HU190803B - Lymphocytes against carcinoma cells, process for producing them, and citostatic active agents containing the said lymphocytes - Google Patents

Abstract

The present invention relates to a method for the production of anti-cancer lymphocytes. Sensitization of lymphocytes is carried out with an antigen containing carbohydrate from cancer cells, which can bind to carbohydrate antigen lectin on the end galactose group of the carbohydrate and the N-acetyl galactose amine of the end of the chain. The invention further relates to a method for producing a carbohydrate-containing antigen which is isolated from a human cancer cell membrane -1

Description

The present invention relates to a process for the production of anticancer cell lymphocytes (hereinafter "killer cells" = "killer cells"). More particularly, it relates to a process for the production of killer lymphocytes which contain carbohydrate-containing antigen (obtained from cancer cells and hereinafter referred to as "GRA") and act on and kill cancer cells. The present invention also relates to the preparation of an anticancer agent comprising a killer cell or GRA as the active ingredient.

The effector cells of the immune response, especially the T-lymphocytes, play an important role in the cell-mediated immune response and cause the rejection of the transplanted tissue by the presence of foreign cell antigens. In contrast to cancerous cells, this activity of lymphocytes is not observed or very small, so that the cancer cells do not die, but survive and, when multiplied in the living organism, eventually cause the death of the cancerous host.

However, the mechanism responsible for the self-recognition of an organization (that is, what an organization recognizes as its own and what is a stranger) is not yet fully understood and various research is ongoing to gain an insight into the material nature of these systems. For example, for cell surface markers, murine leukemia cells or embryonic carcinoma cells use lectins such as Dolichos biflorus agglutinin (DBA) and American hazelnut agglutinin (PNA), see Biochem. Biophys. Gap. Comm. In [89 (2) 448-455 (1979)], ibid. (96 (4), 1547-1553 (1980)), J. Biochem. (59, 473-481 (1981)) and Cell.

To the best of our knowledge, there are no attempts to produce new lymphocytes capable of specifically fighting cancer cells and no anticancer agent that can be used to elicit a lymphocyte immune response.

The method of the invention can be used to prepare killer cells by, for example, using GRA to sensitize lymphocytes.

The GRA used in the above manner can be obtained from human and animal GRA-containing cancer cells, for example, cancer cell cultures, transplanted cancer cells, spontaneous cancer cells, chemical or viral-induced cancer cells, and surgically removed cancerous tissue.

The cell membrane constituents are separated from the cancer cell as described below and treated with lectin which specifically binds to the terminal N-acetylgalactosamine where GRA is attached to lectin and can thus be readily isolated, see JBC [250, 8518-8523 ( 1975)], Biochem. Biophys. Gap. Comm., 1972, 62, 144; Z. Immunitatsforsch., 138, 423-433 (1969), Br. J. Exp. Pathol., 27, 228-236 (1946); Proc. Nat. Acad. Sci. [75, no. 2215-2219 (1978)]; Biochemistry (13, 196-204 (1974)) and Carbohydrate Research (1976, 57, 107-118).

Suitable examples of the coupling between lectin and N-acetylgalactosamine are Dolichos bean (Dolichos biflorus) agglutinin and soybean (Glycine hispida) lectin.

The components of the cancer cell membrane may be isolated by known methods such as homogenization or dissolution, i.e., a solubilization method using a solubilizing agent. More preferably, the method comprises homogenizing the cancer cells in physiological saline or in an appropriate buffer mixture. The precipitated part is collected by centrifugation, dissolved in physiological saline or in a buffer solution using a solubilizer, and the supernatant is separated by centrifugation. Suitable solubilizing agents are surfactants which are capable of solubilizing the cell membrane, such as nonionic surfactants, e.g. TRITON X-100 (manufactured by Wako Pure Chemical Industries Ltd), NP-40 (manufactured by Shell Co. Ltd.), digitonin, urea and anionic surfactants, e.g. sodium dodecyl sulfonate (SDS).

The lectin-binding GRA from the cell membrane components thus obtained can be isolated by conventional physico-chemical or biochemical techniques that utilize the properties of the lectin. Such methods include column chromatography using a lectin-containing packing, immunoprecipitation using GRA antibody or the like, dialysis, gel filtration, electrophoresis, and physical precipitation using sugar and protein precipitants, e.g. polyethylene glycol and • acetone, and combinations thereof.

More preferably, affinity chromatography on a column containing lectin is preferred. The lectin-containing carrier can be readily prepared by binding the lectin to an insoluble carrier. Binding of lectin on an insoluble carrier can be accomplished by known methods used to fix biological materials. Among these methods, the preferred method is the bromocyanic activation of the polysaccharides and the attachment using the N-hydroxysuccinimide ester. The bromocyanic activation of polysaccharides is a method in which an insoluble support is treated with bromocyan and the activated product obtained in the process is used in the coupling reaction with lectin. The pH of the insoluble carrier is treated with bromine

In order to provide a range of 7.5 to 12, basic compounds, e.g. sodium hydroxide, sodium bicarbonate, and the carrier may be treated with water, acetonitrile, or a buffered solution having a pH of 0.1 molar sodium bicarbonate buffer (pH about 8.7) and 0.01 moles of phosphate buffer (pH about 7.7) in the range of 7.5 to 12. The treatment is carried out at room temperature for 1 to 12 minutes.

Preferably, the amount of bromocyano is equal to the amount of insoluble carrier.

Any known insoluble carrier which is non-specifically adsorbed to biological materials, has high porosity functional groups, can be used in the process of the invention.

190 803 which bind biological materials under mild conditions and are chemically and physically stable.

The insoluble carrier may be made from cellulose such as aminomethyl cellulose, carboxymethyl cellulose, bromoacetyl cellulose, p-anilino cellulose, and may be a cross-linked dextran based carrier such as Sephadex and CM Sephadex (product of Farmacia Corp.). , can be an agarose based carrier such as Sepharose 4B, Sepharose 2B and Sepharose 6B (product of Farmacia Corp.).

In the coupling reaction of the bromocyano-activated carrier as described above with the lectin, the activated carrier is 30-80 times the amount of lectin; the compounds are reacted in a suitable solvent, for example, in a 0.1 mol / L aqueous solution of sodium bicarbonate (containing 0.5 mol / L sodium chloride, pH 8.4), the temperature may be from 0 to 40 ° C. . However, it is preferred to hold the solution at 2 to 8 ° C for 10 to 20 hours. In this way, a lectin-containing support for the chromatography is obtained.

During chromatography using a lectin-containing support prepared as described above, the desired GRA is attached to the lectin-containing support and remains in the column. Substances that are capable of binding to lectin may pass through the column via an exchange reaction, or alternatively, an adsorption separator (eluting solution) such as a high potassium thiocyanate aqueous solution and a nitrate buffer may be passed through the column for dissociation. to obtain the desired GRA.

In the exchange reaction, the material sample is capable of binding to lectin using a carrier containing galactose-binding lectin for affinity chromatography, including those suitable for binding to terminal galactose, such as galactose, disaccharides and oligosaccharides containing terminal galactose, a sample of a substance is also suitable for coupling with lectin, even when using a carrier containing N-acetyl-galactosamine-binding lectin for affinity chromatography, including those suitable for coupling with terminal N-acetyl-galactosamine. Examples are N-acetylgalactosamine, disaccharides containing terminal N-acetylgalactosamine and oligosaccharides containing terminal N-acetylgalactosamine.

