NZ201112A - Preparation of a cancer-specific antigen and composition - Google Patents

Description

New Zealand Paient Spedficaiion for Paient Number £0111 £ 2 01112 H® Patents Form No.5 NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION "Cancer cell-combating lymphocytes, process for the production thereof, and anticancer agents containing said lymphocytes" i-WE OTSUKA PHARMACEUTICAL CO., LTD. of No. 9, Kandatsukasa-cho 2 chome, Chiyoda-ku, Tokyo, Japan, a Japanese company hereby declare the invention, for which-f/we pray that a patent may be granted to -me/us, ,and the method by which it is to be performed, to be particularly described in and by the following statement:- Priorjty Date(s): I '.'0.§ L . K'J f; &! Complete Specification Filed: Class: D twurw* Publication Date: P.O. Journal, No: (followed by page I A.) 201112 CANCER CELL-COMBATTING LYMPHOCYTES, PROCESS FOR THE PRODUCTION THEREOF, AND ANTICANCER AGENTS CONTAINING SAID LYMPHOCYTES FIELD OF THE INVENTION The present invention relates to cancer cell-combatting lymphocytes (hereinafter referred to as "killer cells") and a process for the production thereof. More particularly it is concerned with killer cells which act specifically on cancer cells having a glycorelated antigen derived from the cancer cells (this antigen is hereinafter referred to as "GRA") and destroy the cancer cells, and a process for the production of said killer cells. Additionally the present invention relates to a novel anticancer agent, more specifically containing the killer cell or GRA as an active ingredient.

BACKGROUND OF THE INVENTION It is known that immune response effector cells, particularly T lymphocytes playing a main role in cell-mediated immune response, cause rejection of grafts due to foreign cell antigens, but exhibit no appreciable or very limited inhibition against cancer cells. Thus, the cancer cells are not destroyed and multiply iii vivo, finally putting the cancer-bearing host to death.

- U- \ 20111 However, the mechanism responsible to the recognition of self from not-self is not completely clear and various investigations have been made to have insight into the nature of material basis involved in such system. For example, cell 5 surface markers on mouse leukemia cells or embryonal carcinoma cells using lectins such as Dolichos biflorus agglutinin (DBA) / and peanut agglutinin (PNA) as described in Biochem. Biophys. Res^Comm^ 8Sf/(2) 448-455 (1979), Ibid.y96 (4) 1547-1553 (1980), J. Biochem. *o9 473-481 (1981) and Cell 18 183-191 September 10 1979.

However, as far as is known, there have been no appreciable attempts to provide new lymphocytes that can combat cancer cells specifically and also provide anticancer agents' utilizing immune response of lymphocytes.

SUMMARY OF THE INVENTION As a result of extensive studies on the immune response of the host to cancer cells and its application to the treatment of cancer, it has been found that, in a cancer cell specific antigen which is not found in differentiated 20 normal cells, there is GRA which acts as an immunogen for 201112 the host and have very high immunogencity that cause an immune response specific to the cancer cells. Furthermore, it has been found that when GRA is used to sensitize lymphocytes, there can be obtained killer cells which act speci-5 fically on cancer cells containing GRA and if the killer cells are administered to the host, they recognize GRA and act on the cancer cells containing GRA, destroying them, and thus that they exhibit an excellent effect in the treatment and prevention of cancer.

Therefore, the present invention provides killer cells in one embodiment thereof.

In another embodiment, the present invention provides a process for the production of killer cells.

In still another embodiment, the present invention 15 provides an anticancer agent containing a killer cell or GRA as an active ingredient.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a microphotograph of Daudi cancer cells; Fig. 2 is a microphotograph illustrating the forma-20 tion of plaques by GRA-l-K-T of the cancer cell of Fig. 1; Fig. 3 is a microphotograph of KATO-III cancer 201112 cells; Fig. 4 is a microphotograph illustrating the formation of plaques by GRA-l-K-T of the cancer cell of Fig. 3; Fig. 5 is a microphotograph of BT-I cancer cells; Fig. 6 is a microphotograph showing the formation of plaques by GRA-l-K-T of the cancer cell of Fig. 5; Fig. 7 is a microphotograph of MKN-45 cancer cells; Fig. 8 is a microphotograph showing the formation of plaques by GRA-l-K-T of the cancer cell of Fig. 7; 10 Fig. 9 is a microphotograph of MOLT cancer cells; Fig. 10 is a microphotograph showing the formation of plaques by GRA-l-K-T of the cancer cell of Fig. 9; Fig. 11 is a microphotograph of BT-1 treated with a mixture of unsensitized human peripheral blood lymphocytes; 15 Figs. 12 and 13 are each a microphotograph of the cancer cell tissue of cancer-bearing mouce which is administered with GRA-M-1-K; Fig. 14 is a microphotograph showing the state of cancer in a cancer-bearing mouse group which is administered 20 with GRA-M-1; Fig. 15 is a microphotograph showing the state of cancer in a cancer bearing mouse group which is not administered with GRA-M-1; 2011 t 2 Fig. 16 is a microphotograph showing the cancer cell tissue of a cancer-bearing mouse group which is not administered with the GRA-M-1; Fig. 17 is a microphotograph of the cancer cell tissue of the group which is administered with GRA-M-1; Fig. 18 shows an SDS gel electrophoresis diagram of GRA using protein staining by C.B.B. method; Figs. 19 to 21 show SDS gel electrophoresis diagrams of GRA using sugar coloration by PAS method; Fig. 22 is a schematical illustration of the results of SDS gel electrophoresis diagram of GRA using protein staining by C.B.B. method, and Fig. 23 represents a graph showing tumor growth rate in C3H/HE mouse transplanted with X5563 immune mouse spleen cells and X5563 cells.

DETAILED DESCRIPTION OF THE INVENTION .

The killer cells of the invention can be prepared, for example, by a method in which GRA is used to Sensitize lymphocytes.

GRA as used in the above method can be obtained from cancer cells containing GRA of humans or animals, e.g., cultivated cancer cells, transplanted cancer cells, spontaneously-occurring cancer cells, chemical substance or virus-induced cancer cells, and cancer cells derived from operated tissues, by the following procedure. 201112 Cell membrane components are separated from cancer cells as described above and treated with a lectin which combines specifically with a terminal galactose or a terminal galactosamine, whereby GRA is combined with the lectin and N-acety can be easily separated.

Suitable examples of the galactose-binding lectin include peanut lectin, Ricinus communis lectin and Soybean (Glycine max) lectin (see J. B. C., 250, 8518-8523 (1975); Biochem. Biophys. Res. Comm., 62, 144 (1975); Z. Immunitaets-forch, 138, 423-433 (1969); Br. J. Exp. Pathol, 27_, 228-236 (1946); Proc. Nath. Acad. Sci. USA, 75, No. 5, 2215-2219 (1978); Biochemistry, 13, 196-204 (1974) ; and Carbohydrate Research, 51, 107-118 (1976)). Suitable examples of the N-acetyl-galactosamine-binding lectin include Dolichos bean (Dolichos biflorus) agglutinin, braid orange lectin, Helix pomatia lectin, lima-bean (Phaseolus limensis) lectin, soybean (Glycine max) lectin and Bauhinia bean (Bauhinia' purpurea) lectin.

The separation of the cancer cell membrane components can be achieved by known techniques such as a homogeni-zation method and a solubilization method using a dissolving agent. It is more advantageous to employ a method in which 0111 cancer cells are homogenized in physiological saline or in an appropriate buffer, a portion precipitated is collected by a technique such as centrifugal separation and dissolved in physiological saline or a buffer by the use of a dissolving agent, and the supernatant portion is separated by a technique such as centrifugal separation. Dissolving agents which can be used include surface active agents which are generally known to be capable of dissolving cell membrane, such as nonionic surface active agents, e.g., TRITON X-100 (produced by IVako Pure Chemical Industries Ltd.), NP-40 (produced by Shell Co., Ltd.), digitonin, and urea, and anionic surface active agents, e.g., sodium dodecylsulfonate (SDS).

