DE3249568A1 - Glycol-related antigen, process for its preparation and its use for controlling cancer - Google Patents

Abstract

Glycol-related antigen which was isolated from a cancer cell membrane component and which combines with a lectin which links with a terminal galactose or a terminal N-acetylgalactosamine. It is prepared by obtaining cell membrane components from cancer cells by homogenisation or solubilisation, treating the cell membrane component with a lectin which combines specifically with terminal galactose or a terminal N-acetylgalactosamine with the formation of a lectin/cell membrane component complex, the complex obtained is collected, and the lectin is separated off from the complex, and the lectin-free cell membrane component is collected. The invention also relates to the use of a glycol-related antigen for controlling cancer.

Description

HOFFMANN · EITLE ^ 'PARTNER "" "

PATENT AND LAWYERS O 2 4 S ü D ö

PATENTANWÄLTE DIPL.-ING. W. EITLE DR. RER. NAT. K. HOFFMANN DIPL.-ING. W. LEHN DIPL.-ING. K. FDCHSLE DR. RER. NAT. B. HANSEN · DR. RER. NAT. HA. BROWN. DIPL.-iNG. K. GORG DIPL.-ING. K. KOHLMANN · LAWYER A. NETTE

P 32 49 568.4 39 855 o / sm

OTSUKA PHARMACEUTICAL CO., LTD., Tokyo / Japan

Glyco-related antigen, process for its preparation and its use for combating cancer

Immune responsive effector cells, especially T lymphocytes, that play a major role in a cell-triggered immune response cause rejection of grafts due to a foreign cell antigen, however, cause no noticeable or only very limited immune inhibition against cancer cells. Consequently the cancer cells are not destroyed, but multiply in vivo and ultimately cause the Death of the cancerous carrier.

However, the mechanism in place here is not yet

j I

completely clear and various studies have been made to gain a more detailed understanding of the nature of the material base involved in such a system. For example, cell surface markings on mouse dukemia cells or embryonic carcinoma cells are made using lectins such as Dolichos biflorus agglutinin (DBA) and peanut agglutinin (PNA) in Biochem. Biophys. Res. Comm. 89 (2) 448-455 (1979) Ibid. 96 (4) 1547-1553 (1980),

J. Biochem. 89 473-481 (1981) and Cell 18 183-191, September 1979.

\ BELLASTRASSE 4 · D-8000 MÜNCHEN 81 · TELEPHONE CO89J 911OB7 ■ TELEX O5-29619 CPATHEJ ■ TELECOPER 918356

As far as is known, however, no attempts have been made so far has been undertaken to make new lymphocytes available ;: bodies that can specifically fight cancer cells and that are anti-cancer agents using a Lymphocyte immune response.

Thorough research on the. Carrier's immune response to cancer cells and their use for the treatment of cancer have now shown that - in a cancer cell-specific antigen that is not found in differentiated normal cells, GRA is present, which acts as an immunogen for the carrier and has a very high immunogenicity causing an immune response specific to cancer cells. It has also been found that when GRA is used to sensitize lymphocytes, you get killer cells that specifically act on GRA-containing cancer cells and that you contribute to Administration of the killer cells to a carrier recognize these GRAs and act on GRA-containing cancer cells, destroy them and in this way you can have a very good effect in treatment and prevention of cancer.

The invention relates to a glyco-related antigen isolated from a cancer cell membrane component and that- "combines with a lectin which connects with a terminal galactose or a terminal galactosamine.

The invention also relates to a method for the production of a glyco-related antigen, in which cell membrane components are used The cell membrane component is obtained from cancer cells by homogenization or solubilization

-A-

a lectin, which is specific to the terminal Galactose or a terminal N-acetylgalactosamine combined with the formation of a lectin-cell membrane component complex, treated, the complex obtained 5 collects and separates the lectin from the complex and collects the lectin-free cell membrane component.

According to a further embodiment, the invention relates to an anti-cancer agent containing GRA as active 10 component.

Fig. 1 is a photomicrograph of Daudi cancer cells,

Fig. 2 is a photomicrograph showing the

Shows the formation of coatings of GRA-1-K-T on cancer cells according to FIG.

Fig. 3 is a photomicrograph of KATO-III cells,

Fig. 4 is a photomicrograph showing the formation of plaque caused by GRA-I-K-T Shows cancer cells according to FIG. 3,

Fig. 5 is a photomicrograph of BT-I cancer cells,

Figure 6 is a photomicrograph showing plaque formation by GRA-1-K-T

"i shows on the cancer cells of FIG. 5,

Fig. 7 is a photomicrograph of MKN-45 cancer cells,

Fig. 8 is a photomicrograph showing the

Shows the formation of coatings by GRA-1-K-T on cancer cells according to FIG. 7,

Fig. 9 is a photomicrograph of MOLT-

Cancer cells,

Fig. 10 is a photomicrograph showing the formation of GRA-1-K-T deposits on cancer cells according to FIG. 9 shows,

Figure 11 is a photomicrograph of BT-1 exposed to a mixture of unsensitized human peripheral blood lymphocytes., π was treated,

Figures 12 + 13 are each microphotographs of cancer cell tissue a cancerous mouse that GRA-M-IHKtT. \ was obtained,

Fig. 14 is a photograph showing one

Cancer status in a group of cancerous mice given GRA-M-1

Fig. 15 is a photograph showing the cancer state in a group of cancerous mice not given GRA-M-1.

Fig. 16 is a photomicrograph showing cancer cell tissue from a cancerous mouse

group not given GRA-M-1,

Figure 17 is a photomicrograph of cancer cell tissue a group treated with gRA-M-1,

Fig. 18 shows an SDS gel electrophoresis diagram of GRA using protein staining by the C.B.B. method,

Figures 19 to 21 show SDS-gel electrophoresis diagrams

using sugar coloring by means of the PAS method.

22 is a schematic representation of the results of SDS gel electrophoresis

Diagram of GRA using protein staining by the C.B.B. method.

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

to which X5563 immune mouse spleen cells and Y5563 cells were transplanted.

GRA is obtained

transplanted from human or animal cancer cells containing GRA, e.g., from cultured cancer cells Cancer cells, spontaneously occurring cancer cells, induced by chemical substances or by viruses Cancer cells, or cancer cells that have been surgically removed from tissues using the following procedures.

