Health Care

Therapeutic agent for a cancer and method of screening the same, and health-care auxillary food

Health Care Abstract
A therapeutic agent for a cancer comprising a therapeutically effective amount of an active ingredient, wherein the therapeutic agent is used referring to an ability of acting on NK cell antigen receptor NKR-P1 of NKT cell as an index of the ability to activate the NKT cell.

Health Care Claims
What is claimed is:

1. A method for screening therapeutic agents effective against cancer, comprising; administering a potential therapeutic agent in vivo; and determining the ability of the potential therapeutic agent to activate natural killer T cells by assaying natural killer receptor P1 that exists on the cell surface of natural killer T cells.

2. The method of claim 1, wherein said assaying natural killer P1 receptors that exists on the cell surface of natural killer T cells measures CD3 and CD161 cell surface markers.

Health Care Description
BACKGROUND OF THE INVENTION

1. Field of Invention

The present invention relates to a therapeutic agent for cancers intended to activate natural killer T (NKT) cells, to a method of screening such a therapeutic agent and to health-care auxiliary food preparations for oral uptake which are taken up with view to obtaining anti-cancer activity intended to activate NKT cells.

2. Description of the Related Art

Screening of substances useful for the prevention or therapy of cancers has been made laying importance on their direct action onto cancer cells. It has been known that immune activators are useful in the therapy of cancers. However, the compounds obtained as immune activators are all weak in their anti-cancer effect and no sufficient therapeutic effect against cancers has been achieved yet by immune therapy alone or even by immune therapy in combination with chemical therapy.

Dr. Yagita, the inventor or the present invention, paid attention to the usefulness of substances that induce interleukin 12 (IL-12) in vivo and found that AHCC, a processed product of the mycelia of Lentinus edodes (Berk.) Sing. (also called shiitake), has such a function as above and established a method of treating cancers, i.e., a new immunological therapeutic method. IL-12 itself is known to have an anti-cancer effect but cause side effects when administered directly to patients and the patients tend to be intolerable to the therapy. Therefore, IL-12 itself could hardly be used as an anti-cancer agent. However, the preparation containing AHCC which Dr. Yagita reported has achieved remarkable curing and life lengthening effects in the therapy of cancers. In other words, Dr. Yagita has achieved an object of treating cancers by administration of an effective amount for inducing IL-12 in vivo of AHCC (JP 10-139670A).

IL-12 has an activity of increasing production of interferon .gamma. (IFN-.gamma.), an activity of activating and enhancing natural killer (NK) cells, lymphokine activated killer (LAK) cells, and killer T cells, which are competent for cell mediated immunity. INF-.gamma. is a cytokine that induces an immune response of an organism in a state where T helper 1 (Th1) is active. In this state, NKT cells or killer T cells readily exhibit their effects and in other words production of interleukin 2 (IL-2) and IL-12 occurs on a large scale. Killer T cells and LAK cells are known to be cells that participate in cancer immunity. NK cells are also reported to participate in anti-cancer activity of an organism. In the case of NK cells, Dr. Yagita proved that the clinical anti-cancer effect and their activity do not correlate with each other and the amount of induced production of IL-12 and NK activity show a complete inverse correlation therebetween. Therefore, it is concluded that no NK cell participates in anti-cancer activity in humans.

Currently, Dr. Yagita has indicated that a substance that has activity of inducing the production of IL-12 is likely to be a promising carcinostatic substance.

However, in some patients who suffer cancers, the administration of AHCC does not sufficiently induce the production of IL-12 to give no therapeutic effect or even when it induces the production of IL-12, it gives no sufficient therapeutic effect. Accordingly, there has been a keen demand for the development of a novel therapeutic agent for cancers which agent acts in a mechanism other than the anti-cancer effect which AHCC has.

It has been known that in the mechanism of cancer immunity, the amount of cytokine produced or induced in vivo is an important factor and hence a therapeutic method has been proposed and practiced which administers, induces or cause to be produced a cytokine that is believed to have anti-cancer activity to treat the cancer. However, although the relationships between cancers and immunity or between cancers and cytokines have been made clearer, the effect of curing the cancers and of life lengthening have been observed in 50% or less of patients who suffer the cancers. Furthermore, recently, natural killer T (NKT) cells have been found as cells that participate in cancer immunity (Cui J. et al., Science 278, 1623, 1997). The NKT cell is one of various types of cells that participate in the immune system and has, for example, potent cytokine productivity, in particular IFN-.gamma. productivity, and a function of cytotoxicity through Fas or perforin. Therefore, it is anticipated that activation of the cells will further increase curing or life lengthening effects in those patients who suffer cancers.

