, 2 mM CaCl2 and 2 mM MgCl2 and containing 0.1% TRITON X-100. The portion thus eluted was dialyzed against 0.01 M Tris-HCl buffer containing 0.85% NaCl and 011 2 mM MgCl 2 and 2 mM CaCl 2 for 48 hours to give 17 ml of GRA solution. The amounts of protein and sugar in the GRA solution were measured by the Folin-Lowry method and the phenolic sulfuric acid method, respectively, and were found to be 644 μg and 120 pg, respectively. From this solution-V. hereinafter referred to as "GRA-1".

(b) C3H / He mouse breast cancer (MMT) cells (1x10 4) were washed three times with physiological saline and 30 ml of 0.01 M Tris-HCl buffer (pH 7.4) containing 2% TRITON was added. X-100, 0.85% NaCl and was 2 mM for CACl2 and 2 mM MgCl2. The mixture was stirred at 4 ° C for 30 minutes. The mixture was then separated by ultracentrifugation (100,000 xg) for two hours and the supernatant was dialyzed overnight in 0.1 M Tris-HCl buffer containing 0.85% NaCl and 2 mM CaCl 2 and 2 mM MgCl 2. regarding. The dialyzed solution was thus concentrated to 3 ml and a 1 ml portion of 16,771,5 was passed through an affinity chromatography column (0.5 cm diameter, 2 cm length) packed with PNA-Sepharose (same as above) equilibrated with tris. -HCl buffer (pH = 7.4) containing 0.005% TRITON X-100, 0.85% NaCl and 2 mM CaCl 2 and 2 mM MgCl 2.

After washing the column thoroughly with the above buffer, it was eluted with 0.01 M Tris-HCl buffer (pH 7.4) containing 0.85% NaCl and 0.005% TRITON X-100 and was 0.1 M lactose, 2 mM CaCl 2 and 2 mM MgCl 2 and the 10 eluted parts thus obtained were dialyzed for 48 hours in 0.01 M Tris-HCl buffer (pH 7.4) containing 0.85% NaCl and 2 mM CaCl 2 and 2 mM MgCl 2 to give 2 ml of GRA solution.

In the GRA solution thus obtained, the amount of protein was 15,156 and the amount of sugar was 94. This solution is hereinafter referred to as "GRA-M-1".

(c) Approximately 120 g (wet weight) of KATO-III was homogenized in 100 ml of PBS with a Waring mixer. After centrifugation for one hour (100,000 g), the pellet was dissolved in 0.01 M Tris-20 HCl buffer (100 mL) containing 2% TRITON X-100 and 0.15 M NaCl. (pH = 7.6). The supernatant was separated by centrifugation (100,000 g) for one hour and applied to a column packed with PNA-Sepharose 4B (0.8 x 15 cm) equilibrated with 0.01 M Tris-HCl buffer. ) containing 0.015% TRITON X-100 and 0.15 M NaCl. After washing the column with 50 ml of buffer, the GRA was eluted with a buffer of 0.1 M lactose. The eluted GRA was dialyzed against 0.85% NaCl solution, concentrated with Sephadex (Pharmacia Co.) and stored at -20 ° C before use.

The amounts of protein and sugar were determined as in (a) and were found to be 2.0 mg and 0.8 mg, respectively. This solution is hereinafter referred to as "GRA-2".

35 (d) In the same manner as in (c), the following GRA samples were prepared.

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Table 3

GRA sample Material GRA

Source Quantity (g) Protein Sugar content content (mg) (mg) 5 GRA-3 BT-1 33 0.5 0.09 GRA-4 Breast cancer 5 0.24 0.5 (cut) GRA-5 QG- 56 24 0.6 0.38 GRA-6 QG-90 26 1.0 0.54 10 GRA-7 Raji 29 0.78 0.45 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: 15 (e) In the same manner as above (c) but using MKN-45 (about 29 g) instead of KATO-III and DBA-Sepharose prepared in (1) -B instead of PNA-Sepharose 4B, and eluting with N-acetylgalactosamine, a GRA preparation with a protein content of 0.03 mg and a sugar content of 0.01 mg was obtained. This sample is hereinafter referred to as "GRA-8".

(f) GRA-3 (5 ml) prepared above (5 ml) was applied to a DBA-Sepharose column and eluted with Tris-HCl buffer (0.015% TRITON X-100, 2 mM MgCl 2, CaCl 2, 0.85% NaCl). to give 4 ml of fractions 1-12. Fractions 1-3 are abbreviated "GRA-3-A" and fractions 4-12 are abbreviated "GRA-3-B". The column was then eluted with the same buffer but containing N-acetylgalactosamine (0.1 M) to give fractions denoted by the abbreviation "GRA-3-C".

