DD209577A5 - METHOD FOR THE PRODUCTION OF A GLYCOME-RELATED ANTIGEN - Google Patents

Abstract

CANCER CELLS CONCENTRATING LYMPHOCYTES, METHODS FOR THE PRODUCTION THEREOF AND ANTI-CANCER, CONTAINING THE LYMPHOCYTES AS AN ACTIVE INGREDIENT, ARE DESCRIBED. THE CANCER CELLS CONTROLLING LYMPHOCYTES SPECIFICALLY APPLY TO CANCER CELLS CONTAINING AND DISSOLVING A GLYCODIC ANTIGEN DERIVED FROM CANCER CELLS.

Description

241290 L - ^ " Berlin, October 4, 1983

^ AP A 61 K / 241 290

61 066/12

Process for the preparation of a glyc-related antigen Field of application of the invention

The invention relates to a Herstellungsaverfahren of glycated antigen, which can be used to combat cancer cells and thus in medicine.

Well-known technical solutions

Immune-responsive uterine cells, particularly T lymphocytes, which play a major role in a cell-triggered immune response, cause rejection of graft sites due to a tfrem cell antigen, but do not produce appreciable or very limited immuno-inhibition against cancer cells. As a result, the cancer cells are not destroyed but multiply in vivo and eventually cause the fod of the cancerous carrier ·

However, the mechanism involved is not yet fully understood, and various studies have been made to gain further insight into the nature of the material basis found in such a system. For example, cell surface markers on mouse leukemia cells or embryonal carcinoma cells are made using lectins such as Dolichos biflorus agglutinin (DBA) and idiotanagglutinin (PNA) in Biochem. Biophys. Res. Gomm. 89 (2) 448-455 (1979), Ibid. 96 (4) 1547-1553 (1980), J. Biochem. 8J 473 - 481 (1981) and

241230 U ~ ² ~ 4.10.1983

AP A 61 K / 241 290 62 066/12

Cell 18 189-191, September 1979, object of the invention

iüe is the object of the invention to develop a means for combating cancer cells,

Essence of speech

The invention has for its object to provide a method for producing a glycated antigen.

Thorough studies on the immune response of the carrier to cancer cells and their use in the treatment of cancer have now shown that in a cancer cell-specific antigen not found in differentiated hormonal cells, GRA is present, which acts as an immunogen for the carrier and a very has high immunogenicity causing an immune response specific to cancer cells. GRA is the glyco-related antigen that derives from the cancer cells,

Furthermore, it has been found that, when GRA is used to sensitize lymphocytes, one obtains killer cells which specifically act on GRA-containing cancer cells and, upon administration of the killer cells to a carrier, recognize these GRAs and act on GRA-containing cancer cells.

4 1 2 9 0 A -3- 4.10.1983

AP A 61 K / 241 290 61 066/12

destroying them and thus achieving a very good effect in the treatment and prevention of cancer.

The killer cells can be prepared, for example, according to a method in which GRAs are used to sensitize lymphocytes.

2.4 1 29 Ö 4

The GRA used in the above method is obtained from GRA-containing human or human cancer cells

Animals such as cultured cancer cells, transplanted cancer cells, spontaneously occurring cancer cells, chemical substances or virus-induced cancer cells or cancer cells which have been surgically removed from tissues using the following methods. ^;

Cell membrane components are separated from cancer cells of the aforementioned kind and treated with a lectin which binds specifically to terminal galactose or terminal N-acetylgalactose moiety, whereby GRA can be readily separated with the lectin (see JBC, 250 /

8518-8523 (1975); Biochem. Biophys. Res. Comm., B_ £, 144 (1975); 2. Immunology., V38, 423-433 (1966); Br. J. Exp. Pathol., 2Ί_, 228-236 (1946); Proc. Nath. Acad. Be. USA, 75, No. 5, 2215-2219 (1978); Biochemistry, 13 , 196-204 (1974); and Carbohydrate Research, 5J, 107-118 (1976)), whereby GRA combines with the lectin and can be easily separated.

The separation of the cancer cell membrane components can be achieved in a simple known manner, e.g. by a Hogenogenisierungsmethode and a Soiübilisierungsme-. , method under the exclusion of a dissolving agent. It is advantageous to use a method in which cancer cells are homogenized in saline or in a suitable buffer, a portion of the precipitate is collected by, for example, centrifugal separation and in saline or a buffer by means of a solvent

241290 U -5 -

is dissolved, after which the supernatant then separated for example by centrifugal separation. As the dissolution agent, there may be used, for example, surface active agents generally known to dissolve cell membranes, for example, nonionic surfactants such as Triton X-100 (manufactured by Wako Pure Chemical Industries Ltd.), NP-40 (manufactured by Shell Co. _r Ltd.), digitonin and urea, or anionic surfactants, for example Natxiumdodecylsulfonat (SDS).

From the cell membrane components thus obtained, GRA capable of separating with lectin using conventional chemical or biochemical methods and making use of the properties of the lectin can be separated. Examples of such methods are affinity chromatography using a column carrier containing a lectin, an immunoprecipitation method using GRA antibody or the like, a dialysis method, a gel filtration method, an electrophoresis method or a physical precipitation using a sugar protein precipitating agent, e.g. Pclyethylenglykol and acetone, or a combination of such methods. Particularly preferred is affinity chromatography using a column containing a lectin, whereby the column support can be easily prepared by immobilizing lectin on an insoluble support. The immobilization of lectin on an insoluble carrier can be used in the known manner as used for the immobilization of biosubstances

24 1290 4 -

will, perform. Of these. Methods are preferably used the cyanobromide activating polysaccharide method and an immobilization method using N-hydroxysuccimide ester. The Cyanobromidaktivierungs polysaccharide method is a method. In which an insoluble support with cyanogen bromide is treated and the activated product thus obtained is then subjected to mild conditions a coupling reaction with a lectin, the lectin is immobilized. For example, in the treatment of the insoluble carrier with cyanogen bromide, basic compounds such as sodium hydroxide and sodium bicarbonate are used to adjust the pH to 7.5 to 12 and then the carrier is treated in a solvent such as water, acetonitrile or one at pH 7. 5-12 held buffer, eg, a 0, TM sodium bicarbonate buffer (pH about 8.7). and. O ₇ OIM phosphate buffer (pH about 7.7) at room temperature during. about 1 to 12 minutes. In general, it is preferable to use an amount of cyanogen bromide equivalent to the insoluble carrier.

Any known insoluble support exhibiting a low non-adsorbent adsorption to all biosubstances, having a high porosity and having a functional group capable of immobilizing biosubstances under mild conditions and being chemically and physically sufficiently stable, may be used of the present invention.

Useful insoluble carriers are, for example, carriers made from cellulose, e.g.

24 129 0 A -

Aminoethylcellulose, carboxymethylcellulose, bromoacetylcellulose and p-anilino-cellulose, or a carrier crosslinked! Dextran, e.g. Sephadex and CM-Sephadex (manufactured by Farmacia Corp.) and a carrier of agarose, e.g. Sepharose 2B, Sepharose 4B and Sepharose 6B (manufactured by Farmacia Corp.).

In the coupling reaction of the cyanogen bromide activated carrier obtained as above, the cyanogen bromide activated carrier is used in an amount corresponding to 30 to 80 times that of the lectin, and the reaction is carried out in a suitable solvent such as 0.1 mol / l aqueous solution of sodium hydrogencarbonate (containing 0.5 mol / l sodium chloride; pH 8.4) at a temperature between 0 and 40 ⁰ C, preferably 2 and 8 ° C for a period of about 10 to hours. In this way, a lectin-containing support for affinity chromatography is obtained.