Taniguchi et al., have found a specific glycolipid antigen. This antigen is recognized by specific T cell antigen receptor (TCR), V.alpha.24V.beta.11, that NKT cells have. Taniguchi et al. reported that the antigen is an .alpha.-galactosylceramide. Furthermore, Taniguchi et al. proved that in cancer-carrying mice administered with .alpha.-galactosylceramide, NKT cells are activated and metastasis of the cancer is suppressed though the elimination of cancer is not observed.

It has been reported that NKT cells contain NK cell antigen receptor (hereinafter, sometimes referred to also NKR-P10; natural killer receptor P1) as another receptor (Special issue: Basis and Clinic of NKT Cells; Saishin Igaku (CurrentMedicine), Vol. 55, No. 4, 2000, p. 818-823). NKR-P1 also participates in the activation of NKT cells.

SUMMARY OF THE INVENTION

An object of the present invention is to elucidate the mechanism of activating NKT cells and provide a novel and useful therapeutic agent for cancers having an ability of activating NKT cells.

With view to solving the above problems, the present inventor has made extensive research on cancer immunity cascade for the prevention or therapy of cancers and as a result he has found that in the cascade in which activated NKT cells competent for cancer immunity participate, two different antigen receptors, i.e., NKR-P1 and V.alpha.24V.beta.11 have quite different activities from each other. The present invention has been achieved based on this discovery.

That is, the present invention provides the followings:

(1) A therapeutic agent for a cancer comprising a therapeutically effective amount of an active ingredient, wherein the therapeutic agent is used referring to an ability of acting on natural killer receptor P1 of natural killer T cell as an index of the ability to activate the natural killer T cell.

(2) The therapeutic agent for a cancer as described in (1) above, wherein the active ingredient comprises a substance having a capability of selectively acting on natural killer receptor P1 of natural killer T cell to activate the natural killer T cell.

(3) The therapeutic agent for a cancer as described in (2) above, wherein the active ingredient comprises at least one substance selected from products derived from the mycelia of fungi having an ability of selectively acting on natural killer receptor P1 of natural killer T cell to activate the natural killer T cell.

(4) The therapeutic agent for a cancer as described in (2) above, wherein the substance in the active ingredient comprises at least one selected from polysaccharides derived from the filtrate of culture of the mycelium of Shizophyllum commune Fries, processed products of the mycelium of Coriolus versicolor (Fr.) Quel., and processed product of the mycelium of Shiitake.

(5) The therapeutic agent for a cancer as described in (4) above, wherein the substance in the active ingredient comprises a sugar component having .alpha.-1,3-glucoside linkage structure.

(6) The therapeutic agent for a cancer as described in (1) above, wherein the agent comprises a processed product of the mycelium of Shizophyllum commune Fries or a polysaccharide derived from the filtrate of a culture of the mycelium of Shizophyllum commune Fries, a processed product of the mycelium of Shiitake, and a processed product of the mycelium of Ganoderma lucidum (Fr.) Karst.

(7) The therapeutic agent for a cancer as described in (1) above, wherein the agent comprises 20 to 60% by weight of a processed product of the mycelium of Shizophyllum commune Fries or a polysaccharide derived from the filtrate of a culture of the mycelium of Shizophyllum commune Fries, 20 to 60% by weight of a processed product of the mycelium of Shiitake, and 5 to 40% by weight of a processedproduct of the mycelium of Ganoderma lucidum (Fr.) Karst.

(8) The therapeutic agent for a cancer as described in (1) above, wherein the agent comprises 30 to 50% by weight of a processed product of the mycelium of Shizophyllum commune Fries or a polysaccharide derived from the filtrate of a culture of the mycelium of Shizophyllum commune Fries, 30 to 50% by weight of a processed product of the mycelium of Shiitake, and 10 to 0.30% by weight of a processed product of the mycelium of Ganoderma lucidum (Fr.) Karst.

(9) The therapeutic agent for a cancer as described in (1) above, wherein the agent is in the form of a formulation suitable for oral administration.