The GRA produced in this manner contains glycoprotein, glycolipid and / or saccharide containing terminal galactose and / or N-acetylgalactose amine. The GRA thus prepared can be lyophilized and, if desired, even more purified, using conventional purification procedures. For example, GRA compositions isolated with galactose-binding lectin may be subjected to an isolation process using terminal N-acetylgalactosamine-linked lectin, while GRA compositions isolated with N-acetyl-galactosamine-linked lectin may be subjected to a galactose-binding lectin isolation process. We can manage.

The origin of lymphocytes is irrelevant and lymphocytes from intact, healthy or cancerous human or animal organs from peripheral blood, bone marrow, lymph node, spleen, tonsil and thymus can be used. Lymphocytes are isolated by physical or chemical methods, membrane surface method (method described in The Japanese Journal of Chemical Pathology, Vol. 25, 1976, p. 105) or other methods.

GRA-sensitization of lymphocytes is performed by culturing lymphocytes in GRA-containing medium for 1-10 days, preferably

2-7 days.

Any of the conventional media usually used for cell culture can be used to sensitize lymphocytes. Useful Mediums: RPMI 1640 Medium [JAMA-b. 799 (1967), p. 519], the Eagle MEM medium [Science, 130 (1959) p. 432 medium], all supplemented with human serum, fetal calf serum (FCS), calf serum, equine serum or similar sera. The amount of GRA to be added to the culture is 1-1000 mg / ml, preferably 1-500 mg / ml, 1 x 10 ⁶ / ml of lymphocyte, calculated as sugar.

The cultivation is carried out in a conventional manner, e.g. 7.2 at pH 37 ° C.

The killer cells of the present invention are essentially "normal" lymphocytes and have specific anti-GRA activity. For example, GRA-1-KT, one of the killer cells of the invention, has properties similar to human peripheral blood T cells and exhibits specific cell killing activity against GRA-containing cancer cells as described below.

Representative examples of novel killer cells of the invention are GRA-1-KT and GRA-M-1, the preparation of which is illustrated by the following examples. All killer cells of the invention are available and can be obtained from the inventor.

The killer cells of the present invention can be grown indefinitely in medium containing the T cell growth factor (TCGF-IL-2) described above. In this case, selective growth of the clones of the killer cells is accomplished by the ultrasound method. These killer cells can be stored for a long time, for example in liquid nitrogen.

GRA can be used as a single agent and in combination with other antibacterial and anticancer agents. The anti-cancer agents containing the GRA of the invention as active ingredient may be formulated in any form as long as they are present in an effective amount of the GRA. The active ingredient may be administered intravenously, subcutaneously, intradermally or intramuscularly in the form of a solution, suspension or emulsion. The active ingredient may also be formulated as a pre-dry product which may be reconstituted by addition of a suitable vehicle. The liquid in which the solution is made may contain a suspending agent such as methylcellulose, an emulsifying agent such as lecithin, preservatives such as methyl p-hydroxybenzoate, and a stabilizing agent that does not elicit any immune response in humans or animals, buffers. and the like.

An aqueous vehicle can be used as a physiological vehicle

-3190 803.

saline, and as a non-aqueous vehicle, vegetable oil such as sesame oil, mineral oil such as paraffin, animal oil such as squalene or propylene glycol. Suitable adjuvants may be added to enhance the immunological effect, which may be Freund's complete adjuvant, saponin for animals, aluminum hydroxide for humans.

For the treatment of cancer patients, the compositions of the present invention may be administered once or repeatedly, or may be administered prophylactically to individuals at risk of cancer.

The LD _S0 value of GRA (intraperitoneal in mice) is 500 mg / kg of sugar, so that the dosage of the anticancer agent of this invention is of low toxicity and can therefore be varied within wide limits. Although the concentration of GRA in the anticancer compositions of the present invention is not critical, it is preferred that the amount of sugar as a sugar is between 0.001 and 10 µg / ml. Preferably, the anti-cancer agent is administered in an amount of 0.001-1,000 pg / kg / day, administered in one or more small doses, however, depending upon the extent of the disease, the age and sex of the patient.

The anticancer agent prepared in this manner, containing the killer cells as the active ingredient, can be advantageously used in the form of an injection and combined with carriers commonly used in similar blood products. The types of carriers used are not critical, but isotonic carriers with blood are preferred. It is particularly advantageous to use physiological saline as the carrier. In the preparation of the active ingredient, it is advantageous to wash the killer cells with a sufficient amount of physiological saline or the like to remove the medium described above and to kill the killer cells in the vehicle.

The concentration of killer cells is not strictly determined, but it is preferable to use values ranging from 10 'to 10 ⁹ / ml. No toxic lesions were observed when the killer cells were administered intraperitoneally at 10x concentration to mice. Although the dosage of the anticancer composition of the present invention will depend on the severity of the disease, the age and sex of the patient, it is generally preferred to administer the composition in doses of 10 ^s and 10 ® / kg / day, in one or more administrations.

The present invention is further illustrated, without limiting its scope, by way of illustrative, experimental and comparative examples.

Information Example I Localization of GRA (1) - Preparation of FITC (Fluorescein Isothiocyanate) Labeled Lectin (PNA-FITC).

mg of hazelnut lectin (PNA, Califomia product of EY, Laboratories, Inc.) was dissolved in 2 ml of 0.01 M phosphate buffer (pH 7.2) containing 0.85% sodium chloride. 1 ml of 0.5 M hydrogen carbonate buffer (pH 9) was dissolved in 2 mg FITC (product of Sigma Laboratories Inc.) to give

0.5 ml of the solution is added to the above PNA buffer solution. After stirring for 2 hours at room temperature, the mixture was separated on Sephadex G 25 (10mm x 300mm, Farmacia Corp.). The initial peak is collected. FITC / PNA ratio 1.0.

(1) - Preparation of B-FITC-labeled lectin (DBAFITC)

Dolichos bean agglutinin. In a similar manner to DBA-FITC (1) -A, the DBA used is a product of E. Y. Inc. FITC / DBA ratio = 0.9.

(1) - C FITC-labeled soybean agglutinin (SBA-FITC) is available from E. Y. Inc.

FITC / SBA ratio = 1.4.

(2) Location of GRA in various cancer cells.

(a) Cultured human cancer cells (1 × 10 ⁶ )

Washed three times with 0.05 M Tris-hydrogen chloride buffer (pH 7.2) containing 0.85% sodium chloride, centrifuged and then -F1TC-PNA or FITC prepared as described in Example 1 above. 100 ml (200 pg / ml) of -DBA or FITC-SBA is added. The resulting mixture was allowed to stand at room temperature for 30 minutes. After completion of the reaction, the reaction mixture was washed three times with 0.01 M phosphate buffer (pH 7.2) containing 0.85% sodium chloride, and the cells were placed on a slide and examined under a fluorescent microscope.

The results are shown in Table 1. All cancer cells used are known and come from First Pathology Laboratories, Medical Department of Niigata University.