In chromatography using the lectin-containing carrier prepared as described above, the desired GRA combines with the lectin contained on the carrier and is retained in the column. Subsequently, substances capable of combination, e.g. with a lectin, passed through the column, with an exchange reaction taking place, or alternatively an adsorption separation (with an eluting solution), e.g. with a highly concentrated salt, an aqueous solution of potassium thiocyanate and a nitrate buffer to separate and recover the desired GRA.

41290 4

In the exchange reaction, substances capable of binding to a lectin when an affinity chromatography carrier containing a galactose-binding lectin is used, for example, those combining with a terminal galactose-linking lectin, e.g. Galactose, disaccharides containing terminal galactose, and oligosaccharides containing a terminal galactose. Examples of substances capable of combining with a lectin when using as the support for affinity chromatography an N-acetylgalactosamine-binding lectin are those which react with a terminal N-acetylgalactosamine-linking lectin could connect, eg N-acetylgalactosamine, disaccharides / containing a terminal N-acetylgalactosamine and oligosaccharides containing a terminal N-acetylgalactosamine. · "/.

The GRA thus obtained contains glycoprotein containing a galactose and / or N-acetylgalactosamine end group, glycolipids and / or saccharides.

If desired, the GRA produced in this way can be lyophilized and also purified in the usual way using customary separation methods. For example, such GEL preparations isolated with a galactose-binding lectin can be subjected to a separation test using N-acetylgalactosamine-binding lectin and GRA isolated with L-linking N-acetylgalactosamine can be prepared by a separation method treated using a galactose-binding lectin

24129

The lymphocytes used herein are not critical and all lymphocytes from normal or cancerous humans or animals can be used. Examples include lymphocytes obtained from peripheral blood, bone marrow, lymph nodes, spleen, tonsils, or thymus gland. These lymphocytes are isolated by physical or chemical methods, by a surface membrane method or the like.

Sensitization of lymphocytes with GRA. by culturing the lymphocytes on a culture medium containing GRA for a period of 1 to 10 days, preferably 2 to 7 days.

As a culture medium for use in sensitizing the lymphocytes, various common culture media commonly used for such cell culturing can be used. Preferred examples include RMPI 164O medium _r, Eagle MEM culture, etc. to which human serum, fetal calf serum (FCS), calf serum, horse serum or the like has been added. The amount of GRA added to the culture is generally 1 to 1,000 ng / ml and preferably 1 to

24 1290 4-

500 ng / ml, calculated as amount of sugar., Per 1 χ 10 / ml lymphocytes.

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

The killer cells according to the invention thus produced are essentially normal lymphocytes and have a GRA-specific cell-destroying activity. For example, GRA-1-KT, one of the killer cells of the present invention, has properties found in human peripheral blood T cells, and exhibits a cell-fighting activity specific to cancer cells containing GRA, as shown in the examples below.

Typical examples of the new killer cells according to the invention are GRA-1-KZ and GRA-M-I, which are prepared in the examples described below. These killer cells are available from the assignee and deposited with ATGC.

The killer cells according to the invention can be multiplied without restriction / using the vorv / similar culture medium containing a T cell growth factor (TCGFr IL-2). In this case one can carry out a selective cultivation of killer cell clones by means of a customary ultra-dilution method. The killer cells can be stable over long periods of time, in

241 290 k -11-

For example, liquid nitrogen can be stored.

The GRA may be used singly as an active ingredient or may additionally be used in combination with other bactericidal agents and anti-cancer agents. Cancer inhibiting agents containing the GRA of the invention as an active ingredient may be in any form, provided that they contain GRA in effective amounts. Generally, the agent will be administered intravenously, subcutaneously, intrafermally or intramuscularly in the form of a solution, suspension or used as an emulsion. It can also be prepared as a dry product and then converted into a liquid immediately before use by adding a suitable carrier. This liquid agent may be a suspending agent, e.g. Methylcellulose, an emulsifying agent, e.g. Lecithin, preservatives, e.g. Methyl p-hydroxybenzoate, stabilizers and the like, so long as they do not cause adverse effects on immunization function in humans or animals. Buffers can also be included.

Suitable aqueous carriers are, for example, a physiological saline solution, and a suitable nonaqueous carrier may be, for example, a vegetable oil,

241290

For example, sesame oil, a mineral oil, for example, paraffin, or an animal oil, such as Sguallene, or Prcpylenglykol be. To increase the immunological effect, it is possible to incorporate a suitable adjuvant. Suitable adjuvants are for example ¹ friend's Complete Adjuvant, saponin in animals and aluminum in humans.

The anticancer agent of the invention may be administered once or repeatedly for prolonged periods to a cancer patient for the treatment of cancer, or it may be administered to someone who is cancerous, thereby preventing the cancer.

Since LD ₅₀ (mouse, intraperitoneal) of GRA is at least 500 mg / kg, calculated as the amount of sugar, the anti-cancer agent according to the invention exhibits low toxicity and can therefore be administered within a wide dosage range. Although the concentration of GRA in the anticancer agent according to

not critical to the invention, it is preferred to use it in an amount of 0 "001 to 100 μg / ml, calculated as the amount of the sugar. With regard to: Dosage of the anti-cancer agent is generally preferred to administer the agent in an amount of 0.001 to 1000 μg / kg / day at a time, or in different proportions, but this dosage depends on the degree of disease, age and sex depends on the patient.

·

The anticancer agent thus prepared _r which the

24 1 290 4

Containing killer cells as an active ingredient is preferably used as an injectable solution in combination with excipients used in the manufacture of blood medicines. The carriers used herein are not critical, but preferred are carriers having the same tonicity as blood. In particular, physiological saline solution is preferred. In the preparation of the agent, it is preferable to wash the killer cells sufficiently with a physiological saline solution or the like to remove the aforementioned culture medium, and then it is then taken up in a carrier.

The concentration of killer cells in your agent is not particularly limited, but it has merits *

If the killer cells are administered intraperitoneally in one dose, the dose is between 10 and 10 per milliliter

of 10 per mouse, no toxicity is detected.

Although the dose of the inventive anti-cancer agent depends on. the degree of illness, age and sex of the patient, but in general

Preferably, the agent is administered in a dose of 10 to

12

10 / kg / day avif eimnal or in different proportions

, administered. _.

The following examples, reference examples, experimental examples, and comparative examples describe the invention without restricting it.

_ -j 4 _

FIG. 1 is a photomicrograph of Daudi

Cancer cells,

Fig. 2 is a photomicrograph showing the formation of deposits of GRA-1 K-T

Fig. 1 shows cancer cells.

3 is a photomicrograph of KATO-IlI

Lines, 10

Fig. 4 is a photomicrograph showing the formation of deposits by GRA-I-K-T on cancer cells according to Fig. 3,

Fiy. 5 is a photomicrograph of BT-I

Cancer cells,

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

7 is a photomicrograph of MKN-45

Cancer cells,

Fig. 8 is a photomicrograph showing the

Formation of deposits by GRA-1-K-T on cancer cells according to FIG.

9 is a photomicrograph of MOLT cancer cells,

- IB

ig. Fig. 10 is a photomicrograph showing the formation of plaque by GRA-1-K-T on cancer cells of Fig. 9,

Fig. 11 is a photomicrograph of BT-1 which

treated with a mixture of unsensitized human peripheral blood lymphocytes,

Fig. 12 · (· 13) are respectively photomicrographs of cancer

cell tissue of a cancerous. Mouse administered GRA-M-1i ~ K-rT,

14 is a photograph showing a cancerous condition in a cancerous mouse

group administered GRA-M-1,

Fig. 15 is a photograph showing the cancerous condition of a cancerous mouse group;

no GRA-M-I was given.

Fig. 16 is a photomicrograph showing cancer cell tissue of a mouse cancerous group which has not been GRA-M-I-gated;

FIG. 17 is a microfuge graph of cancer cells.

tissue of a group treated with'g RA-M-V,.