From the thus-obtained cell membrane components can be separated GRA capable of combining with lectin by usual physical chemical or biochemical techniques utilizing the properties of lectin. Examples of such techniques include affinity chromatography utilizing a column carrier containing lectin, an immune precipitation method using GRA antibody or the like, a dialysis method, a gel filtration method, an electrophoresis method, a physical precipitation method using a sugar protein-precipitating agent, e.g., polyethylene glycol and acetone, and a combination thereof.

More preferred is affinity chromatography utilizing a column carrier containing lectin, and the column carrier can be 2.01 1 1 easily prepared by immobilization of lectin on an insoluble support. Such immobilization of lectin on an insoluble support can be performed by known techniques which are conventionally employed in immobilization of biosubstances.

Of these techniques, it is preferred to use a cyan bromide activation polysaccharide method and an immobilization method using N-hydroxysuccimide esters. The cyan bromide activation polysaccharide method is a method in which an insoluble support is treated with cyan bromide, and the activated 10 product thus obtained is subjected to a coupling reaction with lectin under mild conditions to immobilize lectin. In treating the insoluble support with cyan bromide, for example basic compounds such as sodium hydroxide and sodium hydrogen-carbonate are used to adjust the pH to from 7.5 to 12, and 15 the support is treated in a solvent such as water, aceto- nitrile, or a buffer maintained at pH 7.5 to 12, for example, a 0.1 M sodium hydrogencarbonate buffer (pH about 8.7) and 0.01 M phosphate buffer (pH about 7.7), at room temperature for about 1 to 12 minutes. Usually the amount of the cyan 20 bromide used is preferred to be equal to that of the insolubl support.

Any known insoluble support which is of low nonspecific adsorption to all biosubstances, has high porosity, contains a functional group capable of immobilizing bio-25 substances under mild conditions, and is chemically and 201 1 physically sufficiently stable can be used in the invention. Insoluble supports which can be used include a support made of cellulose, e.g., aminoethyl cellulose, carboxymethyl cellulose, bromoacetyl cellulose, and p-anilino cellulose, 5 a support of cross-linked dextran, e.g., Sephadex and CM-Sephadex (produced by Farmacia Corp.), and a support of agarose, e.g., Sepharose 2B, Sepharose 4B, and Sepharose 6B (produced by Farmacia Corp.).

In the coupling reaction of the cyan bromide-10 activated support as obtained above with lectin, the cyan bromide-activated support is used in an amount from 30 to 80 times the lectin, and they are reacted in an appropriate solvent such as a 0.1 mol/£ aqueous solution of sodium hydrogencarbonate (containing 0.5 mol/2, of sodium chloride; 15 pH: 8.4) at a temperature of from 0 to 40°C, preferably from 2 to 8°C for a period of from about 10 to 20 hours. In this way, there can be prepared a carrier for affinity chromatography containing lectin.

By chromatography using the lectin-containing 20 carrier as prepared above, the desired GRA combines with the lectin contained in the carrier and is trapped in the column. Subsequently, substances capable of combining with e.g., ,201| |2 lectin can be passed through the column to perform an exchange reaction, or alternatively an adsorption separator (eluting solution), e.g., a high concentration salt, an aqueous solution of potassium thiocyanate, and a nitrate buffer, is passed through the column to dissociate and obtain the desired GRA.

In the exchange reaction, examples of the substances capable of combining with lectin when a carrier for affinity chromatography containing galactose-binding lectin is used include those which can combine with a terminal galactose-combining lectin, e.g., galactose, disaccharides containing a terminal galactose and oligosaccharides containing a terminal galactose, and examples of the substances capable of combining with lectin when a carrier for affinity chromatography containing N-acetylgalactosamine-binding lectin is used include those which can combine with a terminal N-acetyl-galactosamine-combining lectin, e.g., N-acetylgalactosamine, disaccharides containing a terminal N-acetylgalactosamine and oligosaccharides containing a terminal N-acetylgalactosamine.

The thus-obtained GRA contains glycoprotein containing a galactose and/or N-acetylgalactosamine terminus, £lyco-lipid and/or saccharide. 201112 The GRA thus prepared can be lyophilized, if desired and further purified using ordinary separation techniques. For example, those GRA preparations isolated with a galactose-binding lectin can be subjected to a separation method using a 5 N-acetylgalactosamine-combining lectin, and those isolated with a N-acetylgalactosamine-combining lectin can be treated by a separation method using a galactose-binding lectin.

Lymphocytes as used herein are not critical, and any lymphocytes of normal or cancer-bearing human being or 10 animals can be used. Examples include those lymphocytes derived from peripheral blood, bone marrow, lymph node, spleen, tonsils, and thymus. These lymphocytes are isolated by physical or chemical techniques, a surface membrane method, or the like.

The sensitization of lymphocytes with GRA is per formed by cultivating the lymphocytes on a culture medium containing GRA for a period of from 1 to 10 days, preferably from 2 to 7 days.

As culture media for use in the sensitization of 20 lymphocytes, there can be used various common nutrient media which are conventionally used in such cell cultivation. Preferred examples include a RPMI 1640 medium, an Eagle MEM culture, etc. with human serum, calf fetus serum (FCS), calf serum, horse serum or the like added thereto. The amount of 25 GRA being added to the culture is usually from 1 to 1,000 201112 ng/ml and preferably from 1 to 500 ng/ml, as calculated as an amount of sugar, per lxlO^/ml lymphocyte.

The cultivation is performed by the common method, for ex£-.ple, at a pH of about 7.2 and a temperature of about 37cC.

The lciller cells of the invention as prepared above are substantially normal lymphocytes and have a GRA-specific cell-combatting activity. For example, GRA-1-KT, which is one of the killer cells of the invention, has properties common to human peripheral blood T cells and shows a cell-combatting activity vhich is specific to cancer cells containing GRA, vhich is shown in the example as described hereinafter.

Typical examples of the novel killer cells of the invention are GRA-l-KT and GRA-M-1 which are prepared in the examples as described hereinafter. All the killer cells of the present invention are available from the applicant. / * . . . - i. -> The killer cells of the invention can be mutliplied unlimitedly on the above-described culture medium containing a T cell growth factor (TCGF, IL-2). in this case, selective cultivation of cloning of the killer cell may be performed by the conventional ultra-dilution method. These killer cells can be stored stably over a long period of time, for example, in liquid nitrogen. 20H f 2 The GRA can be used singly as an active ingredient and additionally, can be used in combination with other antibacterial agents and cancer-inhibiting agents. Cancer- inhibiting agents containing the GRA of the invention as an active ingredient may be in any form as long as they are in the condition that the GRA is contained in effective amounts.

Usually the agent is administered intravenously, subcuta-intradermally neously,/or intramuscularly as a solution, a suspension or as an emulsion. In addition, it may be provided as a dry product which can be made liquid by the addition of a suitable carrier prior to the use thereof. These liquid agent may contain a suspending agent, e.g., methyl cellulose, an emulsifying agent, e.g., lecithin, preservatives, e.g., methyl-p-hydroxy benzoate, a stabilizer which does not exert as such adverse influences on the immunizing function of humans or animals, a buffer, and the like.