Table 7

cancer cells GRA occurrence rate (%) PNA DBA SBA

Raji Burkitt lymphoma 98.3 1.4 Dauji Burkitt lymphoma 93.1 5.2 BT-L 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 There IgD myeloma 0.6 10.0 QG-56 lung cancer, (squamous cell) 70.4 2.0 PCI lung cancer (squamous cell) 78.4 0.4 PC-3 lung cancer, (Adenocarcinoma) 77.1 0 QG-90 small cell lung cancer 68.0 0 PC-13 large cell lung cancer 17.0 0 MK-2 low differences in gastric cancer 63.7 0.1 KATO-H MKN-45 MKN-1 seal-ring stomachs are little differentiated gastric cancers stomach cancer (squamous cell adenocarcinoma) 57.3 1.0 4.6 0 40.3 0.4 MKN-28 gyomorrok 0.4 0.1 MKN-74 stomach cancer 0.5 0 MGH-U1 bladder cancer 37.4 0 KU-2 bladder cancer 4.5 21.4 T-24 bladder cancer 14.6 0 NBT-2 bladder cancer 13.1 1.0 NRC-12 kidney cancer 23.9 0 KU-1 kidney cancer 3.3 0.6 Kuramochi ovarian cancer 80.0 0

190 803

Continuation of Table 1

cancer cells GRA incidence rate (X) PNA DBA SBA NB-1 neuroblastoma 50.9 1.7 YT-nu neuroblastoma 3.6 0.5 TGW-Nu -1 neuroblastoma 4.1 0 TGW- nu-yl neuroblastoma 2.0 1.0 GOTO neuroblastoma 0.5 0 ITO embryonic carcinoma. 96.9 12.3 NEC-8 embryonic carcinoma 44.6 0 SCH choriocarcinoma, gastric 14.6 3.1 GCH kriokarcinoma, uterus 5.4 0 YN-1 rhabdomyosarcoma 5.7 1.7 Mouse plasmacytoma 92.0 0 90.6 Mouse liver tumors 18.6 0 6.4

Malignant tissue from cancer patients is pressed through a stainless steel mesh (150 mesh) to form a suspension in which the cells are individually placed.

After washing twice with 0.01 M Tris (hydrochloric acid) buffer (pH 7.4) containing 2 mM calcium chloride, 2 mM magnesium chloride and 0.85% sodium chloride, 5 x 10 ⁵ cells were washed. resuspend in 100 μΐ buffer. To the cell suspension was added 100 μC PNA-FITC or DBA-FITC (200 μg / ml), mixed and incubated at room temperature for 20 minutes. It is then washed three times with cold PBS (0.15 M phosphate buffer containing sodium chloride and pH 7.6) and the cells are examined under a fluorescent microscope. The result is shown in Table 2. Malignant tissue from cancer patients was provided by Kansai Medical University.

Table 2

number Cancerous tissue GRA PNA DBA 1 stomach cancer + + 2 stomach cancer + + 3 stomach cancer - - 4 stomach cancer + - 5 stomach cancer + + 6 stomach cancer + - 7 breast cancer + - 8 breast cancer + - 9 breast cancer + - 10 breast cancer + + 11 Colon Cancer + + 12 Colon Cancer - + 13 esophageal cancer + - 14 liver tumors - +

Comment:

+ label: GRA appeared on the cell surface,

- Labeling: GRA did not appear on the cell surface.

Information Example 2 for GRA Preparation (1) - Preparation of Bound Lectin (PNA-Sepharose) 3 g of bromocyano-activated Sepharose ^ 4B (product of Farmacia Corp.) were thoroughly washed with 1 mM hydrochloric acid and 200 ml. It is suspended in 0.1 M sodium bicarbonate, pH 8.5. Then, 5 ml of 0.01 M phosphate buffer (pH 8.5) containing 20 mg of PNA was added and reacted at 25 ° C for 2 hours with occasional stirring to prepare PNA-Sepharose.

(1) - B In a similar manner to (1) -A above, DBA can be used in place of PNA and DBA-Sepharose (Dolichos bean) is obtained.

(2) Preparation of GRA (a) BT-1 (Burkitt's lymphoma) cells (1.3 x 10 ⁸ ) are washed three times with physiological saline and 30 ml of 0.01 M Tris-hydrogen chloride buffer, pH 7.4 ) containing 2% TRITON-X-100 (manufactured by Wako Pure Chemical Industries Ltd.), 0.85% sodium chloride, 2 mM calcium chloride and 2 mM magnesium chloride. The mixture was stirred at 4 ° C for 15 minutes and separated by ultracentrifuge (100,000 xg). A 14 ml portion of the resulting 28 ml supernatant fluid was passed through a column (0.5 cm diameter, 1 cm length) by affinity chromatography; the column was packed with PNA agarose (product of Maruzen Co., Ltd.) containing 0.1% TRITON-X-100, 0.85% sodium chloride, 2 mM calcium chloride and 2 mM magnesium. chloride-containing Tris (hydrogen chloride) buffer (pH 7.4). After washing with the above buffer, eluted with 0.01 M Tris-Hydrochloride pH 7.4 buffer containing 0.1 M lactose, 0.85% sodium chloride, 2 mM containing calcium chloride, 2 mM magnesium chloride and 0.1% TRITON-X-100. The eluted portion was dialyzed against 0.01 M Tris (hydrochloric acid) buffer containing 0.85% sodium chloride, 2 mM magnesium chloride and 2 mM calcium chloride to yield 17 ml. GRA solution was obtained. The amount of protein and sugar in the GRA solution was determined by the Folin-Lowry and the phenolic sulfuric acid method and found to be 644 pg and 120 pg. This solution is hereinafter referred to as "GRA-1".

(b) C3H mouse mammary tumor (MMT) cells (1 x ^{10! O)} was washed three times with saline and 30 ml of 0.01 M Tris (HCl) PCA buffer (pH 7.4) was added containing 2% TRITON X-100 contains 0.85% sodium chloride, 2 mM calcium chloride and 2 mM magnesium chloride. The mixture was stirred at 4 ° C for 30 minutes. The supernatant was then separated by ultracentrifugation (100,000 x g) for 2 hours and the supernatant fluid was added overnight against 0.1 M Tris hydrochloride buffer (pH 7.4) containing 0.85% sodium chloride, 2 mM containing calcium chloride and 2 mM magnesium chloride - dialysis. The dialyzed liquid is concentrated to 3 ml and a 1 ml portion of this is passed through an affinity chromatography column (diameter 0.5 cm, length 2 cm) filled with PNA-Sepharose as described above, previously prepared with 0.005% TRI5.

190 803

TON-X-100, 0.85% sodium chloride, 2 mM calcium chloride and 2 mM magnesium chloride in 0.1 M Tris-hydrogen chloride buffer, pH 7.4 ) balance. After thoroughly washed with the same buffer, 0.1 mol / 1 in 0.1 M tris inhabit ⁵ maltose, 0.85% sodium chloride, 2 mmol / 1 calcium chloride, and 0.005% Triton X-100 (H chloride) buffer (pH =

7.4) was eluted, and the eluate was dialyzed for 48 hours with 0.85% sodium chloride, 2 mmol / 1 Kai triszt- 0.01 M calcium chloride and ¹⁰ 2 mmoles / 1 of magnesium chloride (H chloride) buffer to obtain 2 ml of GRA solution. The GRA solution thus obtained had a protein and a sugar content of 156 pg and 94 pg respectively. This is also referred to as ¹⁵ "GRA-MI" used herein.

(c) About 120 g (aqueous weight) of KATO-III is homogenized in 100 ml PBS (phosphate buffer containing sodium chloride, pH 7.6) in a blender. After centrifugation for one hour (100,000 xg), ²⁰ pellets were dissolved in 100 ml of 0.01 M Tris-hydrogen chloride buffer containing 2% TRITON-X-100at, 0.15 M NaCl pH =

7.6). The supernatant was collected for 1 hour (100,000 x g) centrifugation, loaded into a PNA-Sepharose 4B ősz- Lopra ²⁵ (0.8 x 15 mm) and the column was equilibrated with 0.015% Triton-X-100, 0, 0.01 M Tris (Hydrochloride) buffer, pH 7.6, containing 15 moles of sodium chloride. After washing with 50 ml of a buffer of the GRA is eluted with 0.1 mol / 1 inhabit the same buffer containing ³⁰ maltose. The eluted GRA is again dialyzed with 0.85% sodium chloride solution, concentrated on Sephadex (from Pharmacia Co.) and stored at -20 ° C until use.

