GB2106935A - Cancer cell-combatting lymphocytes process, for the production thereof, and anticancer agents containing said lymphocytes - Google Patents

Abstract

A cancer cell-combatting lymphocyte, a process for the production of such lymphocytes, and an anticancer drug containing the lymphocyte as an active ingredient are described. The cancer cell-combatting lymphocyte acts specifically on cancer cells containing a cancer cell-derived glyco-related antigen and destroys them.

Description

SPECIFICATION Cancer cell-combatting lymphocytes, process for the production thereof, and anticancer agents containing said lymphocytes The present invention relates to cancer cell-combatting lymphocytes (hereinafter referred to as "kil ler 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.

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 in vivo, finally putting the cancer-bearing host to death.

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 surface markers on mouse leukemia cells or embryonal carcinoma cells using lectins such as Dolichos biflorus agglutinin (DBA) and peanut aggiutinin (PNA) as described in Biochem. Biophys. Res. Comm. 89 (2) 448-445 (1979), Ibid. 96(4)1547-1553 (1980), J. Biochem. 89 473-481 (1981) and Cell 18183-191 September 1979.

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

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 normal cells, there is GRA which acts as an immunogen for 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 specifically on cancer cells containing GRA and if the killer cells are administered to the host, they recognise 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 provides an anticancer agent containing a killer cell or GRA as an active ingredient.

Fig. 1 is a microphotograph of Daudi cancer cells; Fig. 2 is a microphotograph illustrating the formation of plaques by GRA-1-K-T of the cancer cell of Fig. 1; Fig. 3 is a microphotograph of KATO-III cancer cells; Fig. 4 is a microphotograph illustrating the formation of plaques by GRA-1-K-T of the cancer cell of Fig.3; Fig. 5 is a microphotograph of BT-1 cancer cells; Fig. 6 is a microphotograph showing the formation of plaques by GRA-1-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-1-K-T of the cancer cell of Fig. 7; Fig. 9 is a microphotograph of MOLT cancer cells; Fig. 10 is a microphotograph showing the formation of plaques by GRA-1 -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; 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 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; 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 cell elctrophoresis diagram of GRA using protein staining by C.B.B. method; and Fig. 23 represents a graph showing tumor growth rate in CSH/HE mouse transplanted with X5563 immune mouse spleen cells and X5563 cells.

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-occuring cancer cells, chemical substance or virus-induced cancer cells, and cancer cells derived from operated tissues, by the following procedure.

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 N-acetyl, whereby GRA is combined with the lectin and 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 Immunitaetsforch, 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-acetylgalactosamine-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 homogenization method and a solubilization method using a dissolving agent. It is more advantageous to employ a method in which cancer cells are homogenized in physiological saline or in an appropriate buffer, a portion precipitated is collected by a technique such as centrifual 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 Wako 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 compo nents can be separated GRA capable of combining with lectin by usual physical chemical of bioche mical 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 easily preprared by immobilisation 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 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 hydrogencarbonate are used to adjust the pH to from 7.5 to 12, and the support is treated in a solvent such as water, acetonitrile, 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 temperatureforaboutl to 12 minutes. Usuallythe amount of cyan bromide used is preferred to be eqdal to that of the insoluble support.

Any known insoluble support which is of low non-specific adsorption to ail biosubstances, has high porosity, contains a functional group capable of immobilizing biosubstances under mild conditions, and is chemically and 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, 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-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 a 0.1 mol/l aqueous solution of sodium hydrogencarbonate (containing 0.5 mol/l of sodium chloride; 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 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., lectin can be passed through the column to perform an exchange reaction, or alternatively an adsorption separator (eiuting 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 ofthe 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-acetylgalactosesamine-binding lectin is used include those which can combine with a terminal N-acetylgalactosamine-combining lectin, e.g., N-acetyigalactosamine, disaccharides containing a terminal N-acetylgalactosamine and oilgosaccharides containing a terminal N-acetylgalactosamine.

The thus-obtained GRA contains glycoprotein containing a galactose and/or N-acetylgalactosamine terminus, glycolipid and/or saccharide.

The GRAthus 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 N-acetyl galactosamine-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 hu man being or 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 mem brane method, or the like.

The sensitization of lymphocytes with GRA is performed 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 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 GRA being added to the culture is usually from 1 to 1,000 ng/ml and preferably from 1 to 500 ng/ml, as calculated as an amount of sugar, per 1 x 106/ml lymphocyte.

The cultivation is performed by the common method, for example, at a pH of about 7.2 and a temperature of about 37"C.

The killer 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 cel!-combatting activity which is specific to cancer cells containing GRA, which is shown in the example as described hereinafter.

Typical examples of the novel killer cells of the invention are GRA-1-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 and a sample of GRA-1-K-Tcell line has been deposited with ATCC on September 1982 and received ATCC No. CRL 8175.

