JPS6229524A - Irritant for remedy of malignant tumor - Google Patents

Abstract

PURPOSE:To obtain the titled irritant by bonding at least one kind of pyrazolo(3,4-d)pyrimidine or its derivative to the surface of an insoluble carrier. CONSTITUTION:The objective irritant for inducing an antineoplastic immunocyte having remarkably improved practicability, operability and safety can be produced by bonding at least one kind of pyrazolo(3,4-d)pyrimidine or its derivative [e.g. pyrazolo(3,4-d)pyrimidine, etc.] to the surface of an insoluble carrier (e.g. a hydrophilic carrier or hydrophobic carrier having the form of granule, fiber, hollow fiber, membrane, etc.) by covalent bond, ionic bond, physical adsorption, etc. The surface of the insoluble carrier has preferably a porous structure having an average pore diameter of preferably 50-3,000Angstrom . USE:It induces strong cancer-breaking cell and antineoplastic helper-T cell, and is useful not only for the remedy of cancer such as gastric cancer, pulmonary cancer, mammary cancer, hepatic cancer, etc., but also for the test of lympho cyte function of cancerous patient and the experiment of animals such as mouse.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to an immune cell stimulating material that has the function of activating immune cells in blood cells and inducing antitumor immune cells.

(Prior Art) As is well known, killer T cells, NK cells, activated macrophages, K cells, etc. play important roles as anti-tumor cells that act as an immune surveillance mechanism against malignant tumors in living organisms. It has been reported.

Therefore, as immunological therapy for malignant tumors,
It is considered that cancer patient immune cells (white blood cells) are activated to efficiently induce these anti-tumor cells. However, it has been reported that the immune function of cancer patients generally decreases as the cancer progresses, and the presence of immunosuppressive factors that suppress the immune response or the presence of suppressor T cells and suppressor macrophages in cancer patients. Induced activation has been reported.

It must be said that it is difficult to efficiently induce antitumor cells in living cancer patients whose immune capacity is in such a suppressed state. Therefore, it is thought that taking patient's leukocytes out of the body, which has been released from the immunosuppressive state, and efficiently inducing and activating antitumor cells outside the body will become a highly effective new cancer immunotherapy.

Killer T cells are thought to play a major role in anti-tumor immunity, especially among anti-tumor cells, and research has been intensively conducted to induce and activate them outside the body. In other words, the patient's tumor cells are sensitized to the cancer patient's peripheral blood white blood cells taken out of the body, and the patient's white blood cells are activated to produce killer T cells that specifically damage only the patient's tumor cells but not the patient's normal cells. This is an attempt to treat cancer by inducing it and returning it to the body of a cancer patient.

However, the killer T cells induced by this method are not strong enough to be expected to have a therapeutic effect, so they are cultured using T cell growth factor, a type of lymphoid, and are grown in large quantities. A method of administering the drug is being considered.

(Problems to be Solved by the Invention) The above method is possible because it has become possible to industrially mass-produce T-cell growth factors through genetic engineering technology, and it has become a reality that large amounts of T-cell growth factors can be used. However, there are problems such as cell degeneration caused by culturing killer T cells outside the body for a long period of time. In addition, there are various other problems that make it difficult to put it into practical use, such as the need for surgery on patient tumor cells to induce killer 1 cells, and the fact that only a small number of cancer patients have been treated with killer 1 cells. There are many problems that need to be solved, such as the ability to induce T cells, but not the gold side, and the extremely complicated operation.

(Means for Solving the Problems) In view of the problems of inducing antitumor immune cells based on the conventional techniques as described above, the present invention has been proposed to be dramatically improved in terms of practicality, operability, and safety compared to the conventional methods. The present invention provides a stimulating material for inducing anti-tumor immune cells that has improved properties.

As a result of extensive research in line with the above objectives, the present inventors have discovered that stimulants containing nucleobases, nucleosides, nucleotides and their chemically modified derivatives covalently bonded to insoluble carriers can be applied to human peripheral blood leukocytes or mouse lung cells. It was discovered that strong tumor-toxic cells were induced when the cells were brought into contact with leukocytes, and a patent application was previously filed (Japanese Patent Application No. 59-208).
066).