(g) SDS gel electrophoresis

Each of the above GRA preparations was electrophoresed by the method described in Fairbanks et al., Biochemistry 10 (1971) 2606.

35 The results obtained are shown in Figures 18-22.

18 771 57

In Figures 18-19, the numbers 1-5 mean the following.

1 = standard; 2 = GRA-M-3; 3 = GRA-7; 4 = GRA-1; and 5 = GRA-2.

In Figure 20, the numbers 1-4 mean the following. 1 = 5 standards; 2 = GRA-M-2; 3 = GRA-6; 4 = GRA-5.

In Figure 21, the numbers 1-3 mean the following. 1 = GRA-M-4; 2 = GRA-M-5; 3 = standard.

In Fig. 22, the numbers 1-4 mean the following. 1 = GRA-3; 2 = GRA-3-A; 3 = GRA-3-B; 4 = GRA-3-C.

Figure 18 shows a GRA stained for protein staining by the C.B.B. method (Fairbanks et al., Biochemistry 10 (1971) 2606).

Figures 19-21 show a GRA subjected to sugar staining by the PAS method /R.M. Zacharius et al., Anal.

15 Biochem. 30 (1962) 148 /.

Figure 22 is a schematic diagram of the results of staining by the C.B.B. method.

The substances listed below, obtained from Biorad, were used as standards. Lab. Calif. USA.

20,200 K Dalton; myosin 116 "/ J-galactosidase 92.5" phosphorylase

66.2 "BSA

45 "ovalbumin 25 21.5" soybean trypsin inhibitor

Reference Example 3 Preparation of TCGF (a) The spleen (4 kg) of a Japanese monkey (obtained from Japan Plymates Co., Ltd.) was excised and washed twice with RPMI-1640-30 medium (manufactured by Flow Laboratory Co., Ltd.).

Cells were filtered through a Mesh screen (manufactured by Milipore Inc., 150 mesh) and subjected to specific gravity centrifugation (specific gravity 1.076) to obtain 2 L of lymphocytes (2 x g 10 / ml). The lymphocytes thus obtained were washed three times with RPMI-35 1640 medium and the number of lymphocytes was adjusted to 7 x 10 10 per milliliter using the same medium as above containing 10% FCS. It was then allowed to stand in a 19,771 57 carbon dioxide fermenter at 37 ° C for one hour. The supernatant lymphocytes were collected and the number of lymphocytes was adjusted to 1 x 10 per milliliter using the same medium as above containing 1% FCS.

Then, 1 μg / ml indomethacin (manufactured by Sigma Laboratories, Inc.) and 0.2% PHA-P (manufactured by Difco Co.) were added, and the cells were cultured in a carbon dioxide fermenter at 37 ° C for 48 hours. Centrifuged (3,000 rpm) for 10 minutes, the supernatant was collected and sterilized by filtration through a 10 Millipore filter (manufactured by Millipore Inc., 0.2) to give 2 liters of TCGF.

(b) The source of TCGF was mixed cultured peripheral circulating lymphocytes from 10 healthy donors.

15 Unadsorbed lymphocytes prepared from C.F. / Conray. Ficol (Japan Immunoresearch Co.,) from J-separated lymphocytes by adsorption on a plastic surface at 37 ° C for one hour was suspended in RPMI medium containing * * g containing 1% FCS (1.5 x 10 cells / ml) and incubated for 20 hours. PHA-P (0.2%), indomethacin (1 μg / ml) and human B cell preparation / 5 (BT-1) pretreated with mitomycin C (50 μg / ml) (1.5 x 10 cells / ml) in the presence of. The culture supernatant was collected after 48 hours and used as a source of TCGF (H.Inoue et al., Scand. J. Immunol. 12 (1980) 25 149-154 /.

Reference Example 4 Preparation of Lymphocytes (1) Human Peripheral Circulatory Lymphocytes Blood samples (50 ml) obtained from healthy 30 adults or several cancer patients by heparinization were separated by centrifugation using a Ficol Pack (manufactured by Farmacia Japan Co., Ltd.). x 10 peripheral circulating lymphocytes.

(2) Mouse spleen lymphocytes The spleen of 35 C3H / He mice (male, six weeks) was dissected and washed twice with RPMI-1640 medium.

The spleen was then softened with a spray needle and pressed through a 20-771 57 stainless steel screen (100 mesh) to remove large pieces. The cells thus filtered were washed twice with the same culture medium and separated by centrifugation (1,200 rpm) for 10 minutes to obtain 4 x 10 6 splenic lymphocytes.