The killer cells of the invention can be multipled unlimitedly without losing their activity on the above-described culture medium containing a T cell growth factor (TCGF, 1 L-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.

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, subcutaneously, intradermally 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 agents may contain a suspend ing 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 physiological saline, and non-aqueous carrers 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, appropriate adjuvants may be incorporated. Adjuvants which can be used include Freund's complete adjuvants, saponin for animals, and aluminium hydroxide for humans.

The anticancer agent of the invention can be 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 cancerforthe prevention of cancer.

Since LD50 (mouce, intraperitoneal) of GRA is at 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 wide range of dosage. Although the concentration of GRA in the anticancer agent of the invention is not critical, it is usually preferred to be from 0.001 to 100 ,ag/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,ag/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.

The thus-prepared anticancer agent containing the 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 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 washed 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 105 to 109 per milliliter. When the killer cell is administered intraperitoneally in a dose of 108 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 agent is administered in a dose of 105 to 1012/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 FlTC-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% NaCI (pH=7.2). In 1 ml of a 0.5 M hydrogen carbonate buffer (pH=9.0) was dissolved 2 mg of FlTC (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 tem per- ature 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 FlTC-Labelled Lectin (DBA-FITC) In the same manner as in (1)-A above DBA-FITC was obtained using DBA produced by EY Co.

FITC/DBA ratio=0.9.

(1)-C Soybean agglutinin FITC (FITC-SBA) is available from EY Co. FlTC/SBAratio=1.4.

t2) Locality of GRA in Various Cancer Cells (a) Human cultured cancer cells (1 x 106) were washed three times with a 0.05 M tris hydrochloric acid buffer containing 0.85 NaCI (pH=7.2) by a centrifugal procedure and then 100 l of PNA-FITC, DBA-FITC or SBA-FITC (200 Lg/ml) as prepared in (1) above was added thereto. The resulting mixture was allowed to stand at room temperature for 30 minutes 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% NaCI (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 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 Positive Ratio of GRA (%) PNA DBA SBA Raji (Burkittlymphoma) 98.3 1.4 Dauji (Burkitt lymphoma) 93.1 5.2 BT-1 (Burkitt lymphoma) 50.1 0 P-12 (T-cell lymphoma) 44.3 6.7 MOLT (T-cell leukemia) 0.6 4.8 Fujimaki (B-cell lymphoma) 19.1 5.3 Oda (IgD myeloma) 0.6 10.0 QG5-56 (lung cancer, squamous) 70.4 2.0 PC-1 (lung cancer, squamous) 78.4 0.4 PC-3 (lung cancer, adenocarcinoma) 77.1 0 QG-90 (lung cancer, small cell) 68.0 0 PC-13 (lung cancer, large cell) 17.0 0 MK-2 (stomach cancer, poor differentiation) 63.7 0.1 KATO-III (stomach cancer, signet ring cancer) 57.3 0 MKN-45 (stomach cancer, poor differentiation) 1.0 40.3 MKN-1 (stomach cancer, adenocarcinoma-squamous) 4.6 0.4 MKN-28 (stomach cancer) 0.4 0.1 MKN-74 (stomach cancer) 0.5 0 MGH-U1 (urinary bladder cancer) 37.4 0 KU-2 (urinary bladder cancer) 4.5 21.4 T-24 (urinary bladder cancer) 14.6 0 NBT-2 (urinarybladdercancer) 13.1 1.0 NRC-12 (renal cancer) 23.9 0 KU-1 (renal cancer) 3.3 0.6 Kuramochi (ovarian cancer) 80.0 0 NB-1 (neuroblastoma) 50.9 1.7 YT-nu (neuroblastoma) 3.6 0.5 TGW-nu-1 (neuroblastoma) 4.1 0 TGW-nu-11 (neuroblastoma) 2.0 1.0 GOTO (neuroblastoma) 0.5 0 ITO (embryonal carcinoma) 96.9 12.3 NEC-8 (embryonal carcinoma) 44.6 0 SCH (choriocarcinoma, stomach) 14.6 3.1 GCH (chriocarcinoma, uterus) 5.4 0 YN-1 (rhabdomyosarcoma) 5.7 1.7 Mouse X5563 (plasmacytoma) 92.0 0 90.6 Mouse MH134 (hepatoma) 18.6 0 6.4 (b) The malignant tissues from cancer patients were passed through a stainless steel mesh (#150) to make single cell suspension. After washing twice with 0.01 M Tris.HCI buffer (pH=7.4) containing 2mM CaCI2, 2mM MgCl2 and 0.85% NaCI, 5x105 cells were resuspended in 100 EI of the buffer. One hundred ,ml of FITC-PNA or FITC-DBA (200,ag/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 fluoescene microscope.