The present inventors conducted intensive research in order to find a stimulating material that induces even more powerful tumor-toxic cells, and as a result, pyrazolo (3
, 4-d) When a stimulant containing pyrimidine and its derivatives covalently bonded to an insoluble carrier was brought into contact with human peripheral blood leukocytes or mouse lung leukocytes, it was surprisingly found that the stimulants were extremely potent tumor-toxic cells. The present inventors have discovered that anti-tumor helper T cells are induced and have completed the present invention.

That is, pyrazolo(3,4-d)pyrimidine derivatives, which do not have cell activation ability when dissolved in a cell suspension, have the ability to activate human and mouse leukocytes when bound to an insoluble carrier. When these activated leukocytes were mixed with tumor cells, most of the tumor cells were damaged and destroyed after 5 hours of culture, and the activated leukocytes were then added to the system of tumor cells and non-activated leukocytes. They found that the ability of leukocytes to damage tumor cells was enhanced, and strong tumor-toxic cells and anti-tumor helper T cells were induced. Based on this finding, the present invention was developed based on pyrazolo (3,4 -d) A stimulating material for inducing anti-tumor immune cells, characterized in that at least one of pyrimidine and its derivatives is bound to the surface of an insoluble carrier.

In the present invention, the surface of an insoluble carrier refers to the material surface of the carrier that can come into contact with proteins, glycoproteins, lipids, and glycans, including cell surface antigens and receptors present on the membrane surface of cells, that is, leukocytes.

In the present invention, white blood cells refer to so-called white blood cells, which are blood cells excluding red blood cells and platelets, but the present invention is not limited to peripheral blood, and also includes white blood cell fractions obtained from lymph vessels, lymph nodes, lungs, and thoracic ducts. included in the concept of white blood cells.

Furthermore, a cell fraction obtained by removing granulocytes or B cells from these leukocytes is also included in the concept of leukocytes in the present invention.

The leukocytes to be activated in the present invention may be, for example, a leukocyte fraction collected from peripheral blood by continuous centrifugation, or a mononuclear cell fraction separated by Ficoll-Paque multilayer centrifugation. Alternatively, strong tumor-toxic cells can be induced using a T cell fraction separated and concentrated from peripheral blood mononuclear cells by rosette formation with known neuraminidase-treated sheep red blood cells.

The tumor-toxic cells to be induced and activated in the present invention belong to the lymphocyte fraction among white blood cells, excluding granulocytes, monocytes, and macrophages, and particularly have T cell properties.

Pyrazolo(3,4-d)pyrimidine derivatives that can be bonded to insoluble carriers that can be used in the present invention include:
You can use anything.

i.e. pyrazolo(3,4-d)pyrimidine, 4-aminopyrazolo(3,4-d)pyrimidine, 6-aminopyrazolo(3,4-d)pyrimidine, 4-amino-6-
Examples include hydroxypyrazolo(3,4-d)pyrimidine, 4-amino-6-mercaptopyrazolo(3,4-d)pyrimidine, and the like. Among them, 4-aminopyrazolo (3
, 4-d) pyrimidine, 4-amino-6-hydroxypyrazolo(3,4-d)pyrimidine gives the most favorable results. Rather than simply binding only one of these, a plurality of types may be bound to the insoluble carrier.

These substances are easy to handle during immobilization, and are extremely safe for living organisms even when eluted from the carrier. In addition, sterilization of the stimulation material can be easily performed.

The insoluble carrier used in the present invention may be either a hydrophilic carrier or a hydrophobic carrier. The shape of the insoluble carrier may be any known shape such as particulate, fibrous, hollow fiber, or membrane. As the granular or spherical insoluble carrier, those having a particle size of I to 3000 microns can be used. If the particle size is 1 micron or less, it is difficult to separate it from activated leukocytes. In particular, if the particle size is 50 microns or more, it is possible to easily separate the activated leukocytes by filtration, whereas if the particle size is 3000 microns or more, the contact area per unit weight of the carrier with the leukocytes decreases, which is not preferable. Particularly preferably, particle size 8
0 to 2000 microns. Further, if the specific gravity of the granular or spherical insoluble carrier is 1.07 or more, it can be easily separated from activated leukocytes by centrifugation or standing still.

In addition, when using a flat membrane-like or hollow fiber-like porous carrier, if the pore size is 0.05 to 10 microns, through which cells cannot pass through but medium components can freely pass through, one side of the membrane can be used. Nutrition can be supplied to the leukocytes bound to the membrane from the other side of the membrane, and it is possible to stimulate and activate a high concentration of leukocytes. In particular, porous carriers in the form of flat membranes or hollow fibers with pore diameters of 0.1 to 5 microns can be used favorably.