(10) The therapeutic agent for a cancer as described in (1) above, wherein the therapeutic agent selectively acts on natural killer receptor P1.

(11) The therapeutic agent for a cancer as described in (1) above, wherein the therapeutic agent induces mass production of interferon .gamma. as a result of selective action on natural killer receptor P1 and shifts the ratio of T helper 1 cell/T helper 2 cell, Th1/Th2, toward a value at which an immune system where mainly Th1 acts operates.

(12) The therapeutic agent for a cancer as described in (1) above, wherein natural killer receptor P1 is determined by measuring CD3 and CD161, cell surface markers, and the ability of activating natural killer T cell.

(13) A method for screening therapeutic agents for a cancer, comprising screening a therapeutic agent for a cancer based on an ability of acting on natural killer receptor P1 of natural killer T cell as an index of the ability to activate the natural killer T cell.

(14) The therapeutic agent for a cancer as described in (1) above, wherein it is used as a health-care auxiliary food preparation for oral uptake.

(15) A method of treating a cancer, comprising administering to a patient suffering a cancer a therapeutically effective amount of an active ingredient, said agent being used referring to an ability of acting on natural killer receptor P1 of natural killer T cell as an index of the ability to activate the natural killer T cell.

(16) A health-care auxiliary food comprising a substance having a capability of selectively acting on natural killer receptor P1 of natural killer T cell to activate the natural killer T cell.

(17) A commercial medium carrying information as described in (1) above.

(18) A commercial method utilizing information as described in (1) above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram illustrating an overview of the correlation among various cytokines.

FIG. 2A is a diagram illustrating the correlation of CD3.times.CD161 with the amount of interferon .gamma. (IFN-.gamma.) (IU/ml).

FIG. 2B is a diagram illustrating the correlation of V.alpha.24V.beta.11 with the amount of IFN-.gamma. (IU/ml)

FIG. 3 is a diagram illustrating the correlation of IL-12 (pg/ml) with CD3.times.CD161.

FIG. 4 is a diagram illustrating the correlation of IL-12 (pg/ml) with V.alpha.24V.beta.11.

FIG. 5 is a diagram illustrating the correlation of Th1/Th2 ratio with IL-12.

FIG. 6 is a diagram illustrating the correlation of V.alpha.24V.beta.11 with CD3.times.CD161.

FIG. 7 is a diagram illustrating the correlation of V.alpha.24V.beta.11 with Th1/Th2 ratio.

FIG. 8 is a diagram illustrating the correlation of CD3.times.CD161 with Th1/Th2 ratio.

DETAILED DESCRIPTION OF THE INVENTION

Hereinafter, the present invention will be described in detail.

The present invention has been practiced by studying the correlation of the clinical effect with cytokines. The present inventor has administered mycelia-derived products to 37 patients suffering cancer diseases and determined the levels of various cytokines (Table 1). The results are shown in FIGS. 2 to 8 as data illustrating the correlation to obtain a diagram for illustrating the correlation of various cytokines as illustrated in Table 1.

As shown in FIG. 1, it has been proved that there are strong positive correlations between Th1/Th2 ratio and IL-12, Th1/Th2 ratio and IFN-.gamma., IFN-.gamma. and IL-12, IL-12 and the ratio of CD3.times.CD161 (NKR-P1) -positive cells (CD3+/CD161+), and IFN-.gamma. and the ratio of CD3.times.CD161 (NKR-P1)-positive cells, respectively and that there is a strong inverse correlation between IL-12 and V.alpha.24V.beta.11-positive cells (TCR V.alpha.24+/TCR V.beta.11+).

Accordingly, in the present invention, it has been demonstrated that NKT cells in which V.alpha.24V.beta.11 T cell antigen receptors are stimulated show a strong inverse correlation with IL-12 and weak inverse correlation with IFN-.gamma. production amount and Th1/Th2 ratio and that the stimulation by NKT cell to V.alpha.24V.beta.11 serves to inhibit the immune function. It has been presumed that the stimulation to V.alpha.24V.beta.11 could lead to mass production of interleukin 4 (IL-4), which causes immune suppression. On the other hand, it has been demonstrated that when an NK cell antigen receptor, NKR-P1, in NKT cell is stimulated, the NKT cell shows strong positive correlation with IL-12 and IFN-.gamma. but a weak positive correlation with Th1/Th2 ratio and that the stimulation to NKR-P1 results in the activation of immune function.

