JPH05239099A - New protein and material for inducing and stimulating antitumor immunocyte - Google Patents

Abstract

PURPOSE:To obtain an inducer and an inducing and stimulating material capable of inducing a safer and stronger antitumor immunocyte than that induced by a conventional method. CONSTITUTION:A purified protein obtained from a pokeweed mitogen and having 35000+ or -5000 molecular weight and 4+ or -1 isoelectric point or an antitumor immunocyte inducer consisting of the above-mentioned protein or a purified protein having an amino acid sequence consisting of Ala-Pro-Glu-Cys-Gly-Arg- Glu-Ala-Ser-Gly-Lys-Val-Cys-Pro-Asp-Leu or an antitumor immunocyte inducer consisting of this protein, and an antitumor immunocyte inducing and stimulating material characterized by having this protein on the surface of a water insoluble carrier. This inducer as well as this inducing and stimulating material activate leukocytes in while blood or body fluid cells to induce an antitumor immunocyte and are useful for treating malignant tumors.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel protein for treating malignant tumor and an antitumor immune cell-inducing stimulant having a function of activating leukocytes in whole blood or body fluid cell components to induce antitumor immune cells. Regarding

[0002]

2. Description of the Related Art In recent years, as a treatment method for cancer patients, leukocytes taken out from the patient are cultured with interleukin-2 (IL-2, hereinafter referred to as IL-2) to activate cells or IL-activated cells. Immunotherapy has been attempted in which treatment is carried out by returning tumor-infiltrating leukocytes cultured in the presence of 2 into the body of a cancer patient (N. Engl. J. Me.
d. , 316 , 889-897, 1987), (NE
ngl. J. Med. , 319 , 1676-1680,
1988).

[0003] This treatment method uses a large amount of white blood cells from cancer patients,
Furthermore, the tumor cells are removed and aseptically as long-term IL-
It is very complicated in terms of operation to inject an infusion solution into a cancer patient after culturing in the presence of 2.
There is a problem that enormous treatment cost is required because it takes 3 to 4 days for each operation, and further, a considerable amount of time and labor is required by a doctor for each patient.

In order to solve these problems, the present inventors have provided a stimulating material and a stimulating method which are more operable than conventional methods and which are capable of inducing stronger antitumor immune cells (Japanese Patent Laid-Open Publication No. Sho. 60-120821). That is,
We found that a type of lectin, pork weed mitogen (hereinafter referred to as PWM), has an activity of inducing antitumor immune cells, and immobilize this protein on a water-insoluble carrier so that it does not enter the body. did.

[0005]

However, the inducer of the antitumor immune cells bound to the stimulant used here is PWM as a lectin and also contains impurities that do not have the ability to induce antitumor immune cells. Has been. When used for treating cancer, it is feared that the inclusion of impurities may not bring about a sufficient effect and may cause side effects. Therefore, a substance capable of inducing stronger antitumor immune cells with a single substance is desired.

In view of the above-mentioned problems of the induction of antitumor immune cells based on the prior art, the object of the present invention is an inducer and an inducer capable of inducing potent antitumor immune cells more safely than conventional methods. Is to provide wood.

[0007]

Means for Solving the Problems As a result of intensive research aimed at achieving the above objects, the present inventors have found that the substances described in claims 1 to 4 have a strong ability to induce antitumor immune cells. Found that there is. Furthermore, on the surface of the water-insoluble carrier,
A strong antitumor immune cell is induced by stimulating leukocytes with an antitumor immune cell induction stimulating material comprising one of the substances according to claims 1 to 4. It was found that the present invention has been completed.

That is, the present invention relates to the first claim.
Item 4. An antitumor immune cell induction stimulating material comprising the substance according to any one of claims 4 to 4 and one of the substances according to claims 1 to 4 on the surface of the water-insoluble carrier. Get involved.