Aqueous carriers which can be used include physio-logocal saline, and non-aqueous carriers which can be used include vegetable oil, e.g., sesame oil, mineral oil, e.g., paraffin, animal oil, e.g., squallene, and propylene glycol. In addition, for the purpose of immunological enhancement, 201112 appropriate adjuvants may be incorporated. Adjuvants which can be used includc Freund's complete adjuvants, saponin for a n i r s 2 ? , and alur.ir.u~: hydro* i dc for hur.ons . administered once or repeatedly over a long period of time to a cancer patient for the treatment of cancer, or can be administered to one who is liable to suffer from cancer for the prevention of cancer. least 500 mg/kg as calculated as an amount of sugar, the anticancer agent of the invention is of low toxicity and, therefore, it can be administered within a vide range of dosage. Although the concentration of GRA in the anticancer agent of the invention is not critical, it is usually preferred to be fron 0.001 to 100 yg/ml as calculated as an amount of sugar. With regard to the dose of the anticancer agent, it is usually preferred that the agent is administered in an amount of 0.001 to 1,000 vg/kg/day, at the same time or in several portions, although it varies depending on the extent of diseas, and the age and sex of the patient. killer cell as an active ingredient is preferably used as an injectable solution in combination with carriers which are used in the preparation of such blood medicines. Carriers as used herein are not critical, but carriers The anticancer agent of the invention can be Since LDj.q (mouse, intraperitoneal) of GRA is at The thus-prepared anticancer agent containing the 14 201112 having a tonicity equal to that of blood are preferred. In particular, physiological saline is preferred. In the preparation of the agent, it is preferred that the killer cell is sufficiently trashed with physiological saline or the like to remove the above-described culture medium and, thereafter, it is floated in a carrier.

The concentration of the killer cell in the agent is not limited specifically, but it is preferably from 10^ 9 to 10 per milliliter. When the killer cell is admmstered O intraperitoneally in a dose of 10 per mouce, no toxity is observed. Although the dose of the anticancer agent of the invention varies depending on the degree of disease, and the age and sex of the patient, it is usually preferred that the 12 agent is administered in a dose of 10 to 10 /kg/day, in one portion or in divided portions.

The following example, reference examples, experiment examples, and comparative examples are given to illustrate the invention in greater detail although the invention is not limited thereto.

REFERENCE EXAMPLE 1 Locality of GRA (1)-A Preparation of FITC-labelled Lectin (PNA-FITC) Ten milligrams of peanut lectin (PNA, produced by EY Co.) was dissolved in 2 ml of a 0.01 M phosphate buffer containing 0.85% NaCl (pH=7.2). In 1 ml of a 0.5 M hydrogen- 201 carbonate buffer (pH=9.0) was dissolved 2 mg of FITC (produced by Sigma Laboratories Inc.), and a 0.5 ml portion of the resulting solution was added to the above prepared PNA buffer. The mixture was then stirred at room temperature 5 for 2 hours and, thereafter, it was separated on Sephadex G25 (10 mm x 300 mm, produced by Farmacia Corp.). The initial peak was collected. FITC/PNA ratio=1.0. (1)-B Preparation of FITC-Labelled Lectin (DBA-FITC) In the same manner as in (1)-A above DBA-FITC was 10 obtained using DBA produced by EY Co. FITC/DBA ratio=0.9. (1)-C Soybean agglutinin FITC (FITC-SBA) is available from EY Co. FITC/SBA ratio=1.4. (2) Locality of GRA in Various Cancer Cells i (a) Human cultured cancer cells (1x10^) were washed 15 three times with a 0.05 M tris hydrochloric acid buffer containing 0.85% NaCl (pH=7.2) by a centrifugal procedure and then 100 yl of PNA-FITC, DBA-FITC or SBA-FITC (200 ug/ml) as prepared in (1) above was added thereto. The resulting mixture was allowed to stand at room temperature for 30 minutes 20 to cause them to react. After the reaction was completed, the reaction mixture was washed three times with a 0.01 M phosphate buffer containing 0.85% NaCl (pH=7.2) and, thereafter, the cells were placed on a glass plate and examined under a fluorescent microscope.

Mouse X5563 and mouse MH134 were treated and 201 1 12 examined in the same manner as above.

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

Table 1 Cancer Cells Raji (Burkitt lymphoma) Dauji (Burkitt lymphoma) BT-1 (Burkitt lymphoma) P-12 (T-cell lymphoma) MOLT (T-cell leukemia) Fujimaki (B-cell lymphoma) Oda (IgD myeloma) QG-56 (lung cancer, squamous) PC-1 (lung cancer, squamous) PC-3 (lung cancer, adenocarcinoma) QG-90 (lung cancer, small cell) PC-13 (lung cancer, large cell) MK-2 (stomach cancer, poor differentiation) KATO-III (stomach cancer, signet ring cancer) MKN-45 (stomach cancer, poor differentiation) MKN-1 (stomach cancer, adenocarcinoma-squamous) Positive Ratio of GRA (%) PNA DBA 98.3 1.4 93.1 .2 50.1 0 44.3 6.7 0.6 4.8 19.1 .3 0.6 .0 70.4 2.0 78.4 0.4 77.1 0 68.0 0 17.0 0 63.7 0.1 57.3 0 1.0 40.3 4.6 0.4 SBA 1 f 1 2.

Positive Ratio of Cancer Cells GRA (%) PNA DBA SBA MKN-28 (stomach cancer) MKN-74 (stomach cancer) MGH-U1 (urinary bladder cancer) KU-2 (urinary bladder cancer) T-24 (urinary bladder cancer) NBT-2 (urinary bladder cancer) NRC-12 (renal cancer) KU-1 (renal cancer) Kuramochi (ovarian cancer) NB-1 (neuroblastoma) YT-nu (neuroblastoma) TGW-nu-1 (neuroblastoma) TGW-nu-11 (neuroblastoma) GOTO (neuroblastoma) ITO (embryonal carcinoma) NEC-8 (embryonal carcinoma) SCH (choriocarcinoma, stomach) GCH (chriocarcinoma, uterus) YN-1 (rhabdomyosarcoma) Mouse X5563 (plasmacytoma) 92.0 0 90.6 Mouse MH134 (hepatoma) 18.6 0 6.4 0.4 O.i 0.5 0 37.4 0 4.5 21.4 14. 6 0 13.1 1.0 23.9 0 3.3 0.6 80.0 0 50.9 1.7 3.6 0.5 4.1 0 2.0 1.0 0.5 0 96.9 12 .3 44.6 0 14.6 3.1 .4 0 .7 1.7 92 .0 0 18.6 0 201112 (b) The malignant tissues from cancer patients were passed through a stainless steel mesh (#150) to make single cell suspension. After washing twice with 0.01M Tris-HCl buffer (pH=7.4) containing 2mM CaC^, 2mM MgC^ and 0.85% 5 NaCl, 5x10^ cells were resuspended in 100 yl of the buffer. One hundred yl of FITC-PNA or FITC-DBA (200 yg/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 cell were observed under a fluorescene micro-10 scope.

The results are shown in Table 2. The malignant tissues from cancer patients have been obtained from Kansai Medical University.

Table 2 GRA No. Cancer Patient Tissue PNA DBA 1 Stomach cancer + + 2 Stomach cancer + + 3 Stomach cancer 4 Stomach cancer + 5 Stomach cancer + + 6 Stomach cancer + 7 Breast cancer + 8 Breast cancer + 9 Breast cancer + 10 Breast cancer + + 201 I GRA No. Cancer Patient Tissue PNA DBA 11 Colon cancer + + 12 Colon cancer - + 13 Esophagus cancer + 14 Hepatoma - + Note: Symbol M+" indicates GRA is expressed on the cell surface.

Symbol indicates GRA is not expressed on the cell surface.