The amount of protein and sugar is determined by the method of (a) ³⁵ , which is 2.0 mg and 0.8 mg, respectively. This is hereinafter referred to as GRA-2.

in (d) (c) can be obtained from samples GRA: ⁴⁰

Table 3

GRA sample The material GRA original quantity (g) fehétje- content (Mg) sugar- ¹ content (mg) GRA-3 BT-1 Breast cancer 33 0.5 0.09 GRA-4 i (excision) 5 0.24 0.5 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 GRA-M-2 MMT 200 11.3 28.4 GRA M-3 LIC 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

(e) The method of (c) may also be used to produce GRA by substituting ATO-45 (about 29 g) for PNA-Sepharose-4B instead of KATO-III according to (1) to B 60. The resulting DBA-Sepharose was used and eluted by the procedure of (c) with 0.01 M Tris-hydrogen chloride buffer containing N-acetylgalactosamine. The resulting GRA formulation contains 0.03 mg of protein and 0.01 mg of sugar. 65

Hereinafter referred to as "GRA-8".

(f) 5 ml of GRA-3 according to (d) was applied to a DBASepharose column and eluted with 0.01 M Tris-Hydrochloride buffer, pH 7.2 (0.015% TRITON-X-100, 2). 1 mM magnesium chloride, 2 mM calcium chloride, and 0.85% sodium chloride) to obtain 4 ml fractions for 1-12. Fractions 1-3 are referred to as GRA-3-A and 4-12 are referred to as GR-3-B. The column was then eluted with the same buffer containing even 0.1 mol of N-acetylgalactosamine to give the fraction GRA-3-C.

(g) SDS gel electrophoresis

All GRA formulations prepared by the above procedures are subjected to Fairbanks electrophoresis. [Fairbanks et al., Biochemistry 10 vol. 2606: 1971]. The results obtained are shown in Figures 17-21.

17-18. In Figures 1 to 5, numbers 1-5 show:

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

In Figure 19, numbers 1 through 4 show:

- standard, 2 - GRA-M-2, 3 - GRA-6, 4 - GRA-5.

In Figure 20, the numbers 1 through 3 show:

- GRA-M-4, 2 - GRA-M-5, 3 - Standard.

In Figure 21, the numbers from 1 to 4 show:

- GRA-3, 2 - GRA-3-A, 3 - GRA-3-B, 4 - GRA-3-C.

Figure 17 shows the GRA status of the protein staining reaction C. Β. B. technique. [Fairbanks et al., Biochemistry Vol. 2606: 1971].

18-20. Figure 4A shows the GRA status of the PAS staining reaction. Zacharius R.M., et al., Anal. Biochem. 30th 148, 1962].

Figure 21 is a diagram of C. Β. B. Schematic representation of the result of the above staining method.

Below is a list of standard materials used [Biorad Foot. Calif. product, United States]

200 K dalton, myosin

116 K dalton, β-galactosidase

92.5 K dalton, phosphorylase

66.2 K dalton, BSA

K dalton, ovalbum

21.5 K dalton, soybean trypsin inhibitor

3. Informative Example (a) Japanese Monkey Spleen (4 kg); (purchased from Japan Plymates Co. Ltd.) and washed twice with RPMI-1640 culture medium (product of Flow Laboratory Co.). The cells were filtered through a sieve (150 mesh from Millipore Inc.) and centrifuged at a density difference of 1.076 to absorb 2 liters of a suspension containing 2 x 19 '/ ml of lymphocyte. The lymphocytes so obtained are washed three times with RPMI-1640 culture medium and the lymphocytes are then washed to 5 x 10 ⁷ / ml.

190,803 with the above culture medium containing 10% FCS. It is then allowed to stand at 37 'C for one hour in a carbon dioxide fermenter.

Supernatant lymphocytes were recovered and adjusted to 1 x 10 * / ml with the above medium containing 1% FCS. Subsequently, 1 pg / ml of indomethacin (obtained from Sigma Laboratories, Inc.) and 2% PHA-P (purified fíthemagglutinin) (Difco Co.) was added and carbon dioxide fermentation apparatus 37 'at ¹⁰ C for 48 hours culturing. It is then separated by centrifugation (3000 rpm) for 10 minutes, the supernatant recovered, and germinated on a Millipore filter (Millipore Inc. 0.2 µm) to obtain 21TCGF (T cell growth factor, interleukin-2).

(b) TCGF is obtained from the supernatant of a mixed lymphocyte culture from peripheral blood of ten healthy donors. According CF (Conray Ficoll Immunoreseardh Japan Co.) separated limfóciták- suspended in 1% FCS-containing RPMI 1640 with ²⁰ made from non-adherent lymphocytes were 37 ° C for 1 hour suspending plastic surface (1.5 χ 10 "cells / ml) and incubated with 0.2% PHA-P, indomethacin (1 pg / ml) and 50 pg / ml) with human B cell line pretreated with mitomycin C. After forty-eight hours, the culture supernatant was harvested and used as a source of TCGF [Inone et al., Scand. J. Immunol. 12, 149-150. (1980). 30

Information Example 4 Preparation of Lymphocytes (1) Human peripheral blood lymphocytes are centrifuged in ml of heparinization blood obtained from healthy adult or various cancer patients using Ficol Pack (product of Farmacia Japan Co. Ltd.) to 40 5 x 10 '/ ml. obtaining peripheral blood lymphocytes.

(2) Mouse spleen lymphocytes

The spleen was excised from C3H / He mice (6 weeks old male) and washed twice with RPMI-1640 culture medium. The spleen is then worked with a needle and passed through a 100 mesh stainless steel sieve to remove larger pieces. The cells so screened were washed three times with the same medium as described above and separated by centrifugation at 1200 rpm for 10 min to obtain 4 x 10 ⁷ / ml alpha lymphocytes.

. 5 ml of medium and placed in a petri dish and cultured at 37 ° C for 2 days. They are cultured on 20% TCGF and 15% FCS-containing RPMI-1640 media obtained in Reference Example (3) for a further 5 days to obtain a 20 ml solution of killer cells at 1 x 10 ⁶ per ml. there is a killer cell. This is hereinafter referred to as "GRA-lKT".

Example 2

The number of mouse spleen lymphocytes obtained by the procedure of Informative Example 4 (2) is adjusted to 5 x 10 .mu.l with RPMI-1640 medium containing 15% FCS. GRA-Ml obtained by the procedure of informative Example 2 (b) is then added so that the final amounts of sugar and protein are 0.9 pg / ml and 1.5 pg / ml, respectively. 5 ml of the resulting mixture was grown on a Petri dish (60 mm x 15 mm, product of Falcon Co.) at 37 ° C for 2 days. Clone formation is monitored. Subsequently, it was further cultured in RPMI-1640 medium containing 15% FCS and 20% TCGF (product of Japan Immuno Research Lab. Co., Ltd.) for 4 days to obtain 50 ml of a solution containing 1 x 10 ⁶ / ml killer cells. This is hereinafter referred to as "GRA-MlKT".

Example 3

Peripheral blood lymphocytes from healthy donors (5 x 10 * / ml) were incubated at 37 ° C in 50 ng / ml (protein content) RPMI-1640 medium containing 15% FCS. On the second day, the above human TCGF is added to the solution until the concentration reaches 20%. The incubation was continued for another 3 days to obtain killer cells. This is referred to below as GRA-2-K-T.