0 cell membrane components are made by cancer cells of the aforementioned Art separated and treated with a lectin, which specifically deals with terminal galactose or terminal N-Acetylgalactosämirr, 'where GRA is associated with the lectin can easily be separated, binds (see J.B.C., 250,

8518-8523 (1975); Biochem. Biophys. Res. Comm., T £ _r 144 (1975); Z. Immunitätsforsch., 1J38. ' 423-433 (1966); Br. J. Exp. Pathol., 27_, 228-236 (1946); Proc. Nath. Acad. May be. USA, 7J5, 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, for example by a Homogenization method and a solubilization method using a dissolving agent. It is advantageous to use a method in which Cancer cells are homogenized in physiological saline solution or in a suitable buffer, a part of the precipitate is collected by, for example, centrifugal separation and in physiological saline solution or a buffer using a solvent

is solved, whereupon the protruding part is then separated for example by centrifugal separation. Surface-active solvents can be used, for example, as dissolving agents Use agents well known to dissolve cell membranes, e.g., nonionic surfactants such as Triton X-100 (manufactured by Wako Pure Chemical Industries Ltd.), NP-40 (manufactured by Shell Co., Ltd.), digitonin and urea, or anionic surfactants, e.g. sodium dodecyl sulfonate (SDS).

From the cell membrane components thus obtained, one can obtain GRA, which is able to interact with lectin using usual chemical or biochemical process and by learning from the properties of the lectin Makes use, detach. Examples of such methods are an affinity chromatography, in which one uses a pillar containing a lectin, an immunoprecipitation method, using GRA antibody or the like, a dialysis method, a gel filtration method, an electrophoresis method or physical precipitation using a sugar protein precipitating agent such as polyethylene glycol and acetone, or a combination of such methods. Affinity chromatography is particularly preferred using a column containing a lectin, making the column support easily can by immobilizing lectin on an insoluble support '. The immobilization of lectin on a

30 insoluble carriers can be obtained in a known manner, such as

they are used for the immobilization of biosubstances

will perform. One of these methods is preferred using the cyanobromide activation polysaccharide method and an immobilization method from N-hydroxysuccimide ester on. The Cyanobromide Activation Polysaccharide Method is a method

in which an insoluble carrier is treated with cyanobromide and the activated product obtained in this way is then subjected to a coupling reaction with a lectin under mild conditions, the lectin being immobilized. In the treatment of the insoluble support with cyanogen bromide, for example, applies basic compounds such as sodium hydroxide and sodium hydrogen carbonate to adjust the pH to 7.5 to 12 and then treated to the carrier in a solu-

15 solvents, such as water, acetonitrile or a

at pH 7.5 to 12 held _r buffer such as a 0.1 M Natriumhydrogenkarbonatpuffer (pH about 8 _r 7) and 0.01M phosphate buffer (pH about 7.7) at room temperature for about 1 to 12 minutes. In general, it is preferred to use an amount of cyanogen bromide which is equivalent to the insoluble carrier.

Any known insoluble carrier that has low non-specific adsorption to all bio-substances shows that has a high porosity and that has a functional group that works under mild conditions is able to immobilize bio-substances and is chemically and physically stable enough, can be used in the present invention.

Insoluble carriers which can be used are, for example, carriers made of cellulose, e.g.

- / 9 -

Aminoethyl cellulose, carboxymethyl cellulose, bromoacetyl cellulose and p-anilino cellulose, or a carrier from networked! Dextran, e.g., Sephadex and CM-Sephadex (manufactured by Farmacia Corp.) and a carrier made of agarose such as Sepharose 2B, Sepharose 4B and Sepharose 6B (manufactured by Farmacia Corp.).

In the coupling reaction of the cyanobromide activated carrier obtained as above, the cyanobromide activated carrier is used in an amount 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 hydrogen carbonate (containing 0.5 mol / l sodium chloride; pH 8.4) at a temperature between 0 and 40 ⁰ C and preferably 2 and .8 ⁰ C for a time of about 10 to hours. In this way, a lectin-containing carrier for affinity chromatography is obtained.

20 In chromatography using lectin

containing carrier prepared as described above, the desired GRA is combined with the lectin contained in the carrier and is retained in the column. Subsequently, substances that lead to a Combination are able to run through the column, e.g. with a lectin, with an exchange reaction takes place, or alternatively an adsorption separation (with an eluting solution) is carried out, e.g. with a highly concentrated salt, an aqueous one

Solution of potassium thiocyanate and a nitrate buffer to Detach the desired GRA and win.

- ve -

In the exchange reaction, substances are capable of are to combine with a lectin when a vehicle for affinity chromatography containing a galactose binding agent Lectin, for example those which are terminated with a galactose-linking lectin combine, e.g. Galactose, disaccharides, containing terminal Galactose, and oligosaccharides containing a terminal galactose. Examples of substances used in the Leagues are to combine with a lectin when as Carrier for affinity chromatography an N-acetylgalactosamine-binding Lectin used are those that bind with a terminal N-acetylgalactosamine Lectin, e.g., N-acetylgalactosamine, disaccharides containing a terminal N-acetylgalactosamine and oligosaccharides containing a terminal N-acetylgalactosamine.

The GEA obtained in this way contains glycoprotein containing a galactose and / or N-acetylgalactosamine end group, glycolipids and / or saccharides.

The GRA produced in this way can, if desired, be lyophilized and are also cleaned in the usual way using conventional separation methods. For example, you can do sohe GRA preparations containing a galactose binding agent lectin were isolated, subjected to a separation method, using an N-acetylgalactosamine connecting Lectin and GRA, which has then been isolated with a lectin combining N-acetylgalactosamine, can be separated according to a separation method using a galactose binding lectin.

The lymphocytes used here are not critical and all lymphocytes are normal or cancerous Humans or animals can be used. Examples of this are lymphocytes, which one can get from peripheral Receives blood, bone marrow, lymph nodes, from the spleen, tonsils or thymus gland. These lymphocytes are obtained by physical or chemical methods, by a surface Meipbran method, or the like isolated.

10

The sensitization of lymphocytes with GRA takes place, by placing the lymphocytes on a culture medium containing GRA for a period of 1 to 10 days, preferably 2 to 7 days, cultured.

Various conventional nutrient media can be used as the culture medium for use in sensitizing the lymphocytes which are commonly used for such cell cultivation can be used. Preferred examples include an RMPI 1640 medium, an Eagle MEM culture, etc., added to human serum, fetal calf serum (FCS), calf serum, horse serum or the like became. The amount of GRA added to the culture is generally 1 to 1,000 ng / ml, and preferably 1 to

²⁵ I. '

-Vi-

500 ng / ml, calculated as the amount of sugar per 1 χ 10 / ml Lymphocytes.

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

The killer cells so produced are essentially normal lymphocytes

0 and have GRA-specific cell fighting activity. For example, GRA-1-KT has one of the inventive Killer cells, properties that occur and display in human peripheral blood T cells anticancer activity specific to cancer cells containing GRA, as in the later the following examples.

Typical examples of the new killer cells

are GRA-1-KZ and -GRA-M-1, which are in the Examples described below are produced. -These killer cells are available from the applicant and a sample of GPA-i-rKT 'cells is with ATCC on September 27, 1982 and has been assigned the number ATCC CRL 8175

Pie «There is no limit to the number of killer cells

duplicate using the aforementioned culture medium, containing a T cell growth factor (TCGF, IL-2). In this case one can choose a selective one Carry out cultivation of clones of the killer cells by means of a conventional ultraviolet dilution method. the Killer cells can remain stable over long periods of time

-1 / 3-

for example, liquid nitrogen can be stored.