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

Table2 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 + + 11 Colon cancer + + 12 Colon cancer - + 13 Esophagus cancer + 14 Hepatoma - + Note: Symbol "+" 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 HCI 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 (a) BT-1 (Burkitt lymphoma) cells (1.3x108) 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"TRITON X-100" (produced by Wako Pure Chemical Industries Ltd.), 0.85% of NaCI, 2 mM of CaCI, and 2 mM of MAC12 was added thereto. The mixture was stirred at 40C for 15 minutes and then was subjected to ultra-centrifugal separation at a 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 (pH=7.4) containing 0.1% TRITON X-100, 0.85% NaCI, 2 mM CaCI2, 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% NaCI, 2 mM CaCI2, 2 mM MgC12, and 0.1% TRITON X-100.

The thus-eluted portion was dialyzed with a 0.01 M tris hydrochloric acid buffer containing 0.85% NaCI, 2 mM MgCl2, and 2 mM CaCI2 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 acid method, respectively, and they were found to be 644,us and 120 clog, respectively. This is hereinafter referred to as "GRA-1".

(b) C3H/He Mouse mammary tumor (MMT) cells (1 x1010) were washed three times with physiological saline, and 30 ml of a 0.01 M tris hydrochloric acid buffer (pH=7.4) containing 2% TRITON X-100, 0.85% NaCI, 2mM CaCI2, and 2 mM MgCl2 was added thereto. The mixture was stirred at 4"C for 30 minutes.Subsequently the mixture was subjected to ultra-centrifugal separation at a rate of 100,000 x g for 2 hours, and the supernatant liquid was dialyzed over-night with a 0.1 M tris hydrochloric acid buffer (pH=7.4) containing 0.85% NaCI, 2 mM CaCI2, and 2 mM MgC12. The thus-dialyzed liquid was concentrated to 3 ml, and a 1 ml portion was then passed through a column (diameter 0.5 cm, length 2 cm) for affinity 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% NaCI, 2 mM CaCI2, and 2 mM MgC12. After being fully 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% NaCI, 2 mM CaCI2, 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% NaCI, 2 mM CaCI2 and 2 mM MgCl2 to obtain 2 ml of a GRA solution. With the GRA solution thus obtained, the amount of protein and the amount of sugar were 156,at and 94,ug, respectively. This is hereinafter referred to as "GRA-M-1".

(c) Approximately 120 g, (wet weight) of KATO-III was a homogenized in 100 ml of PBS by a Waring blender. After 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.HCI buffer(pH=7.6) containing 0.15 M NaCI. The supernatant collected by centrifugation at 100,000 g for 1 hour and applied to a column of PNA-Sepharose 4B (0.8x 15 cm) equilibrated with 0.015% TRITON X-100 in 0.01 M Tris.HCI (pH=7.6) containing 0.15 M NaCI. 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% NaCI, 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.

Table3 GRA GRA Material Protein Sugar Sample Source Amount Content Content (g) (mg) (mg) GRA-3 BT-1 33 0.5 0.09 GRA-4 Breast Ca. 5 0.24 0.5 (excised) GRA-5 QG-56 24 0.6 0.38 GRA-6 QG-90 26 1.0 0.54 GRA-7 Raji 29 0.78 0.45 GRA-M-2 MMT 200 11.3 28.4 GRA-M-3 LLC 14.4 0.06 0.07 GRA-M-4 MH-134 85 0.65 0.35 GRA-M-5 X-5563 25 0.56 0.23 (e) In the same manner as in (c) above except that MKN-45 (about 299) was used instead of Kato-lll 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 ml) was charged in DBA-Sepharose column and eluted with Tris-HCI buffer (0.015% Triton X-100, 2 mM Mac12 CaCI2, 0.85% NaCI) to obtain 4 ml 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 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 method described in Fairbanks et al: Biochemistry vol. 10 p.2606, 1971.

The results obtained are shown in Figs. 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 subjected to protein staining reaction according to C.B.B. method (Fairbanks et al: Biochemistryvol. 10 p.2606, 1971).

Figs. 19-21 shows the state of GRA subjected to sugar color reaction according to PAS method (R.M.

Zacharius et al: Anal. Biochem. vol.30 p.148 (1962)).

Fig. 22 shows schematical illustration of the results of staining according to the C.B.B. method described above.

For the standards substances listed below from Biorad Lab. Calif. U.S.A. were used.

200 Kdalton; Myosin 116 ; ss-galactosidase 92.5 ; phosphorylase 66.2 ; BSA 45 ; ovalbumin 21.5 ; soybeantrypsin inhibitor REFERENCE EXAMPLE 3 Preparation of TCGF (a) The spleen (4kg) 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 obtain 2 liters of 2x109/ml of lymphocytes.