As a result, upon screening substances for the ability of activating NKT cells, it is necessary to perform screening utilizing as an index at least the action to NKR-P1. Moreover, screening must be performed utilizing as an index the fact that in the activation of NKT cells, the action is selective to NKR-P1, which is NK cell antigen receptor. In addition, it is important that the action should not affect V.alpha.24V.beta.11. As a result of selective action of the substance thus screened, mass production of IFN-.gamma. is induced and in immune responses, the immune system can be shifted toward the direction where Th1 operates. Use of such a selected substance can provide a therapeutic agent for a cancer which is very useful for immune therapy for cancers. In the selection of such a useful substance, the ability of activation of a substance when it is administered in an organism can be assayed by determining its stimulation, if any, of cells carrying NKR-P1, i.e., cells having CD3.times.CD161, which are cell surface markers. The screened therapeutic agent may be an oral health-care auxiliary food preparation for oral uptake which comprises, for example, components derived from fungal mycelia.

The therapeutic agent for a cancer of the present invention comprises a therapeutically effective amount of a substance having an ability of selectively acting on NKR-P1 of an NKT cell to activate the NKT cell and preferably is administered orally.

The therapeutic agent for a cancer of the present invention is a health-care auxiliary food preparation which is orally taken in order to obtain anti-cancer effects.

The therapeutic agent for a cancer is effective in the therapy of lung cancer, pulmonary adenomatosis, thymoma, thyroid cancer, bladder cancer, colon cancer, rectal cancer, cecum cancer, ureteral cancer, breast cancer, cervical cancer, brain cancer, lingual cancer, pharynx cancer, nasal cancer, larynx cancer, gastric cancer, hepatic cancer, biliary tract cancer, testicular cancer, ovarian cancer, uterine cancer, malignant melanoma, liposarcoma, esophagus cancer, pancreatic cancer, prostatic cancer, etc. However, the present invention is not limited to these cancers.

The therapeutic agent for a cancer of the present invention comprises at least one substance selected from fungal mycelia processed products as an active ingredient. More specifically, products derived from the mycelium of Scizophyllum commune Fries, such as SPG (sizofiran: Kaken Seiyaku Co., Ltd.), i.e., a polysaccharide obtained from filtrate of the culture of mycelium of Schizophyllum commune Fries and SCP (oral uptake preparation of processed product of the mycelium of Shizophyllum commune Fries (Tozai Iyaku Kenkyusho, Ltd.)), processed product of the mycelium of Coriolus versicolor (Fr.) Quel. (Kawaratake), such as PSK (Krestin), and processed product of the mycelium of Lentinus edodes (Berk.) Sing., such as AHCC and LEM (Noda Shokkin Kogyo Co., Ltd) are effective. SPG (sizofiran: Kaken Seiyaku Co., Ltd.) is used as a carcinostatic agent for only certain types of cancers (Taito Co., Ltd. and Kaken Seiyaku Co., Ltd.). The same will do as to PSK (Krestin).

In the present invention, using the ability of inducing IL-12 production in an organism and the ability of selectively acting on NKR-P1 of NKT cell to activate the NKT cell as indices, usefulness of processed products of the mycelium of Shizophyllum commune Fries, polysaccharides derived from the filtrate of culture of the mycelium of Shizophyllum commune Fries, processed products of the mycelium of Coriolus versicolor (Fr.) Quel., such as PSK (Krestin), and processed product of the mycelium of Lentinus edodes (Berk.) Sing., such as AHCC and LEM, has been discovered and the present invention has been achieved based thereon. That is, the present invention provides a therapeutic agent for a cancer which comprises a composition containing as an active ingredient at least one selected from polysaccharides derived from the filtrate of culture of the mycelium of Shizophyllum commune Fries, such as SPG, processed products of the mycelium of Coriolus versicolor (Fr.) Quel., such as PSK Lentinus edodes (Berk.) Sing. (Krestin), and processed product of the mycelium of Lentinus edodes (Berk.) Sing., such as AHCC and LEM and which selectively acts on NKR-P1 of NKT cell to activate the NKT cell.