When using a fibrous carrier, the fiber diameter is 1 to 300
0 micron, more preferably in the range of 50 to 2000 micron. If the fiber diameter is too large,
If the frequency of contact between leukocytes and fibers decreases, sufficient activation of leukocytes does not occur, and if the leukocytes are too small, nonspecific adhesion of blood cells and clogging are likely to occur. As the fibrous carrier, known carriers such as regenerated cellulose fibers, nylon, acrylic, and polyester can be used.

Insoluble carrier materials include activated carbon, glass, etc., and their derivatives for inorganic-based carriers, and natural polymer-based carriers include simple polysaccharides such as cellulose, sepharose, dextran, starch, and their derivatives. .

Regarding synthetic polymers, vinyl polymers include styrene, vinyl acetate, methacrylate ester, acrylate ester, vinyl halide, vinylidene halide, acrylonitrile, acrylamide, methyl vinyl ketone, vinyl pyrrolidone, - There are polymers and copolymers of vinylpyridine, ethylene, propylene, butadiene, isoprene, etc. and their derivatives. Ring-opening polymers of cyclic compounds include dimethylcyclopropane, spiro-di-xylylene, norbornene, cyclobutene, Polymers and copolymers of trioxane, lactide, cyclopolysiloxane, phosphonitrile chloride, N-carboxy-α-amino acid anhydrides and their derivatives, polyformaldehyde, polyethylene oxide, polypropylene glycol, poly-3,3-bis( Examples include chloromethyl)oxacycloptane, polytetrahydrofuran, polycaprolactam, and their derivatives. .

Examples of resins and others include acrylic resins, methacrylic resins, fluororesins, epoxy resins, urea resins, amine resins, styrene resins, melamine resins, polyurethanes, silicone resins, alkyd resins, and derivatives thereof.

Examples of the polycondensate include polyester, polyamide, polyanhydride, polycarbonate, polyurea, polysulfonamide, polyimide, polybenzimidazole, and derivatives thereof.

The above-mentioned polymer carriers can be made into insolubilized carriers by using appropriate comonomers and crosslinking agents as necessary. , epoxy compounds, dienes, glitaraldehyde, etc. can be selected depending on the functional group of the object to be crosslinked (Taiseisha, "Crosslinking Agent Handbook", p.
3-77°1981).

The insoluble carrier of the present invention having a porous surface is preferably used in view of its antitumor cell inducing activity.

The porous structure on the surface of the insoluble carrier preferably has an average pore size in the range of 50 to 3000 pores, but if the average pore size is too small, proteins present on the cell surface,
If sufficient contact between cell surface molecules such as glycoproteins, lipids, and glycolipids and the surface of the insoluble carrier material cannot be obtained and the pore size is too large, the strength of the insoluble carrier will decrease, making it impractical. The pore size of the carrier that can obtain sufficient contact with cell surface molecules and is used practically is more preferably 100.
The number ranges from 00 to 2000 people.

The average pore size can be measured using a mercury intrusion porosimeter. In this method, mercury is injected into a porous material, and the pore volume is determined from the amount of mercury infiltrated, and the pore diameter is determined from the pressure required for the intrusion, and pores of 40 Å or more can be measured.

The average pore diameter is the value of r when the value of dv/dlogr is the maximum, where r is the pore diameter and V is the cumulative pore volume measured by a porosimeter.

Among insoluble carriers with a porous structure, cross-linked copolymers have heat resistance in terms of physical properties, making heat sterilization possible, and further improving the physical and mechanical strength that is a characteristic of synthetic polymers. Are better.

- For example, it can be produced by copolymerization of a vinyl monomer and a vinyl or allylic crosslinking agent. Examples of the vinyl monomer in this case include carboxylic acid vinyl esters such as vinyl acetate and vinyl propionate, vinyl ethers such as methyl vinyl ether and ethyl vinyl ether, and vinylene carbonates.

Examples of crosslinking agents include allyl compounds such as triallyl isocyanurate and triallyl cyanurate, di(meth)acrylates such as ethylene glycol dimethacrylate and diethylene glycol dimethacrylate, butanediol divinyl ether, diethylene glycol divinyl ether, and tetravinyl. Polyvinyl ethers such as glyoxal, polyallyl ethers such as diarylidene pentaerythritol and tetraallyloxyethane, and glycidyl acrylates such as glycidyl methacrylate can be used. Moreover, copolymerization of other comonomers can also be used, if necessary.

