JPS6193121A - Antitumor immunocyte-inducing material - Google Patents

Abstract

PURPOSE:To provide the titled inducing material having oligosaccharide part at the surface. CONSTITUTION:The objective antitumor immunocyte-inducing material can be prepared by immobilizing an oligosaccharide (composed of three of more compounds selected from N-acetylglucosamine, N-acetylgalactosamine, galactose, fucose, mannose, glucose and sialic acid) to an insoluble carrier (e.g. hydrophilic carrier or hydrophobic carrier; preferably a porous membrane or hollow fiber carrier having a pore size of 0.1-5mu). A strong antitumor immunocyte can be induced by the stimulation and activation of leukocyte with said inducing material. The tumor-injuring cell to be induced and activated belongs to the lymphocyte fraction in the leukocyte excluding the granulocyte, monocyte and macrophage, and has especially the property of T-cell. EFFECT:A strong antitumor immunocyte can be induced safely and in high operability, and the material is useful for the treatment, examination, diagnosis, and research of various cancers such as gastric cancer, pulmonary cancer, mammary cancer, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to an anti-tumor immune cell inducing material that has the function of inducing anti-tumor immune cells by activating white blood cells.

(Prior art) As is well known, killer T cells, NK cells, activated macrophages, K1 cells, etc. play important roles as anti-tumor immune cells that carry out the immune surveillance mechanism against malignant tumors in living bodies. [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) and 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. (S
, Fujimoto etal: J.Immun.
ol+116, 791 ('76)). Such immunosuppressive cells suppress the induced activation of various anti-tumor immune cells that have the function of damaging tumor cells, and therefore suppress tumor AtI.
! It is thought that this allows the proliferation of cells, leading to a further decline in the immune response ability against tumors. 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 (J.A., Rothetal: J.
, Immunol, 128. 1955 ('8
2)) It must be said that it is difficult to efficiently induce and activate antitumor immune cells in a cancer patient's body under such immunosuppressive conditions.

Therefore, optimal conditions for inducing and activating anti-tumor immune cells without immunosuppression are set outside the body, stimulating and activating white blood cells taken from cancer patients, inducing strong anti-tumor immune cells, and redirecting these to the original cancer. It is thought that a method of treating cancer by reintroducing it to the patient has the potential to become a highly effective new cancer immunotherapy.

(Problems to be solved by the invention) There are currently active attempts 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. Although research is currently underway, tumor cells extracted from cancer-bearing organisms are used to stimulate and activate white blood cells, making the procedure extremely complicated.

(Means for Solving the Problem) As a result of intensive research to solve the above problem, the present inventors surprisingly found that white blood cells were stimulated with an inducing material in which oligosaccharides were covalently immobilized on an insoluble carrier. They discovered that activation induces strong anti-tumor immune cells, leading to the completion of the present invention.

That is, the present invention relates to an antitumor immune cell-inducing material characterized by having an oligosaccharide moiety on its surface.

The oligosaccharides on the surface of the rust material of the present invention include N-acetylglucosamine, N-acetylgalactosamine, galactose,
It is composed of three or more of fucose, mannose, glucose, and sialic acid. For example, an oligosaccharide consisting of sialic acid, galactose, N-acetylglucosamine, and fucose, or an oligosaccharide consisting of galactose, N-acetylglucosamine, and fucose, for example,
Galβ1 = 4 GlcN Ac7? 1 = 3
It is an oligosaccharide having the structure Galβ1↑Fucα1. The oligosaccharide on the surface of the induction material used has a molecular weight of 10,000 or less. Preferably, the molecular weight is 5,000 or less, more preferably an oligosaccharide with a molecular weight of 2,000 or less. Furthermore, for example, oligosaccharides obtained from animal mucous membranes bound to insoluble carriers can be used as strong induction materials. The inducing material of the present invention is not limited to the above, and an inducing material having an oligo or the like on the surface of an insoluble carrier can be used as a strong inducing material.

In the present invention, the surface of the inducing material is the part of the insoluble carrier that can be contacted by leukocytes.