The thus-obtained lymphocytes were washed three times with a RPMI-1640 culture medium, and the lymphocyte number was adjusted to 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. The supernatant lymphocytes were recovered and the lymphocyte number was adjusted to lx 106 per milliliter by the use of the same culture medium as used above, containing 1% FCS.Subsequently 1,zbg/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 ism) 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 adsorption of plastic surface at 37"C for 1 hour were suspended in RPMI 1640 medium containing 1 FCS (1.5x106 cells/ml) and incubated with 0.2% PHA-P, indomethacin (1,ag/ml) and human B-cell line (BT-1) (1.5x105 cells /ml) pretreated wjth mitomycin C (50 jag/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)) REFERENCE EXAMPLE 4 Preparation of Lymphocytes (1) Human Peripheral Blood Lymphocytes Blood (50 ml) obtained from a healthy adult or various cancer patients by heparinization was subjected to centrifugal separation by the use of Ficol Pack (produced by Farmacia Japan Co., Ltd.) to obtain 5x107 of peripheral blood lymphocytes.

(2) Mouce Spleen Lymphocytes The spleen of a C3H/He mouce (male, 6w) was excised and washed twice with a RPM 1-1640 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 1200 RPM for 10 minutes to obtain 4x107 of spleen lymphocytes.

EXAMPLE 1 GRA-1 (amount of protein: 40,ag/ml; amount of sugar: 7.5 g/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 (5x106/5 ml) as obtained in Reference Example 4 ((1)) was added to 5 ml of the sensitization culture medium as prepared above which 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 1 x106 killer cells per milliliter. This is hereinafter referred to as "GRA-1-K-T".

EXAMPLE 2 The mouce spleen lymphocytes as obtained in Reference Example 4 ((2)) were adjusted to a number of 5x106/ml bythe use of a RPMI-1640 culture medium containing 15% FCS. Then, GRA-M-1 as obtained in Reference Example 2 ((2))-(B)) was added so that the final amounts of protein and sugar were 1.5,ag/ml and 0.9 g/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.) at37'Cfortwo days. The formation of cloning was observed.The portion was further cultivated on a RPMI-1640 culture medium having 15% FCS containing 20% by volume of TCGF (produced by Japan Immuno Research Laboratories Co., Ltd.) for additonal 4 days to obtain 50 ml of a killer cell colution containing 1 xl 06 killer cells per milliliter. This is hereinafter referred to as "GRA-M-1 -K-T".

EXAMPLE 3 Peripheral blood lymphocytes (5x 106) of healthy donor was incubated in RPMI-1640 medium containing 50 ng/ml (protein content) of GRA-2 and 15% 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-Twere prepared in the same manner as in EXAMPLE 1 using GRA-8, GRA-3-A and GRA-3-C, respectively (protein content: 50 ng/ml) and the TCGF obtained in REFERENCE EXAMPLE 3-(b).

EXAMPLE 5 C3H/He mice (female, 8 weeks) was implanted intradermally with X5563 cells (106) of the same strain and after 7 days the tumor was excised surgically. After another 7 days X5563 myeloma cells (105) of the same strain was inoculated, and mice resistance 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 (5x 1 06/well) was sensitized with 40 ng/ml (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 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 perioheral blood lymphocytes (5x106) were incubated in 5 mi of RPMI-1640 medium containing 50 ng/ml (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/ml (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/ml Initial Day Day 3 Klller Cell 50 0 GRA-1-K-T-1 50 1.6 GRA-1-K-T-2 50 3.2 GRA-1-K-T-3 50 6 GRA-1-K-T-4 50 12.5 GRA-1-K-T-5 50 25 GRA-1-K-T-6 50 50 GRA-1 -K-T-7 50 100 GRA-1-K-T-8 EXAMPLE 7 (a) Peripheral blood lymphocytes (PBL) from various cancer patients after operation were sensitized with GRA-3 to obtain killer cell preparations. PBL (5x 106) from the patients were incubated in RPMI-1640 medium containing 50 ng/ml (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.

Table5 Peripheral Blood Lymphocytes Days after Cancer P-GRA D-GRA Operation Killer Cell Gastric ca. + + 14 GRA-3-K-T-1 " + + 35 GRA-3-K-T-2 Breast ca. + - 21 GRA-3-K-T-3 + + 7 GRA-3-K-T-4 " + 35 G GRA-3-K-T-5 Gastric ca. + - 21 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 (5x 106) collected from breast cancer patients after 21 days from the operation was incubated in RPMI-1640 medium containing 50 ng/ml (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", EXAMPLE 8 GRA-1-K-T (108) 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,ag/ml and 1.6,ag/ml, respectively, to thereby prepare an anticancer agent No.1.

(ii) A tumor lump of C3H/He spontaneously occuring breastcancerwassterilelycutto 5 mm cube clump and transplanted to under the back skin of each often 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 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: Administered group 22.3 mm3 (Fig. 14) Control 162.7 mm3 (Fig. 15) This means that there was a 86.3% reduction in the tumor.