The GRA can be used individually as an active ingredient or it can also be used in combination

be used with other bactericidal agents and cancer inhibiting agents. Cancer-inhibiting agents, the GRA according to the invention as an active ingredient may be in any form provided they contain GRA in effective amounts Generally, the agent is administered intravenously, subcutaneously, intradermally or intramuscularly in the form of a solution, a Suspension or used as an emulsion. It can also be made as a dry product and then immediately Before use, transfer to a liquid by adding a suitable carrier. This liquid Agent can be a suspending agent, e.g. methyl cellulose, an emulsifying agent, e.g. lecithin, preservative, e.g., methyl p-hydroxybenzoate, stabilizers and the like, if they do not contain them

25 adverse effect on immunization function

cause in humans or animals. Buffers can too be included.

Suitable aqueous carriers are, for example, a physiological saline solution and a suitable non-aqueous carrier can, for example, be a vegetable oil,

e.g. sesame oil, a mineral oil, e.g. paraffin, or a animal oil, e.g. squalene, or propylene glycol. To increase the immunological effect one can incorporate a suitable adjuvant. Suitable adjuvants are, for example, Freund's

Complete adjuvants, saponin in animals and aluminum hydroxide in humans.

The anti-cancer agent according to the invention can be used once or administered repeatedly over prolonged periods of time to a cancer patient for the treatment of cancer or it can be given to someone prone to cancer to help prevent cancer.

Since LD ₅₀ (mouse, intraperitoneal) of GRA at least

Is 500 mg / kg calculated as the amount of sugar, the anti-cancer agent according to the invention shows a low one Toxicity and can therefore be administered over a wide range of doses. Although the The concentration of GRA in the anti-cancer agent according to the invention is not critical, but it is preferred it in an amount of 0 "001 to ίΟΟ µg / ml, calculated as the amount of sugar to use. Regarding the dosage of the anti-cancer agent, generally preferably, the agent in an amount of 0.001 to 1,000 µg / kg / day all at once or in various proportions to be administered, however, this dosage depends on the degree of the disease, the age and the sex depends on the patient.

The anti-cancer agent produced in this way, which the

Containing killer cells as the active ingredient is preferably used as an injectable solution in combination with carriers involved in the manufacture of blood medicines Find application, used. The supports used here are not critical, but preference is given to supports which have the same tonicity as blood. Physiological saline solution is particularly preferred. In the The preparation of the .Means is preferably washed Killer cells sufficiently with a physiological saline solution or the like to dissolve the aforementioned culture medium to remove and then it will then recorded in a carrier.

The concentration of killer cells in the agent is

not particularly limited, but it is preferably

5 9
wise between 10 and 10 per milliliter. If the killer cells are administered intraperitoneally at a dose of 10 per mouse, no toxicity is found. Although the dose of the anti-cancer agent according to the invention depends on the degree of the disease, the age and the sex of the patient, in general the agent is preferably used in a dose of 10 to

1 2
10 / kg / day all at once or in different proportions

administered. 25th

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

Reference example 1
Locality_of_GRA

5 (1) ~ ^A Production of FITC-Labeled Lectin

(PNA-FITC)

• 10 mg of peanut lectin (PNA) manufactured by EY Co.) in 2 ml of a Ο, ΟΙΜ phosphate buffer containing

10 dissolved 0.85% NaCl (pH 7.2). In 1 ml of a 0.5M

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

20 (D-B preparation of FITC-labeled lectin

(DBA-FITC)

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

(DC Soybean Agglutinin-FITC (TIFT-SBA) is available from Ey Co.
FITC / SBA ratio = 1.4. 30th

IO

-Yi-

(2) Locality of GRA on various cancer cells

(a) Human cultured cancer cells (V χ 10) were three times with a 0.05 M tris-hydrochloric acid buffer containing Purified 0.85% NaCl (pH 7.2) by centrifugal method and then 100 μΐ PNA-FITC, DBA-FITC or SBA-FXTC, (200 μg / ml), manufactured and according to (1), added. The mixture was allowed to stand at room temperature for 30 minutes to react. After completion the reaction was carried out three times with a 0.01M phosphate buffer containing 0.85% NaCl (pH 7.2) and then the cells were placed on a glass plate and under

15 examined a fluorescence microscope.

The results are shown in Table 1. The cancer cells are all known and were identified by First Pathology Laboratories, Medical Department of Niigata University "

i I

10

25th 30th 35

-W- Cancer cells (Neuroblastoma) 324956 from Table 1 (Neuroblastoma) (Neuroblastoma) positive ver DBA SBA (Burkitt Lymphoma) (Neuroblastoma) ratio 1.4 (Burkitt Lymphoma) (Neuroblastoma) GRA (%) 5.2 (Burkitt Lymphma) PNA 0 (T-cell lymphona 98.3 6.7 Raji (T-cell leukemia) 93.1 4.8 Dauji (B-cell lymphoma) 50.1 5.3 BT-1 (IgD myeloma) 44.3 10.0 P-12 (Lung cancer, squamous) 0.6 2.0 MOLT (Lung cancer, squamous) 19.2 0.4 Fujimaki (Lung cancer, adenocarcinoma) 0.6 0 ODA (Lung cancer, small cells) 70.4 0 QG-5 6 (Lung cancer, large cells) 78.4 0 PC-1 (Stomach cancer, por.) 77.1 0.1 PC-3 (Magenkregs sig.) 68.0 0 QG-90 (Gastric cancer por.) 17.0 40.3 PC-13 (Stomach cancer, adsq.) 63.7 0.4 MK-2 (Stomach cancer, tubI) 57.3 0.1 KATO-III (Stomach cancer, tubI) 1.0 0 MKN-45 (Bladder cancer approx.) 4.6 0 MKN-1 (Bladder cancer approx.) 0.4 21.4 MKN-28 (Bladder cancer approx.) 0.5 ' 0 MKN-74 (Bladder cancer approx.) 37.4 1.0 MGH-U1 (Kidney cancer) 4.5 0 KU-2 (Kidney cancer) 14.6 0.6 T-24 Kuramochi {ovarian cancer) 13.1 0 NBT-2 ' NB-1 23.9 1.7 NRC-12 YT-nu 3.3 0.5 KU-I TGW-nu-1 80.0 0 TGW-nu-11 20.9 1.0 GOTO 3.6 0 4.1 2.0 0.5

1 2 3249568 90.7 96.9 0 , 3 44.6 3 14.6 0 ,1 5.4 1 5.7 0 , 7 92.0

Xb

- 3-9 -

ITO (embryonic carcinoma)

NEC-8 (embryonic carcinoma)

SCH (Choriacarbinoma, stomach)

GCH (Chriocarcinoma, uterus)

5 YN-1 (Rhabdomyosarcoma)

Mouse X 5563 (Plasinacytoma)

Mouse MHI34 (Hepatoma) 18.6 0 6.4

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

Obtaining a single line suspension. After washing twice with 0.01M HCl buffer (pH 7.4) containing 2mmol CaCl ₂ , 2mmol MgCl ₂ and 0.85% NaCl, 5x1O ⁵ cells were resuspended in 100 μl buffer. 100 μΐ FITC-PNA or FITC-DBA (200 ng / ml) were 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.
20th

The results are shown in Table 2. The malignant tissue from cancer patients was tested by Kansai Medical University received.

