JPS6330500A - Antitumor leukocyte inducing material - Google Patents

Abstract

PURPOSE:The titled material activating leukocyte and introducing powerful tumor damaging cell safely and in good operation efficiency, obtained by adding an antigen existing on the surface of a cell in charge of antitumor immunity or an antibody to receptor to the surface of an insoluble carrier. CONSTITUTION:An antigen existing on the surface of killer T cell, helper T cell, NK cell, K cell, monocyte or macrophage, a cell in charge of antitumor immunity, or an antibody to receptor is fixed to the surface of insoluble carrier preferably by covalent bond to give the aimed material. An anti-idiotype monoclonal antibody to antigen bond site of T cell antigen receptor, etc., are preferable as the antibody. A porous carrier of flat membrane or hollow fiber having 0.1-5 micron pore diameter is preferable as the insoluble carrier.

Description

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

(Prior art) As is well known, killer T cells, NK cells, activated human macrophages, and other cells play important roles as anti-inflammation immune cells that carry out the immune surveillance mechanism against malignant attacks in living organisms. It has been reported that Masayo Fukuzawa's history of medicine, 126.420 ('83), therefore, as immunotherapy for malignant tumors, it is necessary to compare the activity of cancer patient's immune cells (white blood cells). It is conceivable to compare the inducing activity of these anti-inflammatory 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 the induction activity of immunosuppressive cells (suppressor T cells, suppressor macrophages, etc.) by tumor cells. (S
, Fujimoto etal: J.Immun.
oi.

116.791 ('76)L immunosuppressive cells are
Suppresses the induction activity of various anti-tumor immune cells that have the function of damaging tumor cells, thereby allowing tumor cell proliferation,
This is thought to lead to a further decline in the ability to respond to tumors. 1\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') CJ
, A. Rothetal: J. Immunol;
128, 1955 ('82) - It must be said that it is difficult to efficiently induce antitumor immune cells in cancer patients under such immunosuppressive conditions. Anti-tumor immune cell inducing activity without immunosuppression 1
The optimal conditions for humans are set outside the body, and white blood cells taken from cancer patients are stimulated to induce strong anti-tumor immune cells, which are then returned to the original cancer patient to treat cancer. The proposed method is thought to have the potential to become a highly effective new cancer immunotherapy.

(Problems to be Solved by the Invention) Attempts to induce and activate anti-tumor immune cells by comparing the stimulating activity of white blood cells removed from the body and administering this to cancer-bearing organisms to cure cancer have been made. Currently, active research is being carried out, but the procedure is extremely complicated, as it involves the use of cells extracted from cancer-bearing organisms to determine the stimulating activity ratio of white blood cells.

(Means for Solving the Problems) In order to solve the above problems, the present inventors conducted intensive research and found that killer cells, helper T cells, NK cells, which are anti-tumor immunocompetent cells, K, IIB bleb.

Antigens present on the surfaces of monocytes and macrophages are produced by contacting human peripheral blood leukocytes with an inducing material in which antibodies against the receptors are covalently bonded to an insoluble carrier.
Surprisingly, we discovered that extremely strong @susceptible cells were induced, which led us to complete the present invention.

In other words, the present invention is directed to the use of antibodies directed against antigens or receptors present on the surface of killer cells, helper T cells, NK cells, monocytes, and macrophages, which are immune-competent cells. The above relates to an anti-tumor leukocyte-inducing material, which is characterized by having it on the surface of an insoluble carrier.

The surface of an insoluble carrier in the present invention refers to a surface capable of contacting cells, ie, leukocytes.

In the present invention, white blood cells refer to blood cells excluding red blood cells and platelets. The so-called white blood cells refer to all white blood cells, but in addition to these white blood cells, there are also granulocytes, B cells are removed, and the next cell fraction is also included. It is included in the concept of white blood cells in the present invention. The leukocytes to be activated in the present invention may be collected from peripheral blood using a continuous centrifugation method and used as a leukocyte fraction, or may be separated using a Ficoll-Birk multilayer centrifugation method and used as a mononuclear cell fraction. often.