Pathohistological Examination: 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 occured, 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-1-K-T (1,gI) 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, 4iLl of FCS (produced by Falcon Corp.) was added and the mixture was allowed to stand at room temperature for 30 minutes. Neuraminidase-treated sheep red blood cells (SRBCN) adjusted in number to lx 109 per milliliter and 5,al of a 0.01 M phosphate buffer (pH=7.2) with 0.85% NaCI 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 SRBCN was removed.A dyeing solution (Brilliant Cresyl Blue, produced by Merck & Co.) was added to dye the lymphocytes, and rosette-forming positivity was examined. As a result, it has been found that at least 98% exhibits rosette-forming positivity (T-cells.) TESTEXAMPLE 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.5 MOLT (T ceil leukemia) On a microplate (produced by Falcon Corp.) were laminated Sx 1 per well of target cancer cells by a ceritrifugal procedure at a rate of 800 rpm for 5 minutes. Then, 4x103 per well of GRA-1-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 in the same manner as in Example 1 except that GRA was not used. The results are produced by the process of the invention have shown in Table 6. 5 strong GRA-specific cytotoxic activity.

It is apparent from Table 6 that the killer T cells Table 6 Target Cancer GRA Positivity Determination of Cell Ratio (%) Plaque-Formation GRA-1-K-T Daudi (Fig. 1) 93.1 ++(Fig.2) Group KATO-III (Fig. 3) 57.3 + (Fig. 4) BT-1 (Fig. 5) 50.1 ++(Fig.6) MKN-45 (Fig.7) 1.0 i (Fig. 8) MOLT (Fig.9) 0.6 - (Fig. 10) Control Group BT-1 50.1 - (Fig. 11) (b) The same target cancer cells as used in (a) 10 medium containing 15% FCS. After one hour, the above (3.2x 106) were mixed with 8x 105 GRA-1-K-T number of remaining cells was counted and the % (cell ratio: 5/1) and the resulting cell mixture (total cytoxicitywas calculated by the following equation.

number: 4x106) was cultivated on a RPMI-1640 number of cells after cultivation % Cytotoxicity = (1 - ( ) x 100) number of cells before cultivation (4x106) The results are shown in Table 7.

Table7 Number of Cells Target Cell Before Cultivation After Cultivation % Cytotoxicity Daudi 4x106 2.9x106 28 KATO-III 4x106 3.7x106 7.5 BT-1 4x106 3.2x106 20 MKN-45 4x106 3.9x106 2.5 MOLT 4x106 4.2x106 -5 (c) The procedure of (b) above was repeated with 15 target cancer cell was changed to 5/3. The results are the exception that the mixing ratio of GRA-1 -K-T to shown in Table 8.

Table 8 Number of Cells Target Cell Before Cultivation After Cultivation % Cytotoxicity Daudi 4x106 3.6x105 91 KATO-III 4x106 3.6x106 24 BT-1 4x106 1.4x106 65 MKN-45 4x106 3.7x106 7.2 MOLT 4x106 4.1x106 -3 In the above test it is observed that GRA-1-K-Tshows a high binding activity to Daudi, KATO-III and BT-1 but only a low binding activity to MKN-45 and MOLT.

TESTEXAMPLE 3 C3H/He spontaneously-occurring breast cancerbearing mice were administered subcutaneously by GRA-M-1 -K-T as obtained in Example 2 at a dosage of 3 x 106/0.3 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 perse 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 lox 106 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 9 that the above-prepared lymphocytes do not have any cytotoxic activitity.

Table 9 Cells for Use in Sensitization of Lymphocytes Target Cell BT-1 Daudi KATO-III MKN-45 BT-l - - - - Daudi - - - - KATO-IlI - - - - MKN-45 - - - - 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 deter mined. The results obtained are shown in Table 10.

Table 10 Target % Cytotoxicity Cell GRA-8-K-T GRA-3-A-K- T GRA-3-C-K-T 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 51Cr release test (J. Immunol. 122, 2527-2533 (1979). That is 50 ,aCi of radioactive 51Cr (Japan Isotope Association) was added to KATO-lli (2x 10') and the cells were incubated at 37"C for 1 hour in RPMI-1640 medium and sufficiently washed by centrifugation to obtain 5'Cr-labeled target cells.Killer cells (effector cells) (2x105) were added to the target cells (1 x103) (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.

Notes: Group 1; medium (Hanks solution) 20 IL Group 2; GRA-M-4, 3.8 pg (protein)/20 l medium Group 3; 3.8 yg (protein)/20 IL1 medium of "C.P." above Group 4; 3.8 IL (protein) of GRA-M-4 and 3.8 g (protein)/20 l CLI medium of "C.P." The specific 51Cr Release (%) which corresponds to the cytolytic activity of effector cells was calculated according to the following equation.

Specific 51Cr Release (%) (Release in Test)- (Spontaneous Release) x 100 (Maximum Release) - (Spontaneous Release) (The maximum release indicates the radioactivity when all the cells are lysed).

The results obtained are shown in Table 11.