In the present invention, PWM means American pokeweed (Fitracca americana Phytolaccc).
a americana) and is a lectin extracted from the root. Conventionally used PWM is a crude refined product prepared by a method exemplified in the following documents, for example, and contains several kinds of proteins (J. Exp. Med.,
124 , 859-872, 1966), (Biochi.
mica et Biophysica Acta, 4
27 , 443-452, 1976). Five types of proteins contained in PWM have been clarified,
Although the constituent amino acid residues of each isolectin have been clarified, the physiological activity exhibited by each is only the mitogenic activity on T cells or B cells (Biochemistry, 13 , 3671 to 367).
7, 1974), (Methods in Enzym
biology, 50 , 354-361, 1978).

The action of PWM on the induction of anti-tumor immune cells is not only known in the presence of IL-2 (Journal of Japan Society of Transfusion, 36 , 309, 1990, The 2nd JBRM Society Annual Meeting). General Assembly Program / Abstracts, p3
3, 1989), PWM alone has been investigated (artificial organs, 18 , 1401-1405, 19).
89). However, both are the results of using the roughly purified PWM. The novel protein of the present invention can be separated and purified as a substance that induces antitumor immune cells from crudely purified PWM.

As a method for separating and purifying from the crudely purified PWM, a method usually used in protein chemistry, for example, an adsorption method by a carrier, an ion exchange method, a fractional precipitation method, a gel filtration method, an electrophoresis method, various affinity chromatography methods. , These can be used alone or in combination. It can be confirmed that the thus obtained protein according to claim 1 or 2 is a single protein on electrophoresis. The ability to induce anti-tumor immune cells is crude P
It is much stronger than WM, and its use is as a drug for direct administration to cancer patients to shrink tumors to treat cancer, and to act on leukocytes taken out from cancer patients, and only leukocytes activated after washing. By returning the drug to the patient's body, the toxicity caused by the heterologous protein can be avoided, and it can be used as a medicine for more desirable treatment of cancer. Furthermore, if it was used as a mitogen in the measurement of the ability of lymphocytes to react with lectin, which measures the function of lymphocytes, there were variations in the measured values even with the same sample when different lots of PWM were used. However, stable measurement results can be obtained.

Claims 1 to 4 of the present invention
An example of a specific method for separating and purifying the substance described in the item will be given. The protein referred to in the present invention may be any substance described in claims 1 to 4, and the purification method is not limited to the following method.

The crudely purified PWM is converted into a phosphate buffer solution (Ca, Mg
(Not containing) (hereinafter referred to as PBS (-)) and dissolved in PBS
Pass through a gel filtration column equilibrated with (-) to give a molecular weight of about 3
Collect 50,000 to 40,000 fractions. Ammonium sulfate was added to the collected solution to make the final concentration 1M,
It is passed through an octyl sepharose (Pharmacia Co., Ltd.) column equilibrated with 1 M ammonium sulfate / PBS (−), and the adsorbed component is recovered with PBS (−). After concentrating the collected components with an ultrafiltration system etc., they are passed through a gel filtration column equilibrated with physiological saline to obtain a molecular weight of 35,000 ±
Collect 5,000 fractions.

The fact that this substance has the ability to induce antitumor immune cells is described in, for example, the new lymphocyte function search method, p187-19.
8, Chugai Medical Co., Ltd., 1987, Clinical Immunity, 19 , 2
50 to 256, 1987 can be confirmed. The physicochemical properties of the thus obtained novel protein will be described below.

1) Molecular weight: 35,000 ± 5,000 Measured by SDS-polyacrylamide gel electrophoresis under the condition of denaturation with SDS and reduction with 2-mercaptoethanol. 2) Isoelectric point: It was measured by an isoelectric focusing method using 4 ± 1 Ampholine. 3) Amino acid analysis: (Table 1) Amino acid analysis was performed after hydrolysis, but the values were different from any of the isolectins Pa-1 to Pa-5 described in the literature (Biochemistry, 13 ,
3671-3677, 1974).

[0016]

[Table 1]

The antitumor immune cell-inducing stimulant of the present invention can be obtained by binding one of the substances described in claims 1 to 4 to the surface of a water-insoluble carrier. .. The induction stimulant may be any one that can contact white blood cells, and the carrier or binding method is not particularly specified. Since this induction stimulant has only one of the substances described in claims 1 to 4 bound to the surface, it can induce strong antitumor immune cells when contacted with leukocytes, The induction of cells that adversely affect other organisms is eliminated. The use of this induction stimulant is to induce white blood cells taken out from a cancer patient with this induction stimulant to induce strong antitumor immune cells, and to return them to the body of a cancer patient to shrink the tumor, It can be used as a therapeutic device for treating.