In the case of vinyl copolymers, triallylisocyanurate crosslinking of polyvinyl alcohol obtained by copolymerizing a vinyl ester of carboxylic acid and a vinyl compound (allyl compound) having an isocyanurate ring and hydrolyzing the copolymer. The body provides a good carrier in terms of strength, chemical stability, and thermal stability.

Although the case of a vinyl copolymer has been exemplified above, the present invention is not limited thereto.

All known methods such as covalent bonding, ionic bonding, and physical adsorption can be used to immobilize pyrazolo(3,4-d)pyrimidine derivatives on the surface of an insoluble carrier. It is desirable to use it by fixing it by bonding. For this purpose, methods commonly used for immobilized enzymes and affinity stomatography can be used.

For example, activating agarose, Sepharose, etc. with hydrogen bromide (CNBr), or activating silica glass beads with γ
A method such as reacting with -aminopropyltriethoxysilane to obtain alkylamino glass, activating it with glutaraldehyde, and bonding can be used. Furthermore, if necessary, a molecule (spacer) of any length may be introduced between the insoluble carrier and the insoluble carrier. For example, by reacting and bonding the hydroxyl group of agarose with the isocyanate group on one side of hexamethylene diisocyanate, the remaining isocyanate group and pyrazolo(3,4-d) are bonded.
This can be carried out by reactively bonding the amino groups of the pyrimidine derivatives.

In the present invention, pyrazolo (3,4-d
) The amount of pyrimidine derivative is determined based on the specific surface area of the insoluble carrier.
(0.05 p no l or 50011 per
IIIo j! more preferably 0.5μ
lll0! and 50μIIIol.

The specific surface area is expressed as the surface area occupied by nitrogen gas adsorbed per unit weight of dry crosslinked copolymer.

The specific surface area of the present invention was determined by the most common nitrogen gas venting method (BET method).

The method for producing the stimulant of the present invention, which consists of the above-mentioned elements, does not specify the order in which the constituent elements are combined.

Specifically, in the method of introducing pyrazolo(3,4-d)pyrimidine derivatives, it is also possible to bind them to monomers and polymerize them, or to bind them to an insoluble carrier after activation. That is, the present invention basically only needs to have one or more pyrazolo(3,4-d)pyrimidine derivatives on the surface of an insoluble carrier, and is not dependent on the production method.

Activation of leukocytes by stimulants can induce strong tumor-toxic cells when performed in a medium containing serum components. That is, a medium containing 2 to 20% of animal serum such as fetal bovine serum, live serum, horse serum, or human serum is prepared. The medium in this case is a medium commonly used for animal cell culture,
For example, RPMI 1640 medium, MEM medium, etc. can be used. Furthermore, RPM11640 medium supplemented with serum components such as serum albumin can also be used.

Also, even if Interleukin 2 is added during the induction period,
More potent tumor-toxic cells can be induced.

Leukocytes collected by various methods were added to the prepared medium at 0.
The cells are suspended at a concentration of 5 to 3 x 106 cells/lIIβ, an appropriate amount of stimulant is added thereto, and culture is carried out at a temperature of 25 to 45°C. At a temperature of 25°C or lower, little effective activation of leukocytes occurs, and at a temperature of 45°C or higher, the survival rate of leukocytes decreases. Cultivation can be easily carried out in a CO2 incubator using a commercially available plastic container for cell culture. After several hours of culture, leukocytes adhere to the stimulant and become activated.

It has been found that the leukocytes activated in this manner contain potent tumor-damaging cells and anti-tumor helper T cells.

That is, when human peripheral blood leukocytes activated with a stimulant were allowed to act on human tumor cells, MKN-1 gastric cancer cells, P
(-10 strongly damaged lung cancer cells, and by adding these activated leukocytes to a mixed system of human tumor cells (MKN-1 gastric cancer cells) and non-activated human leukocytes, the ability to damage tumor cells was enhanced. In addition, white blood cells in the lungs of BAL B/c mice activated with stimulants are Co1
on 26 (BALB/c-derived tumor cells), it had strong tumor-damaging activity and anti-tumor helper T cell activity.