Table ii Specific 51Cr Killer Cell Release (%) GRA-1-K-T-1 13 GRA-1-K-T-2 25 GRA-1-K-T-3 22 GRA-1-K-T-4 20 GRA-1-K-T-5 22 GRA-1-K-T-6 25 GRA-1-K-T-7 27 GRA-1-K-T-8 32 From the results shown in Table 11 above, it can be seen that TCG F 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 51Cr release test in the same manner as in (a) above. Specific 51Cr release of 14.3% was observed on 51Cr labeled KATO-lII as target cell (E/T = 20/1).

TEST EXAMPLE 6 (a) Using KATO-III (ElT = 20/1) as target cell cytotoxicity of killer cells obtained in EXAMPLE 7 - (a) was determined in the same manner as in TEST EXAM PLE 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 G RA-3-K-T-6 20.2 (b) Cytotoxicity of GRA-3-K-T-7 obtained in EXAM PLE 7 - (b) was determined using S1Cr-KATO-III (ElT = 20/1) as target cell in the same manner as in TEST EXAMPLES. Specific 51Cr release of the killer cell was found to be 25.5%.

TEST EXAMPLE 7 Cytotoxicity of the killer cells obtained in EXAM PLE 5 was determined by 5'Cr release test in the same manner as in TEST EXAMPLE 5. The target cell used was S1Cr-labeled X5563 cell. As a control lymphocytes obtained in the same manner as in EXAMPLE 5 except that sensitization of the spleen cells was carried out with 1 x 105/well of mitomycin C-treated X5563 cells (5x5/ml of X5563 cells were treated with 50 ,ag/ml of Mitomycin C for 60 minutes) instead of GRA-M-5 were used.

The results obtained are shown in Table 13 below.

Table 13 Specific 51Cr Release {%) E/Tratio Killer Cell 40:1 20:1 10:1 GRA-M-5-K-T-1 12.7 6.3 5.7 GRA-M-5-K-T-2 18.4 20.6 13.7 Control 0.0 0.0 0.0 TESTEXAMPLE 8 (H-2 Assay) GRA-M-1, GRA-M-3 and GRA-M-4 obtained by REFERENCE EXAMPLE 2 above were serially diluted with PBS (0.85% NaCI) 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 obtained from B10 (H-2b) and B10.BR (H-2k) 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 0.2% 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.

Table 14 GRA Anti-H-2 Serum, Concentration Target Cells GRA-M-1 D-23 (anti-H-2Kk), X80 B10BR (H-2k) or spleen cells D-32 (anti-H-2Dk), X300 GRA-M-3 D-33 (anti-H-2Kb), X600 B10 (H-2b) or spleen cells D-2 (anti-H-2Db), X80 GRA-M-4 D23 , X80 B10.Br (h-2k) or lymph node cells D-32 ,X300 The results obtained are shown in Table 15.

Table 15 % Cytotoxicity Anti-H-2 Dilution of sample GRA Serum X2 X4 X8 X16 X32 X64 X128 X256 X512 0 * I - - 13 14 12 13 14 13 14 13 14 13 D-23 - 97 99 96 97 97 96 97 96 96 14 D-32 - 95 95 95 95 94 95 95 96 95 13 II - 19 15 14 12 15 12 11 12 13 14 D-33 - 98 98 99 99 98 99 99 99 99 15 D-2 - 95 96 95 95 97 95 97 95 95 17 Ill D-23 100 100 100 100 - - - - - 100 10 D32 99 99 99 99 - - - - - 99 10 Notes: GRA : GRA-M-1 GRAIl: GRA-M-3 GRA Ill: GRA-M-4 indicates % cytotoxicity when complement alone was used.

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 C57BU6 mouse was transplanted under S.C. with LLC (2x106) from the strain and after 6 days 1 mg (protein) of GRA-M-3 obtained in REFERENCE EX AMPLE 2 (d) was administered subcutaneously.

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 comprises 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 average tumor weight of about 100 mg.

TEST EXAMPLE 10 C57BL6 mouse was immunized subcutaneously with 1 ng (protein) of GRA-M-3 obtained in REFER ENCE 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 (1 x105) 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.

Table 16 Day 20 Effector Tumor Growth Rate (%) Mortality Cells Day 15 Day 17 Day 18 Day 19 Day20 Group A 5.7 5.5 5.7 3.1 1.4 0/10 B 21.4 39.5 37.1 55.4 76.8 4/10 C 39.3 65.1 61.7 98.1 96.4 4/10 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 (1 x106).

Tumor Growth Rate was calculated according to the following equation: Tumour Growth Rate (%) = TA-TN X 100 TN wherein TA represents the thickness of the foot pad on the side where the tumor was transplanted and TN represents the thickness of the normal foot pad.

TEST EXAMPLE 11 C3H/He mouse was immunized with 4.5 cog (protein) of GRA-M-4 obtained in REFERENCE EXAMPLE 2 (d) and 0.1 ml of 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 response by GRA-M-4.