The molecular weight referred to in the present invention means SDS-polyacrylamide gel electrophoresis (for example, Biochemistry Experimental Course 1, Protein Chemistry I, p 469-492, edited by The Biochemical Society of Japan, published by Tokyo Kagaku Dojin, March 1976). It is the molecular weight in the SDS-denatured state and the 2-mercaptoethanol reduced state, as measured by (issued on March 22).

The isoelectric point in the present invention means the principle of isoelectric focusing (for example, 2117 Multiphor II).
Electrophoresis System Sy
Stem Handbook, p48-95, 1989
Year, Pharmacia LKB Biotechno
It is the isoelectric point measured by logarithm).

The water-insoluble carrier used in the present invention may be either a hydrophilic carrier or a hydrophobic carrier. As the shape of the water-insoluble carrier, any known shape such as a particle shape, a fiber shape, a hollow fiber shape, and a film shape can be used. As the granular or spherical water-insoluble carrier, those having a particle size of 1 to 3000 μm can be used. If the particle size is 1 micron or less, separation from activated leukocytes is difficult. In particular, if the particle size is 50 μm or more, separation from activated leukocytes is easily possible.
If the particle size is 3000 microns or more, the contact area per unit weight of the carrier with the white blood cells decreases, which is not preferable. Particularly preferably, the particle size is 80 to 2000 microns. Further, if the specific gravity of the granular or spherical water-insoluble carrier is 1.07 or more, it can be easily separated from activated leukocytes by centrifugation or standing. When a flat membrane-shaped or hollow fiber-shaped porous carrier is used, its pore size does not allow cells to pass but medium components can pass freely.
If 10 micron is used, leukocytes bound to one side of the membrane can be supplied with nutrients from the other side of the membrane, and high concentration of antitumor immune cells can be induced. In particular, a flat membrane-like or hollow fiber-like porous carrier having a pore diameter of 0.1 to 5 microns can be favorably used.

As the material of the water-insoluble carrier, there are activated carbon, glass and the like and derivatives thereof in the case of inorganic bases, and the natural polymer-derived carriers include polysaccharides such as cellulose, sepharose, dextran and starch and derivatives thereof. There is.

In the case of synthetic polymers, vinyl polymers include styrene, vinyl acetate, methacrylic acid esters, acrylic acid esters, vinyl halides, vinylidene halides, acrylonitrile, acrylamide, methyl vinyl ketone, and vinylpyrrolidone. , 2-vinylpyridine, ethylene, propylene, butadiene, isoprene and the like, and polymers and copolymers of their derivatives. Ring-opening polymers of cyclic compounds include dimethylcyclopropane, spiro-di-o-xylylene, norbornene, cyclobutene,
There are polymers and copolymers of trioxane, lactide, cyclopolysiloxane, phosphonitrile chloride, N-carboxy-α-amino acid anhydride and the like and derivatives thereof. Examples include polyformaldehyde, polyethylene oxide, polypropylene glycol, poly-3,3-bis (chloromethyl) oxacyclobutane, polytetrahydrofuran, polycaprolactam, and derivatives thereof.

Polycondensates include polyester, polyamide, polyanhydride, polycarbonate, polyurea, polysulfonamide, polyimide, polybenzimidazole and the like and derivatives thereof. Examples of the resin and others include acrylic resin, methacrylic resin, fluororesin, epoxy resin, urea resin, amino resin, styrene resin, melamine resin, polyurethane, silicone resin, alkyd resin and the like and derivatives thereof.

The polymer carrier mentioned above can be used as a water-insoluble carrier by using an appropriate comonomer and a crosslinking agent, if necessary. As the cross-linking agent, various ones such as sulfur, organic peroxide, phenol resin, diisocyanate, epoxy compound, diene and glutaraldehyde can be selected according to the functional group of the substance to be cross-linked (Taisei, " Crosslinking Agent Handbook ", p3 to 77, 1981).