Further, the present inventor has found that products derived from the mycelia of fungi having the ability of selectively acting on the NKR-P1 of NKT cell to activate the NKT cell may be sugar components having .alpha.-1,3- and/or .alpha.-1,4-glucoside linkage structure, particularly preferably at least .alpha.-1,3-glucoside linkage structure. Also, he has found that the substance having the ability of activating NKT cells may be a composition derived from the mycelia of fungi containing polysaccharides and/or 2 to 10 oligosaccharides having this structure. The present inventor has studied products derived from various fungi and the ability of activating NKT and their relation with the components thereof. As a result he has found that the existence of the above sugar structure is essential for the ability of the product derived from the mycelia of fungi to selectively act on NKR-P1 of NKT cells to activate the NKT cells. In addition, he has confirmed that the .beta.-1,3- and .beta.-1,6-glucoside linkage structures of the mycelia have the ability of selectively acting on NKR-P1 of NKT cells to activate the NKT cells.

The dose of the therapeutic agent for a cancer of the present invention comprising a composition containing the mycelia of fungi sufficient for selectively acting on NKR-P1 to activate NKT cells is about 1 to about 2,000 mg/kg body weight/day and administered for 10 days to 12 months, preferably by oral administration. Of course, the therapeutic agent of the present invention may be taken up parenterally by decreasing the dose and preparing the therapeutic agent so as to have a quality bearing parenteral administration.

For the therapeutic agent for a cancer of the present invention that selectively acts on NKR-P1 of NKT cells to activate the NKT cells, the processed products of the mycelia or substances derived from the mycelia of fungi may be used alone or two or more of them may be used in combination simultaneously.

Further, the present invention may be a therapeutic agent for a cancer or a health-care auxiliary food preparation for oral uptake intended to exhibit an anti-cancer effect, comprising a substance having the ability of inducing production of IL-12 and a substance having the ability of selectively acting on NKR-P1 to activate NKT cells.

As an example for such a blend composition, at least two substances selected from processed products of the mycelium of Shizophyllum commune Fries, such as SCP, polysaccharides derived from the filtrate of culture of the mycelium of Shizophyllum commune Fries, such as SPG, processed product of the mycelium of Lentinus edodes (Berk.) Sing., such as AHCC and LEM, and processed products of the mycelium of Ganoderma lucidum (Fr.) Karst., such as MAK (Noda Shokkin Kogyo Co., Ltd.), are blended. Most of these have been used as an immune activator so as to obtain anti-cancer effects. In contrast, the present invention has found the relationship between the combination of these and the ability of inducing IL-12 production and the ability of selectively acting on NKR-P1 of NKT cells to activate the NKT cells and established its superiority over the anti-cancer effect of conventional carcinostatic agents (20% availability).

Preferred combination is a ternary composition comprising a processed product of the mycelium of Shizophyllum commune Fries or a polysaccharide derived from the filtrate of a culture of the mycelium of Shizophyllum commune Fries, a processed product of the mycelium of Lentinus edodes (Berk.) Sing., and a processed product of the mycelium of Ganoderma lucidum (Fr.) Karst. More particularly, an optimal combination is a blend composition of 20 to 60% by weight, preferably 30 to 50% by weight of a processed product of the mycelium of Shizophyllum commune Fries or a polysaccharide derived from the filtrate of a culture of the mycelium of Shizophyllum commune Fries, 20 to 60% by weight, preferably 30 to 50% by weight of a processed product of the mycelium of Lentinus edodes (Berk.) Sing., and 5 to 40% by weight, preferably 10 to 30% by weight of a processed product of the mycelium of Ganoderma lucidum (Fr.) Karst. The blend composition is effective as a therapeutic agent for a cancer or a health-care auxiliary food preparation for oral uptake intended to obtain anti-cancer effect.

The amount of oral uptake of the blend composition of the invention is usually about 1 to 2,000 mg/Kg body weight/day for adults. The dosage regimen may be adjusted depending on the amount of induced IL-12 production and/or the degree of selectively acting on NKR-P1 of NKT cells to activate the NKT cells. The period of administration is from 10 days to 12 months.

The polysaccharides obtained from the filtrate of a culture of the mycelium of Shizophyllum commune Fries has already been put on the market as SPG (sizofiran) by Kaken Seiyaku Co., Ltd. and Taito Co., Ltd. The method for producing them includes those disclosed in, for example, JP-B-Sho-52-4634 and JP-B-Sho-52-44634.

