WO2000033069A1

WO2000033069A1 - Substance containing shiitake mushroom hypha extract for screening lak activity and method for screening lak activity by using the same

Info

Publication number: WO2000033069A1
Application number: PCT/JP1999/006615
Authority: WO
Inventors: Kenji Asano; Yukiko Matsuda; Yutaka Tajima
Original assignee: Kobayashi Pharmaceutical Co., Ltd.; Nagaoka, Hitoshi
Priority date: 1998-11-27
Filing date: 1999-11-26
Publication date: 2000-06-08
Also published as: HK1041518A1; JP4308350B2; CN1314963C; KR20010089498A; TWI222515B; GB2360090B; HK1042745A1; CN1332847A; CA2352614A1; JP2000162204A; HK1041518B; GB0112999D0; CA2352614C; GB2360090A

Abstract

An inexpensively available substance for screening an immunotherapeutic agent having antitumor and/or anticancer activities. A screening method for determining in vitro a substance having a LAK activity potentiating effect suitable for a subject which involves the following steps: (a) collecting the peripheral blood of the subject and preparing a lymphocyte fraction therefrom; (b) preparing a LAK-induction sample by adding the above screening substance to the lymphocyte fraction and a control sample containing no screening substance; and (c) measuring the LAK activities of the induction sample and the control sample and comparing the activities to thereby determine the LAK activity potentiating effect of the screening substance in vitro on the subject.

Description

Specification

LAK activity screening substance containing shiitake mycelium extract

And LAK active screening method using the same

The present invention relates to the field of tumor immunology. In particular, the present invention relates to substances and methods for screening for anti-tumor and immunotherapeutic agents with Z or anti-cancer activity. More specifically, the present invention relates to a screening substance for determining whether or not the effect of enhancing the activity of LAK cells (Ly immediate hokine Activated Killer Cell) in vivo can be obtained in vitro. And its method. Background art

As a basic concept in tumor immunology, it is known that tumor cells have tumor antigens. Tumor specific antigens (TSA: Tumor Specific Antigen), which are not expressed on normal cells but exist only on tumor cells, or are present in very small amounts on normal cells There are tumor associated antigens (TAA) whose expression is enhanced. Such a tumor antigen is newly expressed due to a gene mutation caused by canceration of normal cells, or is expressed by a change in the regulation of the expression accompanying the mutation. Immunotherapy is the most common treatment for tumor cells with abnormal antigenicity, such as drugs that immunize a subject with tumor antigens or enhance the immune function of the subject. . Generally, the activity of destroying tumor cells is high in NK (natural killer cells) among immune system cells, and it is known that the activity of NK cells is also enhanced by immunotherapy. NK cells are non-T non-B cytotoxic lymphoid cells that exist in normal individuals, and do not express MHC class I molecules without being restricted by MHC antigens against tumor cells, virus-infected cells, etc. It is known that it shows a damaging activity on cells whose expression is reduced. However, it has now become apparent that there are tumor cells that cannot be killed by NK cells. S. Rosenberg of the US National Cancer Institute (XCI) states that when peripheral lymphocytes are cultured with Inuichi Leukin 2 (IL-2), they can target a wide range of target cancer cells, including autologous cancers. It has been discovered that cells exhibiting cytotoxicity can be induced, and that these cells can also kill cancer cells that cannot be killed by the cells (see JP-A-62-116518). This killer cell was designated as a lymphokine-activated killer cell (LAK cell). It is known that LAK cells are not a homogeneous population in cytology but a cell population of NK cell line and killer T cell line. In recent years, adoptive immunization methods have been attempted in which peripheral lymphocytes derived from a subject are activated using IL-2 in a cell culture system, and then LAK cells exhibiting antitumor activity are re-transferred into the subject. (LAK therapy). It has been reported that adoptive immunization by repeated administration of LAK cells reduces terminal cancer or suppresses growth. However, there are individual differences in the effects of LAK therapy, and in some cases, the effects may hardly be obtained. Also, there are various problems such as separating a large amount of leukocytes from the subject, which imposes a physical burden on the subject, and necessitating large-scale culturing of the separated leukocyte cells, which is economically burdensome. is there. Furthermore, when LAK therapy is administered by direct administration of IL-2, severe side effects of high concentrations of IL-2 occur.