Table 2

GRA

No. Cancer patient tissue _{pNA DBA}

1 Stomach cancer 2 Stomach cancer 3 Stomach cancer 4th Stomach cancer 5 Stomach cancer 6th Stomach cancer 7th Breast cancer 8th Breast cancer 9 Breast cancer 10 Breast cancer 11 Colon cancer 12th Colon cancer 13th Esophageal cancer s 14th Hepatoma

_{U ||} j_

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

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

Reference example 2
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(1) -A production of immobilized lectin (PNA-Sepharose)

3g of CNBr-activated Sepharose 4B (manufactured by Farmacia Corp.) were washed thoroughly with 1 mmol HCl and suspended in 200 ml 0.1M sodium hydrogen carbonate (pH 8.5). 5 ml of a 0.1M phosphate buffer (pH 8.5) containing 20 mg of PNA were added and then carried out the reaction at 25 ° C for 2 hours with stirring several times to obtain PNA-Sepharose.

(1) -B Same as for (D-A above, except that DBA was used instead of PNA, one obtained DBA-Sepharose.

(2) Manufacture of GRA

(a) BT-I (Burkitt Lymphoma) cells (1.3 10 ⁸ ) were washed 3 times with physiological saline and then 30 ml of a 0 _f 01M tris-hydrochloric acid buffer (pH 7.4) containing 2% Triton were added X-100 (manufactured by Wako Pure Chemical Industries Ltd.), 0.85% NaCl, 2 mmol CaCl ₂ and 2 mmol MgCl _{2 were} added. The mixture was stirred for 15 minutes at 4 ° C. and then subjected to ultracentrifuge separation at a rate of 100,000 μg. From 28 of the thus obtained supernatant ml a 14 ml portion through a column (diameter O ₁ .5, length 1 cm) was sent for the affinity chromatography, eluting the column with PNA-agarose beads (manufactured by Maruzen Co., Ltd.) which had previously been brought into equilibrium with a tris-hydrochloric acid buffer (pH 7.4) containing 0.1% Triton X-100, 0.85% NaCl, 2 mmol CaCl ^ and 2 mmol MgCl-. After washing with the same buffer as used above, a 0.01M tris-salsic acid buffer (pH 7.4) containing 0.1M lactose, 0.85% NaCl, 2 mmol CaCl ₂ , 2 mmol MgCl ₂ and IP, 1% Triton X-100 eluted. The part edited in this way

was dialyzed with a 0.1M tris-hydrochloric acid buffer solution containing O ₁ - 85% NaCl, 2 mmol MgCl ₂ and 2 mmol CaCl _{2 for} 48 hours, 17 mg of a GRA solution being obtained. In this GRA solution, the amount of protein and the amount of sugar were determined according to the Folin-Lowry method and the phenol-sulfuric acid method - these amounts were 644 μg and 120 μg, respectively. This product is hereinafter referred to as "GRA-1".

1 0 (b) C3 / HE. _M äuse breast ^{cancer-1> iMT} - Π ^{x 10 cells} were ~ 3 times washed with saline solution, 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 were} added. The mixture was stirred at 4 ° C. for 30 minutes. The mixture was then subjected to centrifugal separation at a rate of 100,000 μg for 2 hours, and the supernatant liquid was overnight with a 0.1M tris-hydrochloric acid buffer (pH 7.4) containing -0.85% NaCl, 2 mmol CaCl ₂ and 2 mmol MgCl ₂ , dialyzed. 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) which had been filled with the same PNA-Sepharose as used before and which was equipped with a tris Hydrochloric acid buffer (pH 7.4) containing 0.05% Triton X-100, 0.85% NaCl, 2 mmol CaCl ₂ and 2 mmol MgCl ₂ , was subjected to affinity chromatography. After washing thoroughly with the same buffer solution as before, co-eluted

- 23-

an O _r 1M tris-hydrochloric acid buffer solution (pH 7.4) containing 0.1M lactose, 0.85% NaCl, 2 nunol CaCl ₂ , 2 mmol MgCl and 0.005% Triton X-100, and the portion thus eluted was then 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".

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

Stored 25 -20 C 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 ii

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

material .11 GRA Candy
hold (mg) source -2A- Protein content
(mg) 0.09 BT-a Table 3 0.5 GRA- More excised 0.5 sample Breast cancer Amount (g) 0.25 0.38 GRA-3 QG-4 6 33 0.6 0.54 GRA-4 QG-90 1.0 0.45 - Raji 5 0.78 28.4 GRA-5 MMT 24 11.3 0.07 GRA-6 LLC 26th 0.06 0.35 GRA-7 MH-135 29 0.65 0.23 GRA-M-2 X-5563 200 0.56 GRA-M-3 14.4 GRA-M-4 85 GRA-M-5 25th

(e) In the same way as in (c), but using NKN-45 (about 29g) instead of Kato-III and using vn DBA-Sepharose, obtained according to (1) -B, instead of PNA-Sepharose 4B and eluting with N-acetylgalactosamine a GRA preparation with a protein content of 0.03 mg and a sugar content of 0.01 mg was obtained. this will

25 hereinafter referred to as GRA-8.

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

whereby 4 ml fractions No. 1-1: 2 were obtained. Fractions 1-3 are designated as "GRA-3-A" and fractions 4-12 as "GRA-3-B". Then the column was filled with the same buffer eluted, but which contained 0.1M N-acetylgalktosamine and whereby fractions were obtained which are designated with "GRA-β-C"

35 become.

(g) SDS gel electrophoresis

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

The results are shown in Figs.

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

In Fig. 20, numerals 1 to 4 mean the following: 1 ... standard; 2 ... GRA-M-2; 3 ... GRA-6; 4 ... GRA-5. 15th

In Fig. 21, numerals 1 to 3 mean the following: 1 ... GRÄ-M-4; 2 ... GRA-M-5; 3 ... Standard:

In Fig. 22, numerals 1 to 4 mean the following: 20 1 ... GRA-3; 2 ... GRA-3-A; 3 ... GRA-3-B, 4 ... GRA-3-C.