The insoluble carrier used in the present invention can be either a lignophilic carrier or a hydrophobic carrier, but when a hydrophobic carrier is used, nonspecific adsorption of serum components to the carrier occurs. gives a more favorable result. 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 1 micron to 3000 micron 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 with the leukocytes per unit weight of the carrier decreases, which is not preferable. A particle size of 80 to 2000 microns is most suitable in the present invention. 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 60.05 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. More preferably, a porous carrier in the form of a flat membrane or hollow fiber having a pore diameter of 0.1 to 5 microns can be used.

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 products include activated carbon,
Glass, etc. and derivatives thereof, and carriers derived from natural polymers include simple polyesters such as cellulose, sepharose, dextran, starch, alginic acid, chitin, and their derivatives, and composites such as agar, pectin, konjac, gum arabic, etc. There are proteins such as fibers and their derivatives, wool, protein and derivatives thereof, and these are used as carriers after being subjected to insolubilization treatment such as cross-linking reaction, if necessary.

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, Trioxane, lactide, cyclopolysiloxane, phosphonitrile chloride, polymers and copolymers of N-carboxy-α-amino acid anhydrides and their derivatives, polyformaldehyde, polyethylene oxide, polypropylene glycol, BoIJ-3.3-bis (chloromethyl)oxacyclobutane, polytetrahydrofuran, polycaprolactam, etc., and derivatives thereof.

Examples of the polycondensate include polyester, polyamide, polyanhydride, polycarbonate, polyurea, polysulfonamide, polyimide, polybenzimidazole, etc., and their 'Gg conductors.

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

Furthermore, an insoluble carrier having a multilayer structure such as activated carbon coated with PHEMA or the like can also be used.

As a method for immobilizing oligosaccharides on the surface of an insoluble carrier,
Any known method such as covalent bonding, ionic bonding, physical adsorption, etc. can be used, but considering the elution properties of oligosaccharides, it is desirable to use covalent bonding. 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 an oligosaccharide is bonded thereto. Furthermore, if necessary, a molecule (spacer) of any length may be introduced between the insoluble carrier and the oligosaccharide.

The method for producing the derivative material of the present invention is not limited to the above method, but includes, for example, a method in which an oligosaccharide is bonded to a vinyl monomer and polymerized, and a method in which a ligand is activated and bonded to a carrier. The present invention is not limited to the method for producing the induction material.

In the present invention, leukocytes refer to so-called leukocytes, which are blood cells excluding red blood cells and platelets, but a cell fraction obtained by removing granulocytes or B111 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 a leukocyte fraction collected from peripheral blood by a known continuous centrifugation method, or a mononuclear cell fraction separated by a known Ficoll-Paque multilayer centrifugation method. Alternatively, strong tumor-toxic cells can be induced by 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 of white blood cells excluding granulocytes, monocytes, and macrophages, and particularly have T cell properties.

Activation of peripheral blood leukocytes by oligosaccharide-bound insoluble carriers can strongly induce tumor-toxic cells when carried out in a medium containing serum components or in a medium to which interleukin 2 is added. That is, a medium containing 2 to 20% of animal serum such as fetal bovine serum, bovine blood fraction, horse serum, or human serum is prepared.

Preferably, a medium containing 2 to 20% human serum is prepared. The medium in this case is a medium commonly used for animal cell culture, such as RP MI 1640 medium, M
EM medium etc. can be used. It is also possible to use RPM 11640 medium supplemented with serum components such as serum albumin.

Peripheral blood leukocytes collected by various methods are suspended in the prepared medium at a cell concentration of 0.5 to 3 x 10 cells/ml,
An appropriate amount of oligosaccharide-bound insoluble carrier is added to this, and culture is performed 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 CO□ incubator using a commercially available plastic container for cell culture. After culturing for one to several days, activated leukocytes are collected.

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

(Effects of the Invention) As described above, the oligosaccharide-bound insoluble carrier of the present invention stimulates and activates leukocytes, and is safe and easy to operate.
It induces powerful anti-tumor immune cells and is intended to be used for cancer treatment, examination and diagnosis, research, etc. of gastric cancer, lung cancer, breast cancer, etc.