Fig. 18 shows an SDS gel electrophoresis slide

of GRA using protein staining according to the C.B.B. method,

Figures 19 to 21 show SDS gel electrophoresis slide strains

using sugar stains using the PAS method.

Fig. 22 is a schematic illustration of SDS gel electrophoresis results.

Diagraxmh 'of GRA using the • protein staining, by means of the C.B.B. »method.

Fig. 23 ' is a graph showing tumor growth rate in C3H / HE / 6 mice,

those X5563 Immunin & use MilSäellen and. , Y5563 cells had been transplanted.

U 4 - 17 -

Reference Example 1

(1) ~ ^A Preparation of FITC-labeled lectin

(PNA-FITC)

10 mg peanut lectin (PNA), manufactured by EY Co.) was dissolved in 2 ml of a 0.01M phosphate buffer containing 0.85% NaCl (pH 7.2). In 1 ml of a 0.5M

Hydrogen carbonate buffer (pH 9.0) was dissolved in 2 mg of FITC (manufactured by Sigma Laboratories Inc.) and a 0.5 ml portion of the resulting solution was added to the previously prepared PNA buffer. The mixture was stirred for 2 hours at room temperature and then separated via Sephadex G25 (10 mm χ 300 mm, manufactured by Farmacia Corp.). The initial peak was collected. FITC / PNA ratio: 1.0.

2Q ^: (DB Preparation of FITC-labeled lectin

(DBA-FITC)

In the same manner as in (1) -A above, DBA-FITC was obtained by using DBA produced by EY Co. EITC / DBA ratio = 0.9.

f -? * (1) -C soybean agglutinin FITC (TIFT-SBÄ) is available from Ey Co.

FITC / SBA ratio = 1.4.

30 '-

(2) locality of GRA on different cancer cells

(a) Human cultured cancer cells (1 × 10) were purified three times with a 0.05M tris-hydrochloric acid buffer containing 0.85% NaCl (pH 7.2) by centrifugal method and then 100 μM PNA-FITC, DBA-FITC or SBA-FITC (200 μg / ml) prepared and added according to (1). The mixture was allowed to stand at room temperature for 30 minutes to react. After finishing the reaction, the reaction mixture was washed three times with a 0.01 M phosphate buffer containing 0.85% NaCl (pH 7.2), and then the cells were placed on a glass plate and examined under a fluorescence microscope.

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

Table 1

cancer cells positive ratio of GRA (%) DBA PNA 1.4 Raji (Burkitt Lymphoma) 98.3 5.2 Dauji (Burkitt Lymphoma) 93.1 O BT-1 (Burkitt Lymphoma) 50.1 6.7 P-12 (T cells Lymphoma 44.3 4.8 MOLT (T-cell Leukemia) 0.6 5.3 Fujimaki (B-cell lymphoma) 2.19 10.0 ODA (IgD myeloma) 0.6 2.0 QG-56 (Lung cancer, sguamös) 70.4 0.4 PC-1 (Lung cancer, sguamös) 78.4 0 PC-3 (Lung cancer, adenocarcinoma) 77.1 0 QG-90 (Lung cancer, small cells) 68.0 0 PC-1 3 (Lung cancer, big cells) 17.0 0.1 MK-2 (Stomach cancer, por.) 63.7 0 KATO-III (Magenkregs sig.) 57.3 40.3 MKN-45 (Stomach cancer por.) 1.0 0.4 MKN-1 (Stomach cancer, adsq.) 4.6 0.1 MKN-28 (Gastric cancer, TUBL) 0.4 0 MKN-74 (Gastric cancer, tübl) 0.5 0 MGH-U1 (Bladder cancer approx.) 37.4 21.4 KU-2 (Bladder cancer approx.) 4.5 0 T-24 (Bladder cancer approx.) 14.6 1.0 NBT-2 (Bladder cancer approx.) 13.1 0 NRC-12 (Kidney cancer) 23.9 0.6 KU-1 (Kidney cancer) 3.3 0 Kuramochi {Ovarian cancer) 80.0 ¹ ' ⁷ NB-1 (Neuroblastoma) 20.9 0.5 YT-nu (Neuroblastoma) 3.6 0 TGW-nu-1 (Neuroblastoma) 4.1 1.0 TGW-nu-11 (Neuroblastoma) 2.0 0 · GOTO (Neuroblastoma) 0.5

- 20 -

96.9 12 3 90 ,1 44.6 0 6 14 14.6 3 /1 5.4 0 5.7 1 , 7 92.0 0 18.6 , 0

ITO (embryonal carcinoma)

NEC-8 (embryonal carcinoma)

SCH (Choriacarbinoma, stomach)

GCH (Chriocarcinoma, uterus)

5 YN-1 (rhabdomyosarcoma)

Mouse X 5563 (Plasmacytoma)

Mouse MH134 (Hepatoma)

(b) The malignant tissue of cancer cells was passed through a sieve. stainless steel (150 mesh), under

Preservation of a single cell suspension. After washing twice with 0.01 M HCl buffer (pH 7.4) containing ₂ mM CaCl ₂ , ₂ mM MgCl ₂ and 0.85% NaCl, ⁵ × 10 ⁵ cells were resuspended in 100 μM buffer. 100μl of FITC-PNA or FITC-DBA (200μg / ml) was added to the cell suspension and the mixture was incubated at room temperature for 20 minutes. After washing three times with cold PBS, the cells were examined under a fluorescence microscope

 20

The results are shown in Table 2. The malignant tissue of cancer patients was obtained from Kansai Medical University.

ί "f"'h A

I. I. "" J U # - 21 -

Table 2

GRA

No cancer patient tissue

T stomach cancer 2 stomach cancer 3 stomach cancer 4 stomach cancer 5 stomach cancer 6 stomach cancer 7 breast cancer 8th breast cancer 9 breast cancer 10 breast cancer 11 colon cancer 12 colon cancer 13 Esophaguskrebs 14 hepatoma

Note: "+" denotes the GRA on the cell surface;

"-" means / that GRA does not occur on the cell surface.

Reference Example 2 25

(1) -A Preparation of immobilized lectin (PNA-Sepharose)

3 g of CNBr-activated Sepharose 4B (manufactured by Farmacia Corp.) were thoroughly washed with 1 mmol HCl and suspended in 200 ml of 0, TM sodium bicarbonate (pH 8.5). To this was added 5 ml of a P, 01 M phosphate buffer (pH 8.5) containing 20 mg of PNA, and then the reaction was carried out at 25 ° C for 2 hours while stirring several times to obtain PNA-Sepharose.

-YJ'E - 22 -

(D-B) In the same manner as in (D-A above except that DBA was used in place of PNA, DBA-Sepharose was obtained.

5 (2) Production of GRA

(a) BT-1 (Burkitt Lymphoma) cells (1.3 × 10 ⁸ ) were washed 3 times with physiological saline and then 30 ml of a 0 _r 01M tris-saline

10 acid buffer (pH 7.4) containing 2% Triton X-100

(manufactured by Wako Pure Chemical Industries Ltd.), 0.85% NaCl, 2mmol CaCl 2 and 2mmol MgCl. The mixture was stirred for 15 minutes at 4 ° C and then subjected to a centrifugal vacuum deposition at a rate of 100,000 g. Of 28 ml of the supernatant liquid obtained, a 14 ml portion was added. was passed through a column (diameter 0.5, length 1 cm) for affinity chromatography, the column being filled with PNA agarose beads (manufactured by Maruzen Co., Ltd.),

2Q previously equilibrated with a tris-hydrochloric acid buffer (pH 7.4) containing 0.1% Triton X-100, 0.85% NaCl, 2 mmol CaCl ₂ and 2 mmol MgCl ₂ .