As the method of immobilizing the ligand on the surface of the above water-insoluble carrier, any known method such as covalent bond, ionic bond, physical adsorption can be used. It is desirable to use. For that purpose, a method usually used for immobilized enzymes and affinity chromatography can be used. For example, agarose, sepharose, etc. are activated with cyanogen bromide (CNBr), or epoxy activated with epichlorohydrin, or silica glass beads with γ-
A method of reacting with aminopropyltriethoxysilane to obtain an alkylamino glass, activating it with glutaraldehyde, and binding it can be used. If necessary, a molecule (spacer) of arbitrary length may be introduced between the water-insoluble carrier and the carrier for use.
For example, it can be carried out by reacting the hydroxyl group of agarose and the isocyanate group on one side of hexamethylene diisocyanate by reaction, and the remaining isocyanate group and the amino group of the antibody by reaction.

The manufacturing method of the induction stimulating material of the present invention comprising the above-mentioned elements does not define the order of connecting the constituent elements. That is, the induction stimulant used in the present invention basically has only one of the substances described in claims 1 to 4 on the surface of the water-insoluble carrier,
It does not depend on the manufacturing method.

The antitumor immune cells in the present invention are, for example, new lymphocyte function search method, p187-198, Chugai Medical Co., Ltd., published in 1987, clinical immunity, 19 , 241-25.
6, 1987 refers to killer cells.

The white blood cells in the present invention refer to so-called white blood cells obtained by removing red blood cells and platelets from blood cells. The cell fraction obtained by removing granulocytes or B cells from the white blood cells is also included in the concept of white blood cells in the present invention. included. The leukocytes to be activated in the present invention may be a leukocyte fraction collected from peripheral blood by a known continuous centrifugation method, or a mononuclear cell fraction separated by a known Ficoll Park overlay centrifugation method. It is also possible to induce a strong antitumor immune cell by using a T cell fraction separated or concentrated from the peripheral blood mononuclear cells by known rosette formation with neuraminidase-treated sheep red blood cells.

The antitumor immune cells that are induced and activated in the present invention belong to the lymphocyte fraction of leukocytes excluding granulocytes, monocytes, and macrophages, and have the characteristics of T cells in particular. The antitumor immune cell inducer according to claims 1 to 4 is a stronger antitumor immune cell inducer than before, and the water-insoluble carrier having the surface bound thereto is stronger than before. It is an anti-tumor immune cell induction stimulant.
The measurement method is specified below.

Molecular weight: SDS-polyacrylamide gel electrophoresis is performed using an antitumor immune cell inducer denatured with SDS and reduced with 2-mercaptoethanol as a sample. As gel, 13% uniform concentration gel or 4-2
A 0% gradient gel is used. Phosphorylase b (94,000), albumin (6
7,000), ovalbumin (43,000), carbonic anhydrase (30,000), trypsin inhibitor (20,000), α-lactalbumin (14,000), LMW kitE (Pharmacia Co., Ltd.) ) Is used.

Isoelectric point: Multiphor II electrophoresis system (Pharmacia KK) and Amphol
ine PAG Plate (Pharmacia Co., Ltd.)
Determine the isoelectric point of the anti-tumor immune cell inducer using pH 3.5 to 9.5 according to the instruction manual. Isoelectric point value is pI
It is determined using a calibration kit 2.5 to 6.5 (Pharmacia Co., Ltd.).

Antitumor immune cell inducing activity: Human leukocytes are prepared as follows. That is, human peripheral blood collected by adding an anticoagulant is centrifuged at 1,600 rpm for 15 minutes, and the supernatant (plasma) is transferred. RPMI164 for precipitation (cells)
0 medium (manufactured by Nissui) was added in an amount of about 2.5 times the amount of blood collected and stirred, and then 10 ml was overlaid on 3 ml of Ficoll Park solution (manufactured by Pharmacia Co., Ltd.) to prepare 1,6
Centrifuge for 25 minutes at 00 rpm. After removing the intermediate layer (white blood cell layer) and washing it with RPMI1640 medium, 2% was added to RPMI1640 medium supplemented with 10% autologous serum.
Suspend at a concentration of × 10 ⁶ cells / ml.