(Effects of the Invention) As described above, the stimulating material of the present invention efficiently activates patient's leukocytes and induces strong tumor-toxic cells and anti-tumor helper T cells in a safe and easy-to-operate manner. It is used not only for cancer treatment such as stomach cancer, lung cancer, breast cancer, and liver cancer, but also for lymphocyte function tests of patients with bile cancer, mice, rats,
It is intended to be used in research on anti-tumor immunity in animal experiments such as rabbits.

(Example) Example 1 A stimulant was prepared as follows. Sunawachi, polyvinyl alcohol gel (copolymer of polyvinyl alcohol and triallyl isocyanate: particle size 140-21
0 μm) using the shrimp chlorohydrin method (affinity chromatography, Chibata Division).

Kodansha Scientific, 1976, P71
) to form an epoxy-activated gel, to which a 5% potassium hydroxide solution of various pyrazolo(3,4-d) pyrimidine derivatives was added, and the mixture was bonded by shaking at 50°C for 24 hours. After repeated washing with acetic acid buffer and pH 8.5 sodium carbonate buffer, the sample was washed with physiological saline, sterilized in an autoclave, and used for experiments. The amount of pyrazolo(3,4-d)pyrimidine derivative retained by the insoluble carrier was calculated by subtracting the amount in the suspension after the binding reaction from the initially added amount.
20μmof to 200μff1o per ml! ! Met. The specific surface area of this insoluble carrier is 2 per ml.
Since it is 0%, the amount retained per specific surface area is 1μmoβ
It corresponds to 10 μmox.

Human leukocytes were obtained as follows. That is, the collected human peripheral blood was diluted 2 times with Hank's solution, layered on Ficoll-Birk's solution (manufactured by Pharmacia), centrifuged at 2000 rpm for 20 minutes, and the white blood cell layer in the middle layer was separated. After washing with 10% autologous serum, 2×1011/
The cells were suspended at a cell concentration of lll1. 1 of this cell suspension
Dispense 1 mA each into 2III wells for cell culture (Fashion 1 m 3047), and add 100 μl of stimulation material to this well.
1 at a time in a CO2 incubator at a temperature of 37°C.
Culture was carried out in After culturing for 60 hours, the activated leukocytes are peeled off from the surface of the stimulator by pipetting the culture solution and allowed to stand.The stimulator will sink to the bottom of the container, so remove the supernatant cell solution and add it to Hank's tube. After washing with solution, add 5 Xl06/m to RPMr1640 medium supplemented with 10% autologous serum.
The cells were suspended at a cell concentration of 6.

Whether or not these activated leukocytes have tumor cytotoxicity was evaluated using the following killer activity measurement method. Various human cancer cell lines that adhere to and proliferate on culture plates were used as target cells at a cell concentration of 5 XIO'/mz.
% beef tallow serum supplemented RPM11640 medium, and 10μβ of each 10μ! Dispense the mixture into Terasaki plates and culture in a CO2 incubator at a temperature of 37°C.

When cultured for 24 hours, cancer cells strongly adhere to the bottom of the culture plate. After washing the plate with a culture solution, 10 μβ of activated leukocyte suspension is added and cultured at 37° C. for 4 hours in a CO2 incubator to damage cancer cells attached to the plate. Damaged cancer cells lose their adhesion to the bottom of the plate and are removed along with activated leukocytes when washed with Hank's salt solution. After fixing the cancer cells that survived and attached to the bottom of the plate with acetone and staining them with Giemsa solution,
Count using a microscope. Killer activity is calculated using the following formula.

Antitumor helper activity was measured by observing the effect of activated leukocytes on enhancing the ability of non-activated leukocytes to damage tumor cells.

That is, evaluation was performed using the following measurement method.

Adherent human cancer cell line as target cell, 1×1057
m1! RPM116 supplemented with 10% tallow serum at a cell concentration of
40 culture medium, and 100μβ of this was placed on a 96-well culture plate (Corning, CELL LLST14).
25860) and cultured in a CO□ incubator at a temperature of 37°C for 3 hours. To this, 2 XIO' /
RP MI with 10% tallow serum added at a cell concentration of mβ
A system in which 50 μβ of human leukocytes suspended in 1640 medium was added and a system in which 50 μβ were not added were established, and the cells were incubated at 37°C for 3 hours.
After culturing under 5% C (h conditions, lXl0' to 1x10
RPM with 10% tallow serum at a cell concentration of 6150μl
Human activated leukocytes suspended in 11640 medium are added to each of the above systems, and further cultured in a CO□ incubator at a temperature of 37°C for 60 hours. During culture, damaged cancer cells lose their attachment to the bottom of the plate. Therefore, similarly to the killer activity measurement method, after washing the white blood cells with Hank's solution, the surviving cancer cells adhering to the bottom of the plate were fixed with acetone, and after staining with Giemsa solution, the presence of surviving cancer cells was determined. I examined it with a microscope.