, the goods, sent. After washing with the same buffer as used above, with a 0.01M tris-salsoic acid buffer (pH 7.4) containing 0.1M lactose, 0.85% NaCl, 2 mmol CaCl ₂ , 2 mmol MgCl ₂ and p, 1% Triton X-100 eluted. The proportion thus eluted

4 1 2 9 O 4

was dialyzed with a 0.1 M tris-hydrochloric acid buffer solution containing 0.85% NaCl, 2 mmol MgCl ₂ and 2 mmol CaCl _{2 for} 48 hours to obtain 17 mg of a GRA solution. In this GRA solution, the amount of protein and the amount of sugar according to the Folin-Lowry method or by the phenol-sulfuric acid method was determined j these amounts were 644 ug or 120 ug. This product is hereinafter referred to as "GRA-1".

(b) -, C3 / HE mice mouse cancers ^{I <iMT} - ^cells d * 10) were washed 3 times with physiological saline and then 30 ml of a 0.01 M tris-hydrochloric acid buffer solution (pH 7.4) containing 2% Triton X-100, 0.85% NaCl, 2 mmol CaCl ₂ and 2 mmol MgCl ₂ /. The mixture was stirred at 4 ⁰ C for 30 minutes. Subsequently, the mixture of an ultrafine centrifugal separation was applied at a rate of 100,000 g for 2 hours

The supernatant was dialyzed overnight with a 0.1 M tris-hydrochloric acid buffer (pH 7.4) containing 0.85% NaCl, 2 mmol CaCl ₂ and 2 mmol MgCl ₂ . The thus-dialyzed liquid was concentrated to 3 ml, and a 1 ml portion was passed through a column (diameter 0.5, length 2 cm) filled with the same PNA-Sepharose as used before and filled with tris Hydrochloric acid buffer (pH 7.4) containing 0.05% Triton X-TOO, 0.85% NaCl, 2 mmol CaCl _2, and 2 mmol MgCl ₂ , was subjected to affinity chromatography. After thoroughly washing with the same buffer solution as before, elution was carried out

, '^; .ϊ J * •1! * \ sw »*?

an O _r 1 M tris-hydrochloric acid buffer solution (pH 7, A) containing 0.1 M lactose, 0.85% NaCl, 2 mmol CaCl ₂ , 2 mmol MgCl _2, and 0.005% Triton X-100, and the portion thus eluted became Dialyzed for 48 hours with a 0.01M tris-hydrochloric acid buffer (pH 7.4) containing 0.85% NaCl, 2 mmol CaCl ₂ and 2 mmol MgCl ₂ . 2 ml of a GRA solution were obtained. In this GRA solution the protein content was 156 μg and the sugar content 94 μg. This solution is hereinafter referred to as "GRA-M-1". 10

(c) Approximately 120 g (wet weight) of KATO-III was homogenized in a Waring blender in 100 ml of PBS. After centrifuging at 100,000 g for one hour, the pellets were dissolved in 100 ml of a 2% Triton X-100 solution in 0.01M Tris-HCl buffer (pH 7.6) containing 0.15M NaCl. The supernatant solution obtained by centrifugation at 100,000 g for 1 h was added to a column of PNA-Sepharose 4B (0.8x15 cm) previously treated with 0.015% Trito X-100 in 0.01M Tris-HCl (pH 7, 6) equilibrated with 0.15M NaCl was applied. After washing with 50 ml of the buffer, GRA was eluted with the buffer containing 0.1M lactose. The eluted GRA was dialyzed against 0.85% NaCl, concentrated by Sephadex Pharmacia Co. and at

25 -20 C stored before use.

The amounts of protein and sugar determined in the same manner as in (a) above were 2.0 mg and 0.8 mg, respectively. This product is hereinafter referred to as "GRA-2".

30 ''

(d) As in (c), the following GRA samples were obtained:

2 3 ϋ 4-25- Amount (g) excised 24 GRA Sugar content (mg) Table 3 33 Breast cancer 5 26 Protein content (mg) 0.09 GRA Material · QG-46 29 0.5 sample source QG-90 200 0.5 GRA-3 BT-a Ra j i 14.4 0.25 0.38 GRA-4 MMT 85 0.6 0.54 LLC 25 1.0 0.45 GRA-5 MH-135 0.78 28.4 GRA-6 X-5563 11.3 0.07 GRA-7 0.06 0.35 GRA-M-2 0.65 0.23 GRA-M-3 0.56 GRA-M-4 GRA-M-5

(e) In the same manner as in (c), but using NKN-45 (about 29 g) in place of Kato-III and using DBA-Sepharose obtained according to (1) -B, instead of PNA-Sepharose 4B and eluting with N-acetylgalactosamine gave a GRA preparation with a protein content

j of 0.03 mg and a sugar content of 0.01 mg. This will be j

hereafter referred to as GRA-8. j

(f) GRA-3 prepared according to (d) (5 ml) was applied to a DBA-Sepharose column and treated with Tris-HCl buffer (0.015% Triton X-100, 2 mM MgCl ₃ , CaCl ₃ , 0, 85% NaCl) eluted,

to obtain 4 ml fractions No. 1-1: 2. Fractions 1-3 are referred to as "GRA-3-A" and fractions 4-12 as "GRA-3-B". Then, the column was eluted with the same buffer but containing 0.1M N-acetylgalactosarine to give fractions designated "GRA-β-C"

become.

(g) SDS gel electrophoresis

Each of the above GRA preparations was electrophoresed using the method of Fairbanks et al: Biochemistry, Vol. 10, p. 2606, 1971.

The results are shown in FIGS. 18 to 22.

In Figs. 18-19, numbers 1 to 5 denote the following: 1. , : Standard; 2. , , GRa-M-3 .; 3 .. .GRA-7; 4 ... GRA-1; and 5 ... GRA-2.

In Fig. 20, numbers 1 to 4 denote:! ... standard; 2 ... GRA-M-2; 3 ... GRA-6; 4 ... GRA-fifth 15.

In Fig. 21, numbers 1 to 3 are as follows: 1 ... GRA-M-4; 2 ... GRAE-M-5; 3 ... Standard:

In Fig. 22, numbers 1 to 4 denote: 20 1 ... GRA-3; 2 ... GRA-3-A; 3 ... GRA-3-B, 4 ... GRA-3-C.

Fig. 18 shows the state of GRA after protein staining according to C.B.B. (Fairbanks et al: Biochemistry, Vol. 10, p. 2606, 1971)

 25

Figures 19 to 21 show the condition of GRA after treatment with a sugar color reaction according to the PAS method (R.M. Zacharius et al: Anal. Biochem., Vol. 30, p.128 (1962)).

Fig. 22 is a schematic representation of the results of staining according to the aforementioned C.B.B. method.

As standard, the following were from Biorad Lab. Calif.USA substances obtained: 35

29 0 4

4 ί Ί y O 4 - 27 -

K Dalton; myosin

"β-galactosidase

92.5 "; phosphorylase

66.2 "; BSA

"Ovalbumin

21.5 β; Soybean trypsin inhibitor

Reference Example 3

(a) The spleen obtained (of the Japanese monkey from the. Plymates Japan Co., Ltd.} was excised and 2 times with a RPMI-1 64 0 culture medium (manufactured by Flow Laboratory Co. Ltd. _f) washed. The Cells were filtered using mesh (manufactured by Millipore, Inc., 150 mesh) and subjected to a specific gravity centrifugal [specific gravity

1.076) to obtain 2 1 of 2 × 10 / ml lymphocytes. The lymphocytes thus obtained were washed 3 times with an RPMI-1640 culture medium and the number of lymphocytes was adjusted to 5 × 10 -5 / ml using the same medium as before containing 10% FCS. Then, they were allowed to stand in a carbon dioxide fermentation apparatus for 1 hour at 37 ° C. The supernatant lymphocytes were recovered

24129 0 4 -μ-

and the number of lymphocytes was adjusted to 1 × 10 / ml using the same culture medium as stated above containing 1% FCS. Subsequently, 1 μg / ml of indomethacin (manufactured by Sigma Laboratories Inc.) and 0.2% of PHA-P (manufactured by Difco Co.) were added, and then cultivation in a carbon dioxide gas fermentation apparatus was carried out at 37 ° C for 48 hours. After centrifuging for 10 minutes (3,000 rpm), the supernatant liquid formed was recovered and sterilized by filtering through a Millipore filter (manufactured by Millipore Inc., 0.2 μπι) and allowing 2 1 TCGF received.