Example 4

Killer cell preparations such as GRA-8-KT, GRA-3-AKT and GRA-3-CKT were prepared according to the procedure of Example 1, GRA-8, GRA-3-A, GRA-3-C ( protein content of 50 ng / ml) and TCGF obtained according to Informative Example 3 (b).

Example 7 55

The GRA obtained in Informative Example (2) (a) (40 pg / ml protein, 7.5 gg / ml sugar) was diluted 1 000 times the original volume with RPMI-1640 medium containing 15% FCS θθ to prepare the sensitizing medium.

Human peripheral blood lymphocytes (5 x 10 ® / 5 ml) of Informative Example 4 (1) are added to the sensitizer prepared as described above.

Example 5

10 ⁶ x 5563 cells from the same strain were implanted into the skin of a C3H / He mouse (female, 8 weeks old). The tumor is surgically removed after seven days. After another seven days, 10 'χ 5563 cells from the same strain were inoculated. The mouse that is resistant to the vaccine is called an immune mouse.

Spleen cells from both the immune mouse and the normal C3H / He mouse are processed by standard techniques. Spleen cells were sensitized with 40 ng / ml con-71

190,803 (protein content) with GRA-M-5 RPMI-1640 medium containing 15% FCS. Sensitization was performed for 5 days to obtain killer cells.

Killer cells derived from normal spleen are referred to below as GRA-M-5-K-T-1 and those derived from immune mouse spleen GRA-M-5-K-T-2.

Example 6

Human peripheral blood lymphocytes (5x10x) are incubated in RPMI-1640 medium containing 50 ng / ml (protein content) GRA-1 for two days at 37 ° C. On the third day, lymphocytes are transferred to RPMI-1640 medium containing 10% lymphocyte donor serum and 0-100 ng / ml (protein content) GRA-1. The incubation was continued for another 5 days to obtain killer cells.

The formulations are shown in Table 4.

Table 4

GRA concentration (nglml)

starting day 3 days Killer cell 50 0 GRA-L-K-T-1 50 1.6 GRA-L-K-T-2 50 3.2 GRA-L-K-T-3 50 6 GRA1KT4 50 12.5 GRA-L-K-T-5 50 25 GRA-L-K-T-6 50 50 GRA-L-K-T-7 50 100 GRA-L-K-T-8

Example 7 (a) Lymphocytes obtained from peripheral blood of patients who have already undergone various cancers are sensitized with GRA-3 to obtain killer cell preparations. Patient-derived PBL (peripheral blood lymphocyte) (5x10®) was incubated in RPMI-1640 medium containing 50 ng / ml (protein content) GRA-3 for two days followed by 20% TCGF and 15% FCS. incubated in RPMI-1640 medium for another five days to obtain killer cells as described in

Table 5 shows.

Table 5

Cancer P GRA D-GRA Days after surgery Killer cell stomach Cancer + + 14 GRA-3-K-T-1 stomach Cancer + + 35 GRA-3-R-T-2 breast Cancer + - 21 GRA-3-K-T-3 breast Cancer + - 7 GRA-3-R-T-4 breast Cancer + - 35 GRA-3-R-T-5 stomach Cancer + 21 GRA-3-R-T-6

In the above table, P-GRA and D-GRA indicate the location of GRA in surgically removed tumor tissue as determined by the procedure described in Reference Examples 1 and 2 (b).

Tumor tissue is derived from patients with prior PBL collection. The results of P-GRA and D-GRA are obtained using FITC-PNA and FITC-DBA respectively. The days after surgery indicate the time at which PBL is collected after surgery.

(b) PBL (5x10®) was collected from breast cancer patients on day 21 postoperatively and incubated in RPMI-1640 medium containing 50 ng / ml (protein content) GRA-3 and 10% of containing serum, thereby sensitizing PBL to obtain killer cell preparations. Hereinafter referred to as GRA-3-K-T-7.

Example 8 (i) GRA-M1 obtained by the procedure of Informative Example 2 (2) - (b) was diluted with physiological saline solution so that the amount of sugar and protein was 1.0 pg / ml and 1.6 pg / ml, respectively. ml to produce anticancer composition # 1.

(ii) C3H / He cancerous tumor derived from spontaneous breast cancer was dissected germ-free into 5 mm cubic sections and transplanted under the dorsal skin of 10 C3H / He mice of the same strain (7-week-old male mice). Seven days after transplantation, the cancer can be confirmed and spread. 5 of the 10 mice above were treated with a subcutaneous dose of 300 μΐ / day of the anticancer composition # 1 (i) at 2-day intervals. The other 5 mice form the untreated control group. Ten days after the first treatment, the cancer is surgically excised and weighed. At the same time, a histological examination is also performed.

Tumor volume:

Treated group 22.3 mm ³ (Figure 14)

Untreated group 162.6 mm ³ (Figure 15)

According to this, the tumor was reduced by 86.3%.

Histological examination:

In the control group (Fig. 15), a tumor with a nest structure was formed, the type of tissue was similar to that of an articular cancer, and the proliferation of tumor cells extended throughout the tissue. On the other hand, in the drug-treated group (Fig. 16), the cancer cells became fluid, died, calcified, and fibrated. Very few cancer cells survived. Thus, the antitumor activity of the anticancer composition of the invention is observed.

1. Test Example

1 μΐ of the GRA-1KT prepared in Example 1 was placed on a slide (Falcon Corp.) and allowed to stand at room temperature for 15 minutes. At this time, 4 μΐ FCS (from Falcon Corp.) was added and the mixture was allowed to stand at room temperature for 30 minutes. 1 x 10 ° ml neuraminidase treated sheep erythrocytes (SRBC ^N ) was added and 5 μΐ 0.01 mol phosphate buffer

190 x 803 (pH 7.2) containing 0.85% sodium chloride was added and the plate was centrifuged (600 rpm for 5 min). The plate is inverted and unreacted SRBC ^N is removed. Dye solution (brillient cresyl blue, Merck ⁵ and Co.) was added to the plate to stain the lymphocytes and assay for rosette positivity. At least 98% shows rosette-like positivity (Cells).

gene. After one hour, the number of cells remaining is determined and the percent cytotoxic activity is calculated from the following equation:

cytotoxicity =% of the number of cells after culturing _ "- (1 - ^{x is} the number of cells before culturing iw (4x ^10-e)

The results are shown in Table 7.

2: Test Example

Specific Cancer Cell Killing Activity (a) Of the cells shown in Table 1, the following 5 cell groups, which showed different GRA positivity, were used as human cancer cells to be tested.

Target cancer cells:

(Burkitt's lymphoma) (Burkitt's lymphoma) (stomach cancer) (stomach cancer) (T-cell leukemia)

Falcon Corp. product)

No. 1 BT-1

Daudi # 2

No. 3 KATO-III

No. 4 MKN-45

MOLT 5 On a slide (x 10 * target cells are layered by centrifugation (800 rpm, 5 min).

At this time, GRA-1KT obtained according to the procedure of Example 1 ( ⁴ x 10 ³ ) was carefully added and incubated for one hour.

The killing effect was determined on the basis of plaque formation and is denoted as follows:

Table 7

target cell Number -% of cytotoxic effect breeding before breeding after Daudi 4x 10 « 2.9 x 10 « 28 KATO-III 4x10 » 3.7 x 10 « 7.5 BT-1 4x 10 « 3.2 x 10 « 20 MKN-45 4x10 " 3.9 x 10 « 2.5 Molt 4x10 ⁶ 4.2 x 10 « -5

(c) The procedure of (b) above is repeated except that the ratio of GRA-1-K-T to target cells is changed to 5/3. The results are shown in Table 8.