Example 1

The GRA-1 prepared in Reference Example 2 ((2) to (a)) (protein amount 40 μg / ml; sugar amount 7.5 μg / ml) was diluted 1000-fold relative to the initial volume in RPMI-1640 medium containing 15% in FCS, to prepare a sensitization medium.

Human peripheral circulating lymphocytes (5 x 10 * V 5 ml) obtained according to Reference Example 4 ((D)) were added to a sensitization medium (5 ml) prepared as described above and placed in a laboratory dish and grown at 37 ° C. 2 days, they were further grown in RPMI-1640 medium containing 20% TCGF and 15% FCS prepared according to Reference Example 3 for another five days to give 20 ml of a killer cell solution containing 1 x 10 4 killer cells per ml. This solution is hereinafter referred to as "GRA-1-KT".

Example 2

G 25 spleen lymphocytes prepared according to Reference Example 4 ((2)) were diluted to a cell number of 5 x 10 / ml using RPMI-1640 medium containing 15% FCS. GRA-M-1 prepared according to Reference Example 2 ((2) to (b)) was then added so that the final amounts of protein and sugar were 1.5 μg / ml and 0.9 μg / ml, respectively. A 5 ml aliquot of the resulting mixture was grown in a laboratory dish (60 x 15 mm, manufactured by Falcon Co.) at 37 ° C for two days. Cloning was observed. The batch was further grown in RPMI-1640 medium containing 15% FCS containing 20% v / v TCGF (manufactured by 35 Japan Immuno Research Laboratories Co., Ltd.) for four days to give 50 ml of a killer cell solution containing 1 x 21 77157 10 ^ killer cells per milliliter. This solution is hereinafter abbreviated as "GRA-M, L-K-T".

Example 3 Peripheral circulating lymphocytes (5 x 10 6) obtained from a healthy donor were incubated in RPMI-1640 medium containing 50 ng / ml (protein content) GRA-2 and 15% FCS at 37 ° C. On the second day, the human TCGF described above was added to the medium to a concentration of 20% and the incubation was continued for another three to 10 days to obtain killer cells. This product is hereinafter referred to as "GRA-2-K-T".

Example 4

The killer cell preparations GRA-8-KT, GRA-3-AKT and GRA-CKT were prepared according to Example 1 using 15 GRA-8, GRA-3-A and GRA-3-C, respectively (protein concentration of 50 ng / ml) and TCGF prepared according to Reference Example 3- (b).

Example 5 C3H / He mice (females, eight weeks) were implanted intradermally with X5563 cells (10) obtained from the same strain and after seven days the tumors were removed by excision. Seven days later, X5563 cells (10 ^) of the same strain were inoculated, and mice resistant to inoculation were designated immune nihouse.

Spleen cells from immune mice and normal C3H / He mice were prepared conventionally.

Each batch of spleen cells (5 x 10 6 / dose) was sensitized with 30 GRA-M-5 (protein concentration 40 mg / ml) in RPMI-1640 medium containing 15% FCS for five days to obtain killer cells.

The killer cell preparation obtained from normal spleen cells is hereinafter referred to as "GRA-M-5-K-T-1" and from immune spleen cells as "GRA-M-5-K-T-2".

35 Example 6

Human peripheral circulating lymphocytes (5 x 10 6) were incubated in RPMI-1640 medium (5 ml) containing 22,771,550 ng / ml (protein content) of GRA-1 at 37 ° C for two days. On the third day, lymphocytes were seeded in RPMI-1640 medium containing lymphocyte donor serum (10%) and 0-100 ng / ml (protein concentration) GRA-1, and incubation was continued for another five days to obtain the killer cell preparations of Table 4.

Table 4 GRA concentration (ng / ml)

Start date Day 3 Killer cell 10 50 0 GRA-lKT-1 50 1.6 GRA-lKT-2 50 3.2 GRA-lKT-3 50 6 GRA-lKT-4 50 12.5 GRA-lKT-5 15 50 25 GRA -lKT-6 50 50 GRA-lKT-7 50 100 GRA-1-KT-8

Example 7 (a) Peripheral circulating lymphocytes (PBL) obtained from several cancer patients after surgery were sensitized with GRA-3 to prepare killer cells. Patient-derived PBL (5 x 10 **) was incubated in RPMI-1640 medium containing 50 ng / ml (protein content) GRA-3 for two days and then for another five days in RPMI-1640 medium containing contained 20% TCGF and 15% FCS to obtain the killer cells of Table 5.