(b) As a source of TCGF, the supernatant of mixed-culture peripheral blood lymphocytes from 10 healthy donors was used. Non-contiguous lymphocytes manufactured by CF. (Conray, Ficol (Japan Immunoresearch Co.)), by adsorption on plastic surfaces at 37 ° C for 1 h were suspended in RPMI 1640 medium containing 1% FCS (1.5x10 ° cells / ml) and 0, 2% PHA-P, INdomethacin (1 μg / ml) and human B cells (BT-1) (1.5 × 10 ⁵ cells / ml), pretreated with mitomycin C (50 μπι / ml) incubated. After 4 8 hours, the supernatant culture was harvested and used as the source of TCGF (H.Inoue et al, Scand J. Immunol 12, pp. 149-145 (1980)).

Reference Example 4

5 (1) human peripheral blood lymphocytes

50 ml of blood obtained from healthy adults or patients with various cancers by heparinization were subjected to centrifugal separation using Ficol Pack (manufactured by Farmacia Japan Co., Ltd.) to obtain 5 × 10 peripheral blood lymphocytes.

15. (2) mouse spleen lymphocytes

The spleen of a C3H / He mouse (male, 6w) was excised and twice with an RPMI-164O culture medium

.- 30 -

·, washed. Then the spleen was loosened by means of a hypodermic needle and passed through a 100 mesh stainless steel sieve to remove large parts. The thus-filtered cells were washed twice with the same culture medium as above, and then subjected to centrifugal separation at a rate of 1,200 rpm for 10 minutes to obtain 4 × 10 6 mm of spleen lymphocytes.

example 1

GRA-1 (amount of protein 40 μg / ml), amount of sugar 7.5 μg / ml, obtained according to Reference Example 2 (2a)) was reduced to 1,000 times the original volume with an RPMI 1640 culture medium containing 15%. FCS _{f to} obtain a sensitizing culture medium.

Human peripheral blood lymphocytes (5 × 10/5 ml) obtained in Reference Example 4 (1) were added to 5 ml of the sensitizing culture medium prepared above, and this was then placed in a laboratory dish and kept at 37 ° for 2 days C cultivated. Subsequently, a further cultivation on an RPMI-1G4 0 culture medium containing 20% TCGF and 15% FGS, obtained according to Reference Example 3 _r for a further 5 days, wherein 20 ml of a killer cell solution containing

1 χ 10 ⁶ killer cells / ml, received. This is referred to below as GRA-1-KT.

90 4

Example 2

The mouse lymphocytes obtained in Reference Example 4 (2) were adjusted to a number of 5x10 / ml using an RPMI-1640 culture medium containing 15% FCS. To this was added GRA-M-1, obtained according to Reference Example 2 (2b), so that the final product contained 1.5 μg / ml protein and 0.9 μg / ml sugar. A 5 ml portion of the obtained mixture was cultured in a laboratory dish (60 mm χ 15 mm, manufactured by Falcon Co.) at 37 ^° C. for 2 days. The formation of clones was observed. The portion was further cultured in an RPMI-164O culture medium containing 15% FCS containing 20% by volume of TCGF (manufactured by Japan Immuno Research Laboratories Co., Ltd.) for 4 days to obtain 50 ml of a cell liver solution containing Received 1 χ 10 killer cells / ml. This is referred to below as GRA-M-1-KT.

Example 3

Lymphocytes (5x10) of peripheral blood of healthy donors were incubated in an RPMI-1640 medium containing 40 ng / ml (protein content) of GRA-2 and 15% FCS at 37 ° C. On the second day, the aforementioned human TCGF was added to the medium until its concentration reached 20%, and incubation was continued for 3 days to obtain killer cells, hereinafter referred to as "GRA-2-K-T". '

./ Ί Ii MJk _ 32 -

Example 4

Killer cell preparations GRA-8-KT, GRA-3-AKT and GRA-3-CKT were prepared in the same manner as in Example 1 using fRA-8, GRA-3-A and GRA-3-C (protein content: 50 ng / ml / and the TCGF obtained in Reference Example 3- (b).

10 Example 5

C3HE / mice (female 8 weeks old) were implanted intradermally with X5563 cells (10) of the same strain and after 7 days the tumor was surgically excised. After another 7 days, X5563 cells (10) of the same strain were inoculated and mice resistant to inoculation were designated as immune mice.

The spleen cells of the immune mice and C3H / HE mice were prepared using commonly used methods.

6 '

Each lot of spleen cells (5x10 / well) was sensitized with 40 ng / ml (protein content) of GRA-M-5 in RPMI 1640 medium containing 15% FCS for 5 days to give killer cells.

Killer cell preparations obtained from normal spleen cells are hereinafter referred to as "GRA-M-5-Kt-1", and those obtained from immune spleen cells are labeled "GRA-M-5-KT-2". designated.

Example 6

Human peripheral blood lymphocytes (5x10) were incubated in 5 ml of RPMI-1640 medium containing 50 ng / ml (protein content) GRA-1 at 37 ° C for 2 days. On the third day, the lymphocytes were transferred to the RPMI 1640 medium containing 10% serum from the lymphocyte donor, and 0 to 100 ng / ml (protein content) of GRA-1, and the incubation was continued after another 5 days the killer cell preparations shown in the table below were obtained.

Table 4 GRA-Kontentration (ng / ml)

First day Day 3 killer cells

50 .20 50

50

50

50

50 25 50

Example 7

(a) Peripheral blood lymphocytes (PBL) of various cancer patients after surgery were sensitized with GRA-3 to obtain killer cell preparations. PBL (5x10) from the patients was in RPMI 1640 medium ,. containing 50 ng / ml (protein content) of lRA-3 was incubated for 2 days and then incubated in RPMI 1640 medium containing 20% TCGF and 15% FCS for a further 5 days to obtain the killer cells shown in Table 5.

0 GRA-1-K-T-1 3.6 GRA-1-K-T-2 3.2 GRA-1-K-T-3 6 GRA-1-K-T-4 12.5 GRA-1-K-T-5 25 GRA-1-K-T-6 50 GRA-1-K-T-7 200 GRA-1-K-T-8

Table 5 Peripheral blood lymphocytes

Cancer P-GRA D-GRA Days after the killer cells

surgery

Gastric cancer + + 14 GRA-3-K-T-1

Gastric cancer + + 35 GRA-3-K-T-2

Breast Cancer + - 21 GRA-3-K-T-3

Breast Cancer + - 7 GRA-3-K-T-4

Breast Cancer + - 35 GRA-3-K-T-5

Gastric cancer + - 21 GRA-3-K-T-6

In the above table, P-GRA and D-GRA show the locality of GRA expressed as malignant tissue

(taken by surgery) of cancer patients from which PBL had been collected in the same manner as in Reference Example 1, 2- (b). P-GRA and D-GRA were obtained by using FITC-PNA and FITC- respectively. DBA. The days after surgery indicate the time when BPL was collected after surgery.

(b) PBL (5x10) collected from breast cancer patients 21 days after surgery was incubated in RPMI 164O medium containing 50 ng / ml (protein content) of GRA-3 and 10% serum from the patients for 7 days to receive the PBL to sensitize and to obtain killer cell preparations. These are referred to below as GRA-3-K-T-7.

y υ 4 - ³⁵ -

Example 8

(i) GRA-M-1 obtained according to Reference Example 2 (2b) was diluted with physiological saline so that the content of sugar was 1.0 μg / Inl and that of protein was 1.6 μg / ml. In this way, an anti-cancer drug No. 1 was obtained.