For this purpose, responder cells (4x105) 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 period 1 1LCi of 3H-thymidine (3H-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 (nglml) (mean cpm + S.E. S.l.

Control 0 5,302 t 1,761 0.5 7,903 + 1,290 1.5 1 10,076+ 936 1.9 Experiment 5 10,686 + 429 2.0 20 10,615 + 1,270 2.0 40 8,565i1,419 1.6 1.6 Note: "S.l." indicates stimulation index in terms of Experi ment/Control TEST EXAMPLE 12 Delayed type hypersensitivity (DTH) response of C3H/He 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 were challenged at the foot pad skin of the hind leg of the animal and swelling ofthefoot pad 24 hours after the challenge was determined.

Degree of DTH response was calculated by subtracting the swelling before challenge from the after challenge (10.2 mm).

The results obtained are shown in Tables 18-22.

Table 18 Mean Foot Pad Increment (10.2 mm) Challenge Normal Mouse MH134 Immune Mouse 1 2.8 13.6 2 12.4 32.0 3 3.6 3.6 4 3.2 22.0 5 6.8 27.6 Notes: Group 1; syngeneic normal spleen 1x106/ 20,awl medium (Hanks solution) Group 2; MH134 cell 1x106/20 I medium Group 3; medium 20 yl Group 4; GRA-M-4, 0.8 y9 (protein)/20 IL1 medium Group 5; GRA-M-4, 0.4 g (protein)/20 ILl medium Table 19 Mean Foot Pad Increment (10-2 mm) Challenge Normal Mouse X5563ImmuneMouse MH 134 Immune Mouse 1 5.4 4.3 1.1 2 0.9 - 24.3 3 2.0 16.9 Notes:Group 1; medium (Hanks solution) 20,al Group 2; GRA-M-4,4ILg (Protein)/20,a1 medium Group 3; GRA-M-5,4,ag (Protein)/20 l l medium Table 20 Mean Foot Pad Increment (10-2 mm) Challenge Normal Mouse GRA-M-4, Immune Mouse 1 1.7 -0.3 2 5.1 24.6 Notes:Group 1; medium (Hanks solution) 20 l Group 2; GRA-M-4, 4 g (protein)/20,al medium Table 21 Mean Foot Pad Increment (102mm) Challenge Normal Mouse GRA-M-4, Immune Mouse 1 -1.8 0.6 2 6.3 20.0 3 6.8 6.3 Notes Group 1; medium (Hanks solution) 20 IL1 Group 2; GRA-M-4, 4 ILg (protein)/20,al medium Group 3; 4, g (protein)/20,al medium of fraction which passed through PNA-column at the time ol GRA-M-4 (hereinafter "C.P.") Table 21 Mean Foot Pad Increment (10.2 mm) Challenge Normal Mouse GRA-M-4, Immune Mouse 1 2.4 -1.0 2 2.6 17.7 3 2.4 1.8 4 5.5 18.9 Notes Group 1; medium (Hanks solution) 20 it Group 2;GRA-M-4, 3.8 yg (protein)/20 IL1 medium Group 3; 3.8 g (protein)/20 IL1 medium of "C.P." above Group 4; 3.8 IL (protein) of GRA-M-4 and 3.8 ,9 (protein)/20 yI medium of "C.P." REFERENCE TEST X5563 Immune mouse was obtained in the same manner as in EXAMPLES. Spleen cells from this immune mouse were used as effector cell and the effector cells (107) and target cells (X5563, 105) 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 absissa indicates days and the coordinate shows mean tumor size (cm2) + S.E. and various marks represent the following.

o--o Group in which effector cells were not added.

O--O Group in which non-treated effector cells were added.

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 rabbitcomplementwere added.

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

I--I 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 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.

Claims (35)

1. A cancer cell-cytotoxic lymphocyte specific to a cancer cell-derived glyco-related antigen.

2. The iymphocyte as claimed in Claim 1, which is prepared by the sensitization of the lymphocyte with the cancer cell-derived glyco-related antigen.

3. The lymphocyte as claimed in Claim 1 or 2, wherein the glyco-related antigen is a cancer cell membrane component which combines with lectin combining specifically with a terminal galactose or N-acetylgalactosamine.

4. The lymphocyte as claimed in Claim 3, wherein the glyco-related antigen is a cancer cell memrane componentwhich combines with lectin combining specifically with a terminal galactose.

5. The lymphocyte as claimed in Claim 3, wherein the glyco-related antigen is a cancer cell membrane component which combines with lectin combining specifically with a terminal N-acetylgalactosamine.

6. A process for producing cancer cell-cytotoxic lymphocytes which comprises sensitizing lymphocytes with a cancer cell-derived glyco-related antigen.