18 shows the state of GRA after protein staining according to CBB (Fairbanks et al: Biochemistry, Volume 10, p. 2606, 1971).
25th

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

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

! ι

The following, from Biorad Lab. Calif.USA used substances obtained: 35

I)

K Dalton; Myosin
"; β-galactosidase
92.5 "; phosphorylase
66.2 "; BSA
45 "; ovalbumin

21.5 β; Soybean trypsin inhibitor

Reference example 3

(a) The spleen of Japanese monkeys (obtained from Japan Plymates Co., Ltd.j was removed and washed 2 times with RPMI-1640 culture medium (manufactured by Flow Laboratory Co., Ltd.). The cells were filtered using mesh (manufactured by Millipore Inc. _f 150 mesh) and a specific gravity centrifuge method (specific gravity

1.076), taking 2 1 of 2 χ 10 / ml lymphocytes received. The lymphocytes thus obtained were washed 3 times with an RPMI-1640 culture medium and the number of lymphocytes was below 5 χ 10 ^ / ml Discontinued use of the same medium as before, containing 10% FCS. Then they were held in one Stand carbon dioxide fermenter at 37 ° C for 1 hour. The supernatant lymphocytes were collected

and the number of lymphocytes was adjusted to 1 × 10 / ml using the same culture medium as mentioned above containing 1% FCS. Subsequently, 1 μ9 / πι1 Indomethacin (manufactured by Sigma Laboratories Inc.) and 0.2% PHA-P (manufactured by Difco Co.) was added and then the cultivation was carried out for 48 hours at 37 ⁰ C in a carbon dioxide gas fermentation device. After performing centrifugal separation (3,000 rpm) for 10 minutes, the formed supernatant liquid was recovered and sterilized by filtering it through a Millipore filter (manufactured by Millipore Inc., 0.2 µm) and adding 2 liters of TCGF received.

(b) The supernatant fluid from mixed-cultured peripheral blood lymphocytes from 10 healthy donors was used as the source of TCGF. Disjoint lymphocytes produced by CF. (Conray. Ficol (Japan Immunoresearch Co.)) separated lymphocytes by adsorption on plastic surfaces at 37 ° C for 1 hour were suspended in RPMI 1640 medium containing 1% FCS (1.5x10 ⁶ cells / ml) and with 0.2 % PHA-P, indomethacin (1ug / ml) and human B cells (BT-1) (1.5x10 ⁵ cells / ml), pretreated with mitomycin C (50 μΐη / ml), incubated. After 48 hours, the floating culture was harvested and used as a 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 centrifugal separator using Ficol Pack (manufactured by Farmacia Japan Co., Ltd) subjected, being 5x10 peripheral blood lymphocytes received.

15 (2) mouse spleen lymphocytes

The spleen of a C3H / He mouse (male, 6w) was excised and twice with an RPMI-1640 culture medium

ORIGINAL INSPECTED

10

washed. Then the spleen was injected using a hypodermic needle loosened and passed through a stainless steel (100 mesh) screen to remove large parts happened. The cells thus filtered were washed 2 times with the same culture medium as above, and then subjected to centrifugal separation at a rate of 1,200 HpM for 10 minutes, 4x10 Spleen lymphocytes received.

example 1

GRA-1 (amount of protein 40 ug / ml - amount of sugar 7.5 ug / ml, obtained according to reference example 2, (2a)) were increased to 1,000 times the original volume with an RPMI-1640 culture medium containing 15% FCS, diluted, to give a sensitizing culture medium.
20th

Lymphocytes of human peripheral blood (5 × 10/5 ml) obtained in Reference Example 4 (1) were added to 5 ml of the sensitizing culture medium prepared as above, and this was then placed in a laboratory dish and kept at 37 ° for 2 days C cultivated. This was followed by a further cultivation on an RPMI-164 0 culture medium containing 20% TCGF and 15% FCS, obtained according to reference example 3 _r for a further 5 days, where 20 ml of a killer cell solution containing 1 × 10 ⁶ killer cells / ml, ■
referred to as GRA-1-KT.

1x10 killer cells / ml. This is shown below

Example 2

The mouse spleen lymphocytes obtained in Reference Example 4 (2) were adjusted to a number of 5x10 / ml using an RPMI-1640 culture medium containing 15% FCS. GRA-MI, obtained according to reference example 2 (2b), was added to this so that the end 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 15 mm, manufactured by Falcon Co.) at 37 ^° C. for 2 days. The formation of clones was observed. The portion was further prepared in a RPMI-1640 culture medium with 15% FCS containing 20% by volume TCGF (

15 by Japan Immuno Research Laboratories Co., Ltd.)

cultivated for 4 days, 50 ml of a Killer cell solution, containing 1x10 killer cells / ml. This is referred to below as GRA-M-1-K-T .

Example 3

Lymphocytes (5x10) from peripheral blood of healthy donors were in a RPMI-1640 medium containing 40 ng / ml (protein content) of GRA-2 and 15% FCS at 37 ^? C incubated. On the second day, the aforementioned human TCGF was added to the medium until its concentration reached 20%, and the incubation was continued for 3 days to obtain killer cells, hereinafter referred to as "GRA-2-KT".

Example 4

Killer cell preparations GRA-8-K-T, GRA-3-A-K-T and GRA-3-C-K-T 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) was intradermal X5563 cells (10) of the same strain were implanted and after 7 days the tumor was surgically excised.

-5 th. After a further 7 days, X5563 cells (10) inoculated with the same strain and mice resistant to the inoculation were identified as immune mice designated.

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

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

Killer cell preparations obtained from normal spleen cells are hereinafter referred to as "GRA-M-5-K-t-1", and those obtained from immune spleen cells are referred to as "GRA-M-5-K-T-2".

35

Example 6

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

Table 4 Killer cells GRA contentration (ngVml) GRA-1-K-T-1 First day Day 3 GRA-1-K-T-2 50 0 GRA-I-K-T-3 50 1.6 GRA-1-K-T-4 50 3.2 GRA-1-K-T-5 50 6th GRA-1-K-T-6 50 12.5 GRA-1-K-T-7 50 25th GRA-1-K-T-8 50 50 • 50 200 Example 7

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

- 10 Peripheral 36
-IA- + + 14th 3249 cancer Table 5 + + 35 Blood lymphocytes + 21 Stomach cancer + - 7th Killer cells Stomach cancer + - 35 5 Breast cancer + 21 GRA-3-K-T-1 Breast cancer P-GRA D-GRA days after the GRA-3-K-T-2 Breast cancer surgery GRA-3-K-T-3 Stomach cancer GRA-3-K-T-4 GRA-3-K-T-5 GRA-3-K-T-6

In the table above, P-GRA and D-GRA denote the 15 localities of GRA, expressed as malignant tissue

(extracted by surgery) from cancer patients from whom PBL was collected in the same manner as in the reference example 1, 2- (b). P-GRA and D-GRA were obtained using FITC-PNA and FITC-DBA, respectively. The days post-operation indicate the time when BPL was collected after the operation.