REFERENCE EXAMPLE 2 Preparation of GRA (1)-A Preparation of Immobilized Lectin (PNA-Sepharose) Three grams of CNBr-activated Sepharose 4B (produced by Farmacia Corp.) was fully washed with 1 mM HC1 and suspended in 200 ml of 0.1 M sodium hydrogencarbonate (pH= 8.5). Then, 5 ml of a 0.01 M phosphate buffer (pH=8.5) containing 20 mg of PNA was added, and they were reacted at 25°C for 2 hours while sometimes stirring to prepare PNA-Sepharose. (1)-B In the same manner as in (1)-A above except that DBA is used instead of PNA, DBA-Sepharose was obtained. (2) Preparation of GRA O (a) BT-1 (Burkitt lymphoma) cells (1.3x10 ) were washed three times with physiological saline, and 30 ml of a 0.01 M tris hydrochloric acid buffer (pH=7.4) containing 2% of • 201112 "TRITON X-100" (produced by Wako Pure Chemical Industries Ltd.), 0.85% of NaCl, 2 mM of CaCl2, and 2 mM of MgCl2 was added thereto. The mixture was stirred at 4°C for 15 minutes and then was subjected to ultracentrifuga! reparation at a 5 rate of 100,000 x g. Of 28 ml of the thus-obtained supernatant liquid, a 14 ml portion was passed through a column (diameter 0.5, length 1 cm) for affinity chromatography, packed with PNA-agarose beads (produced by Maruzen Co., Ltd.) which had been equilibrated by a tris hydrochloric acid buffer 10 (pH=7.4) containing 0.1% TRITON X-100, 0.85% NaCl, 2 mM CaCl2, and 2 mM MgCl2. After being washed with the same buffer as used above, it was eluted with a 0.01 M tris hydrochloric ) acid buffer (pH=7.4) containing 0.1 M lactose, 0.85% NaCl, 2 mM CaCl2, 2 mM MgCl2, and 0.1% TRITON X-100. The thus-15 eluted portion was dialyzed with a 0.01 M tris hydrochloric acid buffer containing 0.85% NaCl, 2 mM MgCl2, and 2 mM CaCl2 for 48 hours to obtain 17 ml of a GRA solution. * With the GRA solution, the amount of protein and the amount of sugar were , measured by the Folin-Lowry method and the phenol-sulfuric 20 acid method, respectively, and they were found to be 644 yg and 120 yg, respectively. This is hereinafter referred to as "GRA-l". (b) C3H/He Mouse mammary tumor (MMT) cells (1x10"^) were washed three times with physiological saline, and 30 ml 25 of a 0.01 M tris hydrochloric acid buffer (pH=7.4) containing M 12 2% TRITON X-100, 0.85% NaCl, 2 mM CaCl2, and 2 mM MgCl2 was added thereto. The mixture was stirred at 4°C for 30 minutes. Subsequently the mixture was subjected to ultracentrifugal separation at a rate of 100,000 x g for 2 hours, and the 5 supernatant liquid was dialyzed over-night with a 0.1 M tris hydrochloric acid buffer (pH=7.4) containing 0.85% NaCl, 2 mM CaCl2, and 2 mM MgCl2. The thus-dialyzed liquid was concentrated to 3 ml, and a 1 ml portion was then passed through a column (diameter 0.5 any length 2 cm) for affinity 10 chromatography, packed with the same PNA-Sepharose as used above which had been equilibrated with a tris hydrochloric acid buffer (pH=7.4) containing 0.005% TRITON X-100, 0.85% NaCl, 2 mM CaCl2, and 2 mM MgCl2. After being fully washed with the same buffer as used above, it was eluted with a 15 0.01 M tris hydrochloric acid buffer (pH=7.4) containing 0.1 M lactose, 0.85% NaCl, 2 mM CaCl2, 2 mM MgCl2, and 0.005% TRITON X-100, and the thus-eluted portion was dialyzed for 48 hours with a 0.01 M tris hydrochloric acid buffer (pH=7.4) containing 0.85% NaCl, 2 mM CaCl2 and 2 mM MgCl2 to obtain 20 2 ml of a GRA solution. With the GRA solution thus obtained, the amount of protein and the amount of sugar were 156 yg and 94.yg, respectively. This is hereinafter referred to as "GRA-M-1". 201 1 1 (c) Approximately 120 grams (wet weight) of KATO-III was a homogenized in 100 ml of PBS by a Waring blender. After 1 centrifugation at 100,000 g for 1 hour, the pellet was dissolved with a in 100 ml of 2% TRITON X-100 in 0.01 M Tris* HC1 buffer (pH=7.6) containing 0.15 M NaCl. The supernatant collected by centrifugation at 100,000 g for 1 hour and applied to a column of PNA-Sepharose 4B (0.8x15 cm) equilibrated with 0.015% TRITON X-100 in 0.01 M Tris-HCl (pH=7.6) containing 0.15 M NaCl. After washing with 50 ml of the buffer, the GRA were eluted with the buffer containing 0.1 M lactose. The eluted GRA dialysed against 0.85% NaCl, concentrated by Sephadex Pharmacia Co. and stocked at -20°C before use.

The amounts of protein and sugar determined in the same manner as in (a) above were found to be 2.0 mg and 0.8 mg, respectively. This is hereinafter referred to as "GRA-2 (d) In the same manner as in (c) above, the following GRA samples were obtained. r'i7S*r%7''-- r :■,"Tr.iwvw/v« 2011 1 Table 3 GRA Material GRA Protein Sample Source Amount (g) Content (mg) Content (mg) GRA-3 BT-1 33 0.5 0.09 GRA-4 Breast Ca. (excised) 0. 24 0.5 GRA- 5 QG-56 24 0.6 0.38 GRA-6 QG-90 26 1.0 0.54 GRA- 7 Raj i 29 0.78 0.45 GRA-M-2 MMT 200 11.3 28.4 GRA-M-3 LLC 14.4 0.06 0.07 GRA-M-4 MH-134 85 0.65 0.35 GRA-M-5 X- 5563 0. 56 0.23 (e) In the same manner as in (c) above except that MKN-45 (about 29 g) was used instead of Kato-III and DBA-Sepharose obtained in (1)-B above was used instead of PNA-Sepharose 4B and elution was carried out with N-acetylgalactosamine, a GRA preparation having a protein content of 0.03 mg and a sugar content of 0.01 mg was obtained. This is hereinafter referred to as "GRA-8". (f) GRA-3 prepared in (d) above (5 mil) was charged in DBA-20 Sepharose column and eluted with Tris-HC& buffer (0.015% Triton X-100, 2 mM MgC&2 CaCi^, 0.85% NaC£) to obtain 4 mZ fractions Nos. 1-12. Fractions Nos. 1-3 are termed "GRA-3-A" and Nos. 4-12 "GRA-3-B". Then, the column was eluted with the same buffer but containing 0.1 M N-acetylgalactosamine 2011 1 to obtain fractions, which is termed "GRA-3-C". (g) SDS Gel Electrophoresis Each of the GRA preparations according to the above procedures was subjected to electrophoresis according to the 5 method described in Fairbanks et al: Biochemistry vol. 10 p.2606, 1971.

The results obtained are shown in Figs. 18 to 22. In Figs. 18-19, numerals 1 to 5 indicate 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 indicate the following. 1 standard; 2 GRA-M-2; 3 GRA-6; 4 --- GRA-5 In Fig. 21 numerals 1 to 3 indicate the following. 1 GRA-M-4; 2 GRA-M-5; 3 standard In Fig. 22 numerals 1 to 4 indicate the following. 1 --- GRA-3; 2 --- GRA-3-A; 3 --- GRA-3-B; 4 --- GRA-3-C Fig. 18 shows the state of GRA subejcted to 20 protein staining reaction according to C.B.B. method (Fairbanks et al: Biochemistry vol. 10 p.2606, 1971).