Processed products of the mycelium of Lentinus edodes (Berk.) Sing., such as LEM, and processed products of the mycelium of Ganoderma lucidum (Fr.) Karst., such as MAK, have already been put on the market by Noda Shokkin Kogyo Co., Ltd.

They can be produced, for example, by the following method. That is, rice bran is added to bagasse (sugar cane crush residue) and blended well. After adjusting the water content, the mixture is filled in a certain container to form a solid culture medium, which is then subjected to high pressure steam sterilization. Then, a preliminarily cultured mycelium of each fungus is inoculated on the medium and incubated in a culture chamber at 23.degree. C. for 4 months to grow mycelium. The culture medium on which the mycelium has proliferated is crushed and subjected to autolysis treatment. Thereafter, the thus treated culture medium is extracted with warm water for 15 hours in the case of the processed product of the mycelium of Lentinus edodes (Berk.) Sing. and the processed product of the mycelium of Ganoderma lucidum (Fr.) Karst. The extract is filtered through a membrane filter to remove microorganisms and the filtrate is concentrated and dried to obtain powder (cf. JP-B-Hei-7-1435, JP-A-Hei-1-312980, JP-B-Sho-51-19013, and JP-B-Sho-53-18591).

The processed products of the mycelium of Shizophyllum commune Fries are one of the components of the mycelium and are oil-soluble. Therefore, they can be extracted by extraction treatment with a suitable organic solvent, for example, acetone. The extract is filtered to remove microorganisms to obtain a filtrate, which is then concentrated and dried to obtain powder. In the present invention, this oral uptake preparation (SCP (Tozai Iyaku Kenkyusho, Ltd.)) can be used. The processed products of the mycelia of other fungi may be prepared by similar treatments depending of the solubility characteristics (water-soluble or oil-soluble).

The oral uptake preparation may be formulated into tablets, powders, capsules, syrups, etc. The preparation may also be formulated by blending one or more of additives such as conventional excipients, disintegrating agents, binders, and lubricants using conventional means. Further, if needed, one or more of corrigents, colorants, perfumes, stabilizers, antimicrobial agents, and antiseptics may be added.

The therapeutic agent for a cancer of the present invention may be administered in the form of a composition that selectively acts on NKR-P1 of NKT cells to activate the NKT cells, optionally containing an effective amount of a composition that induces production of IL-12 orally or intravenously or intramuscularly, preferably by an oral route that enables continuous self-control by patients themselves.

The health-care auxiliary food preparation for oral uptake of the present invention that comprises a composition that selectively acts on NKR-P1 of NKT cells to activate the NKT cells, optionally containing an effective amount of a composition that induces production of IL-12, is a health-care auxiliary food preparation for oral uptake that is expected to give anti-cancer effects when it is taken up.

As described above, the present invention provides a novel composition that selectively acts on NKR-P1 of NKT cells to activate the NKT cells and makes it clear the relationship between the ability of inducing production of IL-12 and the ability of selectively acting on NKR-P1 of NKT cells to activate the NKT cells and hence a material carrying the information on the above in a commercial medium is very useful. In addition, commercial utilization of the information can provide means for distinguishing the value of the product and commercial methods using the information are very useful.

EXAMPLES

Examples of the present invention will be described below in order to describe the present invention more specifically. However, the present invention should not be limited thereto and various modification may be made without departing from the scope of the present invention.

Example 1

One (1) kg of the composition (IL-X) consisting of 40% by weight of a processed product of the mycelium of Shizophyllum commune Fries (SCP: Tozai Iyaku Kenkyusho, Ltd.), 40% by weight of processed product of the mycelium of Lentinus edodes (Berk.) Sing. (LEM (registered trademark): Noda Shokkin Kogyo Co., Ltd.), 20% by weight of a processed product of the mycelium of Ganoderma lucidum (Fr.) Karst. (MAK: Noda Shokkin Kogyo Co., Ltd.) was uniformly blended and ternary blended granules were prepared by a fluid granulation method using a spraying-drying process. The granules were orally administered to patients in a dose of 6 g/day/body for 3 months. NKT cell and IL-12 were measured before, after 1 month and after 3 months from the onset of the treatment, respectively, in order to confirm the effect of the granules. About 85% of the patients for whom the activation of NKT cells by selective action on NKR-P1 of the NKT cells and induction of production of IL-12 were confirmed showed significant cancer regression effect and about 20% of them showed complete regression of cancer.