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

Example 8

(i) GRA-M-1, obtained according to the reference example 2 (2b) was diluted with physiological saline solution so that the sugar content was 1.0 ug / ml and Protein was 1.6 µg / ml. This is how you got sin anti-cancer drug No. 1.

c t

(ii) A cancerous tissue of C3H / He appears spontaneously Tending breast cancer became sterile to a 5 mm cubic Lumps cut and placed under the dorsal skin of 10 C ^ H / He mice of the same strain as transplanted above (7 weeks old, male). 7 days after the transplant was the fixation and multiplication of cancer confirmed. 5 of these mice received the anti-cancer agent prepared according to (i) subcutaneously No. 1 at a dose of 300 μΙ / day at 2-day intervals. The remaining 5 mice were used as untreated control used. The tumor was surgically removed 10 days after the first administration and its mean weight measured. At the same time, a pathohistological examination was carried out.

Tumor volume

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%. 3 0

In the control group (FIG. 16) there were similar bird nests che cancerous bodies formed, the type of tissue

J *

corresponded to a marrow-like canal-shaped cancer and a multiplication of cancer cells over the entire tissue was found. On the other hand caused in the group that was treated with the agent i (FIG. 17) the cancer cells developed a liquefaction necrosis

in the places where the cancer cells formed . and. a calcification and firosis was.

that was found, leaving only a very small amount of cancer cells. This confirmed the anti-cancer properties of the anti-cancer agent of the present invention.

Test example 1

GRA-1-K-T (1 μΐ), obtained according to Example 1, was put on a microplate (manufactured by Falcon Corp.) and allowed to stand at room temperature for 15 minutes. Then 4 μΐ FCS (manufactured by Falcon

20 Corp.) was added and the mixture was allowed to stand at room temperature for 30 minutes. Neuraminidase

N treated sheep red blood cells (SRBC), discontinued to a number of 1 χ 10 / ml, and 5 μΐ one 0.1M phosphate buffer (pH 7.2) was added to the 0.85% NaCl were added and then the plate was centrifuged at a rate of 6,000 rpm for 5 minutes. Then the plate was turned over and unreacted SRBC became removed. A coloring solution (Brilliant Cresyl Blue,

30 '' manufactured by Merck & Co.) was used for dyeing the

Lymphocytes added and a rosette-shaped one

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

Test example 2

if i§ £ i} eCancer cell killing activity

(a) Of the cancer cells shown in Table 1, the following 5 cell strains were different GRA positivity ratios as to be combated Human Cancer Cells Uses:

15 cancer cells to fight:

No. 1 BT-1 (Burkitt Lymphoma) No. 2 Daudi (Burkitt Lymphoma) No. 3 KATO-III (Stomach cancer) No. 4th MKN-45 (Stomach cancer) No. 5 MOLT (T-cell leukemia)

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

Then 4 × 10 per well of the GRA-1-KT obtained according to Example 1 were carefully added and incubation was then carried out for 1 hour.
30th

The killing activity was depending on the ¹ degree of

The formation of deposits was determined and assessed as follows:

++ a noticeable killing activity is established - - -

posed

+ a killing activity is determined + a low killing activity is determined
- a killing activity cannot be determined

will. 10

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

From Table 6 it can be seen that the invention Killer T cells formed a strong GRA-specific Have eytotoxic activity. 20th

Table 6.

to be combated (Fig. 1) GRA positive judgement the loading Cancer cell : (Fig. 3) ratio (%) lagging GRA-1-K-T- Daudi (Fig. 5) 93.1 ++ (Fig. 2) group ΚΛΤΟ-ΙΙ] (Fig- 7) 57.3 + (Fig. 4) BT-1 (Fig. 9) 50.1 ++ (Fig. 6) MKN-45 1rO + (Fig. 3) · MOLT 0.6 - (Fig. 1 0) Control BT-1 50.1 - (Fig. 1 1) group

- ys -

(b)

The same cancer cells to be fought as

they were used in (a) (3.2 χ 10) were using 8x10 GRA-1-K-T (cell ratio 5/1) mixed and the cell mixture formed (total number 4 × 10) was cultivated in an RPMI-1640 medium containing 15% FCS. After 1 hour, the number of remaining ropes was counted and the inhibition ratio became calculated according to the following equations:

Cytotoxicity (%) =

Number of cells (1 _ ₍ after cultivation _{) y /] QQ)} Number of cells before cultivation (4 χ 106)

The results are shown in Table 7.

Table 7

to fight number 10 ⁶ of cells the KuI- x 10 ⁶ TCyto- de cells in front of the Kul 10 ⁶ after activation χ 10 ⁶ :toxicity activation 10 ⁶ 2.9 χ 10 ⁶ U) Daudi 4 χ 10 ⁶ 3.7 χ 10 ⁶ 28 KATO-III Δ ν 10 ⁶ 3.2 χ 10 ⁶ 7.5 BT-1 4 χ 3.9 20th MKN-45 4 χ 4.2 2 ', 5 MOLT 4 χ -5

(c) The procedure of (b) was repeated except that the mixing ratio of GRA-1-K-T .. ' to the cancer cells to be combated was changed to 5/3. The results are shown in Table 4.

Table 8

to fight
de cells Number <
in front of the Kul
activation χ 10 ⁶ outer cells
after the Kul
activation χ 10 ⁶ Cyto-
toxicity
. (%) Daudi 4th χ 10 ⁶ 3.6 χ 10 ⁶ 91 - KATO-III 4th χ 10 ⁶ 3.6 χ 10 ⁶ . 24 BT-1 4th χ 10 ⁶ 1.4 χ 10 ⁶ 65 MKN-45 4th χ 10 ⁶ 3.7 χ 10 ⁶ ■ 7.2 MOLT 4th 4.1 -3

Test example 3

C3H / He-spontaneously breast cancerous mice received subcutaneously GRA-M-1-KT-, obtained according to Example 2, in a dose of 2 × 10 / 0.3 ml maize 3 times a week every second Day. After 10 days, the focus was taken out and examined. In this experiment it is found that GRA-1-K-T has a high binding activity towards Daudi, KATO-III and BT-1, but only a low binding activity opposite MKN-45 and MOLT shows.

As shown in Fig. 12, infiltration of lymphocytes into the cancer cells took place and disruption of the tumor area was determined. From Fig. 13 it can be seen that a calcification of the tumor area has taken place and from this it can be seen that the cancer cells of the present invention have anti-tumor activity exhibit.

Comparative example 1

In this example, cancer cells per se were used as specific antigens instead of GRA for the procedure I i ι

used according to the invention.

Cancer cell-sensitized lymphocytes were obtained in the same manner as in Example 1, with the exception that used instead of GiRA, BT-I, Daudi, KATO-III or MKN-45 in an amount of 1x10 per laboratory dish became.

With these lymphocytes, the cytotoxic 35

HS

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

It can be seen from Table 9 that the lymphocytes prepared above have no cytotoxic activity.