Table 8 + + Significant destruction effect observed + Destruction effect observed ± Destruction effect observed slightly

- no destructive effect is observed.

. In the control group, non-sensitized human peripheral blood lymphocytes prepared according to the procedure of Example 1 were used, except that GRA was not used. The results are shown in Table 6.

It is striking (Table 6) that the killer T cells prepared by the method of the invention have potent GRA specific cytotoxic activity.

target

% Of cytotoxic activity after culture before culturing

Daudi 4x10 " 3.6 x 10 « 91 KATO-III 4x 10 « 4.6 x 10 « 24 BT-1 4x10 " 1.4 x 10 « 65 MKN-45 4x 10 « 3.7 x 10 « 7.2 Molt 4x 10 « 4.1 x 10 « -3

In the example above, it can be observed that GRA-1-K-T is highly bound to Daudi, KATO-III and BT-1, and less active to MKN-45 and MOLT.

Table 6

Cancerous GRA plaque target Positivity (%) formation GRA-ICT _{Daudi (} Figure ₁ ) group 93.1 + + (Figure 2) KATO-III (Figure 3) 57.3 + (Figure 4) BT-1 (Figure 5) 50.1 + + (Figure 6) MKN-45 (Figure 7) 1.00 ± (Figure 8) MOLT (Figure 9) 0.6 - (Figure 10) Inspection BT — I group 50.1 - (Figure 11)

3. Test Example

C3H / He spontaneous breast cancer mice were challenged subcutaneously with GRA-M-1-K-T prepared in Example 2 at a dose of 3 x 10 10 / 0.3 ml / mouse for one week every other day. After ten days, the nucleus is exposed and examined.

As shown in Figure 12, lymphocytes are infiltrated between cancer cells and a break in the cancerous tissue is observed. Figure 13 also shows the calcification of the cancer and it can be considered that the killer cells of the invention have indeed anti-cancer activity.

(b) The same tumor target cells, which are used in (a) test compound, (3.3 x 10 ®) for 8xl0 ⁵ GRA LKT, with a (cell ratio: 5: 1) and the resulting cell mixture (the total number 4x 10®) RPMI-1640 15% FCS Media- 65

Comparative Example 1

In this example, the cancer cells themselves are used as specific antigens instead of GRA in the method of the invention.

-9190 803

As in Example 1, cancer cell-sensitized lymphocytes are obtained, except that BT-1, Daudi, KATO-III or ΜΚΝ-45 are used in 1 x 10 ^s per Petri dish instead of GRA.

The cytotoxic activity of the above lymphocytes is assayed according to Test Procedure 2 (a). The results are shown in Table 9.

Table 9 shows that lymphocytes are produced by this process do not have any cytotoxic effect ^0.

Table 9

Cells used in the lymphocytes of sensitizing ⁵

Target cell BT-1 Daudi ΚΑΤΟ-Ι11 MKN-45

KATO-III

MKN-45

Test Example 4

Assays 2 to 2 (b) determine the cancer cell killing activity of GRA-8-K-T, GRA-3-A-K-T and GRA-3-C-K-T prepared in Example 4. The results are shown in Table 10.

Table 70

% Of cytotoxic effect

target GRA-8-R-T GRA-3-A R-T GRA-3-C- R-T KATO-III 8.0 5.0 5.0 BT-1 4.3 5.0 4.0 MKN-45 20 5.0 20.0 Molt 0 0 0

Test Example 5 (a) All cytotoxic killer cell preparations prepared according to Example 6 are determined by the ⁵ Cr release assay [J. Immunol. 122: 2577-2553 (1979)].

That is, 50 µCi of radioactive ^sl Cr (Japan Isotope Assciation) is added to KATO-III (2x10 ⁷ ) and the cells are incubated at 37 ° C for one hour in RPMI-1640 medium and washed by centrifugation to obtain ⁵¹ Cr. cells. Killer cells (effector cells) (2x10®) are added to the target cell.,, _Λ . \ ,, τ-rr. effector cell tekhez (χ 1 10 ⁵⁾ (for the E / T · = * = 1) and the mixture was incubated at 37 ° C for 4 hours in RPMI-1640 medium. The supernatant was collected by centrifugation and radioactivity was determined by liquid scintillation.

The specific ⁵¹ Cr release (%) corresponding to the cell lysis rate of effector cells is calculated by the following equation:

Specific ^sl Cr release (%) = (release in the assay) (maximum release) (Maximum release refers to the radioactivity when all cells are already lysed.)

The results obtained are all. Table.

z _ Table 77 (spontaneous release) _χ ^ θθ (spontaneous release)

Test Example 6 (a) Using KATO-III (E / T = 20/1) as the target cell ⁴⁰ and the cell toxicity of the killer cells (obtained by the procedure of Example 7- (a)) is shown in Figs. test example.

The results are shown in Table 12.

Killer cells Specific ^J1 Cr Release (%) 45 Table 72 GRA-L-K-T-1 13 Killer cells Cell Toxicity% GRA-L-K-T-2 25 GRA-L-K-T-3 22 GRA-3-K-T-1 38.0 GRA-L-K-T-4 20 cn GRA-3-K-T-2 18.2 GRA-L-K-T-5 22 GRA-3-K-T-3 20.9 GRA-L-K-T-6 25 GRA-3-R-T-4 23.2 GRA-L-K-T-7 27 GRA-3-R-T-5 24.5 GRA-L-K-T-8 32 GRA-3-R-T-6 20.2

The results in Table 11 show that there is no connection ⁵⁵ between the TCGF, the FCS and the formation of killer cells.

(b) The cellular toxicity of GRA-2-KT obtained in Example 3 was determined by the specific ^sl Cr release assay as described in (a). ⁶⁰ 14.3% of the specific ¹ Cr release was ⁵¹ Cr labeled for KATO-III as for the target cell.

(E / T = ^{eff <, t} ; ° ^{rse 1-20/ 1} ) (b) The cell toxicity of GRA-3-KT of Example 7- (b) was labeled with ⁵¹ Cr KATO-III (E / T). = 20/1) as the target cell in the same manner as in Test Example 5. The specific ^sl Cr release of the killer cells was found to be 25.5%.

7. Test Example

The cell toxicity of the killer cells of Example 5 was determined by the ^sl Cr release assay.

-101

190,803 as in Test Example 5. The target cells are ⁵¹ Cr. were labeled X5563 cells. Control limfocitákként used lymphocytes obtained in Example 5, except that X5563 cells Sensitization of spleen cells 1 x 10 ⁵ / unit mitomycin C treated ⁵ is carried out (5 x 10® / ml X5563 cells were treated with 50 pg / ml mitomycin C. 60 minutes) instead of GRA-M-5.

The result is shown in Table 13.

Table 13

Specific release of ⁵¹ Cr (%)

Killer Cell - E / T ratio 40: 1 20: 1 10: 1 GRA-5-M-K-T-1 12.7 6.3 5.7 GRA-5-M-K-T-2 18.4 20.6 13.7 control 0.0 0.0 0.0

Test Example 8 (H-2 Test)

GRA-M1, GRA-M-3, and GRA-M-4 of Example 2, Information Sheet 2, were serially diluted in 0.15 M phosphate buffer (0.85% sodium chloride, pH 7.6). ) to create samples. The anti-H-2 serum obtained from the National Institute of Genetic Research ₃₀ and the above sample were mixed and incubated at 4 ° C for 2 hours. The target cells are thus contacted with anti-2-serum. Spleen or lymph node cells used as target cells can be obtained from B10 (H-2 ^b ) and BIO.BR (H-2 ^k ) mice in the usual manner. After washing the cells with PBS, rabbit complement was added to the cells and incubated at 37 ° C for 1 hour and stained with 0.2% trypan blue in PBS to determine% cell toxicity. Anti-H-2 Serum is used at maximum dilution so that it ultimately exhibits 95% cellular toxicity in the absence of GRA.