Tables 1 and 2 show the ability of the various stimulating materials evaluated in this way to induce antitumor immune cells.

Table 2 Comparative example 1 of anti-tumor helper T induction ability of each Rimune 1 wood. Evaluation was performed in exactly the same manner as in Example 1, but tumor-toxic cells,
Neither antitumor helper cells were induced.

Comparative Example 2 RP MI 1640 medium supplemented with 10% tallow serum,
Dissolve 2mM of various pyrazolo (3,4-d) pyrimidine derivatives, and add 2X human peripheral blood leukocytes to 1ml of these medium.
Although 106 cells were suspended and cultured for 60 hours, neither tumor-toxic cells nor anti-tumor helper cells were induced.

Example 2 A stimulant was prepared as follows. 120/20
CPG-10 (manufactured by Funakoshi Pharmaceutical Co., Ltd.), which is a porous glass bead with various pore diameters of 0.0 mS. A cyclic epoxy group is introduced, and 4-aminovirazolo (3,4-d
) A 0.1 M carbonate buffer solution (pH 9,0) of pyrimidine was added and the mixture was bonded by shaking at 50°C for 24 hours.
After washing in the same manner as above, it was sterilized in an autoclave and used for experiments. The retained amount was also determined by the same method as in Example 1, and it was found to be 10 μmol to 70 μm per 1 g of porous glass beads.
It was ob. Since the specific surface area of these porous glass beads is 20 to 140 M/g, the amount retained per specific surface area corresponds to about 0.5 μte+ol.

Mouse leukocytes were obtained as follows. That is, B A L
Lungs removed from B/c mice (4 to 6 weeks old) were loosened with a stainless steel mesh, suspended in Hank's solution and allowed to stand, separated from organ fragments, and centrifuged at 800 rpm for 10 minutes. The obtained cell pellet was suspended in a 0.85% ammonium chloride aqueous solution to remove red blood cells.
After incubating for 2 minutes at a temperature of 37°C, immediately
Mix with twice the volume of Hank's solution, centrifuge at 800 rpm for 10 minutes, and add 10% fetal bovine serum to RPM I.
1640 medium) at a cell concentration of 5 x 106/ml. 2 ml of this cell suspension was dispensed into 2 ml wells for cell culture (Falcon M 3047), 0.5 g of the stimulant was added thereto, and the cells were cultured in a CO2 incubator at a temperature of 37°C. After culturing for 3 days, remove the activated leukocytes from the surface of the stimulator by pipetting the culture solution and let it stand. The stimulator will sink to the bottom of the container, so take the supernatant cell solution and add it to the container. After washing with Hank's solution, add 10% fetal bovine serum RP M I 164
The cells were suspended in 0 medium at a cell concentration of 1077 ml.

Whether this activated leukocyte has tumor cytotoxicity and antitumor helper activity was investigated using the mouse cancer cell line Co1on.
No. 26 was used as the target cell, and evaluated using the same measurement method as in Example 1.

Tables 3 and 4 show the ability of various stimulating materials to induce antitumor immune cells.

Table 4! Comparative example 3 of antitumor helper TwvA of lumber lumber 2 m of RP M I 1640 medium supplemented with 10% tallow serum
i, B A L B / obtained in the same manner as in Example 2.
c 1 x 10 7 mouse leukocytes were suspended, and mixed with polypropylene glass beads of various pore sizes (120, 350, 700, 1400), CPG-10 (manufactured by Funakoshi Pharmaceutical Co., Ltd., particle size 75). ~125 microns) to 0.5
Co1 of mouse leukocytes was added in 1 g and cultured for 3 days.
The killer activity against on26 was 10% or less in all cases, and no antitumor helper activity was found. The killer activity and antitumor helper activity were measured in the same manner as in Example 2.

Claims (2)

[Claims] (1) A stimulating material for inducing anti-tumor immune cells, characterized in that at least one of pyrazolo(3,4-d)pyrimidine and its derivatives is bound to the surface of an insoluble carrier. (2) The stimulating material according to claim 1, wherein the surface of the insoluble carrier has a porous structure.