Specifically, when LAK adoptive immunotherapy using IL-2 is performed, symptoms such as general malaise, chills, fever, low albumin, anemia, and eosinophilia are produced as side effects. 2 is known to be stronger than when used alone. Of further note, the occurrence of several important side effects has been attributed to the possibility that LAK cells have cytotoxic activity against normal cells. It has been reported that such hematopoietic stem cell damage by LAK cells causes anemia and thrombocytopenia, as well as in vitro damage to lymphocytes and macrophage ゃ endothelial cells. In addition, IL-12 has poor absorption by oral administration, and at present, injection is the mainstream for direct administration.

Therefore, it has been desired to screen in vitro in advance whether direct administration can enhance LAK activity, rather than performing LAK therapy that may cause side effects in a state where the effect is not clear. However However, screening in vitro using IL-2 has the disadvantage of being too expensive.

Conventionally, it is known that bacteria or foods have an anticancer effect. Since these bacteria or foods having an anticancer effect have few side effects and are safe, search for anticancer substances from these bacteria or foods has been attempted. Many attempts have been made to control cancer with bacteria, such as Coley's toxin (1964) using culture filtrates of Serratia and streptococci, and treatment of leukemia with BCG (Mathe, G. et al. Cancer Res., 14, 1, 1971) and regression of cancer masses in guinea pigs (Zbar, B., et al., J. Natl. Cancerlnst., 48, 831, 1971), and yeast wall polysaccharide. Has been reported to be effective against transplanted cancers such as sarcoma 180 by administration of sarcoma.

In particular, with regard to polysaccharides, much research has been conducted on the anticancer effects of yeast dalcan, yeast mannan, polysaccharides of other bacterial cells, lichens, and polysaccharides of basidiomycetes. Of these, currently marketed as anti-cancer immunopotentiators include krestin (Kureha Chemical, Sankyo Pharmaceutical: host immune function enhancer) derived from the culture mycelium of the basidiomycete, Agaricaceae; There are polysaccharide lentinan and Suehiro Yuga polysaccharide.

Shiitake mushrooms (Lentinus edodes) are edible mushrooms that represent Japan and China and have been artificially cultivated in Japan for about 300 years, but their pharmacological effects and medicinal components have recently been elucidated. For example, the growth inhibitory effects of transplanted tumor cells such as large intestine and liver in rats and mice (Sugano, N, et al., Cancer Letter, 27: 1, 1985; Yasumasa Suzuki et al., Journal of the Japanese Colon Anal Society, 43 : 178, 1990) and mitogen fruit (Tabata, T. et al., Immunopharmacology, 24: 57, 1992; Hibino, et al., Immunopharmacology, 28: 77, 1994).

The present inventors focused on the LAK activity enhancing effect (anti-tumor and Z or anti-cancer activity) of Shiitake, and examined the in vivo LAK activity enhancing effect when Shiitake mycelium extract was directly administered to a living body. It is an object to provide a screening substance and a method thereof for screening in vitro and in vitro.

In the conventional method, the LAK activity enhancer is actually administered in vivo, Alternatively, prepare a large amount of in vivo lymphocytes, activate them in vitro with a LAK activity enhancer, and then return the activated lymphocytes to the living body to determine if they have the LAK activity enhancement effect. Had been confirmed. In addition to the drawback that this method is too expensive for treatment, there is a drawback that the physical burden on the subject becomes a problem. Therefore, if the LAK activity enhancer can be simply screened in vitro as to whether or not it actually has an effect in a living body, the physical and financial burden on the subject can be greatly reduced. Disclosure of the invention

The present inventors have found that components extracted from hyphae, which is a form in front of the edible body in the life cycle of shiitake mushrooms, have an immunostimulatory activity far superior to that of the body, and have antitumor activity and Z or anticancer activity. I found it to be active. In addition, by using the extract as a substitute for IL-2 as a LAK activity inducer in vitro, the antitumor effect and / or anti-tumor effect in vivo when the extract is directly administered to a living body. The present inventors have found that a cancer action, particularly an LAK activity enhancing action, can be squelched in vitro, and have completed the present invention.