The induction of antitumor immune cells is performed as follows. [Induction with anti-tumor immune cell inducer] Take 2 ml of leukocyte suspension and centrifuge at 1,200 rpm for 7 minutes to remove the supernatant. 2 ml of RPMI1640 medium containing 10% autologous serum containing an antitumor immune cell inducer is added to the precipitate (white blood cells) to suspend the cells. After standing in a CO ₂ incubator (37 ° C) for 15 minutes, 1,200r
The supernatant (antitumor immune cell inducer liquid) is removed by centrifugation at pm for 7 minutes. RPMI 1 with 10% autologous serum added to the precipitate
After repeated washing 3 times with 640 medium, autologous serum 10%
1 ml of the RPMI1640 medium added is added to make it float and transferred to a 48-well plate for culture. After standing in a CO ₂ incubator (37 ° C.) for 20 hours, the induced antitumor immune cells are collected in RPMI1640 medium supplemented with 10% fetal bovine serum and suspended at a cell concentration of 10 ⁷ cells / ml.

[Induction with Antitumor Immune Cell Inducing Stimulant] 1.5 ml of leukocyte suspension is added to 150 μl of the inducing stimulant and centrifuged at 800 rpm for 5 minutes. After standing still in a CO ₂ incubator (37 ° C) for 10 minutes, 1 ml
RPMI1640 medium containing 10% autologous serum is added and pipetting is carried out 30 times, and after the induction stimulant is submerged, the supernatant containing white blood cells is collected. 2 ml of induction stimulant again
RPMI1640 medium supplemented with 10% of autologous serum is added, and the supernatant containing white blood cells is recovered in the same manner. Both were combined and centrifuged at 1,200 rpm for 7 minutes, and then the precipitated white blood cells were added at a concentration of 2 × 10 ⁶ cells / ml with 10% autologous serum added RP.
Float in MI1640 medium. 48 ml of suspension 1 ml
The cells were transferred to a well culture plate and allowed to stand in a CO ₂ incubator (37 ° C.) for 20 hours, and the induced antitumor immune cells were collected in RPMI1640 medium supplemented with 10% fetal bovine serum, and the concentration was 10 ⁷ cells / ml. Float with.

The antitumor immune cell activity exhibited by these antitumor immune cells is evaluated by the following assay method. That is,
A human cancer cell line, which is a target cell, was suspended at a concentration of 10 ⁶ cells / ml, 5 ml of the suspension and 1 mg of carboxyfluorescein diacetate (cFDA) dissolved in acetone were placed in a brown glass bottle and shaken in a 37 ° C water bath for 1 hour. Then, the target cells are fluorescently labeled. 10% fetal bovine serum added R
After adding 5 ml of PMI1640 medium and stirring, 800
Centrifuge at rpm for 5 minutes. After removing the supernatant, resuspend in RPMI1640 medium containing 10% fetal bovine serum and
Prepare to a concentration of 10 ⁵ cells / ml and place 10 μl in a microtest plate. Antitumor immune cells 10% here
Recovered in RPMI1640 medium supplemented with fetal bovine serum, 10
^The cells are suspended at a concentration of ⁷ cells / ml, 10 μl of the cells are placed in a micro test plate, and mixed with target cells. C
Let stand for 3 hours in an O ₂ incubator (37 ° C).
During this period, the target cells killed by the anti-tumor immune cells release fluorescence to the outside of the cell. Therefore, 5 μl of ink was added to quench the fluorescence outside the cells, and fluorescence was detected with an automatic fluorescence measurement device (Lights MPV-MT2). To measure. Antitumor immune cell activity is calculated by the following formula. Antitumor immune cell activity = {1- (fluorescence when antitumor immune cells were added-NP
40) / (fluorescence when leukocytes that did not induce antitumor immune cells were added-fluorescence when NP40 was added)} × 100% (NP40: Noni
det P-40 (surfactant))

[0036]

EXAMPLES Next, the present invention will be described in more detail by way of examples. Example 1 First, a specific example of a method for preparing an antitumor immune cell inducer will be described. (1) Manufacturing method PWM (manufactured by Honen Corporation) 20 m
It was dissolved in PBS (-) at a concentration of g / ml. High load 26/60 Superdex 75pg column (2.6x
60 cm, Pharmacia Co., Ltd.) is equilibrated with PBS (-), 20 ml of PWM solution is applied, and PBS is applied.
Gel chromatography was performed with (-). 150-1
The fraction eluted at 80 ml was collected (Fig. 1).