(ii) A cancerous tissue of C3H / He spontaneously occurring breast cancer was sterilized into a 5 mm cubic lump and transplanted under the dorsal skin of 10 C-H / He mice of the same strain as above (7 weeks old, male). Seven days after the transplantation, the fixation and multiplication of the cancer was confirmed. 5 of these mice received the subcutaneously prepared according to (i) anti. No. 1 cancer drug at a dose of 300 μΙ / day every two days. The remaining 5 mice were used as untreated control. Ten days after the first administration, the tumor was surgically removed and its mean weight measured. At the same time, a pathohistological examination was performed.

25 treated group: 22.3 mm ³ (Fig. 14) control: 162.7 mm ³ (Fig. 15)

This means that the tumor decreased by 86/3%.

In the control group (FIG. 16), bird's nest-like cancerous bodies were formed, the nature of the tissue

O 4

corresponded to a mark-like canal-shaped cancer and a multiplication of the cancer cells over the entire tissue was determined. On the other hand, in the group treated with the agent (Figure 1), the cancer cells caused liquefaction crosstica at the sites where the cancer cells had formed, and calcification and firosis were noted with only a very small amount of cancer cells remaining , This confirmed the anti-cancer properties of the anticancer agent according to the invention,

Test Example 1

GRA-1-KT (1 μΐ) obtained in Example 1 _# was placed on a microplate (manufactured by Falcon Corp.) and allowed to stand at room temperature for 15 minutes. Then, 4 μl of FCS (manufactured by Falcon Corp.) was added, and the mixture was allowed to stand at room temperature for 30 minutes. Neuraminidase-treated sheep red blood cells (SRBC) adjusted to a number of 1χ 10 / ml, and 5 μΐ of a 0.1M phosphate buffer (pH 7.2) to which 0.85% NaCl had been added were added and then became subjected the plate to centrifuge separation at a rate of 6,000 rpm for 5 minutes. Then the plate was inverted and unreacted SRBC was removed. A staining solution (Brilliant Cresyl Blue, manufactured by Merck & Co.) was added for staining the lymphocytes and a rosette-shaped

9 0 4 -37-

Positivity was detected. At least 98 % showed the rosette-shaped positivity (T cells).

Test Example 2

(a) Of the cancer cells shown in Table 1, the following 5 cell strains having different GRA positivity ratios were used as human cancer cells to be controlled:

15 cancer cells to fight:

No. 1 BT-1 (Burkitt Lymphoma)

No. 2 Daudi (Burkitt Lymphoma)

No. 3 KATO-III (stomach cancer)

20 No. 4 MKN-45 (stomach cancer)

# 5 MOLT (T-cell Leukemia)

On a microplate (manufactured by Falcon Corp.), 5 × 10 5 of the cancer cells to be controlled were applied per well by centrifugal deposition at a rate of 8,000 rpm for 5 minutes.

Then, 4 χ 10 per well of the GRA-I-K-T obtained in Example 1 was carefully added and then

was incubated for 1 hour. 30

The killing activity was according to the degree of the

L · J U U - 38 -

, Fouling determined and / or evaluated as follows:

++ a noticeable kill activity is detected - '

posed

+ a kill activity is detected + a low kill activity is detected - a kill activity can not be determined

become. 10

In a control group, unsensitized human peripheral blood lymphocytes prepared in the same manner as in Example 1 but without the use of GRA were used. The results are shown in Table 6.

From Table 6 shows that the invention

killer T cells formed a strong GRA-specific

have a cytotoxic activity. 20

Table 6

to be controlled cancer cell (Fig. D GRA positivity (%) Assessment lag formation the GRA-1-K-T Daudi : (Fig. 3) 93.1 ++ (Fig. 2) group KATO-II3 (Fig. 5) - 57.3 + (Fig. 4) BT-1 (Fig- 7) 50.1 ++ (Fig. 6) MKN-45 (Fig. 9) 1.0 + (Fig. 3) MOLT 0.6 - (Fig.1 0) Control group BT-1 50.1 - (Fig.1 D

- 40 -

(b) The same cancer cells to be used as used in (a) (3.2 × 10) were mixed with 8 × 10 ⁵ GRA-1-KT (cell ratio 5/1) and the cell mixture formed (total number 4 × 10) was cultured in RPMI-164O medium containing 15% FCS. After 1 hour, the number of remaining cells was counted and the inhibition ratio was calculated according to the following equations:

Cytotoxicity (%) logo CNRS logo INIST

Number of cells M / after cultivation > number of cells before cultivation (4 χ 6

The results are shown in Table 7.

Table 7

to combat de cells Number before cultivation 10 ⁶ the cells after the Kul tivierung x 10 ⁶ 'Cyto- toxicity (%).' Daudi 4 χ 10 ⁶ 2.9 ⁶ 10 ⁶ 28 KATO-III 4 χ 10 ⁶ 3.7 ⁶ 10 ⁶ 7.5 BT-1 4 χ 10 ⁶ 3.2 ⁶ 10 ⁶ 20 MKN-45 4 χ 10 ⁶ 3.9 ⁶ 10 ⁶ 2.5 MOLT 4 χ 4.2 -5

(c) The procedure of (b) was repeated except that the mixing ratio of GRA-1-K-T. was changed to 5/3 to be combated cancer cells. The results are shown in Table 4.

Table 8

to combat de cells Number <before cultivation 10 ⁶ Another cell after the Kul tivierung ⁶ 10 ⁶ Cytotoxicity. (%) Daudi 4 χ 10 ⁶ 3.6 ⁶ 10 ⁶ 91 KATO-III 4 χ 10 ⁶ 3.6 ⁶ 10 ⁶ .24 BT-1 4 χ 10 ⁶ 1.4 ⁶ 10 ⁶ 65 MKN-45 4 χ 10 ⁶ 3.7 ⁶ 10 ⁶ 7.2 MOLT 4 χ 4.1 -3

Test Example 3 '

C3H / He spontaneously bifustkrebsige mice received subcutaneously GRA-M-1-KT-, obtained according to Example 2, in a dose of 2 χ 10 / 0.3 ml corn 3 times a week every other day. After 10 days, the focus was taken out and examined. In this experiment, it is found that GRA-1 KT has a high binding activity against Daudi, KATO-III and BT-1, but only a low binding activity against MKN-45 and MOLT.

As shown in Fig. 12, infiltration of lymphocytes into the cancer cells took place and breakage of the tumor area was detected. From Ig Fig. 13 it is apparent that calcification of the tumor surface has taken place, and it can be seen from this that the cancer cells of the present invention have antitumor activity.

Comparative Example 1

In this example, cancer cells were used per se as specific antigens instead of GRA for the method according to the invention.

Cancer cell-sensitized lymphocytes were obtained in the same manner as in Example 1 except that instead of GRA, BT-1, Daudi, KATO-III or MKN-45 were used in an amount of 1 χ 10 per laboratory dish.

With these lymphocytes became the c.ytotoxische

c 9 O

Activity was examined in the same manner as in Test Example 2 (a), and the results are shown in Table 9.

From Table 9 it can be seen that the lymphocytes prepared above have no cytotoxic activity.

Table 9

to be controlled cell

BT-1 Daudi KATO-III MKN-45

Cells used to sensitize lymphocytes

BT-1

Daudi

KATO-III

MKN-45

Test Example 4

In the same manner as in Test Example 2- (b), the anticancer activity of GRA - (- K-T, .GRA-3-A-K-T and GRA-3-C-K-T obtained in Example 4 was determined, and the results are shown in Table 10.