Figs. 19-21 shows the state of GRA subjected to sugar dolor reaction according to PAS method (R.M. &charius et al: Anal. Biochem. vol. 30 p.148 (1962)). 25 Fig. 22 shows schematical illustration of the results of staining according to the C.B.B. method described above. 201f12 For the standards substances listed below from Biorad Lab. Calif. U.S.A. were used. 200 K dalton 116 " myosin " ; 3-galactosidase 5 92.5 " ; phosphorylase 66.2 " ; BSA 4 5 M ; ovalbumin 21.5 " ; soybean trypsin inhibitor REFERENCE EXAMPLE 3 Preparation of TCGF (a) The spleen (4 kg) of Japanese monkey (obtained from Japan Plymates Co., Ltd.) was excised and washed twice with a RPMI-1640 culture medium (produced by Flow Laboratory Co., Ltd.). Cells were filtered by the use of Mesh (produced by Milipore Inc., 150 mesh) and were subjected to a specific gravity centrifugal method (specific gravity, 1.076) to 9 obtain 2 liters of 2x10 /ml of lymphocytes. The thus-obtained lymphocytes were washed three times with a RPMI-1640 culture medium, and the lymphocyte number was adjusted to 7 5x10 per milliliter by the use of the same medium as used above, containing 10% FCS. It was then allowed to stand in a carbon dioxide fermentation apparatus at 37°C for 1 hour. 201112 The supernatant lymphocytes were recovered and the lymphocyte number was adjusted to 1x10^ per milliliter by the use of the same culture medium as used above, containing 1% FCS. Subsequently 1 yg/ml of indomethacin (produced by Sigma Laboratories, Inc.) and 0.2% PHA-P (produced by Difco Co.) were added, and they were cultured in a carbon dioxide gas fermentation apparatus at 37°C for 48 hours. A centrifugal separation procedure (3,000 rpm) was applied for 10 minutes, and the resulting supernatant liquid was recovered and sterilyzed by filtering with a Millipore filter (produced by Millipore Inc., 0.2 ym) to obtain 2 liters of TCGF. (b) The source of TCGF was the supernatant of mix-cultured peripheral blood lymphocytes of 10 healthy donors. Non-adherent lymphocytes prepared from C.F. (Conray. Ficol (Japan Immunoresearch Co.)) separated lymphocytes by adsorp-tion on plastic surface at 37°C for 1 hour were suspended in RPMI 1640 medium containing 1% FCS (1.5x10^ cells/ml) and incubated with 0.2% PHA-P, indomethacin (1 yg/ml) and human B-cell line (BT-1) (1.5x10^ cells/ml) pretreated with mitomycin C (50 yg/ml). After 48 hours, the culture supernatant was harvested and used as TCGF source. (H. Inoue et al, Scand. J. Immunol. 12 p.149-154 (1980)) 2011 B Z REFERENCE EXAMPLE 4 Preparation of Lymphocytes (1) Human Peripheral Blood Lymphocytes Blood (50 ml) obtained from a healhty adult or various cancer patients by heparinization was subjected to centrifugal separation by the use of Ficol Pack (produced by Farmacia Japan 7 Co., Ltd.) to obtain 5x10 of peripheral blood lymphocytes. (2) Mouce Spleen Lymphocytes The spleen of a C3H/He mouce (male, 6w) was excised and washed twice with a RPMI-1640 culture medium. The spleen was then loosened by a syringe needle and passed through a stainless steel screen (100 mesh) to remove large pieces. The thus-filtered cells were washed twice with the same culture medium as used above and was subjected to centrifugal separation at a rate of 1,200 rpm for 10 minutes to obtain 7 4x10 of spleen lymphocytes.

EXAMPLE 1 GRA-1 (amount of protein: 40 yg/ml; amount of sugar: 7.5 yg/ml) as obtained in Reference Example 2 (2)-(a)) was diluted to 1,000 times the original volume with a RPMI-1640 culture medium containing 15 % FCS to prepare a sensitization culture medium.

Human peripheral blood lymphocytes (5x10^/5 ml) as obtained in Reference Example 4 ((1)) was added to 5 ml of the sensitization culture medium as prepared above which - 28 201t12 was placed on a laboratory dish, and cultivated at 37°C for 2 days. They were further cultivated on the RPMI-1640 culture medium containing 20% TCGF and 15% FCS, as obtained in Reference Example 3, for additional five days to obtain 20 ml of a killer cell solution containing 1x10^ killer cells per milliliter. This is hereinafter referred to as "GRA-l-K-T".

EXAMPLE 2 The mouce spleen lymphocytes as obtained in Reference Example 4 ((2)) were adjusted to a number of 5xl0^/ml by the use of a RPMI-1640 culture medium containing 15% FCS.

Then, GRA-M-1 as obtained in Reference Example 2 ((2)-Qgi)) was added so that the final amounts of protein and sugar were 1.5 yg/ml and 0.9 yg/ml, respectively. A 5 ml portion of the resulting mixture was cultivated on a laboratory dish (60 mm x 15 mm, produced by Falcon Co.) at 37°C for two days. The formation of cloning was observed. The portion was further cultivated on a RPMI-1640 culture medium having 15% FCS containing 201 by volume of TCGF (produced by Japan Immuno Research Laboratories Co., Ltd.) for additional 4 days to obtain 50 ml of a killer cell solution containing 1x10^ killer cells per milliliter. This is hereinafter referred to as "GRA-M-l-K-T".

EXAMPLE 3 Peripheral blood lymphocytes (5x10^) of healthy donor was incubated in RPMI-1640 medium containing 50 ng/m£ (protein content) of GRA-2 and 151 FCS at 37°C. On day 2 the human TCGF described above was added to the medium until the concentration reached 20% and incubation was continued for further 3 days to obtain killer cells. This is hereinafter referred to as "GRA-2-K-T".

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 GRA-8, GRA-3-A and GRA-3-C, respectively (protein content: 50 ng/mi.) and the TCGF obtained in REFERENCE EXAMPLE 3-(b).

EXAMPLE 5 C3H/He mice (female, 8 weeks) was implanted intradermally with X5563 cells (10^) of the same strain and after 7 days the tumor was excised surgically. After another 7 days X5563 " cells (10**) of the same strain was inoculated, and mice resistant to inoculation were named immune mouse.

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

Each lot of spleen cells (5xl0^/well) was sensitized with 40 ng/mJl (protein content) of GRA-M-5 in RPMI-1640 medium containing 15% FCS for 5 days to obtain killer cells.

Killer cell preparation obtained from normal spleen cells 201112 is hereinafter referred to "GRA-M-5-K-T-1" and that obtained from immune spleen cells is hereinafter referred to "GRA-M-5-K-T-2".

EXAMPLE 6 Human peripheral blood lymphocytes (5x10^) were incubated in 5 m£ of RPMI-1640 medium containing 50 ng/m£ (protein content) of GRA-1 at 37°C for 2 days. On day 3, the lymphocytes were transferred to RPMI-1640 medium containing 10% serum from the donor of the lymphocytes and 0 to 100 ng/m£ 10 (protein content) of GRA-1 and incubation was continued for further 5 days to obtain killer cell preparations shown in Table 4 below.

Table 4 GRA Concentration (ng/mJl) Initial Day Day 3 Killer Cell 50 0 GRA-l-K-T-1 50 1.6 GRA-l-K-T-2 50 3.2 GRA-l-K-T-3 50 6 GRA-1-K-T-4 50 12.5 GRA-l-K-T-5 50 25 GRA-l-K-T-6 50 50 GRA-l-K-T-7 50 100 GRA-l-K-T-8 EXAMPLE 7 (a) Peripheral blood lymphocytes (PBL) from various cancer patients after operation were sensitized with GRA-3 to obtain *112 killer cell perparations. PBL (5x10 ) from the patients were incubated in RPMI-1640 medium containing 50 ng/mS, (protein content) of GRA-3 for 2 days and then in RPMI-1640 medium containing 20% TCGF and 15% FCS for further 5 days to obtain killer cells shown in Table 5.

Table 5 Peripheral Blood Lymphocytes Cancer P-GRA D-GRA Gastric ca. + + " + + Breast ca. + Gastric ca.