More specifically, the present inventors have developed an in vivo cytotoxic activity when an antitumor substance or an anticancer substance containing a shiitake mycelium extract, particularly a LAK activity enhancer is directly administered to a living body. Has a positive correlation with the cytotoxic activity when lymphocytes prepared from a subject are activated in vitro with the aforementioned LAK activity enhancer. The present invention comprises the following steps:

(a) collecting peripheral blood of a subject and preparing a lymphocyte fraction therefrom;

(b) preparing a LAK-inducing sample in which the screening substance of the present invention is added to the lymphocyte fraction and a control sample in which no screening substance is added;

(c) determining the in vitro LAK activity enhancing effect of the screening substance on the subject by measuring the LAK activity of the induced sample and the control sample and comparing the results with each other;

A method for in vitro determining a substance having a LAK activity enhancing action suitable for a subject, comprising: The present invention also relates to the in vitro screening method described above. The present invention provides a screening substance containing a shiitake mycelium extract, which can screen whether LAK activity can be enhanced in vivo. Therefore, the present invention provides a mycelium extract of shiitake mushroom, which can be determined in vitro before administration of a LAK activity enhancer, whether or not the LAK activity enhancer can be expected to have an in vivo LAK activity enhancer effect. The present invention relates to a contained screening substance and a screening method thereof. The screening substance and the screening method of the present invention can be used not only for humans but also for livestock.

As used herein, “LAK activity” refers to the antitumor activity of cytotoxic T lymphocytes that can attack tumors that cannot be recognized by lymphocytes that carry NK activity and have little effect on normal cells of the self. Means that. “Enhancement of LAK activity” refers to enhancing such LAK activity, which means inducing LAK cells from lymphocytes or further increasing the antitumor activity of already existing LAK cells.

By enhancing LAK activity, the antitumor activity of LAK cells can be increased. This leads to an enhancement of the cellular immune system. Therefore, it can be used for treatments aimed at improving the immune system, in addition to treatments expected to improve antitumor activity. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a bar graph showing the results of Table 1 showing the results of LAK activity enhancement screening using the shiitake mycelium extract of the present invention. BEST MODE FOR CARRYING OUT THE INVENTION

The present invention relates to whether an antitumor or anticancer agent containing a Shiitake mycelium extract, particularly a preparation for enhancing LAK activity, can be expected to enhance LAK activity by directly administering it to a living body. A screening substance containing a mycelium extract of Shiitake mushroom, which can be determined in vitro before administration of the preparation, and a method for screening the same. In the present invention, the “screening substance” refers to a substance used for in vitro testing the effect of enhancing LAK activity in vivo when administered to a living body. In the present invention, “Shitake mushroom cell extract” used as a screening substance is obtained by cultivating Shiitake fungi on a solid medium. It refers to an extract obtained by crushing and decomposing mycelium or a solid medium containing mycelium obtained in the presence of water and enzymes.

The shiitake mycelium extract is preferably obtained by the following method. The force is not limited to this. That is, after inoculating shiitake mushroom on a solid medium based on bagasse (sugar cane squeezer) and defatted rice bran and growing the mycelium, the solid medium in which the mycelium had grown was passed through 12 mesh. Unbundle to 30% by weight or less. Water is added to the dissociated solid medium, and then carbohydrate-degrading enzyme, proteolytic enzyme, or enzyme (s) comprising a combination thereof are added, and the temperature is maintained at 30 to 35 ° C. The solid medium is ground and crushed. Enzymes used in this step include, but are not limited to, cellulases, proteases, and dalcosidases. The solid medium crushed and crushed in the above step is prepared so that at least 70% by weight or more of the bagasse fiber can pass through the 12 mesh, and then the enzyme is heated to a temperature of up to 95 ° C to reduce the enzyme. Inactivate and sterilize at the same time. Finally, the obtained suspension liquid is filtered to obtain a shiitake mycelium extract.