(Example) Example 1 2g of commercially available pig gastric mucosal mucin (Sigma) was mixed with 0.2N-N
aOH, (37°C, 24 hours) or trypsin (3
7° C. for 24 hours) for decomposition, ethanol is added to a concentration of 60% to precipitate undegraded mucin, and the supernatant is concentrated to dryness. 5ml of 0.2M phosphate buffer (pH 8,0)? Container and Sephadex G-5
Gel chromatography was carried out at
Collect and concentrate the oligosaccharide fractions.

Through this operation, 20 mg of oligo tFi was obtained from mucin.

10 mg of this oligo F' was bonded to 2 ml of commercially available epoxy-activated Sepharose (manufactured by Pharmacia) by a known method to prepare an induction material.

Human leukocytes were obtained as follows. That is, the collected human peripheral blood was diluted 2 times with Hank's solution, layered on Ficoll-Paque solution (manufactured by Pharmacia), centrifuged at 200 rpm for 20 minutes, the white blood cell layer in the middle layer was separated, and this was layered with Hank's solution. After washing with 10% autologous serum, transfer to RPM11640 medium containing 10% autologous serum.
Suspend at a cell concentration of ml. 1ml of this cell suspension
2 ml wells for cell culture (Falcon Nl13)
047), and add 50% of the oligosaccharide-bound insoluble carrier to this.
Add 0 μβ and incubate at temperature 3 in a CO2 incubator.
Cultivate at 7°C. After culturing for 3 days, pipetting and leaving to stand will cause the carrier to settle to the bottom of the container. Take the supernatant cell solution, wash it with Hank's solution, and add RPMI medium supplemented with 10% autologous serum. 5 x 10'
Suspend cells at a cell concentration of /ml.

Whether or not these activated leukocytes have tumor cytotoxicity was evaluated using the following killer activity measurement method. The cells were suspended in RP MI 1640 medium supplemented with lθ% tallow serum at a cell concentration of '/ml,
This is dispensed into 10 μl Terasaki plates in 10 μl portions, and cultured at a temperature of 37° C. in COz incubator-9. When cultured for 24 hours, cancer cells strongly adhere to the bottom of the culture plate. After washing this with culture solution, 10 Itl of activated leukocyte suspension was added and incubated at 37°C for 4 hours with COz.
The cells are cultured in an incubator to damage cancer cells attached to the plate. Damaged cancer cells lose their adhesion to the bottom of the plate, and when washed with Hank's solution, their activity and leukocytes are removed. The cancer cells adhering to the surface of the -C7' and -to bodies except for 1 are fixed with acetone, stained with Giemsa solution, and then counted under a microscope. Killer activity is calculated using the following formula.

When the tumor cytotoxic activity of immune cells activated with the oligosaccharide-bound insoluble carrier was measured using such a method, it was found that the tumor cytotoxic activity was 50% against MKN-1 human gastric cancer cells.

Example 2 An oligosaccharide-bound carrier prepared in the same manner as in Example 1 was collected from a BALB/C mouse in the same manner as in Example 1, and was added to lung cells (5X 10 h/ml, RPMI medium containing 10% FCS). floating), 50 μl per ml of medium
and cultured for 4 days. activated leukocyte B
When the killer activity against ALB/C-derived syngeneic tumor colon 26 was measured, it showed a 58% damaging activity.

Example 3 Galβl-” 4 GlcN Acβ1-3Gal
An oligosaccharide having the structure β1↑Fucα1 was synthesized by a known method, and in the same manner as in Example 1, 10 mg of the oligosaccharide was bound to 2 ml of epoxy-activated Sepharose to prepare an inducing material, and human leukocytes were stimulated for 3 days. PC of the obtained activated leukocytes
When the killer activity against -10 human lung cancer cells was measured, it showed a 62% damaging activity.

Claims (1)

[Claims] An anti-tumor immune cell-inducing material characterized by having an oligosaccharide moiety on its surface.