Table 5 Peripheral circulating lymphocytes Cancer P-GRA D-GRA Days of surgery- Killer cell from 30 seasons

Gastric cancer + + 14 GRA-3-K-T-1 "+ + 35 GRA-3-K-T-2

Breast cancer + - 21 GRA-3-K-T-3 "+ 7 GRA-3-K-T-4 35 + - 35 GRA-3-K-T-5

Gastric cancer + 21 GRA-3-K-T-6 23 7 7 1 57

In the table above, P-GRA and D-GRA indicate the location of GRA in a malignant cell (tumors removed by dissection) obtained from patients collected from PBL according to Reference Example 1, 2- (b). P-GRA 5 and D-GRA are results obtained using FITC-PNA and FlTC-DBA, respectively. Postoperative days indicate the time at which PBL was collected after surgery.

(b) PBL (5 x 10 6) collected from breast cancer patients 21 days after surgery was incubated in RPMI-1640 medium containing 50 ng / ml (protein concentration) GRA-3 and patient serum ( 10%), seven days to obtain a killer cell preparation. This is hereinafter referred to as "GRA-3-K-T-7".

Example 8

O

GRA-1-K-T (10) :: prepared according to Example 1 was diluted with physiological saline (10 ml) to obtain an injectable solution.

Example 9 (i) GRA-M-1 prepared according to Reference Example 2 ((2) to (b)) was diluted with physiological saline to give sugar and protein concentrations of 1.0 μg / ml and 1.6 ug / ml, respectively, to give the anticancer agent No. 1.

(ii) 5 mm cubic pieces of spontaneously occurring rin-25 flatulence tumor in C3H / He mice were sterile cut and transferred to the dorsal fin of less than ten C3H / He mice of the same strain as the above seven-week-old males ). Seven days after implantation, tumor attachment and division were confirmed. Five mice were each given subcutaneous anti-cancer agent No. 1 prepared according to (i) at 300 μl / day at two-day intervals. Another set of five mice was used as an untreated control group. Ten days after the first 35 administrations, tumors were removed by excision and their average weight was measured. At the same time, a pathohistological study was performed.

24 7 7 1 57

Tumor volume: 3 Drug group 22.3 mm (Figure 14) 3

Control group 162.7 mm (Figure 15) This means that there was an 86.3% reduction in tumor size.

Pathohistological examination:

In the control group (Fig. 16), nested cancerous tumors developed, the tissue type was similar to nuclear canal cancer, and tumor cell distribution was observed throughout the tissue. On the other hand, in the drug-treated group (Fig. 17), the cancer cells caused liquid necrosis at their sites of formation and hardening and connective tissue formation occurred, leaving only a very limited number of cancer cells. Thus, it was found that the anti-cancer agent prepared according to the invention has anti-tumor properties.

Experimental Example 1

GRA-1-K-T (1) prepared according to Example 1 was placed on a microplate (manufactured by Falcon Corp.) and allowed to stand at room temperature for 15 minutes. 4 FCS (manufactured by Falcon Corp.) was then added and the mixture was allowed to stand at room temperature for 30 minutes. Neuraminidase-treated sheep erythrocytes N 9 (SRBC) were added in an amount of 1x10 per milliliter and 5 μl of 0.01 M phosphate buffer (pH = 7.2) supplemented with 0.85% NaCl and separated on a plate. centrifugation at 600 rpm for five minutes. Then the record

OF

was inverted and unreacted SRBC was removed. Staining solution (Brilliant Cresyl Blue, manufactured by Merck & 30 Co.) was added to stain lymphocytes and a bow formation was examined. As a result, it was found that at least 98% of the cells are bow-forming positive (T cells).

Experimental Example 2

Specific Cancer Cell Killing Activity 35 (a) From the cells of Table 1, the following five cell strains with different GRA positivity were used as target human cancer cells.

25 77157

Target cancer cells: No. 1 BT-1 (Burkitt's lymphoma) No. 2 Daudi (Burkitt's lymphoma) No. 3 Kato-III (stomach cancer) No. 5 MKN-45 (stomach cancer) No. 5 MOLT (T-cell leukemia)

4 target cancer cells were laminated to a microplate (manufactured by Falcon Corp.) at 5 x 10 wells by centrifugation at 800 rpm for five minutes. 4 x 10 GRA-1-K-T of Example 1 per well was then carefully added and incubated for one hour.

Killing activity was determined by the degree of spot formation using the following scale: ++ Clearly detectable killing activity 15 + Observable killing activity ± Low detectable killing activity - No detectable killing activity Unsensitized human peripheral circulating lymphocytes were used as a control group, : a. The results are shown in Table 6.