Alternatively, it is possible to induce highly toxic cells by using cell fractions obtained by separating and concentrating peripheral blood mononuclear cells by forming rosettes with known neuraminidase-treated sheep red blood cells.

The tumor-induced cells to be 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.

Antibodies that bind to insoluble carriers that can be used in the present invention include anti-Leu-1 monoclonal antibodies, anti-Leu-2 monoclonal antibodies, and anti-Leu-2 monoclonal antibodies that are antibodies against antigens and receptors present on the surface of T cells.
3 monoclonal antibody, anti-Leu-4 monoclonal antibody, and anti-Leu-5 monoclonal antibody.

Anti-Leu-8 monoclonal antibody, anti-Leu-9 monoclonal antibody, anti-Leu-15 monoclonal antibody.

Anti-T3 monoclonal antibody, anti-T4 monoclonal antibody, anti-T6 monoclonal antibody, anti-T8 monoclonal antibody, anti-9 monoclonal antibody, anti-T10 monoclonal antibody, anti-'l'tl monoclonal antibody, anti-Le
u-1 monoclonal antibody, anti-Leu-2 monoclonal antibody, anti-Leu-3 monoclonal antibody, anti-Le
u-a monoclonal antibody, anti-Leu-5 monoclonal antibody, anti-Leu-8 monoclonal antibody, anti-L
eu-9 monoclonal antibody, anti-Leu-15 monoclonal antibody, anti-human transferrin receptor monoclonal antibody, anti-T cell antigen receptor antibody, anti-IL-
2 receptor monoclonal antibody, anti-Leu-7 monoclonal antibody that is an antibody against antigens and receptors present on the surface of NK cells/K cells, anti-Leu-11 monoclonal antibody,
Anti-Leu-Mj monoclonal antibody, anti-Leu-M3 monoclonal antibody, anti-HLA-
Examples include DR monoclonal antibody and anti-M1 monoclonal antibody.

Inside 4. Are antibodies against antigens and receptors present on the surface of T cells a positive result? give. More preferably,
Anti-T cell antigen and receptor antibodies, such as anti-T3 monoclonal antibody, anti-11 monoclonal antibody, monoclonal antibody against the antigen binding site of T cell antigen receptor, anti-I
When L-2 phosphorylated pig antibodies are bound alone or to the surface of two or more antibodies, more powerful anti-cancer immune cells can be induced.

The most powerful anti-coagulant immune cell-inducing material is a carrier having anti-cell antigen receptor antibodies and anti-IL-2 receptor antibodies singly or simultaneously on the surface.

As the anti-T cell antigen receptor antibody, an anti-idiotype monoclonal antibody directed against the antigen-binding site of the T cell antigen receptor is particularly preferred from the standpoint of selective activation.

As the antibody that binds to the insoluble carrier, polyclonal antibodies such as antiserum can also be used.

Monoclonal antibodies are preferred from the viewpoint of purity as an antibody and uniformity of binding ability.

The insoluble carrier used in the present invention is a hydrophilic carrier.

Any hydrophobic carrier can be used. The shape of the insoluble carrier is 1
Any known shapes such as particulate, fibrous, hollow fiber, and membrane shapes can be used. As the granular and #i spherical insoluble carriers, those having a particle size of 1 micron 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 active leukocytes by filtration, but if the particle size is 3,000 microns or more, the contact area with the leukocytes per unit weight of the carrier decreases, which is not preferable. Particular preference is given to particles having a particle size of 80 to 2000 microns. ``Also, the specific gravity of one granular or spherical insoluble carrier is 1.
07 or higher, it can be easily separated from activated human leukocytes by centrifugation or standing still. When using a primary, flat membrane or hollow fiber porous carrier, the pore size is 0, 0, which does not allow cells to pass through but allows the medium fraction to freely pass through.
If 5-10 microns are used, they can bind to one side of the membrane and feed the leukocytes to the other side of the membrane.

It is possible to stimulate high concentrations of white blood cells. In particular, hollow fiber-like porous carriers can be used favorably for flat membrane-like carriers with pore diameters of 0.1 to 5 microns.

9. Insoluble carrier materials include activated carbon, glass, etc. and their derivatives in the case of inorganic base materials. Natural polymer-derived carriers include simple polysaccharides such as cellulose, sepharose, dextran, starch, and derivatives thereof.