The shiitake mycelium extract may be used as it is as the screening substance or the immunotherapeutic agent of the present invention, but it is convenient to concentrate, freeze-dry and store it as a powder and use it in various forms when used. It is. The powder obtained by freeze-drying is a brown powder, hygroscopic and has a unique taste and odor.

The shiitake mycelium extract of the present invention can be added directly to a lymphocyte fraction obtained from peripheral blood. When added directly to the lymphocyte fraction, the concentration of the shiitake mycelium extract contained in the screening substance of the present invention is preferably 1 ng / ml to 100 mg / ml, more preferably 1 ng / ml to 100 mg / ml. zg / ml to 100; g / ml, particularly preferably 10 ig / ml to 50 g / ml. The shiitake mycelium extract of the present invention is preferably subjected to sterile treatment with acetone before being added to cultured cells or directly added to peripheral blood.

The method of screening using the screening substance of the present invention is according to the method of Takagi et al. (Clinical Immunity, 19: 245-249, 1987), but instead of IL-2, a screening substance, for example, This method differs from the conventional method in using the shiitake mycelium extract of the present invention. That is, the screening method for enhancing LAK activity of the present invention comprises the following steps:

This is a method for determining in vitro a substance having a LAK activity enhancing action suitable for a subject.

Lymphocytes are isolated from the subject's peripheral blood to induce LAK cells. Heparin is added to peripheral blood collected from the subject, and mononuclear cells at the interface are separated by specific gravity centrifugation using Ficolt Conary solution (sg = 1.077). The separated mononuclear PBS After washing 2-3 times with (pH 7.4, free of Ca and Mg), medium (preferably so that the lxi0 ⁶ Zm l, in RPMI 1640 medium (Gibco) Suspended in FBS (inactivated serum) or antibiotics). This is treated with autologous serum (plasma) for 15 minutes at 37, transferred to a coated Petri dish, and cultured at 37 ° C for 1 hour. Non-adherent cells are collected and used as a lymphocyte fraction.

The LAK induction sample is prepared, for example, by the following method. That is, the lymphocyte fraction obtained by way described above, the final concentration of cells is prepared so as to ^{^{IX 10 5 / ml~ixi0 6 / ml}} , suspended in the culture solution, the cells per Ueru Adjust the cell count to between 1 × 10 ⁴ / 数 to 1 × 10 ⁵ / ゥ by adding 100 1 of the suspended culture. The number of cells per well can be appropriately determined by those skilled in the art according to the activity of the effector cells to be used, the sensitivity of the target cells to effecuta cells, and the like. The suspension is supplemented with a shiitake mycelium extract at a final concentration of 1 ng / ml to 100 mg / ml as a screening substance according to the experimental design.

In this way, the subject's lymphocytes are cultured in the presence of various concentrations (including zero concentration) of the shiitake mycelium extract of the present invention to obtain effector cells. As used herein, the term effector cells refers to cells that have been subjected to the above-described culture treatment for 3 days, and are lymphocytes cultured with a LAK activity enhancer (reagents treated with an LAK activity enhancer). Lymphocytes cultured in culture medium without LAK activity-enhancing substances only (lymphocytes treated without induction).

Control samples are prepared in the same manner as for the preparation of LAK-derived samples, except that sterile recombinant IL-2 (rIL-2; 2000 l / ml) is added instead of adding the screening substance.