Table 9

cell to be combated

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 cancer cell killing activity of GRA - (- K-T, GRA-3-A-K-T and GRA-3-C-K-T obtained in Example 4 were determined. the Results are shown in Table TO.

cell

Table 10 % Cytotoxicity

GRA-8-K-T

GRA-3-A-K-T

GRA-3-C-K-T

KATO-III 8th , 0 BT-1 4th , 3 MKN-45 20th MOLT 0

Test example 5

5.0 5.0 5.0 0

5.0 4.0 20 0

(a) The cytotoxicity of the killer cell preparations obtained according to Example 6 was determined by means of a Cr staining test (J. Immunol., 122, 2527-2533 (1979)). For this purpose 50 \ iCi of radioactive Cr (Japan Isotope Association) were added 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) (2x10) were added to the target cells (1x10) (the E / T ratio was 20/1) and the mixture was incubated for 4 hours at 37 ° C. in RPMI-1640 medium. The supernatant was collected by centrifugation and the radioactivity was counted by a liquid filter.

The specific Cr release (%) corresponding to the cytolytic activity of the effector cells was determined as follows

Equation determines:

51
Specific CR approval (%)

(Release in the attempt) - (Spontaneous release)

(Maximum share)

- (spontaneous release)

- 4Ä -

The maximum release shows the radioactivity though all cells are dissolved.

The results are shown in Table 11:

Table 11 Specific approval
⁵¹ Cr: (%) Killer cells 13th GRA-1-K-T-1 25th GRA-1-K-T-2 22nd GRA-1-K-T-3 20th GRA-1-K-T-4 22nd GRA-1-K-T-5 25th GRA-1-K-T-6 27 GRA-1-K-T-7 32 GRA-1-K-T-8

It can be seen from the results in Table 11 that TCGF and FCS have no relation to 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-

51 release test determined. 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 was obtained according to the 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 % Cytotoxicity Killer cells 38.0 GRA-3-K-T-1 18.2 GRA-3-K-T-2 20.9 GRA-3-K-T-3 23.2 GRA-3-K-T-4 24.5 GRA-3-K-T-5 20.2 GRA-3-K-T-6

(b) The cytotoxicity of GRA-3-K-T-7, obtained according to

51 Example 7- (b) was prepared using Cr-KATO-III

(E / T = 29/1) was determined as target cells in the same way as in Test Example 5. The specific Cr release | of the killer cells was 25.5% ..
25th

Test example 7

The cytotoxicity of killer cells, obtained in Example 5,

51
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 was carried out at 1x10 / well of

35 mitomycin C-treated X5563 cells (5x10 / ml of

- 4-6 -

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

The results obtained are shown in Table 13:

Tabel 4th 13th 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-KT-1
GRA-M-5-KT-2
control

Test example 8 (H-2 assay )

GRA-M-1, GRA-M-3 and GRA-M-4, obtained according to the reference example 2 were subjected to serial dilution with PBS (0.85% NaCl) to obtain samples.

Anti-H2 serum from the National Institute of Genetic Research and the above samples were mixed and incubated at 4 ° C for 2 hours, and then target cells corresponding to the anti-H2 serum used were added. 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 hour at 37 ° C. and stained with 0.25 trypan blue PBS to determine the percentage of cytotoxicity. The anti-H-2 serum was used in a maximum dilution so that there was at least 95% cytotoxicity in

35 was shown by GRA.

so ■ '■■■' ■ - '· ■■ "

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

Table 14 GRA Anti-H-2 Serum, target cells congestion

5 GRA-MI D-23 fAnti-H-2K ^k ), X80 BlOBR (H-2 ^k )

or, spleen cells

D-32 (Anti-H-2D ^K ), X300

GRA-M-3 D-33 (Anti-H-2K ^b ), X600 BIO (H-2 ^b 1

or, spleen cells

D-2 (Anti-H-2D ^D ), X80

10 GRA-M-4 D-23, X80 BlO-BR (h-2 ^k )

or lymph node cells

D-32, Χ300

The results are shown in Table 15. 15 table

Anti-H-2 X2 X4 I. % Cytotoxicity X64 - X256 X512 0 * serum - 13th II Dilution of the sample 14th X128 14th 13th 14th 13th GRA - 97 III X8 X16 X52 97 13th 97 96 96 14th I. D-23 - 95 14 12 13 94 96 95 96 95 13th D-32 - 19th 99 96 97 15th 95 11 12th 13th 14th - - 98 95 95 95 98 12th 99 99 99 15th II D-33 - 95 15 14 12 97 99 97 95 95 17th D-2 100 100 98 99 99 - 95 - ■ - 100 10 D-23 99 99 96 95 95 - - - - 99 10 III D32 GRA 100 100 - Annotation -.
II GRA 99 99 I. GRA : GRA-M-I : GRA-M-3 : GRA-M-4

"*" means% cytotoxicity if.Jiurr_-w-U . 'Complement was used.

From the results in Table 15 it can be seen that that GRA-M-1, GRA-M-3 and GRA-M-4 have no H-2.

Test example 9 5

C57BL / 6 mice were injected with S.C. LLC (2x10) of the same Strain transplanted and after 6 days 1ng (protein) GRA-M-3, obtained according to Reference Example 2- (d), administered subcutaneously. This administration was carried out once a day in the same dosage repeated on 4 days. The day after the last administration, the tumor cells were excised and weighed. Animals given only physiological saline solution were used as controls had been. Each test group consisted of 5 animals.

As a result, it was found that the average cancer weight in the control group was about 500 mg. at of the GRA-M-3-treated group, 3 showed a disappearance of the tumors and 2 showed an average tumor

20 weight of 100 mg.

Test example 10

C57BL / 6 mice were administered subcutaneously with 1 ng (protein) GRA-M-3, obtained in Reference Example 2 (d), once a day during Immunized 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 30 was prepared and a portion (1x10) of it was transplanted into mice of the same strain and a Winn assay Was carried out (J. Immunol. 86, pp. 228-239 (1961)).

fa

The results obtained by Elie are shown in Table 16. 35

Sl
-VS- 16 Day 17 Day 18 Day Cancer (%) 324956 Tabel Growth rate of the 5.5 5.7 3.1 19 day 20 Effector Day 15 39.5 37.1 55.4 1.4 Day 20 Mortali cells 5.7 65.1 61.7 98.1 76.8 ity / fruppe A. 21.4 96.4 0/10 B. 39.3 '4/10 C. 4/10

Note: Group A are the spleen cells of GRA-M-3-immune

mice;

Group B are the spleen cells ναι normal mice; Group C is the spleen cells of mice 10 days post-transplant from LLC (1x10);

The cancer growth rate was calculated using the following equation:

rn -T

Ά Ν

Cancer Growth Rate (%) = —— = —- χ 100

where T means the thickness of the sole of the foot on the side where the cancer was transplanted and T- the Thickness of a normal sole of the foot.