In terms of polymers, vinyl polymers include styrene, vinyl acetate, methacrylic esters, acrylic esters, and vinyl halides.

There are polymers and copolymers of halogen ratio vinylidene, acrylonitrile, acrylamide, methylvinylketo/1vinylpyrrolid, 7.2-vinylpyridine, ethylene, propylene, butadiene, inoprene, etc. and their derivatives. Ring polymers include dimethylsiloxane, spiro-di-xylylene, norbornene, cyclobutene, trioxane, lactide, cyclopolysiloxane, phosphonitrile chloride, N-carboxy-α
- Polymers and copolymers of amino acid anhydrides and derivatives thereof, polyformaldehyde, polyethylene oxide, polypropylene glycol, poly-3,3-bis(chloromethyl)oxacyclobutane, polytetrahydrofuran, polycaprolactam, etc., and derivatives thereof.

[ζM condensates include polyester, bolide, polyanhydride, polycarbonate, polyurea, polysulfonamide, polyimide, polybenzimidazole, etc., and derivatives thereof.

Regarding trees and other matters. Acrylic tree pattern.

Examples include methacrylic resin, fluororesin, epoxy resin, urea resin, amine resin, styrene resin, melamine resin, polyurethane, silicone resin, alkyd resin, and derivatives thereof.

The above-mentioned polymeric carriers can be made into insolubilized carriers by using suitable comonomers and crosslinking agents as necessary. , diisocyanates, epoxy compounds, dienes,
Various types such as glutaraldehyde can be selected depending on the functional group of the object to be crosslinked (Taiseisha, 5 Crosslinking Agent Handbook, P3-77, 19131).

Further, a carrier having a sublong layer structure obtained by applying a coating to the above-mentioned insoluble carrier can also be used. for example.

A carrier such as activated carbon or glass beads coated with polyhydroxylethyl methacrylate can be used.

Various known methods such as covalent bonding, ionic bonding, and physical adsorption can be used to immobilize antibodies on the surface of an insoluble carrier, but from the viewpoint of dissolution, it is preferable to immobilize antibodies using covalent bonds. desirable. For example, hydrogen bromide (CNB
r) by comparing the activities of 7galose, Sepharose, etc., or by reacting silica glass beads with γ-aminopropyltriethoxysilane to obtain alkylamino glass, which is activated with glutaraldehyde and bonded. can be used. It was a cabinet. Once the KF is required, a molecule (spacer) of any length can be completely introduced between the insoluble carrier and the insoluble carrier. This can be carried out by reaction-bonding the remaining incyanato groups and the amino groups of the antibody.

The manufacturability of the induction material of the present invention is based on the above factors.

It does not specify the bonding order of its constituent elements.

That is, 1. The present invention basically uses killer cells and helper T cells, which are anti-lying immunocompetent cells on the surface.

NK cells, monocytes, and macrophages (as long as they have one or more antibodies directed against antigens or receptors present on the surface of cells, monocytes, and macrophages), they are not dependent on artificial methods.

When used, the leukocyte-inducing material of the present invention can be used to bind one or more types of antibodies to the surface of a single insoluble carrier. It is also possible to use a combination of multiple.

Activation of leukocytes by inducing agents [Humans can strongly induce susceptible cells when carried out in a serum-containing medium. That is, a medium containing 2 to 20% of animal serum such as fetal bovine serum, bovine serum, horse serum, or human serum is expressed as fA.

The medium in this case is, for example, medium 1 commonly used for animal cell culture.

RPMI 1640 medium, M]IEM medium, etc. can be used.

RPM with temperature controlled serum components such as serum albumin
11640 medium can also be used. Well done.

Even if Interleukin 2 is added during the induction period.

It can induce strong tumor-causing cells.

White blood cells collected by various methods were collected into the ground as described in the next section.
Suspend at a concentration of 5-5 x 10'/lll10 cells.