The measurement of LAK activity is carried out by a method such as ⁵¹ Cr-release Atssay method and [] lysine method. In the present invention, from the viewpoint of simplicity and objectivity, it is preferable to use a ^5l Cr release Atsusi. ^{The 51} Cr-releasing assay is a method for measuring in vitro the target cytotoxic activity of LAK cells derived from a lymphocyte treated with a LAK activity enhancer. ^The 5'Cr release technology involves the following steps:

(0 labeling the target cells by adding ⁵¹ Cr labeled sodium chromate to the target cells;

(ii) reacting the target cells with effector cells (eg, killer T cells or LAK cells) stimulated with rIL-2 as a screening substance or control;

(iii) determining the amount of ⁵¹ Cr released into the cell culture supernatant when the target cell is destroyed by the efecta cell;

And measuring the cytotoxic activity of the effector cells on target cells.

As a subcultured cell which is a target cell used in the 5 ^l Cr-releasing assay, a Daudi cell or a Raji cell is preferably used. Culture the target cells in a culture flask, collect them, label them with ⁵¹ Cr, and dispense them into a microtiter plate. As the culture solution of the target cells, use a culture solution suitable for the growth of the cells to be used. For example, use a culture solution such as RPMI 1640 to which serum, antibiotics, etc. are appropriately added.

Labeled target cells were added to ⁵¹ Cr- chromic acid Natoriumu of 100 to 150 ^ Ci to target cells 10 per ^six cultured, carried out by 1-2 hours Inkyubeto be Rukoto at 37 ° C for after stirred well . Cultured cells were washed 3 times with PBS, and used those suspended in 10% FBS added RPMI 1640 medium so as to ixi0 ⁶ / ml. After labeling, Wash with the culture medium or phosphate buffer (PBS) used at the time of feeding, and finally in a culture medium containing 10% fetal calf serum (FBS) or fetal calf serum (FCS) to a final concentration of IX 10 Prepare ⁶ / ml and use it for Atsushi. Dispense 50 xl of target cells into each well of a microtiter plate so that the cell number will be 5 × 10 ⁴ / ml.

In the assay for measuring cytotoxic activity, add 100 x 1 of 1N-HC1 to each well into which the target cells were dispensed, for maximum dissociation, and only culture medium for spontaneous dissociation. 100/1 was added, and for experimental dissociation, 1 × 10 ⁵ / ml of efecta cells stimulated with various concentrations of Shiitake mycelium extract of the present invention or a control, 2000 U / ffll rIL-2. further adding LX10 ⁶ / ml containing broth 100 1. Then, the microtiter evening plate centrifuge one plate, 800 after a 5 minute centrifugation to cells collected in Ueru bottom in rpm, you have to be cultured for 3.5 hours 37 ° C with 5% C0 ₂ incubator .

The target cytotoxic activity in ⁵¹ Cr-releasing assays is expressed by the following formula: Experimental dissociation (cpm) —Spontaneous dissociation (cpm)

% LAK activity = X100

Maximum dissociation (cpm) — Calculated by spontaneous dissociation (cpm). By comparing the values of the induced sample and the control sample with respect to the LAK activity calculated by this equation, the ability of the screening substance to induce LAK activity in Vitra 0 by the screening substance can be measured.

Maximum dissociation described above, in the step of obtaining the natural dissociation and experimental dissociation culture of target cells is carried out in 5% C0 ₂ at 37 ° C, the incubation time, experimental purposes, the number of cells used in accordance with other conditions Although it can be appropriately determined by those skilled in the art, in the present invention, the cells are cultured for 3.5 hours.

The radiation dose due to ^5lCr released into the culture supernatant can be measured by using a scintillation counter or the like.

In a preferred embodiment of the present invention, each step is performed as follows, but those skilled in the art will recognize that appropriate changes and modifications may be made. Collect the culture supernatant of each well from the cultured plate, and measure the radioactivity by using a set-up counter.

The Shiitake mushroom mycelium extract, which was confirmed to have LAK-inducing activity in inViIro by the above screening method, was administered to a living body at 3600 mg / day for 7 days to induce LAK activity in invitro. Using the lymphocyte fraction collected by the lymphocyte collection method described above, the LAK activity% was measured under the same conditions as for the LAK-induced sample described above. It was found to show a positive correlation with the results obtained in.