Table 6 shows that the killer T cells prepared by the method of the invention have a potent GRA-specific cytotoxic effect.

25 Table 6

Target cancer cell GRA-positive- Spot formation ... visibility (%).-X GRA-1K-DAUDI (Fig. 1) 93.1 ++ (Fig. 2) T-group KATO-III (Fig. 3) 57.3 + (Fig. 4) 30 BT-1 (Fig. 5) 50.1 ++ (Fig. 6) MKN-45 (Fig. 7) 1.0 ± (Fig. 8) MOLT (Fig. 9) ) 0.6 - (Fig. 10)

Comparative BT-1 50.1 - (Fig. 11) group 35 (b) The same target cancer cells as used in (a) were (3.2-10) mixed with GRA-1-KT (8 x 10 ^) (cell ratio: 5/1) and the resulting cell mixture (total number: 4 x 10 ^) 26 7 7 1 57 were grown in RPMI-1640 medium containing 15% FCS. After one hour, the number of remaining cells and the percentage of cytotoxicity were calculated by the following formula.

number of cells c. . . . after rearing v lnn.

5 cytotoxicity (%) = (1 - number of strands x 100) before growth (4 x 106)

The results are shown in Table 7.

Table 7 10 Number of cells

Target cell Before Culture Cytotoxicity (%) after culture

Daudi 4 x 10® 2.0x10® 28 KATO-III 4 x 106 3.7 x 106 7.5 15 BT-1 4 x 106 3.2 x 106 20 MKN-45 4 x 106 3.9 x 106 2, 5 MOLT 4 x 10® 4.2 x 10® -5 (c) The procedure was as in (b), but the mixing ratio of GRA-1KT / target cancer cells was changed to 5/3. The results 20 are in Table 8.

Table 8

Number of cells

Target cell Before Culture Cytotoxicity (%) after culture 25 Daudi 4 x 10® 3.6 x 105 91 KATO-III 4 x 10® 3.6 x 10® 24 BT-1 4 x 10® 1.4 x 10® 65 MKN -45 4 x 10® 3.7 x 10® 7.2 MOLT 4 x 10® 4.1 x 10® -3

30 In the above experiment, it is found that GRA-1-K-T

binds very actively to Daudi, KATO-IIIre and BT-1re, but only weakly to MKN-45re and MOLTre. Experimental Example 3

35 C3H / He mice with spontaneous breast cancer were subcutaneously administered GRA-M-1-K-T of Example 2 at a dose of 3 x 10® / 0.3 ml / mouse three times a week every other day. After ten days, the colony was detached and examined.

As shown in Figure 12, there was an absorption of lymphocytes into the cancer cells and a decrease in the surface area of the tumor 5 was observed. It can be seen from Figure 13 that hardening occurred in the tumor area and thus it can be seen that the killer cells according to the invention have an antitumor effect.

Comparative Example 1 In this example, cancer cells per se were used as specific antigens instead of GRA in the method of the invention.

Cancer cell-sensitized lymphocytes were obtained according to Example 1, except that 15 BT-1, Daudi, KAT0-II or MKN-45 were used instead of GRA in amounts of 1 x 10 6; Laboratory chalice.

: ·; The cytotoxic activity of these lymphocytes was examined according to Experimental Example 2 ((a)). The results are shown in Table 9.

20 It can be seen from Table 9 that lymphocytes prepared as mentioned above do not have any cytotoxic effect.

Table 9

Cells used for lymphocyte sensitization

Target cell BT-1 Daudi KATO-III MKN-45. BT-1 -

Daudi - KATO-III - 30 MKN-45 -

Experimental Example 4

According to Experimental Example 2- (b), the cancer cell killing effect of GRA-8-K-T, GRA-3-A-K-T and GRA-3-C-K-T prepared according to Example 4 was determined. The results 35 are in Table 10.

28 771 57

Table 10

Cytotoxicity (%)

Target cell GRA-8-K-T GRA-3-A-K-T GRA-3-C-K-T

KATO-III 8.0 5.0 5.0 5 BT-1 4.3 5.0 4.0 MKN-45 20 5.0 20 MOLT 000 (a) The cytotoxicity of each of the killer preparations of your killer 10 obtained according to Example 6 was determined by Cr- release test / J. Immunol. 122 (1979) 2527-2533 /. S.O. 50 μCi of radioactive Cr (Japan Isotope Association) was added to KATO-III (2 x 10 4) and the cells were incubated at 37 ° C for one hour in RPMI-1640 medium and then washed

e -I

By centrifugation to obtain Cr-labeled target g cells. Killer cells (effector cells) (2 x 10) were added to c target cells (1 x 10) (thus effector cell / target cell ratio = 20/1) and the mixture was incubated at 37 ° C for four hours in RPMI-1640 medium. The supernatant was collected by centrifugation and its radioactivity was determined with a liquid scintillation counter.

