JPH053855B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a leukocyte stimulating material for cancer treatment that has the function of activating leukocytes and inducing antitumor immune cells.

(Prior art) As is well known, killer T
It has been reported that cells, NK cells, activated macrophages, K cells, etc. play important roles [Masahiro Fukuzawa: History of Medicine, 126, 420 (,
83)]. Therefore, as an immunological therapy for malignant tumors, it is possible to activate cancer patient immune cells (white blood cells) to efficiently induce and activate these anti-tumor immune cells. However, in actual cancer patients, tumor cells proliferate despite the existence of such an immune surveillance mechanism against malignant tumors.

One of the main mechanisms has been reported to be induction and activation of immunosuppressive cells (suppressor T cells, suppressor macrophages, etc.) by tumor cells. [Shigeyoshi Fujimoto etal, Journal of Immunology (S.Fujimoto etal, J.
Immunol.) 116, 791 (, 76)].

Such immunosuppressive cells suppress the induction and activation of various anti-tumor immune cells that play a role in damaging tumor cells, thereby allowing tumor cells to proliferate, further leading to a decline in the immune response ability against tumors. Conceivable. In addition, as another mechanism, it has been reported that the immune response to tumor cells may be suppressed by the production of immunosuppressive factors by tumor cells [JARoth et al., Journal of Immunology]. etal：J.
Immunol.) 128, 1955 (, 82)], it must be said that it is difficult to efficiently induce and activate antitumor immune cells in cancer patients under such immunosuppressive conditions.

Therefore, optimal conditions for inducing and activating anti-tumor immune cells without immunosuppression are set outside the body, and white blood cells taken from cancer patients are stimulated and activated to induce strong anti-tumor immune cells, which can then be used as a base. A method that attempts to treat cancer by restoring cancer to patients is considered to have the potential to become a highly effective new cancer immunotherapy.

(Problems to be Solved by the Invention) At present, active research is underway to stimulate and activate white blood cells taken out of the body to induce and activate antitumor immune cells, and to administer this to cancer-bearing organisms to treat cancer. However, tumor cells extracted from cancer-bearing organisms are used to stimulate and activate leukocytes, and the procedure is extremely complicated.

(Means for Solving the Problems) As a result of intensive research to solve the above-mentioned problems, the present inventors surprisingly found that white blood cells can be stimulated using a stimulant in which OK432 is covalently immobilized on an insoluble carrier. We have discovered that strong anti-tumor immune cells can be induced by stimulation activation, and have completed the present invention.

That is, the present invention relates to a leukocyte stimulating material for cancer treatment, characterized in that OK432 is covalently bonded to an insoluble carrier.

The insoluble carrier used in the present invention can be either a hydrophilic carrier or a hydrophobic carrier, but when a hydrophobic carrier is used, non-specific adsorption of serum components to the carrier occurs. gives better results. The shape of the insoluble carrier may be any known shape such as particulate, fibrous, hollow fiber, or membrane.

The material of the insoluble carrier may be any material as long as the carrier can be activated to immobilize the ligand and is physically stable throughout the entire process including carrier activation reaction, immobilization reaction, etc. Specifically, inorganic-based carriers include activated carbon, glass, etc. and their derivatives, and natural polymer-derived carriers include cellulose, Cephalose, dextran, starch,
There are simple polysaccharides and their derivatives such as alginic acid and chitin, complex polysaccharides and their derivatives such as agar, pectin, konjac, and gum arabic, and proteins such as wool and silk proteins and their derivatives. After undergoing insolubilization treatment such as reaction, it is used as a carrier.

Regarding synthetic polymers, vinyl polymers include styrene, vinyl acetate, methacrylate ester, acrylate ester, vinyl halide,
Examples include polymers and copolymers of vinylidene halides, acrylonitrile, acrylamide, methyl vinyl ketone, vinyl pyrrolidone, 2-vinyl pyridine, ethylene, propylene, butadiene, isoprene, and derivatives thereof.

Methods for immobilizing OK432 as a ligand on the surface of an insoluble carrier include covalent bond, ionic bond,
Any known method such as physical adsorption can be used, but from the viewpoint of elution properties, it is preferable to use it by immobilizing it with a covalent bond. For this purpose, known methods commonly used in immobilized enzymes and affinity chromatography can be used. For example, a method can be used in which an insoluble carrier is activated with epoxy and a ligand is bonded to this.
Furthermore, if necessary, a molecule (spacer) of arbitrary length can be introduced between the insoluble carrier and the ligand.

The method for producing the stimulant of the present invention is not limited to the above method, but may include, for example, a method in which a ligand is bonded to a vinyl monomer and polymerized, and a method in which the ligand is activated and bonded to a carrier. The present invention is not limited to the method of making the stimulant.