An appropriate amount of inducing material is added to this, and culture is carried out at 25 to 45C. At temperatures below 250 degrees Celsius, there is almost no effective activation of leukocytes, and at temperatures above 45 degrees Celsius, the survival rate of leukocytes decreases. The culture can be carried out using a commercially available plastic container for cell culture in a CO2 incubator. After several hours of culture, leukocytes adhere to the inducing material and become activated.

It was discovered that the leukocytes whose activity was determined in this way contained 1 strongly tumor-damaged cells. Namely.

After activating human peripheral blood leukocytes with an inducing agent, human peripheral blood leukocytes were allowed to act on human @x cells, resulting in MKN-1 gastric cancer cells.

It strongly damaged PC-10 lung cancer cells.

(Effects of the Invention) As mentioned above, the inducing material of the present invention effectively and safely activates peripheral blood leukocytes of cancer patients.

And one powerful tumor with easy operation! It induces harmful cells, and is intended to be used in cancer treatment, examination and diagnosis, research, etc., such as gastric cancer, lung cancer, breast cancer, liver cancer, and gastric cancer.

(Example) Example 1 0KT3 monoclonal antibody (Oats Diagnostic Systems Co., Ltd. table) was purified by Affinity chromatography using Protein A-Sepharose 4B (Pharmacia M), and this n Antibody 1
CNB using 00 μi as a ligand by a known method.
r active f Hisepharose 6ME (Pharmacia M)
'Im to prepare an inducing material.

In addition, in order to determine the retention t' of these ligands, the ligand Jl in the supernatant and washing solution after the binding reaction is determined,
As calculated by subtracting from the amount added, 95 ligands were retained.Human leukocytes were obtained as follows. Namely.

Blood was collected and the human peripheral blood was diluted 2 times with sour liquid.

Layered with Ficoll Park solution (Pharmacia), 2
After centrifugation at 000 rpm for 20 minutes, the white blood cell layer in the middle layer was separated, washed with Nox's solution, and then transferred to RPMI 1640 medium supplemented with 10% autologous serum. Float at a degree.

This cell suspension was added 1-well to 2-wells for cell culture (
7Alco7A50d7).

Add 50 μt of induction material to this and culture at 37C in a CO incubator. 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 serum, wash it with Hank's solution, and add RPMI 164 with 10% autologous serum.
0 medium at a cell concentration of 5XiO6/-.

Whether this activity ratio leukocyte has 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 suspended in RPMI 16dO medium supplemented with 10% tallow serum at a cell concentration of 5 x 10'/mt, and 10 μt of this is placed on Terasaki plates in 10 μl volumes. Dispense, C
O, culture in an incubator at a temperature of 37C. 24
When cultured for a certain period of time, cancer cells are strongly attached to the bottom of the culture plate.

After washing this with culture solution, 10μ of active f leukocyte suspension was added.
m1 and cultured in a CO incubator at 37C for 4 hours to damage cancer cells adhering to the plate. Damaged FC 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. Cancer cells that remained alive and attached to the bottom of the plate were fixed with 77 tons.

Stain with Giemsa solution and count using a microscope. Killer activity is calculated using the following formula.

Killer activity = (1-C (number of surviving cells when activated leukocytes are added)/(number of surviving cells when activated leukocytes are not added))) x 100 (%) The method was used to measure the tumor cytotoxic activity of immune cells activated by the inducing material.

As shown in Table 1 (, MKN-1 human gastric cancer cells, PC-1
0 human lung cancer cells.

Example 2 0KT5 monoclonal antibody and 0KIa1 monoclonal antibody (Off Diagnostic Systems Co., Ltd.) were prepared in the same manner as in Example 1, and 100 μm of these antibodies were prepared. As all the ligands, CNBr active f Hisepharose 6MB (Pharmacia JjJ) 11
A special consideration is given to the induction material bonded to Rt.

The tumor-damaging activity of immune cells activated with this inducing material was measured in the same manner as in Example 1, and the results are shown in Table 1. It showed strong damaging activity against cells.

Example 3 The ability to induce anti-tumor immune cells by the inducing material mixed with equal amounts of fcOKT5 monoclonal antibody fixed ratio Sepharose 6MB and monoclonal antibody fixed ratio Sepharose 6MB obtained by the same procedure as in Example 1 was examined. As in Example 2, it was found that leukocytes having strong damaging activity against MKN-1 cells and PC-10 cells could be induced.