The specific β-Cr release (%) corresponding to the cytotoxic activity of the effector cells was calculated by the following equation.

51 25 Specific Cr release (%) (release in experiment) - (spontaneous release) χ (maximum release) - (spontaneous release) (Maximum release is radioactivity when all 30 cells are degraded.)

The results are shown in Table 11.

51

Table 11 77157 29

Killer cell Specific Cr release (%) GRA-lKT-1 13 5 GRA-lKT-2 25 GRA-lKT-3 22 GRA-lKT-4 20 GRA-lKT-5 22 GRA-lKT-6 25 10 GRA-lKT -7 27 GRA-1KT-8 32

From the results given in Table 11, it can be seen that TCGF and FCS have no effect on the induction of killer cells.

(B) The cytotoxicity of GRA-2-K-T obtained according to Example 3 was determined by a Cr release assay in the same manner as in (a) above. A specific Cr release of 14.3% • · 51 was observed using Cr-labeled KATO-III as target cells (or cell / target cell ratio = 20/1).

Experimental Example 6 (a) Using KATO-III (effector cell / target cell ratio = 20/1) as the target cell, the cytotoxicity of the killer cells obtained according to Example 7- (a) was determined in the same manner as in Experimental Example 2- (b).

25 The results are shown in Table 12.

Table 12

Killer cells Cytotoxicity (%) GRA-3-KT-1 38.0 GRA-3-KT-2 18.2 30 GRA-3-KT-3 20.9 GRA-3-KT-4 23.2 GRA-3- KT-5 24.5 GRA-3-KT-6 20.2 (b) The cytotoxicity of GRA-3-KT-7 obtained according to Example 7- (b) was determined using ^ Cr-KATO-III (effect and cell / target cell ratio = 20/1) as the target cell in the same manner as in Experimental Example 5. The specific Cr release of the killer cell was 25.5%.

77157 30

Experimental Example 7

The cytotoxicity of the killer cells obtained according to Example 5 was determined by a ^1 Cr release assay in the same manner as in Experimental Example 5. A Xr63-labeled X5563 cell was used as a target cell. Instead of GRA-M-5, lymphocytes obtained in the same manner as in Example 5 but using sensitization of spleen cells with mitomycin C-treated X5563 cells (1 x 10 6 / well) / 5 x 10 6 / L were used as a control. ml of X5563 cells were treated with mitomycin at 10 ° C (50 μg / ml) for 60 minutes.

The results obtained are shown in Table 13.

Table 13 51

Specific Cr release (%)

Killer cell E / T ratio 15 40: 1 20: 1 10: 1 GRA-M-5-KT-1 12.7 6.3 5.7 GRA-M-5-KT-2 18.4 20.6 13, 7

Comparison 0.0 0.0 0.0

Experimental Example 8 (H-2 Study) 20 GRA-M-1, GRA-M-3 and GRA-M-4 obtained according to Reference Example 2 were serially diluted in PBS (0.85% NaCl) to prepare samples.

The anti-H-2 serum (National Institute of Genetic Reserach) and the above sample were mixed and incubated at 4 ° C for two hours and the target cell corresponding to the anti-H-2 serum used was added. A spleen cell or a lymph node cell obtained from BIO (H-2 °) and] r was used as a target cell.

Bl0 * Br (h-2) mice by a conventional method. After washing the cells with PBS, supplement (ka-30in) was added to the cells and incubated at 37 ° C for one hour and stained with 0.2% trypan blue-PBS to determine the percentage of cytotoxicity in the absence of GRA.

The GRA-induced inhibitory effect was determined for the systems in Table 14.

31 77157

Table 14 GRA Anti-H-2 serum, concentration Target cells GRA-M1 D-23 (anti-H-2Kk), X80 B10BR (H-2k) spleen or lut 5 D-23 (anti-H-2Dk) , X300 GRA-M-3 D-33 (anti-H-2Kb) f X600 BIO (H-2b) spleen cells or D-2 (anti-H-2Db), X80 GRA-M-4 D-23 X80 B10 BR (h-2k) lymph node 10 or mu cells D-32 X300

The results are shown in Table 15.