In the present invention, leukocytes refer to so-called leukocytes, which are blood cells excluding red blood cells and platelets, but cell fractions obtained by removing granulocytes or B cells from these leukocytes are also included in the concept of leukocytes in the present invention. The leukocytes to be activated in the present invention may be a leukocyte fraction collected from terminal blood by a known continuous centrifugation method, or mononuclear cells separated by a known Ficoll-Paque multilayer centrifugation method. It is possible to induce strong tumor-toxic cells by using a cell fraction, or by using a T cell fraction separated and concentrated by rosette formation with known neuraminidase-treated sheep red blood cells from terminal blood mononuclear 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.

Activation of terminal blood leukocytes by stimulants can be achieved by adding serum component-containing medium or interleukin 2 to this medium.
It is possible to induce strong tumor-toxic cells using a medium supplemented with . That is, animal serum such as fetal bovine serum, bovine serum, horse serum, or human serum is
Prepare a medium containing 20%. Preferably, a medium containing 2 to 20% human serum is prepared. As the medium in this case, a medium commonly used for animal cell culture, such as RPMI1640 medium or MEM medium, can be used. Furthermore, RPMI1640 medium supplemented with serum components such as serum albumin can also be used.

Terminal blood leukocytes collected by various methods are suspended in the prepared medium at a cell concentration of 0.5 to 3 x 10 ⁶ cells/ml, an appropriate amount of stimulant is added to this, and the temperature is maintained at 25 to 25 ml.
Culture at 45°C. At temperatures below 25°C, there is almost no effective activation of leukocytes, and at temperatures above 45°C, the survival rate of leukocytes decreases. Cultivation can be easily carried out in a CO ₂ incubator using a commercially available plastic container for cell culture. After culturing for one to several days, activated leukocytes are collected.
Alternatively, long-term culture may be performed in a medium containing interleukin 2.

The activated leukocytes obtained in this way were found to potently kill tumor cells.

(Effects of the Invention) As described above, the stimulating material of the present invention stimulates and activates white blood cells to induce strong antitumor immune cells in a safe and easy-to-operate manner, and is effective against gastric cancer, lung cancer, and breast cancer. It is intended to be used for cancer treatment, examination and diagnosis, research, etc.

Example A stimulant was prepared by binding 1 mg of OK432 (Chugai Pharmaceutical Picibanil) as a ligand to 1 ml of commercially available CNBr-activated Sepharose (manufactured by Pharmacia) by a known method. In order to determine the amount of retained ligand, the amount of ligand in the supernatant and washing solution after the binding reaction was determined and calculated by subtracting it from the amount added, and 95% of the ligand was retained.

Human leukocytes were obtained as follows. That is,
The collected terminal human blood was diluted 2 times with Hank's solution,
After layering with Ficoll-Paque solution (manufactured by Pharmacia) and centrifuging at 2000 rpm for 20 minutes, the white blood cell layer in the middle layer was separated and washed with Hank's solution, followed by RPMI1640 medium supplemented with 10% autologous serum. (Nitsui) at a cell concentration of 2 x 10 ⁶ /ml.
Dispense 1 ml of this cell suspension into 2 ml wells for cell culture (Falcon No. 3047), add 50 μl of stimulant each, and culture at 37° C. in a CO ₂ incubator. After culturing for 3 days, pipette and leave to stand, the carrier will settle to the bottom of the container, so take the supernatant serum, wash it with Hank's solution, and add 10% autologous serum.
The cells are suspended in RPMI1640 medium at a cell concentration of 5×10 ⁶ /ml.

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 are used as target cells, and 5×10 ⁴ /ml
The cells were suspended in RPMI1640 medium supplemented with 10% fetal bovine serum at a cell concentration of 10%, aliquoted into 10μ volume Terasaki plates, and cultured in a CO ₂ 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, 10μ of an activated leukocyte suspension is added and cultured at 37°C for 4 hours in a CO ₂ incubator to damage cancer cells adhering to the plate. Damaged cancer cells lose their adhesion to the bottom of the plate, and when washed with Hank's solution, their activity is removed along with white blood cells. Cancer cells that remain alive and adhere to the bottom of the plate are fixed with acetone, stained with Giemsa solution, and then counted using a microscope. Killer activity is calculated using the following formula.

Killer activity = {1 - [(number of viable tumor cells when activated leukocytes are added)/(number of surviving tumor cells when activated leukocytes are not added)]} x 100 (%) Using this method When we measured the tumor cytotoxic activity of immune cells activated with stimulants, we found that
50% harmful activity against MKN-1 human gastric cancer cells
showed strong damaging activity against MKN-1 human gastric cancer cells.

Comparative Example When an experiment was conducted in the same manner as in the example without the addition of a stimulant or with the addition of only an insoluble carrier (cephalose), almost no antitumor immune cells were induced.

Claims (1)

[Claims] 1. A leukocyte stimulating material for cancer treatment, comprising OK432 covalently bonded to an insoluble carrier.