Days after Operation 14 21 7 21 Killer Cell GRA-3-K-T-l GRA-3-K-T-2 GRA-3-K-T-3 GRA-3-K-T-4 GRA-3-K-T-5 GRA-3-K-T-6 In the above table, P-GRA and D-GRA indicate locality of GRA expressed on the malignant tissue (excised by operation) from the cancer patients from whom PBL was collected as determined in the same manner as in REFERENCE EXAMPLE 1, 2-(b). P-GRA and D-GRA are results obtained using FITC-PNA and FITC-DBA, respectively. The days after operation indicate the timing when PBL was collected after the operation. (b) PBL (5x10^) collected from breast cancer patients after 21 days from the operation was incubated in RPMI-1640 medium containing 50 ng/m£ (protein content) of GRA-3 and 10% serum from the patient for 7 days to sensitize PBL and obtain killer cell preparation. This is hereinafter referred to "GRA-3-K-T-7", 2011 H 2 EXAMPLE 8 O GRA-l-K-T (10 ) as obtained in Example 1 was dissolved in 10 ml of physiological saline to prepare an injectable solution.

EXAMPLE 9 (i) GRA-M-1 as obtained in Reference Example 2 ((2)-(b)) was diluted with physiological saline so that the amounts of sugar and protein be 1.0 yg/ml and 1.6 yg/ml, respectively, to thereby prepare an anticancer agent No. 1. (ii) A tumor lump of C3H/He spontaneously occurring breast cancer was sterilely cut to 5 mm cube clump and transplanted to under the back skin of each of ten C3H/He mice of the same strain as above (7-week-age, males). Seven days after the transplantation, the fixation and multiplication of the tumor were confirmed/ To five of the mice were each administered subcutaneously the anticancer No. 1 as prepared in (i) above in a dosage of 300 yl per day at two day intervals. .The remaining five mice were used as untreated controls. Ten days after the first administration, the tumor was excised by operation, and the mean weight was measured. At the same time, pathohistological examination was performed.

Tumor Volume: 3 Adminstered group 22.3 mm (Fig. 14) Control 162.7 mm^ (Fig. 15) This means that there was a 86.3% reduction in the tumor. Pathohistological Examination: I 1 12 In the control group (Fig.16), birds' nest bodies of cancer were formed, the type of tissue was like medullary canalicular cancer and the multiplication of tumor cells was observed all over the tissue. On the other hand, in the agent administered group (Fig- 17), the cancer cells caused liquefaction necrosis at the sites where the cancer cells were formed, and calcification and fibrosis occurred, leaving only a very limited amount of cancer cells. Thus, the anti-tumor properties of the anticancer agent of the invention were observed.

TEST EXAMPLE 1 GRA-l-K-T (1 yl) as obtained in Example 1 was placed on a microplate (produced by Falcon Corp.) and was allowed to stand at room temperature for 15 minutes. Then, 4 yl of FCS (produced by Falcon Corp.) was added and the mixture was allowed to stand at room temperature for 30 N minutes. Neuraminidase-treated sheep red blood cells (SRBC ) 9 adjusted in number to 1x10 per milliliter and 5 yl of a 0.01 M phosphate buffer (pH=7.2) with 0.85% NaCl added were added, and the plate was subjected to a centrifugal separation procedure at a rate of 600 rpm for 5 minutes. The plate was then reversed, and unreacted SRBC^ was removed. A dyeing solution (Brilliant Cresyl Blue, produced by Merck § Co.) was added to dye the lymphocytes, and rosette-forming posi-tivity was examined. As a result, it has been found that 201112 at least 981 exhibits rosette-forming positivity (T-cells.) TEST EXAMPLE 2 Specific Cancer Cell-Killing Activity (a) Of the cells shown in Table 1, the following five cell strains having different GRA positivity ratios were used as target human cancer cells.

Target Cancer Cells: No. 1 BT-1 (Burkitt lymphoma) No. 2 Daudi (Burkitt lymphoma) No. 3 KATO-III (stomach cancer) No. 4 MKN-45 (stomach cancer) No.

MOLT (T cell leukemia) On a microplate (produced by Falcon Corp.) were 4 laminated 5x10 per well of target cancer cells by a centrifugal procedure at a rate of 800 rpm for 5 minutes. Then, 3 4x10 per well of GRA-l-K-T as obtained in Example 1 was gently added and incubated for 1 hour.

The killing activity was determined according to the degree of plaques-formation, and was rated as follows: ++ . Killing activity is significantly observed. + Killing activity is observed.

^ Killing activity is slightly observed.

Killing activity is not observed. In a control group, there were used unsensitized human peripheral blood lymphocytes which had been prepared * 1 w ■■ ■■ it ■ i i— v t s-ai i ■! • ' 201I12 in the same manner as in Example 1 except that GRA was not used. The results are shown in Table 6.

It is apparent from Table 6 that the killer T cells produced by the process of the invention have strong GRA-5 specific cytotoxic activity.

Table 6 Target Cancer GRA Positivity Determination of Cell Ratio (1) Plaque-Formation GRA-l-K-T Daudi (Fig. 1) 93.1 ++ (Fig. 2) Group KATO-III (Fig. 3) 57.3 + (Fig. 4) BT-1 (Fig. ) 50.1 ++ (Fig. 6) MKN-45 (Fig. 7) 1.0 + (Fig. 8) MOLT (Fig. 9) 0.6 - (Fig.

) Control BT-1 50.1 - (Fig.

ID Group (b) The same target cancer cells as used in (a) above (3.2x10^) were mixed with 8x10^ GRA-l-K-T (cell ratio: 5/1) and the resulting cell mixture (total number: 4x10^) was cultivated on a RPMI-1640 medium containing 15% FCS. After one hour, the number of remaining cells was counted and the % cytotoxicity was calculated by the following equation. number of cells after on ft.ou.j_ . A f i ^-cultivation ^ i a a ^ 4 Cytotoxicity - (1 - Cnumber of cells befoTe) * 100) cultivation (4x10^) The results are shown in Table 7.

I 201 | f 2 Table 7 Number of Cells Before Culti- After Culti- Target Cell vation vation % Cytotoxicity Daudi 4xl06 2.9xl06 28 KATO-III 4xl06 3.7xl06 7.5 BT-1 4xl06 3.2xl06 20 MKN-45 4xl06 3.9xl06 2.5 MOLT 4xl06 4.2xl06 -5 (c) The procedure of (b) above was repeated with the exception that the mixing ratio of GRA-l-K-T to target cancer cell was changed to 5/3. The results are shown in Table 8.

Table 8 Number of Cells Before Culti- After Culti-Target Cell vation vation % Cytotoxicity Daudi 4xl06 3.6xl05 91 KATO-III 4xl06 3.6xl06 24 BT-1 4xl06 1.4xl06 65 MKN-45 4xl06 3.7xl06 7 MOLT 4xl06 4.lxlO6 -3 In the above test it is observed that GRA-l-K-T shows a high binding activity to Daudi,: KATO-III and BT-1 but only a low binding activity to MKN-45 and MOLT.

TEST EXAMPLE 3 C3H/He spontaneously-occurring breast cancer-bearing mice were administered subcutaneously by GRA-M-1-K-T as obtained |f12 in Example 2 at a dosage of 3x10^/0.3 ml/mouse three times per week every other day. After ten days, the focus was taken out and examined.

As shown in Fig. 12, the infiltration of lymphocytes into cancer cells occurred, and the breakage of the tumor area was observed. Also, from Fig. 13, it has been observed that the calcification of the tumor area occurred, and thus it can be seen that the killer cells of the invention have antitumor activity.

COMPARATIVE EXAMPLE 1 In this example, cancer cells per se were used as specific antigens in place of GRA for use in the process of the invention.

Cancer cell-sensitized lymphocytes were obtained in the same manner as in Example 1 except that, in place of GRA, BT-1, Daudi, KATO-III, or MKN-45 was used at a level of 1x10^ per laboratory dish.

With these lymphocytes, the cytotoxic activity was examined in the same manner as in Test Example 2 ((a)). The results are shown in Table 9.

It can be seen from Table 5 that the above-prepared lymphocytes do not have any cytotoxic activity.

Table 9 Cells for Use in Sensitization of Lymphocytes Target Cell bTTT Daudi KATO-III MKN-45 BT-1 - - Daudi - KATO-III - MKN-45' - - 38 201112 TEST EXAMPLE 4 In the same manner as in TEST EXAMPLE 2-(b), the cancer cell-killing activity of GRA-8-K-T, GRA-3-A-K-T and GRA-3-C-K-T obtained in EXAMPLE 4 was determined. The results obtained are shown in Table 10.

Table 10 Target % Cytotoxicity Cell GRA-8-K-T GRA-5-A-1CT GRA-3-C-lTT KATO-III 8.0 5.0 5.0 BT-1 4.3 5.0 4.0 MKN-45 20 5.0 20 MOLT 0 0 0 TEST EXAMPLE 5 (a) Cytotoxicity of each of killer cell preparations obtained in EXAMPLE 6 was determined by ^Cr release test (J_^ Immunol. 122, 2527-2533 (1979 ). That is, 50 yCi of radioactive *^Cr - 7 (Japan Isotope Association) was added to KATO-III (2x10 ) and the cells were incubated at 37°C for 1 hour in RPMI-1640 medium and sufficiently washed by centrifugation to obtain 9 1 Cr-labeled target cells. Killer cells (effector cells) (2x10^) were added to the target cells (1x10^) (thus E/T = 20/1) and the mixture was incubated at 37°C for 4 hours in RPMI-1640 medium. Supernatant was collected by centrifugation and its radioactivity was determined by liquid scintillation counter. 20111 The specific ^Cr Release (%) which corresponds to the cytolytic activity of effector cells was calculated according to the following equation.

Specific ^Cr Release (%) _ (Release in Test) - ("Spontaneous Release) , (Maximum Release) - (Spontaneous Release) x (The maximum release indicates the radioactivity when all the cells are lysed).

The results obtained are shown in Table 11.

Table 11 Specific ^Cr Killer Cell Release GRA-l-K-T-1 13 GRA-l-K-T-2 GRA-l-K-T-3 22 GRA-1-K-T-4 GRA-1-K-T-5 22 GRA-1-K-T-6 GRA-1-K-T-7 27 GRA-1-K-T-8 32 From the results shown in Table 11 above, it can be 20 seen that TCGF and FCS have no relation with the induction of killer cells. (b) Cytotoxicity of GRA-2-K-T obtained in EXAMPLE 3 was determined by ^Cr release test in the same manner as in (a) above. Specific ^Cr release of 14.3% was observed on ^Cr-25 labeled KATO-III as target cell (E/T = 20/1). fj TEST EXAMPLE 6 (a) Using KATO-III (E/T = 20/1) as target cell 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 below.

Table 12 Killer Cell % 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) Cytotoxicity of GRA-3-K-T-7 obtained in EXAMPLE 7-(b) was determined-using 51Cr-KATO-111 (E/T = 20/1) as target cell in the same manner as in TEST EXAMPLE 5. Specific ^Cr release of the killer cell was found to be 25.51.

TEST EXAMPLE 7 Cytotoxicity of the killer cells obtained in EXAMPLE 5 was determined by "*^Cr release test in the same manner as in TEST EXAMPLE 5. The target cell used was 51Cr-labeled X5563 cell. As a control lymphocytes obtained in the same manner / of spleen cells as in EXAMPLE 5 except that sensitization/was carried out with lxlO^/well of mitomycin C-treated X5563 cells (5xl0^/m& of X5563 cells were treated with 50 yg/mil of Mitomycin C for 60 minutes) instead of GRA-M-5 were used. 2011 1 The results obtained are shown in Table 13 below.

Table 13 Specific 5lCr Release (%) E/T ratio Killer Cell 40:1 :1 : 1 GRA-M-5-K-T-l 12.7 6.3 . 7 GRA-M-5-K-T-2 18.4 .6 13. 7 Control 0.0 0.0 0. 0 TEST EXAMPLE 8 (H-2 Assay) GRA-M-1, GRA-M-3 and GRA-M-4 obtained in REFERENCE EXAMPLE 2.above were serially diluted with PBS (0.85% NaC£) to prepare samples.

Anti-H-2 serum from National Institute of Genetic Research and the above sample were mixed and incubated at 4°C for 2 hours and a target cell corresponding to the anti-H-2 serum used was added thereto. Spleen cell or lymph node cell h V obtained from BIO (H-2 ) and B10-BR (H-2 ) mice by conventional method were used as target cell. After washing the cells with PBS, complement (rabbit) was added to the cell and the cells were incubated at 37®C for 1 hour and stained with 0.21 trypan blue-PBS to determine % cytotoxicity. The anti-H-2 serum was used in a maximum dilution such that it showed at least 95% cytotoxicity in the absence of GRA.

The blocking effect by GRA was determined for systems shown in Table 14 below.

-TTTT-TTTST 201112 Table 14 GRA GRA-M-1 GRA-M-3 Anti-H-2 Serum, Concentration D-23 (anti-H-2Kk), X80 or k D-32 (anti-H-2D ), X300 D-33 (anti-H-2K ), X600 or cb. ,b- D-2 (anti-H-2D ), X80 GRA-M-4 D-23 , X80 or D-32 , X300 The results obtained are shown in Table 15.

Table 15 Target Cells B10BR (H-2k) spleen cells BIO (H-2 ) spleen cells B10-BR (h-2*) lymph node cells Anti-H-2 % Cytotoxicity Dilution of Sample GRA Serum X2 X4 X8 XI6 X32 X64 X128 X256 X512 £ * I - - 14 12 13 14 13 14 13 14 13 D-23 - 97 99 96 97 97 96 97 96 96 14 D-32 - 95 95 95 95 94 95 95 96 95 13 II - - 19 14 12 12 11 12 13 14 D-33 - 98 98 99 99 98 99 99 99 99 D-2 - 95 96 95 95 97 95 97 95 95 17 III D-23 100 100 100 100 - - - - - 100 D32 99 99 99 99 - - - - - 99 Notes: GRA I GRA-M- 1 GRA II GRA-M- 3 GRA III GRA-M- 4 indicates % cytotoxicity when complement alone was used. 201112 From the results shown in Table 15 above it can be seen that GRA-M-1, GRA-M-3 and GRA-M-4 lack H-2.

TEST EXAMPLE 9 C57BL/6 mouse was transplanted under S.C. with LLC (2x10^) nanogram from the same strain and after 6 days 1/ (protein) of GRA-M-3 obtained in REFERENCE EXAMPLE 2 '(d) was administered subcuta-neously. Thereafter administration was repeated for 4 days once a day at the same dosage. The day next the final administration tumor cells were excised and weighed. As a control physiological saline administered animals were used. Each test group comprised 5 animals.

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

TEST EXAMPLE 10 C57BL/6 mouse was immunized subcutaneously with 1 ng (protein) of GRA-M-3 obtained in REFERENCE EXAMPLE 2(d) once a day for 3 days and on day 5 spleen cells were collected from the animal to obtain effector cells. A mixture of the effector cells and Lewis lung carcinoma (LLC) as target cell in a ratio of E/T = 50:1 was prepared and a portion (1x10^) thereof was transplanted to mouse of the same strain and Winn assay was performed (J. Immunol. 86, p.228-239 (1961)).

The results obtained are shown in Table 16 below. ^01 U 2 Table 16 Day 20 Effector Tumor Growth Rate (I) Mortality/ Cells Day 15 Day 17 Day 18 Day 19 Day 20 Group A .7 5.5 .7 3.1 1.4 0/10 B 21.4 39.5 37 .1 55.4 76.8 4/10 C 39.3 65.1 61 . 7 98.1 96.4 4/10 Notes: Group A represents spleen cells from GRA-M-3 immune mouse.

Group B represents spleen cells from normal mouse.

Group C represents spleen cells from mouse after 10 days from the transplantation of LLC (lxlO6).