The present invention will be described in more detail with reference to the following Examples, which are merely illustrative and not intended to limit the scope of the present invention. It will be understood by those skilled in the art that various changes or modifications can be made to the present invention without departing from the spirit of the invention. Example

Example 1: Preparation of shiitake mycelium extract

After adding pure water appropriately to a solid medium consisting of 90 parts by weight of bagasse and 10 parts by weight of rice bran, inoculated with Shii-yuga seeds and allowed to stand in a temperature and humidity-controlled culture room to allow the mycelium to grow. Was. After the mycelium was densely packed in the solid medium, the fibrous material of the bagasse base material was unbundled so that the amount passed through the 12 mesh became 24% by weight or less. To 1.0 kg of the unbound medium, 3.5 L of pure water and 2.0 g of purified cellulase were added while maintaining the solid medium at 40 ° C to obtain a medium-containing mixture.

Next, while circulating the mixture containing the culture medium with a pump equipped with a transmission gear, a crushing and crushing action is applied to a portion of the solid culture medium for about 200 minutes so that about 80% by weight of the bagasse fiber passes through the 12 mesh. I made it. The pulverization and crushing of the medium-containing mixture were performed while gradually increasing the temperature of the mixture. Thereafter, the mixture containing the medium was further heated to 90 ° C. to inactivate the enzyme, and at the same time, sterilized, and left at 90 ° C. for 30 minutes. The obtained medium-containing mixture was filtered through a 60-mesh filter cloth to obtain a shiitake mushroom mycelium extract, which was concentrated to obtain a lyophilized powder.

The shiitake mushroom mycelium extract obtained in this way has a phenol-sulfuric acid method 25.3% of carbohydrate (w / w) by quality analysis, 19.7% (w / w) of protein by Raleigh protein analysis, and 2.6% (w / w) of polyphenol by Folon-Denis method based on gallic acid Weight) included. The Shiitake mycelium extract also contained 8% crude fat, 22% crude ash, and about 20% soluble nitrogen-free substances other than carbohydrates. Of these, the constituent sugar composition in the shiitake mycelium extract was as follows. Xyl: 15.2; Ara: 8.2; Man: 8.4; Gul: 39.4; Gal: δ.4; GlcN ": 12.0; GLuUA: 11.3 Example 2: Measurement of LAK activity

First, peripheral blood before taking Shiitake mycelium extract from subjects A, B, and C, and peripheral blood after oral administration of 1200 mg of Shiitake mycelium extract to each subject three times daily for one week Blood was collected respectively. Using the lymphocyte fraction separated from the peripheral blood by the following method, the ability of the extract of the present invention to activate lymphocytes in vivo and the use of the extract to It can be screened for correlation with its ability to activate in vitro.

First, heparin was added to these collected peripheral blood, and mononuclear cells at the interface were separated by specific gravity centrifugation using a FicoH-Conary solution (sg = 1.077). After washing twice with PBS (H 7.4, not containing Ca and Mg), RPMI 1640 medium (Gibco®) supplemented with 10% FBS (inactivated fetal bovine serum) at a concentration of 1 × 10 ⁶ / ml was added to the culture solution. ). The cells separated by the above method were transferred to a culture dish that had been previously treated with autologous serum (plasma) at 37 ° C for 15 minutes, and cultured at 37 ° C for 1 hour. Collected as fractions.

Supplemented with 10% FBS RPMI 1640 subculture cells are the target cells cultured in culture in (Daudi cells) were harvested by centrifugation, 10 ⁶ cells per 100~150μ (Μ of ⁵¹ Cr- sodium chromate (New England Nuclear) was added, incubated for one hour at 37 ° C with 5% C0 ₂ incubator. ⁵ 'after three times washing the labeled cell cultures with PBS by Cr, 10% so as to 1X10 ⁶ / ml The cells were suspended in RPMI 1640 culture medium supplemented with FBS.