The GRA inhibitory activity of the systems is determined in Table 14.

Table 14

GRA Anti-H-2 Serum, concentration rationality target GRA-M-1 D-23 (anti-H2K ^k ), X80 obsession B10BR (H-2 ^k ) spleen cells D-32 (anti-H- ^20k ), X300 GRA M-3 D-33 (anti-H-2K ^b ), X600 obsession B10 (H-2 ^b ) spleen cells D-2 (anti-H-2D ^b ), X80 GRA-M-4 D-23 X80 obsession BIO.Br (H-2 ^k ) lymph node cells D-32 X300

The results are shown in Table 15.

% Of cell toxicity

Table 15

-.- / - anti- Z ^ T> A TT Dilution of samples n-x serum X2 X4 X8 X16 X32 X64 x128 X256 X512 0 * 13 14 12 13 14 13 14 13 14 13 I D-23 - 97 99 96 97 97 96 97 96 96 14 D-32 - 95 95 95 95 94 95 95 96 95 13 II - - 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

Notes: GRA I: GRA-M-1 GRA II: GRA-M-3 GRA III: GRA-M-4

The cellular toxicity shown is when complement is used alone.

The results of Table 15 show that HRA is deficient in GRA-M-1, GRA-M-3 and GRA-M-4.

9. Test Example

Lewis Lung Cancer (LLC) (2x10®) from the same strain was transplanted into C57BL / 6 mice and, after six days, 1 ng of protein (GRA-M-3 as in Example 2- (d)) was subcutaneously administered. Repeat for 4 days, once daily, at the same dose.

Results: The average tumor weight in the control group was about 500 mg. In the GRA-M-3 treated group, tumor disappearance was observed in 3 cases, and twice the mean tumor weight was 100 mg.

-1 110. Test example

C57BL / 6 mice were immunized with a subcutaneous solution of 1 ng (protein) GRA-M-3 obtained by the procedure described in Reference Example 2 (d). A bypass from the Work at ⁵ is carried out once on day 5 and the spleen cells were collected from the animals to obtain the effector cells per day for 3 days. A mixture of effector cells and Lewis lung cancer (LLC) cells as target cells was prepared at a ratio of ^{1 n} E / T = 50: 1, of which a portion (1 × 10 ⁵ ) was transplanted into mice from the same strain and a Winn test was performed. . [J. Immunol. 86, p. 228-239 (1961)].

Table 16

effector Tumor growth rate On the 20th, the Moorish cell Day 15 Day 17 Day 18 Day 19 Day 20 talitás csopor- tions for

THE 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

Note: Group A represents spleen cells from the GRA-M-3 immune mouse.

Group B represents spleen cells from a normal mouse.

Group C represents spleen cells 10 days after transplantation of LLC (1 x 10 *) from mouse.

190,803 i

Table 17

GRA-M-4 concentration rationality (Ng / ml) 3 ³ H-TdR incorporation (mean stroke / min ++ standard error) S. I. 0 5,302 ± 1,761 control 0.5 7,903 ± 1,290 1.5 First 10,076 ± 936 1.9 5th 10,686 ± 429 2.0 Experiment 20 10.615 ± 1.270 2.0 40 8.565 ± 1.419 1.6

Note: S.I. represents the stimulation index in proportion to the experimental control.

12. Test Example

The C3H / He normal mouse, the X5563 immune mouse, is the MH134 im ²⁰ munis mouse, prepared as in Example 5, all. Delayed Type Hypersensitivity Response (DTH) of GRA-M-4 Immune Mouse and GRA-M-5 Immune Mouse, Prepared as Exemplified by Test Example 3, was determined by the footprint reaction (FPR). That is, tumor cells treated with GRA or MMC (mito25 micin c) are introduced into the foot pad of the animal. Swelling of the sole is determined 24 hours after treatment. The degree of DTH response was calculated by subtracting the pre-swelling of the footpad treatment level (10 ^'2 mm) ^{of 30} degrees to swelling after cure.

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

Tumor growth is calculated from the following equation: ³⁵

T-T

Tumor Growth Rate (%) = -4 = —- ^x 100, where T _{A represents} the thickness of the sole of the foot where the tumor was implanted and T _{N represents} the thickness of the healthy sole of the foot.

11. Test Example ₄₅

C3H / He mice 4.5 pg (protein) GRA-M-4, (obtained according to Example 2 (d) information) and 0.1 ml complete Freund's adjuvant-immunized ol _5Q TASS in the sacral region. After two weeks, lymph node cells were harvested by conventional means and used as responding cells to determine GRA-M-4-induced cell proliferation.

For this reason, responding cells (4 x 10 ⁵ ) were incubated in RPMI-1640 containing 15% FCS- ₅₅ .

In the presence of GRA-M-4 for 5 days. During the last 18 hours of incubation, 1 pCi ³ H thymidine ( ³ H-TdR) was periodically added to the medium and the number of cells into which thymidine was incorporated was counted. The results obtained are shown in Table '17: ⁶⁰

Table 18

Treatment The average number of degrees of swelling of the soles of the soles (10 ^-2 mm) Normal mouse MH134 immune mouse 1 2.8 13.6 2 12.4 32.0 3 3.6 3.6 4 3.2 22.0 5 6.8 27.6

Comment:

Group 1, normal spleen cell 1 x 10 ″ / 20 μΙ medium (Hanks' solution)

Group 2, MH134 cells 1 x 10 10/20 µl medium

Group 3, 20 μΐ medium

Group 4, GRA-M-4, 0.8 pg (protein) / 20 μΐ medium

Group 5, GRA-M-4, 0.4 µg (protein / 20 µl medium)

Table 19

Treatment The swelling of the soles is the average of the degree of swelling (10 ' ² mm) Normal mouse X5563 Immune mouse MH134 Immune Mouse 1 5.4 4.3 1.1 2 0.9 - 24.3 3 2.0 16.9 -

Comment:

Group 1, 20 µl medium (Hanks' solution)

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

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

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190 803

Table 20

Treatment The mean of the degree of swelling of the soles of the feet Normal mouse GRA-M-4, Immune Mouse 1 1.7 -0.3 2 5.1 24.6

Comment:

Group 1, 20 μΐ medium (Hanks' solution)

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

Table 21

The soles of the soles are the average of the degree of swelling

Treatment Normal mouse GRA-M-4, Immune Mouse 1 -1.8 0.6 2 6.3 20.0 3 6.8 6.3.

Notes:

Group 1, 20 g medium (Hanks' solution)

Group 2, GRA-M-4, 4 µg (protein) / 10 µl medium

Group 3, 4 µg (protein) / 20 µl of nutrient, in this case the fraction of GRA-M-4 passing through the PNA column (hereinafter referred to as "C.P.").