Table 15

Cytotoxicity (%) 15 GRA Anti-H-2- Sample dilution serum X2 X4 X8 X16 X32 X64 X128 X256 X512 O x 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 li - - 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 D-32 99 99 99 99 - - - - - 99 10: .V 25 Note: GRA I: GRA-Ml GRA II: GRA-M-3 GRA III: GRA-M-4 "x": cytotoxicity (%) with complement alone 30 From the results shown in Table 15, it can be seen that GRA-M1, GRA-M-3 and GRA -M-4 lacks H-2. Experimental Example 9 C57BL / 6 mice were subcutaneously inoculated with LLC (2 x 10 6) from the same strain, and after six days, GRA-M-3 according to Reference Example 2- (d) was administered subcutaneously. ng (protein). A similar dose was then given once daily for four days. The day after the last 32,771 5 7 administrations, the tumor cells were detached and weighed. Mice given physiological saline were used as a control group. There were five animals in each experimental group.

5 As a result, the mean tumor size in the control group was found to be about 500 mg. In the group given GRA-M-3, three animals had lost their tumors and two had an average tumor weight of about 100 mg.

Experimental Example 10 10 C57BL / 6 mice were immunized by subcutaneous administration of 1 ng (protein) of GRA-M-3 obtained according to Reference Example 2- (d) once a day for three days, and on the fifth day, spleen cells were collected from the animals to obtain influenza cells. A mixture of 15 effector cells and Lewis lung cancer cells (LLC) as target cells was prepared at a 50: 1 effector / target cell ratio, and a portion of 5 (1 x 10) was transferred to a mouse of the same strain and subjected to a Winn test. Immunol. 86 (1961) 228-2397 ·

The results obtained are shown in Table 16.

20 Table 16

Effect- Tumor growth rate (%) Deaths / cells Day Day Day Day Day group 20.

15 17 18 19 20 A 5.7 5.5 5.7 3.1 1.4 0/10 25 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

Note: Group A is GRA-M-3 immune mouse spleen cells.

Group B is normal mouse spleen cells.

Group C are spleen cells obtained from mice transfected with 30 (10 x 10) mice 10 days after transplantation.

Tumor growth rate was calculated by the following formula.

TA - TN

Tumor growth rate (%) = - ^ - x 100

35 N

where TA is the thickness of the footpad as measured from the side when the tumor has been transferred and TN is the thickness of the normal footprint.

33 771 5 7

Experimental Example 11 C3H / He mice were immunized by administering 4.5 g (protein) of GRA-M-4 (Reference Example 2- (d)) and 0.1 ml of Freund's Complete Adjuvant to the sacrum region. After two vii-5 weeks, lymph node cells were harvested conventionally and used as response cells to determine the rapid response elicited by GRA-M-4.

For this purpose, response cells (4 x 10 4) were incubated in RPMI-1640 medium containing 15% FCS in the presence of 10 GRA-M-4 for five days. During the last 3 hours of the incubation period, 1 Ci of H-thymidine (H-TdR) was added to the medium and its uptake into cells was calculated.

The results are shown in Table 17.

Table 17 • 15 GRA-M-4 Concentration 3H-TdR Absorption S.I.

(ng / ml) (mean cpm + S.E.)

Comparison 0 5,302 ί 1,761 0,5 7,903 + 1,290 1,5: 1 10,076 ± 936 1,9 20 Experiment 5 10,686 ± 429 2,0 20 10,615 ± 1,270 2,0 40 8,565 ± 1,419 1,6

Note: "S.I." is the stimulation factor test / comparison. Experimental Example 12 Delayed hypersensitivity (D) of hypersensitivity (C3H / He normal mouse, X5563 immune mouse and MH134 immune mouse obtained according to Example 5 and GRA-M-4 immune mouse and GRA-M-5 immune mouse obtained according to Experimental Example 11) response was determined by a footprint test (FPR). That is, GRA-30 or MMC-treated tumor cells were placed in the skin of the hind paw foot and the foot swelling was determined after 24 hours of treatment. The degree of DTH response was calculated by subtracting the pre-treatment swelling from the post-treatment (10 mm).