0 4 - 44 - GRA-3-C-K-T Table 10 5.0 k Cytotoxicity 4.0 cell GRA-8-K-T GRA-3-A-K-T 20 KATO-III 8.0 5.0 0 BT-1 4.3 5.0 MKN-45 20 5.0 MOLT 0 Ό

Test Example 5

(a) The cytotoxicity of the killer cell preparations obtained in Example 6 was determined by Cr staining test (J. Immunol., 122, 2527-2533 (1979)). To this was added 50 μCi of radioactive Cr (Japan Isotope Association) to KATO-III (2x10) and the cells were incubated for 1 h at 37 ^° C. in RPMI-1640 medium and then washed by centrifugation to obtain Cr-labeled target cells , The killer cells (effector cells) (2x1O ⁶⁾ were added to the target cells (1x10) (the ratio of E / T was 20/1) and the mixture wur.d 4 h at 37 ⁰ C in RPMI-1640 medium incubated. The supernatant was collected by centrifugation and the radioactivity was counted by a liquid intiator.

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

¹ Specific ⁵¹ CR release (%)

- (Release in Trial) - (Spontaneous Release) iqo (Maximum Release) - (Spontaneous Release)

The maximum release shows the radioactivity when all cells are resolved.

The results are shown in Table 11: 5

Table 11

Killer cells Specific release

· ⁵¹ Cr (%)

GRA-1-K-T-1 13

GRA-1-K-T-2 25

GRA-1-K-T-3 22

GRA-1-K-T-4 20

GRA-1-K-T-5 22

GRA-1-K-T-6 25

GRA-1-K-T-7 27

GRA-1-K-T-8 32

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

(b) Cytotoxicity of GRA-2-K-T obtained in Example 3 was determined in the same manner as in (a) above by means of the Cr release test. The specific Cr release was 14.3% for a Cr-labeled KATO-III as target cell (E / T = 20/1).

Test Example 6

(a) Using KATO-III (E / T = 20/1), the cytotoxicity of killer cells obtained in Example 7- (a) was determined in the same manner as in Test Example 2- (b).

The results obtained are shown in Table 12:

  '.-.

Table 12 10

* Killer cells % Cytotoxicity GRA-3-K-T-1 38.0 GRA-3-K-T-2 18.2 GRA-3-K-T-3 20.9 GRA-3-K-T-4 23.2 GRA-3-K-T-5 24.5 GRA-3-K-T-6 20.2

(b) The cytotoxicity of GRA-3-KT-7 obtained in Example 7- (b) was determined using Cr-KATO-III (E / T = 29/1) as target cells in the same manner as in the Test Example 5 determined. The specific ⁵¹ Cr release of the killer cells was 25.5%.

Test Example 7

The cytotoxicity of killer cells obtained in Example 5 was determined by the Cr release test in the same manner as in Test Example 5. The target cells used were Cr-labeled X5563 cells. As a control, lymphocytes obtained in the same manner as in Example 5 except that the sensitization of the spleen cells had been carried out with 1x10 ⁵ / well of mitomycin C-treated X5563 cells ( ⁵ × 10 ⁶ / ml of

\ Z J U «♦ - 47 -

X5563 cells were treated with 50 μg / ml mitomycin C for 60 minutes) and place of GRA-M-5.

The results obtained are shown in Table 13!

table 4 13 0: 1 20: 1 1 0: 1 specific 1 1 2.7 8.4 0.0 6.3 20.6 1 0.0 5.7 3.7 0.0 ⁵¹ Cr release (%) killer cells E / T ratio GRA-M-5-K-T -1. GRA-M-5-K-T-2 control

Test Example 8 (H-2 Assay )

GRA-M-1, GRA-M-3 and GRA-M-4 obtained in Reference Example 2 were subjected to serial dilution with PBS (0.85% NaCl) to obtain samples.

National Institute of Genetic Research anti-H2 serum and the above samples were incubated and incubated at 4 ° C for 2 hours and then target cells were added according to the anti-H-2 serum used. Spleen cells or lymph node cells obtained from B10 (H-2 ^b) and B10-BR (H-2 ^k) mice by conventional methods were used as target cells. After washing the cells with PBS, complement (rabbit) was added to the cells and the cells were incubated for 1 h at 37 ° C and stained with 0.2% trypan blue PBS to determine percent cytotoxicity. The anti-H-2 serum was used at maximum dilution so that it exhibited at least 95% cytotoxicity in absence of GRA. *

j 29 0 4

The blocking effect of GRA in the systems is shown in Table 14:

Table 14

GRA GRA-MI

Anti-H-2 serum, concentration

D-23 ΓΑηίί-Η-2Κ ^Λ ), or

X80

D-32 (Anti-H-2D *), Χ300

GBA-M-3 D-33 (anti-H-2K ^b), Χ600

o <OCE _K D-2 (anti-H-2D), X80

GRA-M-4 D-23

D-32

The results are shown in Table 15.

Table 15

or

, Χ80, Χ300

Target cells

BlOBR (H-2 ^k) splenic cells

BIO (H-2 ^D 1 spleen cells

BIO-BR (h-2 ^K) lymph node lines

Aiiti-H-2 X2 X4 T % Cytotoxicity X16 the  1 sample  X128 X256 X512 0 * serum - 13 dilution 12 X32 X64 13 14 13 14 13 GRA - ... - 97 X8 96 13 14 96 97 96 96 14 I D-23 - 95 14 95 97 97 95 95 96 95 13 D-32 - 19 99 14 95 94 12 11 12 13 14 , - - 98 95 99 12 15 99 99 99 99 15 II D-33 - 95 15 95 99 98 95 97 95 95 17 D-2 100 100 98 100 95 97 - - 100 10 D-23 99 99 96 99 - - - - 99 10 III D32 GRA 100 : GRA-M - - Annotation -. 99

GRA II: GRA-M-3 GRA III: GRA-M-4

ii * n means% cytotoxicity when only complement was used.

9 0 4

From the results in Table 15, it can be seen that GRA-M-1, GRA-M-3 and GRA-M-4 do not have H-2.

Test Example 9

5. ·. - .. '

C57BL / 6 mice were tested under S.C. LLC (2x10) of the same strain and after 6 days 1ng (protein) GRA-M-3 obtained according to Reference Example 2- (d) was subcutaneously administered. This administration was repeated once daily at the same dosage for 4 days. The day after the last administration, the tumor cells were excised and weighed. As a control, animals were used, to which only physiological saline had been administered. Each test group consisted of 5 animals.

is _ '

As a result, it was found that the average cancer weight in the control group was about 500 mg. In the GRA-M-3 treated group, 3 showed disappearance of the tumors and two showed an average tumor weight of 100 mg.

Test Example 10

C57BL / 6 mice were immunized subcutaneously with 1 ng (protein) GRA-M-3 obtained in Reference Example 2 (d) once daily for 3 days, and on the 5th day, spleen cells were collected from the animals to obtain effector cells , A mixture of effector cells and Lewis lung carcinoma (LLC) as target cells in a ratio of E / T = 50/1 was prepared and a portion (1x10) thereof was transplanted into mice of the same strain and a Winn assay was performed (J. Immunol., 86, pp. 228-239 (1961)).

The results obtained are shown in Table 16. 35

/ M if /.

awake table 16 Day cancer (%) Day 20 Mortali effector Day speed of the 3.1 55.4 98.1 19 day 20 t / fruppe cell 5.7 21.4 39.3 15 day 17 day 18 1, 76, 96, 4 8 4 0/10 4/10 4/10 A B C 5, 39, 65, , 5 5,7, 5 37,1 rl 61,7

Note: Group A is the spleen cells of GRA-M-3 immune mice;

Group B are the spleen cells of normal mice; Group C is the spleen cells of mice, 10 days after the transplantation of LLCCIxIO);

'' ^:

The cancer growth rate was calculated according to the following equation:

TT Cancer growth rate (%) = - ^A - = - χ 100

^N

where T is the thickness of the sole on the side where the cancer was transplanted and T is the thickness of a normal sole.