Tumor Growth Rate was calculated according to the following equation: Tumor Growth Rate (%) = ~ x 100 TN wherein T^ represents the thickness of the foot pad on the side where the tumor was transplanted and T^ represents the thickness of the normal foot pad.

TEST EXAMPLE 11 C3H/He mouse was immunized with 4.5 yg (protein) of GRA-M-4 obtained in REFERENCE EXAMPLE 2 (d) and 0.1 mft.iiof Freund's Complete Adjuvant at the sacrococcygeal portion. After two weeks lymph node cells were collected by conventional method and were used as responder cell for determining proliferative 201112 response by GRA-M-4.

For this purpose, responder cells (4xl05) were incubated in RPMI-1640 medium containing 15% FCS in the presence of GRA-M-4 for 5 days. During final 18 hours of the incubation 3 3 period 1 yCi of H-thymidine ( H-TdR) was added to the medium and its incorporation into the cells was counted.

The results obtained are shown in Table 17 below.

Table 17 Concentration 3H-TdR Incorporation GRA-M-4 (ng/m£) (mean cpm + S.E.) S.I.

Control 0 5,302 +_ 1,761 0.5 7,903 + 1,290 1.5 1 10,076 + 936 1.9 Experiment 5 10,686 +_ 429 2.0 10,615 + 1,270 2.0 40 8,565 + 1,419 1.6 Note: S.I. indicates stimulation index m terms of Experiment/Control.

TEST EXAMPLE 12 Delayed type hypersensitivity (DTH) response of C3H/He 20 normal mouse, X5563 immune mouse and MH134 immune mouse obtained in the same manner as in EXAMPLE 5, and GRA-M-4 immune mouse and GRA-M-5 immune mouse obtained in the same manner as in TEST EXAMPLE 11 was determined by foot pad reaction (FPR). That is, GRA or MMC-treated tumor cells 25 were challenged at the foot pad skin of the hind leg of the 201t12 animal and swelling of the foot pad 24 hours after the challenge was determined. Degree of DTH response was calculated by subtracting the swelling before challenge from that _ 2 after challenge (10 mm).

The results obtained are shown in Tables 18-22.

Table 18 Challenge 1 2 3 4 Notes: _ 2 Mean Foot Pad Increment (10 mm) Normal Mouse 2.8 12.4 3.6 3.2 6.8 MH134 Immune Mouse 13.6 32.0 3.6 22.0 27.6 Group 1; syngeneic normal spleen 1x10 /20 y£ medium (Hanks solution) Group 2; MH134 cell 1x10^/20 y£ medium Group 3; medium 20 y£, Group 4; GRA-M-4, 0.8 yg (protein)/20 y% medium Group 5; " 0.4 yg " Table 19 -2 Challenge 1 2 3 Notes: Mean Foot Pad Increment (10 mm) Normal Mouse X5 563 Immune Mouse MH134 Immune Mouse 4.3 1.1 24.3 .4 0.9 2.0 16.9 Group 1; medium (Hanks solution) 20 y£ Group 2; GRA-M-4, 4 yg (protein)/20 yS, medium Group 3; GRA-M-5, " " 201112 Table 20 Challenge 1 2 Notes: Challenge 1 2 3 Notes: Challenge 1 2 3 4 -2 Mean Foot Pad Increment (10 mm) Normal Mouse GRA-M-4, Immune Mouse 1.7 -0.3 .1 24.6 Group 1; medium (Hanks solution) 20 y£ Group 2; GRA-M-4, 4 yg (protein)/20 yJi medium Table 21 -2 Mean Foot Pad Increment (10 mm) Normal Mouse -1.8 6.3 6.8 GRA-M-4, Immune Mouse 0.6 20.0 6.3 Group 1; medium (Hanks solution) 20 y£ Group 2; GRA-M-4, 4 yg (protein)/20 y£ medium Group 3; 4 yg (protein)/20 y£ medium of fraction which passed through PNA-column at the time of GRA-M-4 (hereinafter "C.P.") Table 22 -2 Mean Foot Pad Increment (10 mm) Normal Mouse GRA-M-4, Immune Mouse 2.4 2.6 2.4 .5 -1.0 17.7 1.8 18.9 01112 Notes: Group 1; medium (Hanks solution) 20 y£ Group 2; GRA-M-4, 3.8 yg (protein)/20 y£ medium Group 3; 3.8 yg (protein)/20 y£ medium of "C.P." above Group 4; 3.8 y (protein) of GRA-M-4 and 3.8 yg (protein)/20 y£ medium of "C.P." REFERENCE TEST X5563 Immune mouse was obtained in the same manner as in EXAMPLE 5. Spleen cells from this immune mouse were used as 7 effector cell and the effector cells (10 ) and target cells (X5563, 10^) were together transplanted to mouse of the same strain. Winn assay was performed in the same manner as in TEST EXAMPLE 10.

The results obtained are shown in Fig. 23, in which the abscissa indicates days and the coordinate shows mean tumor 2 size (cm ) +_ S.E. and various marks represent the following.

• ©: Group in which effector cells were not added. o o: Group in which non-treated effector cells were added.

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

□ □; Group in which effector cells treated with anti-Lyt 1 (New England Nuclear Co., U.S.A) 201 1 1 and rabbit complement were added.

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

From the results shown in Fig. 23 it can be seen that Lyt 1 type T-cells play an important role in the mechanism of in vivo effector in tumor immunity.

Further, it is known that DTH response is mediated by Lyt-1 T-cells, (J. Exp. Med. 143 p.1534-39 (1976)).

While the invention has been described in detail and with reference to specific embodiment thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof. 201112 - - V *

Claims (13)

WHAT WE CLAIM IS:

1. A process for preparing a glyco-related antigen which is a cancer specific antigen, which comprises isolating, from cancer cells, a glyco-related antigerr which is a cancer cell "'.eiiibrane component capable of combining with a lectin which car. combine specifically with a terminal . galactose or a terminal •' | ~ - N-acetylgalactosamine.

2. The process as claimed in Claim 1, wherein said cancer cells are human cancer cells.

I 3. The process as claimed in Claim 1, wherein said isolation comprises preparing a cell membrane component from human cancer cells by homogenization or solubilization and separating a component capable of combining with a lectin by using said lectin.

4. The process as claimed in Claim 3, wherein said lectin is one which can combine specifically with a terminal galactose.

5. The process as claimed in Claim 4, wherein said lectin is peanut lectin.

6. The process as claimed in Claim 3, wohrcin said lectin is one which can combine specifically with a terminal N-acetyl-galactosamine.

7- The process as claimed in Claim 6, wherein said lectin is Dolichos bean agglutinin.

8. The process as claimed in Claim 1, wherein said isolation comprises preparing a cell membrane component from human cancer cells by homogenization or solubilization, separating - 51 - .ml] -,-iV 201112 a component capable of combining with a lectin by using a first lectin which can combine specifically with a terminal galactose and then separating a component capable of combining with a lectin from the resulting component by using a second lectin which can combine specifically with a terminal N-acetylgalactosamine.

9. The process as claimed in Claim 8, wherein said first lectin is peanut lectin, and said second lectin is Dolichos bean agglutinin.

10. The process as claimed in Claim 1, substantially as specifically described herein in any one of the Examples.

11. A pu.ro crlyco-related antigen which is obtained by the process as claimed in any one of Claims 1 to 10.

12. A pharmaceutical composition comprising a pharmaceutically effective amount of a glyco-related antigen prepared by the process as claimed in any one of claims 1 to 10, and a pharmaceutically acceptable carrier.

13. A method of treating (non-human animals) for cancer comprising applying an effective amount of a glyco-related antigen prepared by the process as claimed in any one of claims 1 to 10. BALDWIN, SON & CAREY (&.4U. ATTOm iE'i- 3 FOP. THE APPUC^SjTS - 52 -