7. The process as claimed in Claim 6, wherein the glyco-related antigen is a cancer cell membrane component which combines with lectin combining specifically with a terminal galactose or N-acetylgalactosamine.

8. The process as claimed in Claim 6, wherein the glyco-related antigen is a cancer cell membrane component which combines with lectin combining specifically with a terminal galactose.

9. The process as claimed in Claim 6, wherein the glyco-related antigen is a cancer cell membrane component which combines with lectin combining specifically with a terminal N-acetylgalactosamine.

10. An anticancer agent containing as an active ingredient a cancer cell-cytotoxic lymphocyte specific to a cancer cell-derived glyco-related antigen.

11. The anticancer agent as claimed in Claim 10, wherein the cancer cell-cytotoxic lymphocyte is prepared by the sensitization of the lymphocyte with a cancer cell-derived glyco-related antigen.

10. The anticancer agent as claimed in Claim 10 or 11, wherein the glyco-related antigen is a cancer cell membrane component which combines with lectin combining specifically with a terminal galactose or N-acetylgalactosamine.

13. The anticancer agent as claimed in Claim 12, wherein the glyco-related antigen is a cancer cell membrane component which combines with lectin combining specifically with a terminal galactose.

14. The anticancer agent as claimed in Claim 12, wherein the glyco-related antigen is a cancer cell membrane component which combines with lectin combining specifically with a terminal N-acetylgalactosamine.

15. An anticancer agent containing a cancer cell-derived glyco-related antigen as an active ingredient.

16. The anticancer agent as claimed in Claim 15, wherein the glyco-related antigen is a cancer cell membrane component which combines with lectin combining specifically with a terminal galactose or N-acetylgalactosamine.

17. The anticancer agent as claimed in Claim 16, wherein the glyco-related antigen is a cancer cell membrane component which combines with lectin combining specifically with a terminal galactose.

18. The anticancer agent as claimed in Claim 16, wherein the glyco-related antigen is a cancer cell membrane component which combines with lectin combining specifically with a terminal N-acetylgalactosamine.

19. A method for the treatment of cancer which comprises administering a cancer-bearing patient with a cancer cell-cytotoxic lymphocyte specific to a cancer cell-derived glyco-related antigen, or a cancer cell-derived glyco-related antigen.

20. A glyco-related antigen which is isolated from human cancer cell membrane component and can combine with a lectin which can combine with a terminal galactose or a terminal N-acetylgalactosa mine.

21. The glyco-related antigen as claimed in Claim 20, wherein the glyco-related antigen is prepared by isolating a glyco-related antigen from human cancer cell membrane component with a lectin which can combine specifically with a terminal galactose or a terminal N-acetylgalactosamine.

22. The glyco-related antigen as claimed in Claim 21, wherein the lectin is one which combines ;specifically with a terminal galactose.

23. The glyco-related antigen as claimed in Claim 22, wherein the lectin is peanut lectin, Ricinus communis lectin or Soybean lectin.

24. The glyco-related antigen as claimed in Claim 23, wherein the lectin is peanut lectin..

25. The glyco-related antigen as claimed in Claim 21, wherein the lectin is one which combines specifically with a terminal N-acetylgalactosamine.

26. The glyco-related antigen as claimed in Claim 25, wherein the lectin is selected from the group consisting of Dolichos bean agglutinin, braid orange lectin, Helix pomatia lectin, lima-bean lectin, soybean lectin and Bauhinia bean lectin.

27. The glyco-related antigen as claimed in claim 26, wherein the lectin is Dolichos bean agglutinin.

28. A glyco-related antigen prepared by isolating a glyco-reiated antigen from human cancer cell membrane component firstly with a first lectin which combines specifically with a terminal galactose, and then with a second lectin which combines specifically with a terminal N-acetylgalactosamine.

29. The glyco-related antigen as claimed in Claim 28, wherein the first lectin is peanut lectin and the second lectin is Dolichos bean agglutinin.

30. A process for preparing a glyco-related antigen comprising isolating a glyco-related entigen with can combine with a lectin that can combine specifically with a terminal galactose or a terminal N-acetylgalactosamine.

31. The process as claimed in Claim 30, wherein the process comprises isolating a glyco-related antigen from human cancer cell membrane compo nent with a lectin which combines specifically with a terminal galactose or a terminal N-acetylgalactosa mine.

32. The process as claimed in Claim 31, wherein the lectin is one which combines specifically with a terminal galactose.

33. The process as claimed in Claim 31, wherein the lectin is one which combines specifically with a terminal N-acetylgalactosamine.

34. A process for preparing a glyco-related antigen comprising isolating a glyco-related antigen from human cancer cell membrane component firstly with a first lectin which combines specifically with a terminal galactose, and then with a second lectin which combines specifically with a terminal N-acetylgalactosamine.

35. The process as claimed in Claim 32, wherein the first lectin is peanut lectin and the second lectin is Dolichos bean agglutinin.