35 The results obtained are shown in Tables 18-22.

Table 18 34 771 57 -2

Trajectory average thickening (10 mm)

Group Normal mouse MH134 immune mouse 1 2.8 13.6 5 2 12.4 32.0 3 3.6 3.6 4 3.2 22.0 5 6.8 27.6

Note: Group 1; normal spleen cells 1 x 10 ^ / 20 ^ ul ul-10 aa (Hank's solution)

Group 2; MH134 cells in 1 x 10 4/20 μl medium Group 3; 20 ul of medium

Group 4; GRA-M-4, 0.8 g (protein) / 20 μl of medium Group 5; GRA-M-4, 0.4 g (protein) / 20 μl medium 15 Table 19 -2

Trajectory average thickening (10 mm)

Group Normal mouse X5563 immune mouse MH132 immune mouse 1 5.4 4.3 1.1 2 0.9 to 24.3 20 3 2.0 16.9

Note: Group 1; 20 μl of medium (Get the solution)

Group 2; GRA-M-4, 4 μg (protein) / 20 μl medium

Group 3; GRA-M-5, 4 μg (protein) / 20 μl medium

Table 20 _2 25 Path footprint av. thickening (10 mm)

Group Normal mouse GRA-M-4, Immune mouse 1 1.7 -0.3 2 5.1 24.6

Note: Group 1; 20 μl of medium (Hank's solution) 30 Group 2; GRA-M-4, 4 μg (protein) / 20 μl medium

Table 21 -2

Trajectory average thickening (10 mm) Group Normal mouse GRA-M-4, Immune mouse 1 -1.8 0.6 35 2 6.3 20.0 3 6.8 6.3 35 771 57

Note: Group 1; 20 jil medium (Hank's solution)

Group 2; GRA-M-4, 4 μg (protein) / 20 μl medium Group 3; fraction passing through the PNA column simultaneously with GRA-M-4 (hereinafter 5 "C.P."), 4 μg (protein) / 20 μl medium

Table 22 -2

Trajectory average thickening (10 mm) Group Normal mouse GRA-M-4, Immune mouse 1 2.4 -1.0 10 2 2.6 17.7 3 2.4 1.8 4 5.5 18.9

Note: Group 1; 20 jil medium (Hank * solution)

Group 2; GRA-M-4, 3.8 μg (protein) / 20 μl medium 15 Group 3; said "C.P.", 3.8 μg (protein) / 20 μl of medium

Group 4; 3.8 μg (protein) of GRA-M-4 and 3.8 μg (protein) of "C.P." / 20 μl of medium Reference Experiment 20 X5563 mice were obtained in the same manner as in Example 5. Spleen cells from this immune mouse were used as effector cells, and the effector cells (10) and target cells (X5563, 10 ^) were co-transferred to a mouse of the same strain. A Winn experiment was performed according to Experimental Example 10.

25 The results are shown in Figure 23, where the abscissa shows the days and the coordinate of the tumor mean. size (cm) ί S.E. and wherein the entries mean the following: • - ·: A group to which no influencer cells were added.

o-o: Group to which untreated effector cells were added.

Δ-Δ: Group to which rabbit complement-treated effector cells were added.

x-x: Group to which anti-Thy 1 was added (New England

Nuclear Co., USA) and rabbit complement-treated effector cells.

O-O: Group to which anti-Lyt 1 was added (New England

Nuclear Co., USA) and rabbit complement-treated effector cells.

36 7 7 1 57 -: Group to which anti-Lyt 2 was added (New England

Nuclear Co., USA) and rabbit complement-treated effector cells.

The results shown in Figure 23 show that Lyt-1-5 type T cells play an important role in the mechanism of action of influencer cells in vivo upon tumor immunization.

In addition, it is known that the DTH response is mediated through Lyt-1 cells by /J.Exp. Med. 143 (1976) 1534-39 /.

Claims (7)

A process for producing glycobonded antigen which is separated from a human or animal cancer cell membrane component and which associates with lectin which binds to terminal galactose or terminal N-acetyl galactosamine, characterized in that the cell membrane components of human or animal cancer cells are obtained. by homogenizing or dissolving, the cell membrane components are treated with lectin which binds specifically to terminal galactose or terminal N-acetylgalactosamine to form a lectin cell membrane component complex and the lectin-bound glycobonded antigen is separated and hence the glucobond. : 15 2. A method according to claim 1, characterized in that the cancer cell membrane component is a human cancer cell membrane component. Method according to Claim 1, characterized in that the lectin is the peanut lectin. 20 4. A process according to claim 1, characterized in that the lectin is Dolichos bean glutin. 5. A method according to claim 1, characterized in that the glycobonded antigen is separated from a human cancer cell membrane component first with a first lectin, which specifically binds to a terminal galactose and then to a second lectin, which specifically binds to a terminal N. acetylgalactosamine. 6. A process according to claim 5, characterized in that the first lectin is peanut lectin and the second lectin is Dolichos bean agglutinin. A process for the preparation of lymphocyte-toxic lymphocytes specific to a glycobonded antigen which has been secreted from a human or animal cancer cell membrane component and associates with lectin, which