Dispense 50 μl (5 × 10 ⁴ / well) of target cells labeled as above into each well of the microtiter plate, and add 1N-HC1 to the maximum dissociation group (positive control). 100 l each, the spontaneous dissociation group (negative control) was dispensed with 100 l of RPMI 1640 culture medium supplemented with 10% FBS, and the experimental dissociation group was dispensed with 10 xg / ml of the present invention. Effector cells (100/1 (1 × 10 ⁴ / well) each) stimulated with bamboo mycelium extract or rIL-2 at a concentration of 2000 μl / ml as a control were dispensed. After collecting the cells Ueru bottom and centrifuged for 5 minutes at 800 rpm by a plate centrifugal separator, and cultured for 3.5 hours at 37 ° C with 5% C0 ₂ incubator.

The culture supernatant of each well was collected from the cultured plate using S0KEN-PET No.-96, and the radioactivity was measured by a scintillation counter.

LAK activity was calculated according to the following table. '' Experimental dissociation (cpm) Natural dissociation (cpm)

LAK activity% X 100

Maximum dissociation (cpm)-spontaneous dissociation (cpm) The results are shown in Table 1 and Figure 1. table 1

LAK activity

Subject A Subject B—Subject C Before taking 13% 27% 14% Screening with extract

(Final concentration: 10wg / ml) 21% 34% 15% After taking the extract 40% 43% 15% Industrial availability

Oral administration of shiitake mycelium extract to subjects A and B confirmed that LAK activity was enhanced in vivo (see Table 1, Experiment 3). On the other hand, in in vitro screening using the extract of the present invention, the lymphocytes collected from the peripheral blood of the subjects A and B were stimulated with the extract of the present invention. Obtained when the extract of the invention is directly orally administered to subjects A and B PT / JP99 / 0 15

An LAK activity enhancing effect that was positively correlated with the LAK activity enhancing effect was observed (see Table 1, Experiment 2). Therefore, it was found that the effect of enhancing LAK activity upon oral administration of the shiitake mycelium extract of the present invention can be predicted by in vitro screening.

On the other hand, when the substance containing shiitake mycelium was orally administered to subject C, it was confirmed that LAK activity was not enhanced even in vivo (see Table 1, Experiment 3). In contrast, in an in vitro screening using the extract of the present invention, the extract of the present invention can be obtained by stimulating lymphocytes collected from the peripheral blood of the subject C using the extract of the present invention. The effect of enhancing LAK activity obtained by direct oral administration was not observed (see Table 1, Experiment 2). Therefore, it was also found from this example that the effect of enhancing the LAK activity in vivo when the Shiitake mushroom mycelium extract of the present invention was orally administered can be predicted by screening with in Vitr0. Therefore, it was found that the LAK activity enhancing effect in vitro when the LAK activity enhancing substance of the present invention is orally administered to a living body can be more accurately predicted from the screening results in vitro. Thereby, the LAK activity enhancing effect in vivo when the Shiitake mycelium extract of the present invention is directly administered can be easily determined in vitro before directly administering the LAK activity enhancing substance, In addition to being able to rapidly administer the LAK activity-enhancing substance effectively to a subject that can be expected to enhance LAK activity, LAK activity can be enhanced to a subject that cannot be expected to enhance LAK activity. Useless administration of the substance can be prevented.

In addition, the method of the present invention allows the in vivo therapeutic effect of the LAK activity enhancer to be screened in vitro without collecting a large amount of lymphocytes from the blood of the subject. The burden is also significantly reduced.

Claims

The scope of the claims

1. The following steps:

(b) preparing a LAK induction sample in which the screening substance of the present invention is added to the lymphocyte fraction and a control sample in which no screening substance is added;

A method for determining in vitro a substance having a LAK activity enhancing action suitable for a subject.

2. The method according to claim 1, wherein the screening substance is a Shiitake mycelium extract.

3. A screening substance comprising a Shiitake mushroom mycelium extract, which can be used to screen the LAK activity in vivo by using the in vitro screening method according to claim 1.

4. A method for treating a tumor in a subject by administering the screening substance according to claim 2 to a living body.