Test Example 11

C3H / He mice were immunized with 4.5 μg (protein) of GRA-M-4 obtained in Reference Example 2- (d) and 0.1 ml Freund's Complete Adjuvant sacrococcygeal. After 2 weeks, lymph node cells were collected in the usual manner and used as responder cells for the determination of proliferative response by GRA-M-4.

ί · j

For this purpose, responder cells (4x10) were incubated with RPMI 1640 medium containing 15% FCS in the presence of GRA-M-4 for 5 days. During the last 18 hours of the incubation period, IuCl of H-thymidine (H-TdR) was added to the medium and incorporation into the cells was counted.

The results are shown in Table 17.

Table 17

GRA-M-R

Konzentra 3-H-TdR incorporation

tion (ng / ml) (mean cpm + standard error)

S.I.

control

attempt

0.5

5 20 40

5,302 + 1,761

7,903 _ + 1,290

10.076 _ + 936

10,686 ± 429

10,615 _ + 1,270

8,565 + 1,419

1/5 1/9 2.0 2.0 1/6

Note: "S.I." is the stimulus index expressed by experiment / control

Test Example 12

Delayed hypersensitization (DTK) response of C3H / HE normal mice, X556 3 immune mice and MH134 immune mice, obtained in the same manner as in Example 5, and of GRA-M-4 immune mice and GRA-M-5 immune mice Obtained in the same manner as in Test Example 11 was determined by the footpad reaction (FPR). For this purpose, GRA- or MMC-treated tumor cells were introduced into the skin of the footpad in the

The hind leg of the animals was entered and the swelling of the sole of the foot 24 h after the treatment was detected. The DTH response was calculated by checking the swelling

- 2

before treatment of the after treatment (10 mm) withdrawn.

The results obtained are shown in Tables 18-22.

Table 18

_2

Medium foot growth (10 mm)

Experiment normal mice MH134 immune mice

1 2.8 13.6 2 12.4 32; o 3 3.6 3.6 4 3.2 22. 0 5 6.8 27.6

Note: Group 1: syngenic normal spleen 1x10 / 20 μΐ-

Medium (Hanks solution)

Group 2: MH134 cells 1x10 ^6/20 μΐ medium Group 3: medium 20 μΐ Group 4: GRA-M-4, 0.8 \ xq (protein) / 20 μΐ medium Group 5: GRA-M-4, 0, 4 μg »

Table 19

Medium footed ananas (10 mm)

Normal Mouse X5563 Immune Mouse MH134 Immune Mouse

1 2 3

5.4 0.9 2.0

4.3 16.9

1.1 24.3

Note: Group 1; Medium (Hanks solution) 20μ1

Group 2; GRA-M-4, 4 μg (protein) / 20 μΐ medium, group 3; GRA-M-5, "

Table 20 GRA-M-4 immune mouse -0.3 24.6 attempt _2 Medium foot growth (10 mm) 1 2 normal mouse 1.7 5.1

Note: Group 1; Medium (Hanks solution 20 μΐ

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

Table 21

Experiment 1 2 3

-2

Medium foot growth (10 mm)

Mormal mouse -1.8 6.3 6.8

GRA-M-4 immune mouse

0.6

20.0

6.3

Notitie:

Group 1; Medium (Hanks solution) 20 11

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

241290 4

Group 3; 4 μg (protein) / 20 μΐ medium of

Part passing through a PNA column at the time of GRA-M-4 (hereinafter "CP.")

table 22 middle -2 foot growth (10 mm) attempt normal mouse GRA-M-4 immune mouse 1 2 3 4 2.4 2.6 2.4 5.5 17.7 1.8 18.9

Note: Group 1; Medium (Hanks solution 20 μΐ

Group 2: GRA-M-4, 3.8 μΐη (protein) / 20 μΐ medium

Group 3: 3.8 μg (protein) / 20 μΐ of CP. (siehie

above). ; ^s

, '''.\' .

Group 4: 3.8 μg (protein) of GRA-M-4 and 3.8 μg

(Protein) / 20μΐ medium of CP. i

reference test

X5563 immune mice were obtained in the same manner as in Example 5. The spleen cells of these immune mice were used as effector cells and the effector cells (10) and the i i target cells. (X5563, 10) were transplanted together on mice of the same strain. Then, a Winn assay was performed in the same manner as in Test Example 10.

24 129 0

The results obtained are shown in Fig. 23, in which the abscissa indicates the days and the coordinate the mean cancer size (cm ² ) + ^ standard error, and in which the different labels have the following meaning:

-. , ^: _ :. , ''. · _ ': " ^: .'; - -" - '.. f * ;.'.;' · '

m · · : group to which no effector cells are given

were; ο · --- o: group to which untreated effector

Cells were given; ' M :

^. . ^: Group to which effector cells treated with rabbit complement were added; x- c: group to which effector cells treated with Anti-Thy 1 (New England Nuclear Co., USA). were, and given to the rabbit complement;

has been; , /. '.' · ·. ';''','-' ϊϊί, -]. ' "" D "group, to the effector cells treated with Ariti-Lytj 1 (New England Nuclear Co., USA); were, and to the rabbit complement _s

2Ö \. '.' ^: .. ^ 'was; , '' . '. ^: ''''.'':^'''.'^{'^]':} \. ^: ' ^:: ' B 'B: group to which effector cells treated with anti-Lyt (New England Nuclear Co., USA) and too. the rabbit complement was given.

From the results of Fig. 23, it can be seen that Lyt 1 type T cells play an important role in the mechanism of ±τ ± vivo effect in tumor immunity .

Furthermore, it is known that the DTH response is caused by Lyt-1 T cells (J. Exp. Med. 143, pp. 1543-39 (1976)).

Claims (8)

INVENTORY CLAIM :! 1. A method for producing a glykoverwandten antigen ge ke η η ζ e-Λ ch net characterized in that one prepares the glykoverwandte antigen by producing by homogenizing or solubilization of cancer cells cell membrane components, characterized in that the cell membrane components with a lectin which specifically binds to a terminal galactose or N-acetylgalactosamine, combines to collect the complex formed by separating the lectin from the complex to obtain a lectin-free cell membrane component and, if desired, adding a pharmaceutically acceptable carrier. ! j 2. The method according to item!, Characterized in that the cancer cells are human cancer cells. 3. The method according to item 2, characterized in that the lectin is one which combines specifically with terminal galactose. 4. Method according to item 2, characterized in that the lectin is one which specifically binds with terminal N-acetylgalactosamine. 5. Method according to points 1 or 2, characterized characterized by the fact that the glycologically related antigen from the Krebszellmembrankomponente first with a first Lectin specifically with 35, 25) G, 1983 * il234 ;; 24 1 2 9 0 4 - * - 4.10.1983 AP A 61 K / 241 61 066/12 terminal galactose and then isolated with a second lectin that combines specifically with terminal N-acetyl galactosamine 6, method according to item 5 », characterized in that the first lectin is turpentine lectin and the second lectin is dolicho-bean agglutinin. 7. Method according to item 1 or 2, characterized in that the solubilization is carried out using a dissolving agent. S. Method according to point 7 », characterized in that the dissolution agent is a surfactant. 'Is. , , - '· .'. ' } ι; - Method according to items 1 or 2, characterized in that the separation is carried out using affinity chromatography using a column support containing an insoluble support and a lectin mobilized thereon, 10. The method according to item 3, characterized in that the lectin is ürdnußlektin. 11. The method according to item 4, characterized in that the lectin Dolichosbohnenagglutinin istο For this. J H. Pages drawings