Ammonium sulfate was added to the collected fractions to a final concentration of 1M. A column packed with 100 ml of Octyl Sepharose (Pharmacia Co., Ltd.)
It equilibrated with ammonium sulfate / PBS (-), and the above-mentioned collected fractions were applied. Rinse off the non-adsorbed components with 500 ml of 1M ammonium sulfate / PBS (-), 200 ml
The adsorbed component was recovered with PBS (-). The recovered components were concentrated with an ultrafiltration system NM-3 mini module (manufactured by Asahi Kasei Kogyo KK).

4 ml of the concentrated solution was applied to a high load 26/60 Superdex 75 pg column (2.6 × 60 cm, manufactured by Pharmacia Co., Ltd.) equilibrated with physiological saline (manufactured by Otsuka Pharmaceutical Co., Ltd.), and the gel was applied. Chromatography was performed. The substance eluted in the fraction of 180 to 200 ml was used as an antitumor immune cell inducer (Fig. 2). SDS-
As a result of polyacrylamide gel electrophoresis and staining with Coomassie Brilliant Blue, only a single band was detected.

(2) Measurement of molecular weight The molecular weight of the antitumor immune cell inducer was 35,000 ±.
It was 5,000. (3) Isoelectric point measurement The isoelectric point of the antitumor immune cell inducer was 4 ± 1. (4) Amino acid analysis Amino acid analysis of the antitumor immune cell inducer was performed, and the results shown in Table 1 were obtained. Isolectin Pa- described in the literature
It was a value different from any of 1 to Pa-5.

(5) N-terminal amino acid sequence analysis The N-terminal amino acid sequence of the antitumor immune cell inducer was applied.
Edman degradation was performed with a gas phase protein sequencer 470A manufactured by Lied Biosystems, and PTH-amino acid was identified using PTH analyzer 120A. As a result, Ala-Pro-Glu-X-Gly
-Arg-Glu-Ala-Ser-Gly-Lys-
Val-Y-Pro-Asp-Asp-Leu- (X,
Y was an arbitrary amino acid sequence.

Further, the antitumor immune cell inducer was pyridylethylated (PE) and sequenced. As a result, PE-Cys was detected in the 4th and 13th cycles.
And the unidentified amino acid was Cys. From this, the N-terminal amino acid sequence of the antitumor immune cell inducer was Ala-Pro-Glu-Cys-Gl.
y-Arg-Glu-Ala-Ser-Gly-Lys
-Val-Cys-Pro-Asp-Asp-Leu-
Met.

(6) Antitumor Immune Cell Inducing Activity The cells induced by the antitumor immune cell inducer have a final concentration of 1 ng / m using human gastric cancer cell line MKN-1 as target cells.
The activity was 62% when induced by 1 antitumor immune cell inducer, and 83% when induced at a final concentration of 10 ng / ml. Compared with Comparative Example 1, the antitumor immune cell inducer showed an equivalent antitumor immune cell inducing activity in an amount of 1/10 or less that of the crude purified PWM.

Comparative Example 1 In the same measurement of antitumor immune cell activity as in Example 1, conventional crude purified PWM showed 71% antitumor immune cell inducing activity when induced at a final concentration of 100 ng / ml.