ι '!

25 Test example 11

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

S3

- Bff -

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

The results are shown in Table 17.

Table 17

Concentration 3-H-TdR incorporation GRA-MR tion (ng / ml) (mean cpm + standard error)

S.I.

Control attempt

0 5,302 + _ 1 .761 0.5 7,903 _ + 1 .290 1 10,076 + 936 5 10,686 + _ 429 20th 10,615 + 1 .270 40 8,565 + 1 .419

1.5 1.9 2.0 2.0 1.6

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

Test example 12

The delayed hypersensitization (DTH) response of CSH / HE normal mice, X5563 immune mice and MH134 immune mice, obtained in the same manner as in Example 5, and obtained from GRA-M-4 immune mice and GRA-M-5 immune mice In the same way as in Test Example 11, was by the Sole reaction (FPR) determined. For this purpose, GRA- or MMC-treated tumor cells were introduced into the skin of the sole of the foot

-Si-

Hind leg of the animals entered and the swelling of the sole of the foot It was found 24 hours after the treatment. The DTH response was calculated by looking at the swelling before treatment from after treatment (10 mm) withdrew.

The results obtained are shown in Tables 18 shown.

Table 18

_2 central foot sole growth (10 mm)

Experiment normal mice MH134 immune mice

1 2.8 13.6 2 12.4 32.0 3 3.6 3.6 4th 3.2 22.0 5 6.8 27.6

Note: Group 1: Syngeneic 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 μg (protein) / 20 μΐ medium Group 5: GRA-M-4, 0.4 µ9 ν

see p

-Vl-

Table 19

Middle sole of the foot (10 mm)

Experiment normal mouse X5563 immune mouse MH134 immune mouse

1 5.4 4th , 3 1 ,1 2 0.9 - 24 , 3 3 2.0 16 , 9 -

10 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-Immunmau s -0.3
24.6 attempt _2
Medium foot sole 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

Tabel 21 Middle _2
Growing on the sole of the foot (10 mm) attempt Normal mouse GRA-M-4 immune mouse ε 1
2
3 -1/8
6.3
6.8 0.6
20.0
6.3

Note: Group 1; Medium (Hanks solution) 20 11

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

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

Part obtained by a PNA column at the time of GRA-M-4 (hereinafter "C.P.") went through

Tabel 22nd Middle _2
Growing on the sole of the foot (10 mm) attempt Normal mouse GRA-M-4 immune mouse 1
2
3
4th 2.4
2.6
2.4
5.5 -1.0
17.7
1.8
18.9

Note: Group 1; Medium (Hanks solution 20 μΐ

Group 2: GRA-M-4, 3.8 μm (protein) / 20 μm medium Group 3: 3.8 μg (protein) / 20 μΐ of CP. (please refer above)

Group 4: 3.8 µg (protein) of GRA-M-4 and 3.8 µg (Protein) / 20 μΐ medium from CP.

25 reference attempt

X5563 immune mice were prepared in the same manner as in Example 5 obtain. The spleen cells of these immune mice were used as effector cells used and the effector cells (10) and the Target cells (X5563, 10) were collected together on mice of the transplanted from the same tribe. Then it did a Winn assay carried out in the same manner as in Test Example 10.

The results obtained are shown in FIG. 23, in which the abscissa ^{indicates the days and the coordinate indicates the mean cancer size (cm 2} ) jf standard error, and in which the various markings have the following meanings: 5

• ·: Group to which no effector cells were added

became;

ο ο: group to which untreated effector cells -were given;

Λ A '· group to which effector cells treated with rabbit complement were added;

χ -x: group to which effector cells that have been infected with

Thy 1 (New England Nuclear Co., USA) and added to the rabbit complement

became;

D D: Group to the effector cells with anti-Lyt

(New England Nuclear Co., USA) and added to the rabbit complement became;

K fi: group belonging to the effector cells with anti-Lyt

(New England Nuclear Co., USA) and added to rabbit complement.

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

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

Claims (15)

PATENT AND LAWYERS PATENTANWÄLTE DIPL.-ING. W. EITLE DR. RER. NAT. K. HOFFMANN DIPL.-ING. W. LEHN DIPL.-ING. K. FCICHSLE. DR. RER. NAT. B. HANSEN. DR. RER. NAT. H -A. BRAUNS DIPL.-ING. K. GDRG DIPL.-ING. K. KOHLMANN · LAWYER A. NETTE Claims / 1J Glyco-related antigen that was isolated from a cancer cell membrane component and that combines with a lectin which combines with a terminal galactose or a terminal N-acetylgalactosamine. 2. Glyco-related antigen according to claim 1, characterized in that cancer cells are human cancer cells. 3. Glyco-related antigen according to claim 1, characterized in that that the lectin binds specifically to a terminal galactose. 4. Glyco-related antigen according to claim 3, characterized in that that the lectin is peanut lectin. 5. Glyco-related antigen according to claim 1, characterized in that that the lectin binds specifically with a terminal N-acetylgalactosamine. 6. Glyco-related antigen according to claim 5, characterized in that that the lectin is dolichos bean agglutinin. 7. Glyco-related antigen according to claim 1, characterized in that that the cancer cell membrane component is one that binds with a galactose terminated Lectin or a combined N-acetylgalactosamine binding lesson. ORIGINAL INSPECTED VRABELLASTRASSE 4. D-8O0O MUNICH 81. TELEPHONE CO 89) 91IO 87 TELEX 5-29619 CPATHE) TELECOPIER 9183 8. Glyco-related antigen according to claim 7, characterized in that that the cancer cell membrane component is one that deals with peanut lectin or dolichos bean agglutinin combined. 9. A process for the preparation of a glyco-related antigen, characterized in that cell membrane components from cancer cells by homogenization or solubilization, the cell membrane component with a lectin, which specifically with terminal galactose or a terminal N-acetylgalactosamine to form a Lectin-cell membrane component complex combined, treated, collects the complex obtained and separates the lectin from the complex and removes the lectin-free cell membrane components 15 te collects. 10. The method according to claim 9, characterized in that the cancer cells are human cancer cells. 11. The method according to claim 9, characterized in that a lectin is used which specifically deals with terminal galactose connects. 12. The method according to claim 9, characterized in that a lectin is used which specifically combines with a terminal N-acetylgalactosamine. 13. The method according to claim 9, characterized in that iman a glyco-related antigen from a cancer cell membrane component first with a first lectin that specifically combines with terminal galactose and then with a second lectin, which specifically combines with a terminal N-acetylgalactosamine, isolated. 14. An anti-cancer agent containing, as an active ingredient, a glyco-related antigen obtained from cancer cells from a cancer cell membrane component that deals with a lectin which specifically binds with terminal galactose or an N-acetylgalactosamine connects. 15. Use of a glyco-related antigen according to claim 1 for combating cancer.