Table 22

Treatment The average number of degrees of swelling of the soles of the soles (10 ^-3 mm) Normal mouse GRA-M-4, Immune Mouse 1 2.4 -1.0 2 2.6 17.7 3 2.4 1.8 4 5.5 18.9

Notes:

Group 1, 20 g medium (Hanks' solution)

Group 2, GRA-M-4, 3.8 µg (protein) / 20 µl medium

Group 3, 3.8 gg (protein) / 20 g of the above "C. P. " broth

Group 4, 3.8 µg (protein) / 20 µl of GRA-M-4 and 3.8 µg (protein) / 20 µl of “C. P. " medium

Informative test

The spleen cells of the immune mouse X5563 obtained by the procedure of Example 5 were used as effector cells and the effector cells (10 ⁷ ) and target cells (X5563.10 ^sec ) were implanted into a mouse from a similar strain. The Winn probe of Test Example 10 is used.

The results obtained are shown in Figure 22, in which the abscissa shows the days and the ordinate represents the mean tumor size (cm ^J ) ± standard error, and the various signals are as follows:

• _ · The group to which effector cells are not added.

o_o The group to which untreated effector cells are added.

δ_δ The group in which effector cells treated with rabbit complement are administered.

csoport_χ The group in which effector cells are treated with anti-Thy-1 * (New England Nuclear Co., USA) and rabbit complement.

□ __o The group in which effector cells are treated with anti-Lyt-1 * (New England Nuclear Co., USA) and rabbit complement.

__ Az is the group in which cells treated with anti-Lyt-2 * (product of New England Nuclear Co., United States of America) and rabbit complement are added.

(* Anti-Thy-1, anti-Lyt-1, and anti-Lyt-2 are monoclonal antibodies that are specific for a subset of mouse T cells and have no effect on cancer therapy.)

The results of Figure 22 show that anti-Lyt-1 type T cells play an important role in the tumor immune response mechanism in vivo.

Furthermore, it is known that the DTH response is through anti-Lyt-1 T cells. [J. Exp Med 143 p. 1534-39 (1976)].

The invention has been described in detail, with particular regard to its particular features, and it will be understood that it will not be difficult for one of ordinary skill in the art to make various changes or modifications thereto without departing from the spirit and scope of the invention.

Brief Description of the Drawings

Figure 1. Microscopy of Daudi cancer cells.

Figure 2 Microscopic image showing plaque formation by GRA-1-K-T of cancer cells in Figure 1.

Figure 3 Microscopy of KATO-III cancer cells.

Figure 4 A microscopic view showing the plaque formation induced by the GRA-1-K-T of the cancer cells of Figure 3.

Figure 5 Microscopy of BT-I cancer cells.

. Figure 6: Microscopic image showing plaque formation induced by GRA-1-K-T of the cancer cells of Figure 5.

Figure 7 Microscopy of MKN-45 cancer cells.

Figure 8 Microscopic image showing plaque formation induced by GRA-1-K-T of the cancer cells of Figure 7.

Figure 9 Microscopy of MOLT cancer cells.

Figure 10 A microscopic image showing the formation of tarfol [GRA-1-K-T induced by the cancer cells of Figure 9.

Figure 11: Microscopic view of BT-1 treated with a mixture of non-sensitized human peripheral blood lymphocytes.

Figures 12 and 13 Microscopy of both cancerous mouse tissues. The cancerous mouse was given GRA-M-1-K.

Figure 14 Microscopic imaging of cancerous mice in groups of cancerous mice bearing GRA-M-1.

Figure 75. Tissue taken from cancerous mice from a group to which no GRA-M-1 was added.

Figure 16 Microscopic image of cancerous tissue treated with GR-M-1.

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Figure 77. Graph of GRA SDS gel electrophoresis of GRA by CBB wood CBB method coomassie brilliant blue (4-hydroxy-5 - [(4-phenylamino) -5-sulfo-1-naphthalenazo] -2,7-naphthalenedisulfonic acid) disodium salt, product of Ayerst Co.), stained using protein staining according to the method described in Biochemistry 10 (1971) 2606-2617).

18-20. Graphs of GRA SDS gel electrophoresis using sugar staining by PÁS. (According to the PÁS method, the protein is stained with Schiff's acid reagent, Biochemistry 10: 2606-2617 (1971)). side].

Figure 27 is a schematic diagram of the GRA SDS gel electrophoresis diagram using protein staining by C. B. B.

Figure 22 shows the extent of tumor growth after implantation of X5563 immune mouse spleen cells and X5563 cells into C3H / He mouse.

Claims (13)

Claims !. A method for producing cytotoxic lymphocytes for cancer cells comprising sensitizing the lymphocytes to a carbohydrate-containing antigen derived from cancer cells, which is a constituent of the carbohydrate-containing antigen on the cancerous membrane and an N-terminal galactose galactose of a lectin. (Priority June 28, 1982) The method of claim 1, wherein the sensitization is performed with a carbohydrate-containing antigen that is a constituent of a cancerous cell membrane and which is capable of specifically binding to a lectin via its terminal galactose moiety. (Priority: 10/01/1981) 3. The method of claim 1, wherein the sensitization is performed with a carbohydrate-containing antigen that is a constituent of a cancerous cell membrane and which is capable of specifically binding to a lectin via its N-acetyl-galactosamine moiety. (Priority: June 6, 1982) 28) 4. A process for the preparation of an anticancer composition, wherein said agent is a cytotoxic agent for cancer cells, as defined in any one of claims 1-3. A lymphocyte produced according to any one of claims 1 to 6, which is converted into a pharmaceutical composition specific for an antigen containing a carbohydrate derived from a cancer cell, in admixture with excipients conventionally used in pharmaceutical technology. (Priority: June 28, 1982) 5. A method for producing a carbohydrate-containing antigen, comprising isolating an antigen from a membrane of a human cancer cell that is capable of specifically binding to a lectin through its terminal galactose group or its N-acetylgalactosamine terminal end, by specific binding to lectin. (First ⁵ ge: June 28, 1982) 6. The method of claim 5, wherein the lectin is specifically capable of binding to the galactose group at the terminal end of the carbohydrate of the antigen. (Priority: ⁰ 1981-10-01) 7. The method of claim 6, wherein the pectin is derived from a peanut or soybean which is capable of binding to the galactose group of the terminal carbohydrate. (Priority: 10/01/1981) 8. The method of claim 7, wherein said peanut is a lectin capable of binding to a galactose moiety of a carbohydrate antigen. (First ²⁰ : 01.10.1981) 9. The method of claim 5, wherein the lectin is specifically capable of binding to the N-acetylgalactosamine group of the carbohydrate of the antigen. ³⁵ (Priority: June 28, 1982) 10. The method according to claim 9, characterized in that use is obtained from Dolichos bean agglutinin lectin, which is able to connect the terminal ³⁰ of the carbohydrate antigen N-acetyl-galactosamine-group. (Priority: June 28, 1982) 11. The method of claim 5, wherein the carbohydrate-containing antigen is eluted. first separate session coupled to a first lectin that specifically connect to the carbohydrate antigen of galaktózcsoportjához terminal ^35, subsequently turned on a second lectin that can connect the terminal carbohydrate of the antigenic N-acetilgalaktóz amine group, is carried out. (Priority: June 28, 1982) 40 12. The method of claim 11, wherein the first lectin is coupled to peanut-derived lectin to the terminal galactose group of the antigen carbohydrate, and the second lectin is coupled to agglutinin derived from Dolichos bean. 45 antigen carbohydrate to the N-acetylgalactosamine amine group. (Priority: June 28, 1982) 13. A process for the preparation of an anticancer composition, wherein the active ingredient is as defined in claims 5-12. A carbohydrate-containing antigen from a cancer cell prepared according to any one of claims 1 to 5, which is capable of specifically coupling to a lectin via a terminal galactose group or a N-acetyl-galactosamine group of the carbohydrate-containing antigen in admixture with excipients commonly used in pharmaceutical technology.