Example 2 (1) Preparation of induction stimulant In the same manner as in Example 1, an antitumor immune cell inducer was prepared. As a water-insoluble carrier, methylmethacrylate-
Particles of divinylbenzene copolymer AXT-31 (manufactured by Tokyo Organic Co., Ltd.) were sphere-separated to prepare 500 to 710 μm. 100 ml of AXT-31 was reacted with 100 ml of a 2% (w / v) methanol solution of a random copolymer of 2-hydroxyethyl methacrylate and diethylaminoethyl methacrylate to coat it.
100 ml coated AXT-31, 120 m
1 dimethylsulfoxide (DMSO), 80 ml epichlorohydrin, 10 ml 50% NaOH solution 5
The mixture was shaken at 30 ° C. for an hour and an epoxy group was introduced into AXT-31. 100 ml of epoxy activated AXT-31,
The antitumor immune cell inducer was bound to AXT-31 by shaking 100 ml of 0.1 M phosphate buffer (pH 9.0) containing a predetermined amount of the antitumor immune cell inducer for 20 hours. After washing, the remaining epoxy groups were blocked by shaking in a 1 M ethanolamine solution, and after washing, 100 ml of an antitumor immune cell induction stimulant was obtained.

(2) Antitumor immune cell inducing activity The antitumor immune cell inducing activity was measured by measuring the human gastric cancer cell line MKN.
-1 was used as a target cell. The antitumor immune cells induced by the induction stimulant prepared by adding 10 mg and 80 mg of the antitumor immune cell inducer during the binding reaction exhibited 51% and 89% activity, respectively. Compared with Comparative Example 2, the induction stimulant having the antitumor immune cell inducer bound thereto exhibited an antitumor immune cell inducing activity equivalent to that of the induction stimulant having roughly 8 times the concentration of the crude purified PWM.

Comparative Example 2 In the same measurement of antitumor immune cell inducing activity as in Example 2, 10 mg and 8 of conventional crude purified PWM were used in the binding reaction.
Induction stimulants combined with 0 mg added each had 19%
And showed 48% antitumor immune cell inducing activity.

Example 3 In the same measurement of antitumor immune cell inducing activity as in Example 2, human chronic myelogenous leukemia cell line K-562 and human Burkitt lymphoma cell line Daudi were used as target cells to induce antitumor immune cell induction. The induction stimulant bound with 80 mg of the substance showed antitumor immune cell inducing activity of 78% and 69%, respectively.

Comparative Example 3 In the same measurement of antitumor immune cell inducing activity as in Example 3, the induction stimulant to which 80 mg of conventional crude purified PWM was added was used to induce antitumor immune cell induction of 42% and 23%, respectively. It showed activity.

Example 4 Four BALB / c mice were administered with 25 mg of an antitumor immune cell inducer per kg of mouse body weight.
No death was observed even after 4 days. Also 3 BAL
When 50 mg of the antitumor immune cell inducer was administered to B / c mice per 1 kg of mouse weight, one animal died on the 10th day. As compared with Comparative Example 5, it can be seen that the toxicity is far lower.

Comparative Example 4 When three BALB / c mice were administered with 25 mg of crude purified PWM per 1 kg of mouse weight, all deaths were observed by the 8th day. Also, three BALB
When 50 mg / kg of mouse body weight of crude purified PWM was administered to the / c mouse, all deaths were observed by the 5th day.

[0051]

INDUSTRIAL APPLICABILITY As described above, the novel protein and antitumor immune cell-inducing stimulant of the present invention efficiently and safely activate human peripheral blood leukocytes to produce antitumor immune cells stronger than ever before. It induces gastric cancer, lung cancer,
It is intended to be used for treating cancers such as breast cancer and liver cancer.

[Brief description of drawings]

FIG. 1 is a graph obtained by fractionating PWM by performing gel chromatography on crudely purified PWM in PBS (−).

FIG. 2 is a graph in which fractionated PWM was subjected to gel chromatography in physiological saline to fractionate an antitumor immune cell inducer.

Claims (5)

[Claims] 1. A purified protein having a molecular weight of 35,000 ± 5,000 and an isoelectric point of 4 ± 1 obtained from Pork Weed Mitogen. 2. An antitumor immune cell inducer comprising the claim 1. 3. Ala-Pro-Glu-as the amino acid sequence obtained from Pork Weed Mitogen
Cys-Gly-Arg-Glu-Ala-Ser-G
ly-Lys-Val-Cys-Pro-Asp-As
A purified protein according to claim 1 having a sequence consisting of p-Leu. 4. An antitumor immune cell-inducing substance according to claim 3. 5. An anti-tumor immune cell induction stimulating material, characterized in that it has one of the substances according to claims 1 to 4 on the surface of a water-insoluble carrier.