Comparative Example 1 Insoluble carrier (Sepharose 6MB) with no inducer added
) was added and all experiments were carried out in the same manner as in Example 1, but almost no antitumor cells could be induced.

Example 4 The preparation of an anti-tumor leukocyte-inducing material by binding an anti-T cell antigen receptor monoclonal antibody and the ability to induce anti-tumor immune cells will be described.

(H) Culture of T cells and preparation of solubilized peritoneal skin fraction) H) Specific gravity centrifugation of IJ cells (Jun Yada and Michio Fujiwara I, Lymphocyte Function Search Method, p 1y, t' 9ao year.

R obtained from Chugai Pharmaceutical Co., Ltd. and added with 10% total beef tallow serum.
PMI 1640 medium) K2 x 1 oa/- cells! I made it float in one go. Add to this PHA-P (Difco 1
)=i0. i% concentration, transferred to a culture bottle, and
C151CO, which is cultured for 48 hours, and P
The lymphocytes activated by HA and O interleukin 2
(manufactured by Peringer Mann Im) Contains RPMI 1
Cultured in 640 medium (containing 20 rounds of tallow serum) for 14 days to obtain 2×10 cells. From these cells, the T cell antigen receptor fraction was obtained as follows. 7 cells
Wash 3 times with Nax solution and PBS containing 1% Triton-100.
(1mM phenylmethylsulfonyl fluoride,
The membrane was suspended in 2 ml of water containing 1 mM EDTA and 10 mM NaF, pH 7, and stirred for 1 hour to make the membrane soluble. 10.00 solubilized
Centrifuge at 07° for 20 minutes to remove cell debris.

Obtain the solubilized membrane t exposure fraction. This is Cefakreel S
-200 (Pharmacia), gel chromatography was performed with PBS, fractions with a molecular weight of 70,000 to 100,000 were collected, and ultra-condensed to 9 (0.51 nt).

(Table of anti-T cell antigen receptor monoclonal antibodies)
Mix an equal amount of the concentrate with Freund's complete adjuvant and immunize BALB/C mice. After 10 and 20 days, 100 μt of the concentrated solution was administered intraperitoneally to Kirani.
After 0 days, 100μ of the concentrate was injected intravenously, and after 3 days, the pancreas of the immunized mice was excised.

Common method C (G, Kohler & C, Milstein
, Nature 256□ La 49, (1975)], the hybridoma was fused with mouse myeloma P3UI to create a hybridoma. Hybridoma supernatants are screened for binding to human T cells and whether they contain antibodies that bind to synthetic human T cell antigens and receptor β chain constant region peptides, and finally T cell membrane protein fractions and immunizations are performed. The hybridoma was precipitated and subjected to SDS polyacrylamide gel electrophoresis to obtain an anti-T cell antigen receptor monoclonal antibody producing hybridoma.
The culture supernatant obtained by culturing in 1640 medium was purified by affinity chromatography using Protein A-Sepharose CL-4B (Pharmacia).

10-17) Anti-TM cell antigen receptor monoclonal antibodies were obtained.

(Table of anti-cell antigen receptor monoclonal antibody-bound Sepharose 4B) Anti-cell antigen receptor monoclonal antibody 1η 11
Next, the CNBr activity 1 Hisephalose 6MB (manufactured by Pharmacia) was bound by the method of K pasting.

(Induction of anti-tumor immune cells by anti-T cell antigen and receptor monoclonal antibody-bound insoluble carrier) The above antibody immobilized f
Using all human insoluble carriers, human leukocytes were stimulated in the same manner as in Example 1, and cytotoxicity was measured as shown in Table 1 (MKN-1 human gastric cancer cells, PC-10 human It showed strong damaging activity against lung cancer cells.

Claims (1)

[Claims] The insoluble carrier has one or more antibodies against antigens or receptors present on the surface of anti-tumor immunocompetent cells such as killer T cells, helper T cells, NK cells, K cells, monocytes, and macrophages on the surface of the insoluble carrier. An anti-tumor leukocyte-inducing material characterized by: