JPH01221326A - Cytotoxic agent against malignant tumorous cell - Google Patents

Abstract

PURPOSE:To obtain a cytotoxic agent for malignant tumorous cells containing a monoclonal antibody against a factor binding to a receptor of the malignant tumorous cells as an active ingredient. CONSTITUTION:A cytotoxic agent for malignant tumorous cells containing a monoclonal antibody against a factor binding to a receptor of the malignant tumorous cells, e.g., growth factor or activating factor, such as growth factor TAG, insulin, insulin-like growth factor or epidermal growth factor (EGF), as an active ingredient. A monoclonal antibody against the growth factor TAG obtained from TAGI-1 strain (FERM P-9851), etc., are cited as the above- mentioned antibody. The afore-mentioned antibody is reacted wit malignant tumorous cells in the presence of a complement and a factor binding to the receptor of the malignant tumorous cells to damage the malignant tumorous cells. The malignant tumorous cells, factor binding to the receptor thereof and complement can be detected by removing one of components participating in the above-mentioned reaction, using the residual components as required components and detecting the malignant tumorous cells damaged by the reaction.

Description

Detailed Description of the Invention The present invention provides a malignant tumor cytotoxic agent containing as an active ingredient a monoclonal antibody against a factor that binds to a receptor of malignant tumor cells, and a receptor for malignant tumor cells. A method of damaging malignant tumor cells using a monoclonal antibody against a factor that binds to a receptor, a method of detecting a malignant tumor cell, a method of detecting a factor that binds to a receptor of a malignant tumor cell, a method of detecting complement, a malignant tumor This invention relates to a method for detecting cellular receptors.

<Prior Art> As is well known, it has been reported that anti-tumor cells such as killer cells, NK11 cells, and activated macrophages play an important role as an immune surveillance mechanism against malignant tumor cells in vivo.

Therefore, immunotherapy for various malignant tumor cells involves activating immune cells such as killer cells, NK cells, activated macrophages, and other white blood cells or their undifferentiated cells to efficiently kill these antitumor cells. It is thought that it can differentiate and induce.

However, in patients with malignant tumors, it has been reported that as the malignant tumor progresses, the immune capacity decreases, and that suppressive factors that suppress the immune response or induction of suppressor T cells and suppressor macrophages are present.

It is difficult to efficiently differentiate and induce antitumor cells in the body of a malignant tumor patient whose immune capacity is suppressed.
Efficient immunotherapy has been difficult to achieve, and recently methods have been developed in which patients' NK cells are cultured in a medium containing interleukin 2 and/or interleukin 3 and then administered to the patient, and patients' leukocytes are removed outside the body to achieve effective antitumor treatment. A method of activating and reducing cells (Japanese Patent Application Laid-Open No. 62-243562) has been reported.

In addition, for the purpose of treating malignant tumor cells, monoclonal antibodies against malignant tumor cell surface antigens (Japanese Patent Publication No. 58-454
Missile therapy is known that uses monoclonal antibodies (No. 07) or this monoclonal antibody combined with an antitumor agent. However, monoclonal antibodies against such malignant tumor cell surface antigens are difficult to identify, and only unstable effects can be expected, as they may become ineffective due to mutations in the surface antigen.

B. In order to detect horoscopes, the present inventors have, quite surprisingly, discovered antibodies, particularly preferably monoclonal antibodies, against factors capable of binding to receptors of malignant tumor cells, such as growth factors or activating factors for malignant tumor cells. It has been discovered that the malignant tumor cells can be lysed in the presence of complement and its factors, or that the growth of the malignant tumor cells can be inhibited, thereby exhibiting a cytotoxic effect on the tumor cells. Since monoclonal antibodies are directed against factors that can bind to receptors on malignant tumor cells, monoclonal antibodies can be easily obtained by selecting this factor, and can be used to efficiently target malignant tumor cells. Furthermore, based on the above invention, the present inventors used a monoclonal antibody against a factor that binds to this receptor on malignant tumor cells to detect complement and receptor receptors on malignant tumor cells. A method for damaging malignant tumor cells; a method for detecting malignant tumor cells; reacting with malignant tumor cells in the presence of a factor that binds to the body; We have discovered methods for detecting factors that bind to receptors on malignant tumor cells, methods for detecting complement, and methods for detecting factors that bind to receptors on malignant tumor cells.

The present invention was completed based on the above findings, and includes a malignant tumor cytotoxic agent containing as an active ingredient a monoclonal antibody against a factor that binds to a receptor on a malignant tumor cell, and a factor that binds to a receptor on a malignant tumor cell. A method for damaging malignant tumor cells, the method comprising reacting a monoclonal antibody against the malignant tumor cells with complement and a factor that binds to the receptors of the malignant tumor cells. A monoclonal antibody against a factor that binds to a receptor is reacted with a malignant tumor cell in the presence of complement and a factor that binds to a receptor on a malignant tumor cell, and the malignant tumor cells damaged by the reaction or existing malignant tumor cells are A method for detecting malignant tumor cells, comprising: detecting a monoclonal antibody against a factor that binds to the receptor of the malignant tumor cell in the presence of complement and a factor that binds to the receptor of the malignant tumor cell; A method for detecting a factor that binds to a receptor of a malignant tumor cell, the method comprising reacting with the malignant tumor cell and detecting the malignant tumor cell damaged by the reaction or the existing malignant tumor cell; reacting a monoclonal antibody against a factor that binds to the body with a malignant tumor cell in the presence of a factor that binds to complement and a receptor of the malignant I1M tumor cell;
A method for detecting complement characterized by detecting malignant tumor cells damaged by a reaction or existing malignant tumor cells. A receptor for a malignant tumor cell, which reacts with the malignant tumor cell in the presence of a factor that binds to the receptor of the tumor cell, and detects the malignant tumor cell damaged by the reaction or the existing malignant tumor cell. Concerning how to detect.

First, monoclonal antibodies in the present invention include monoclonal antibodies against factors that bind to receptors of target malignant tumor cells, such as growth factors or activation factors of malignant tumor cells. The method for producing this monoclonal antibody is not limited in any way;
Dissolve lN or a protein conjugate such as albumin bound to this factor in 0.1 to 5 ml of a buffer solution or physiological saline, and if necessary, add the same amount of Complete Freund's Adjuvant to emulsify it. The antibody is immunized by subcutaneous injection or by injection several times to several dozen times every 1 to 3 weeks to obtain antibody-producing cells against the target factor, and these antibody-producing cells and myeloma cells are fused. The desired monoclonal antibody-producing fused cells may be selected to produce antibodies, or cells producing antibodies against factors that bind to receptors on malignant tumor cells may be infected with a tumor virus such as EB virus. Antibody production may be carried out by selecting target monoclonal antibody-producing cells that can be subcultured. Regarding the above cell fusion method, for example, a method using a mouse as a mammal is often used, and BA
Using LB/C mice and C57BL/b mice, a target factor that binds to the receptor of malignant tumor cells or a protein conjugate such as albumin that binds this factor is dissolved in a buffer solution or physiological saline, and then If necessary, add the same amount of Complete Freund's Adjuvant and emulsify.
It was administered subcutaneously or subcutaneously, 4 to 5 times about every two weeks, and then 3 to 5 days after the stimulation, 11 visceral cells containing antibody-producing cells against the target factors used for fusion were collected. This is then fused with myeloma cells that can be subcultured. Such myeloma cells may be human cells, chimeric cells of human cells and other animal cells, or animal cells other than human cells, and established cell lines that produce immunoglobulins or related proteins. Any tumor cell line that normally proliferates is used, preferably myeloma 111.
As a single cell (mini I coma cell) line, U266AR+
, GMI 500 6TG A12, P3-NS r
/1-Ag-1, P3-X63-Ag8U1, SP2/
Examples include cell lines such as U-Ag14, MPCII-45,6, TG, and 1.7. These cell lines are cultured and preserved in a normal cell culture medium. For example, culture is D M E M
This may be carried out using a medium, a medium to which glutamine, penicillin, streptomycin, etc. are added, or a RPMI medium supplemented with 10% FC3 (fetal calf serum). In cell fusion, about 1 to 5×10' myeloma cells may be used for one fusion. In addition, lung cells, which are antibody-producing cells used for fusion, are obtained by dissecting the sensitized animal as described above, removing the lungs, cutting them into as many pieces as possible, washing thoroughly, and preparing a lung cell suspension. do. For one fusion, it is usually sufficient to use about 1 to 5" of these lung cells.Myeloma cells and tile H in fusion
The ratio of 1:3 to 10 is preferable. For cell fusion, conventional cell fusion methods can be used, such as a method using an appropriate medium such as RPM 1 medium using a fusion promoter such as Sendai virus or 40-50% polyethylene glycol (molecular weight 1000-6000), or a method using electrical Fusion methods can be used.

The fused cells thus obtained are usually selectively cultured in RPMI medium (HAT medium) supplemented with hypoxanthine, aminopterin, thymidine, and Fe2 in order to select the desired antibody-producing fused cells, and then fused. select a stock,
Next, this fusion strain may be a mixture of two or more cells or may also contain a fusion strain that does not produce the desired antibody, and is a single desired antibody with the same properties. Cloning is performed in order to obtain a fusion strain that produces monoclonal antibodies, and the cloning methods include, for example, the limiting dilution method or the soft agar method using mouse trunk cells as feeder cells. The monoclonal antibody-producing fused cell line obtained in this manner is obtained by adding the cells to a small freezing test tube or ampoule containing DMSO or glycerin, freezing in a -80°C freezer, and then It may be stored under nitrogen. In addition, such a monoclonal antibody-producing fusion cell line can be used as FC3.
The desired monoclonal antibody may be produced by culturing in a RPMI medium containing RPMI or Dulbecco's modified Eagle's medium, or a monoclonal antibody containing Pristane (Pristan; 2+6) may be cultured in advance.
+10+14-tetramethylpentade
The above-mentioned fused cell line may be administered into the peritoneal cavity of a pristane-treated mouse that has been intraperitoneally administered with Pristane (C. cane: manufactured by Aldrisochi), and the ascites fluid may be collected after about 10 days to mass-produce the desired monoclonal antibody. Furthermore, this fused cell line may be grown as a tumor in an animal containing the same histocompatibility antigen or an athymic nude mouse, and monoclonal antibodies may be collected from this tumor. The obtained antibodies can be collected and purified using commonly used methods such as ammonium sulfate fractionation or niglobulin precipitation of the antibody-containing solution, followed by ion exchange chromatography, gel filtration, affinity chromatography, etc. to obtain immunoglobulin fractionation and further To obtain the above monoclonal antibody, human cells or chimeric cells of human cells and other animal cells may be used for therapeutic purposes as a malignant tumor cytotoxic agent. It is preferable. Further, for detection, any cell such as human cells, chimeric cells of human cells and other animal cells, or animal cells other than human cells may be used.

In addition, factors such as growth factors or activators that bind to receptors of malignant tumor cells include, for example, growth factors TAG,
Insulin, insulin-like growth factor, EGF (epidermal growth factor)
, PDGF (platelet-derived growth factor), FGF (
fibroblast growth factor), TGF (transforming growth factor),
Vasopressin, somatomedin C1 bombesin, prostaglandin Et, prostaglandin F2α, G-
C3F.

Examples include M-C3FXG/M-C3F, calcitonin, PTH, etc., and these factors are not limited in any way as long as they bind to receptors of malignant tumor cells.

Furthermore, as the complement, it is convenient to use complement components present in the blood of the target animal. Therefore, when using complement in the detection system of the present invention, the target animal itself is used to utilize complement components C1 to C9 present in the blood of the target animal, or the complement components C1 to C9 present in the blood of the target animal are used, or Existing complement components may be isolated by a conventional method, conveniently using serum, and commercially available complement reagents may also be used.

Furthermore, the target malignant tumor cells may be any malignant tumor cells that bind the above-mentioned growth factors or activators, and are not limited in any way. Target malignant tumor cells that exhibit damaging properties can be easily identified from the selected combination of antibodies and complement.

Examples of malignant tumor cells and factors that bind to the receptors of malignant tumor cells include human lung adenocarcinoma A349 cells, human signet ring cell carcinoma KAT 0-Ill, and human gastric cancer cell BM3.
14. Human nasal cavity carcinoma KYB cells, human epidermoid carcinoma A-, which bind to colorectal cancer cells AZ521 and modified forms of these cells, such as growth factors TAG, and further bind factors such as insulin and EGF. 431 cells and human chorioadenocarcinoma B e
W o cells, etc., and epithelial malignant tumors such as lung cancer, gastric cancer, colon cancer, breast cancer, prostate cancer, cervical cancer, and renal cancer. Examples include combinations of factors such as and the malignant tumor.

Furthermore, in the malignant tumor cytotoxic agent of the present invention, which contains as an active ingredient a monoclonal antibody against a factor that binds to a receptor of malignant tumor cells, for example, the monoclonal antibody is added per 0.1 to 5 ml of distilled water for injection or buffer solution. 1-5
Prepared by dissolving ~10-2 g of , 0.1% or more of amino acids such as glutamic acid, asparagine, aspartic acid, glycine, alanine,
Preferably, it is added at a concentration of 0.2 to 10%, and can be prepared by ordinary drying means such as freeze drying or vacuum drying, and may be formulated as an injection to be dissolved before use. Also, use the above stabilizers or polyhydric alcohols such as propylene glycol, glycerin, ethylene glycol, polyethylene glycol, sorbitol, etc., in 5-30% distilled water for injection or in the evening. As a liquid, this 0.5 to 5
1-5X10-'-10 of this monoclonal antibody per ml
-"g may be added to prepare an injection. Furthermore, to prepare an in vitro reagent using this monoclonal antibody, for example, distilled water for injection, buffer solution, or cell culture medium 0.5 ~1 monoclonal antibody per 5 ml
It is convenient to dissolve and use ~5 x 10-'~10-''g.Furthermore, the factor that binds to malignant tumor cells is prepared separately from the monoclonal antibody formulation, and is used in the above-mentioned method. Preferably, it is prepared in the same manner as the injection form.In this case, compared to the amount of monoclonal antibody used,
The amount of this factor may be at least equimolar, preferably 2 to 10 times the molar amount. These preparations can be conveniently adjusted for permeability by appropriately adding tonicity agents such as sodium chloride or glucose, and then injected intravenously or directly into the vicinity of the tumor-affected area.

Furthermore, in carrying out the detection method of the present invention, the number or amount of malignant tumor cells, the amount of the factor that binds to the receptor of the malignant tumor cell, the amount of complement, and the amount of monoclonal antibody against this factor are all determined. It is sufficient to use an equimolar amount or more, for example, 0.1 to 1 mj! 103 malignant tumor cells per solution
~108 pieces may be used. Furthermore, the amount of factors, complement, and monoclonal antibody may be appropriately determined based on the number of receptors on the surface of the malignant tumor cells, and may be used in an amount several times the molar amount of the malignant tumor cells. These necessary components are added to a solution of 0.05 to 51 II The number of tumor cells or remaining malignant tumor cells may be measured.

<Examples> Next, examples of the present invention will be described, but the present invention is not limited by these in any way.

Example 1 [Confirmation and purification of growth factor TAG] (11 Targeting human lung adenocarcinoma A349 cells, human signet ring cell carcinoma KATO-■, and rat ascites hepatoma cells A37974F using human amniotic fluid at 14 and 32 weeks of pregnancy) The cell proliferation promoting effect was investigated.

The target cells were cultured for 4 days using 1×104 cells and adding 10% human amniotic fluid at 14 and 32 weeks of pregnancy, respectively. In addition, when culturing, in the case of human lung adenocarcinoma A349 cells, DM
EM medium was used, and for other cells RPM11640 medium was used.

The results are shown in Figure 1 (showing the case using human amniotic fluid at 14 weeks of pregnancy) and Figure 2 (showing the case using human amniotic fluid at 132 weeks of pregnancy). Figure 2 shows the growth curve of human lung adenocarcinoma A349 cells in a medium supplemented with amniotic fluid, M shows a growth curve of human signet ring cell carcinoma KATO-1 in a medium supplemented with amniotic fluid, and ■ shows a growth curve of human signet ring cell carcinoma KATO-1 in a medium supplemented with amniotic fluid. The growth curve of Al7974F is shown. In the figure, O indicates the growth curve of human lung adenocarcinoma A349 cells in amniotic fluid-free medium as a control, and Δ
shows the growth curve of human signet ring cell carcinoma KATO-111 in amniotic fluid-free medium as a control, and rat ascites hepatoma cell A3797 in amniotic fluid-free medium as a control.
The growth curve of 4F is shown.

As a result, the growth of malignant tumor cells was promoted in both human amniotic fluids, and from Figures 1 and 2, pregnant tS
Amniotic fluid at 14 weeks was found to have a higher effect on promoting malignant tumor cell proliferation.

In addition, amniotic fluid from 14 weeks of pregnancy was added to the culture medium, and human lung adenocarcinoma A3
Cell growth promoting activity against 49 cells was measured. first,
The target cells were grown to a confluent state in serum-added DMEM1 medium and used in experiments. Cells were collected by centrifugation (1, 20 rpm, 5 minutes), the serum-added medium was removed, and the cells were washed with serum-free DMEM medium and centrifuged repeatedly. The cells were resuspended in serum-free DMIF and M medium, adjusted to a cell density of 2.5 x 104 cells/ml, and distributed in 2IIl portions into 24-well tissue culture plates. Amniotic fluid or PBS (phosphate buffered saline, for control experiments) was added to a final concentration of 1.0 to 12.5%, and the mixture was incubated at 37°C.
After 4 days of culture in a CO2-incubator, cell numbers were determined by Coulter Counter a+odel
It was measured with ZM (Coulter Electronics). As a result, as shown in FIG. 3, the effect of promoting cell proliferation was observed at extremely low concentrations.

Therefore, this growth factor was isolated and purified using amniotic fluid from the 14th week of pregnancy as a material.

(2) Next, 5 tails of amniotic fluid at 14 weeks of pregnancy were taken.
Sephadex G75 (manufactured by Pharmacia) column (diameter 2.6C) equilibrated with PBS (pH'y, 4)
1lX100am: Phar+++acia Co
lumn C26/100) and 1 fraction 2 ca at a flow rate of 0.5a+1/min using PBS as eluent.
11 fractions were collected, and the effect of promoting malignant tumor cell growth in each fraction was evaluated in human lung adenocarcinoma A349 cells and human signet ring cell carcinoma KATO-III cells.
were measured.

The results are shown in Figure 4, where the solid line indicates 280
The amount of protein measured in nm is shown, and the malignant tumor cell proliferation promoting effect in each fraction is shown in a bar graph.

From this bar graph, we can see that mainly the fraction N [L45~
46, strong proliferation-promoting activity was observed in both human lung adenocarcinoma A349 cells and human signet ring cell carcinoma KATO-11 cells.

Therefore, this active fraction was further purified.

(3) The above fraction N [active fraction of L45 211
1 was added to a heparin-Sepharose CL-6B (manufactured by Pharmacia) column (diameter 1.6CJ1.
After washing and removing non-adsorbed white matter with a Cl aqueous solution at a flow rate of 0.1 ml/min, it was eluted with a continuous concentration gradient solution of NaCl concentration from 0.15 to 5.0 M as 1 fraction and 2 ysl, and 100 fractions were collected. These eluted fractions from heparin-Sepharose column chromatography were desalted by gel filtration. Sephadex G-
25 (Pharmacia) was equilibrated with PBS and packed into a Pharmacia column C16/40. To this, 2t of each elution fraction of heparin-Sepharose column chromatography was added.
sl was applied and eluted with PBS. The growth promoting activity of each of these fractions against human lung adenocarcinoma A349 cells was measured.

As a result, as shown in Fig. 5, it was confirmed that the active ingredient exhibiting growth promoting activity was adsorbed on the column and eluted at a NaCl concentration of 2.0M (bar graph in the figure, - - indicates NaC/ concentration), most of the protein appeared in the flow-through fraction (-〇-).

Furthermore, the degree of purification of this active fraction was examined by SDS-PAGE, and staining was performed by silver staining. The results are shown in FIG.

In Figure 6, lane 1 shows the band of the active fraction, and lane 2 shows the blank, but since a silver band appears, one band on the low molecular side of lane 1 shows the desired activity. Recognized as a component and has a molecular weight of 35,000±5,000
Therefore, this active ingredient was named growth factor TAG.

[Creation of monoclonal antibody against growth factor TAG]

Sephadex G of human amniotic fluid obtained from 4-week-old C57BL/b mice (female) at 14 weeks of gestation as described above.
The active fraction obtained by -75 column chromatography was used as an antigen for immunization.

At this time, l1popolysaccaride-KO
3 (LPS-KO3) 100ug to 100uj2 PB
Using the solution dissolved in S as an adjuvant, it was mixed with 100 μl of the eluted active fraction of Sephadex G-75 and added to C.
It was administered subcutaneously to 57BL/6 mice. This operation was performed four times every two weeks, and the tiles were removed aseptically on the third day after the final immunization.
Lung cells were isolated. Next, this lung is made into a single cell,
The contaminated red blood cells were thawed by adding 5 ml of 0.83% ammonium chloride Tris-Salt M buffer (pH 7,65), washed several times with Hank's salt solution, and then combined with the lung cells and BAL B/c. Mouse-derived myeloma cell P3-X63-Ag8U1-87 Zabuanin-resistant strain (
P2O3-88ZG") was mixed at a ratio of 45% polyethylene glycol 400 in DMEM medium.
Fusion was carried out at 37° C. for 2 minutes in the presence of 0 (average molecular weight: 3000). The fused cells were sown on four 96-well microplates, and 100 μM of HAT medium (RPM11640 medium containing 100 μM hypoxanthine, 0.4 μM aminopterin, 16 μM thymidine, and 10% FC3) was added.
1 was added to perform HAT selection culture. Thereafter, half of the medium was replaced with the HAT medium at intervals of 1 to 3 days to obtain fused cells.

Next, the monoclonal antibodies produced by the culture were subjected to EL.
The fused cells producing monoclonal antibodies against the growth factor TAG were detected by ISA method. Furthermore, mouse thymocytes were added at 2XIOb cells/well.
Eight strains with excellent antibody production ability were obtained by cloning using the conventional limiting dilution method by dispensing 0.2 ml/well into well microplates, one of which was TAGI-1.
strain (deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as FERM P-9851). This TAGI
-1 strain was administered intraperitoneally to FI of BALB/C mice and C57BL/6 mice, and after 18 days, the ascites was collected, crudely purified by the nee globulin precipitation method, and further purified by column to obtain the monoclonal antibody TAG 1-1. 1 was obtained, and this monoclonal antibody was a single 1 gM type antibody that specifically recognized the growth factor TAG.

Furthermore, the tight region curve of the monoclonal antibody TAG I-1 against the growth factor TAG measured by ELISA was as shown in FIG.

Detection of monoclonal antibody-producing fused cells is as follows.

The fused cells producing antibodies that specifically react with growth factor TAG were detected by ELISA using a 96-well microtiter plate immobilized with purified growth factor TAG. The procedure of the ELISA method is as follows.

Growth factor T per well in a 96-well microplate.
50 μl of AG-containing solution was added and incubated for 120 minutes at room temperature, then washed three times with PBS, and then 100 μl of PBS containing 10% horse serum was added per well and incubated for 60 minutes at room temperature.

It was further washed three times with PBS. To this, 50 μl of the fused cell culture solution was added per well, incubated at room temperature for 60 minutes, and then washed 5 times with PBS. Next, 50 μl of a peroxidase-labeled anti-IgG mouse antibody-containing solution was added per well and incubated at room temperature for 60 minutes.
Washed 5 times with PBS.

Next, add 100μ of 0-phenylenediamine-containing solution to the well.
After reacting at room temperature for 15 minutes, INH! 100 μl of SO, solution was added.

Thereafter, absorbance was measured at a wavelength of 500 nm using an automatic reader.

[Confirmation of malignant tumor cytotoxicity using monoclonal antibodies]

First, human lung adenocarcinoma A349 cells (
IXIO' individual cells/III in Oyohi DMEM medium
Growth factor TAG solution diluted to 0% 200μIi'l'
Incubate for 20 minutes in a carbon dioxide incubator at 37°C, and centrifuge twice in PBS (1200 rpm
5 minutes) After washing, incubate with serum-free DMHM medium at 1/l 00
Monoclonal antibody TAGI-1-containing solution diluted to 200
IIj! and guinea pig complement (manufactured by Kyokuto Pharmaceutical Industries) 1
0 μl was added and reacted at 37° C. for 2 hours. At this time, the binding of the monoclonal antibody TAGl-1 to the receptor of the cell via the growth factor TAG was confirmed by reacting with an anti-mouse IgM antibody labeled with FITC as a secondary antibody. It was proven that the membrane was stained with fluorescence. In addition, after the above reaction, the supernatant was centrifuged (1200 rpm+
x 5 minutes) and 0.1% trypan blue 100μ
1 was added and the % dead cells were measured on a hemocytometer to determine cell lysis.

In addition, using human lung adenocarcinoma A349 cells in the same manner as above,
The monoclonal antibody TAG I was cultured at different times of addition of the growth factor TAG and diluted to 1/100 in the same manner.
-1-containing solution and complement dried guinea pig complement (Kyokuto Pharmaceutical Industries) 1 vial was dissolved in green solution 11I11 and 20 μl was added (to make 10% TAGI-1
added to the solution. ) was added to observe the damage to human lung adenocarcinoma A349 cells.

The results are shown in Fig. 8, where .
This figure shows a cell lysis curve due to cell reaction, and the binding of growth factor TAG to the receptor of human lung adenocarcinoma A349 cells reaches a peak in about 20 minutes, indicating the highest value of cell lysis. This cell lysis phenomenon is caused by the binding of the growth factor TAG to receptors on the cell surface, and the action of antibodies and complement on this, and it exhibits cytotoxicity such as cell lysis or cell proliferation inhibition. , was due to cytotoxic effects on cells.

In FIG. 8, 0 indicates the case where PBS was used instead of the growth factor TAG as a control.

Furthermore, regarding cytotoxicity due to cytotoxic effects on malignant tumor cells, human gastric cancer cells BM314 and colon cancer cells AZ521 were used for reaction time with growth factor TAG for 20 minutes, and compared to human lung adenocarcinoma A349 cells. Observed.

As a result, human gastric cancer cells BM31
4. It was possible to kill about 60% of both malignant tumor cells, including colon cancer cells AZ521 and human lung adenocarcinoma A349 cells, by cell lysis. In FIG. 9, the hatched graph indicates the cytolysis of malignant tumor cells, and the unmarked graph indicates the case where PBS was used instead of the growth factor TAG as a control.

From this, the factor T that binds to the receptor of malignant tumor cells
The monoclonal antibody against AG binds a factor that binds to the receptor of malignant tumor cells, such as growth factor TAG, to the receptor, and the monoclonal antibody TAGI-1 of interest binds to the receptor through this bound growth factor TAG. binding and further the action of complement, e.g. some monoclonal antibodies TAG
I-1 binds to the surface of malignant tumor cells, activating C1, and C1q binds to the complement binding site on the antibody molecule, resulting in CI
The enzymatic activity of the complex emerges and activates C4, which contacts C1s. C4 is divided into two parts, C4a and C4b.C4b
binds to the surface of malignant tumor cells.

Activated C1s and 02 come into contact and become C2a and C2b. C2a and C4b combine to produce enzymatic activity and C3
Divide. C3b binds to the membrane surface, and C4bC2a and C
3b forms a complex C4bC2aC3b and acts on C5. C5b binds C6 and C7, and 08 acts on the C5b and 6.7 complex to form a pore on the cell membrane surface, and the pore becomes larger due to the further action of C9, allowing water and ions to enter the tumor cell. It is presumed that the cells swell and rupture due to the flow, which lyses the malignant tumor cells, thereby exhibiting harmful effects such as suppressing the proliferation of the malignant tumor cells, making it an effective therapeutic agent for malignant tumor cells. This is the result.

Furthermore, from these results, human gastric cancer cells B M314, colon cancer cells AZ521, and human lung adenocarcinoma A349 cells,
Both have growth factor TAG as their cell surface receptors.
It was determined that the cells possessed receptors that could bind to malignant tumor cells, and the receptors of malignant tumor cells could be detected. Therefore, by using the above malignant tumor cells, the desired monoclonal antibody, and complement, a test solution containing a factor that binds to the receptor of the malignant tumor cells is added to lyse or proliferate the malignant tumor cells. By measuring the inhibitory effect, it was possible to detect the receptor that binds to this factor.

Furthermore, by using the above malignant tumor cells, the desired monoclonal antibody, and complement, a test solution is added to determine whether or not it contains a factor that binds to the receptor of the malignant tumor cells, and the malignant tumor cells are lysed. The factor could be detected by inhibiting proliferation and measuring cell damage.

Furthermore, by using the above-mentioned malignant tumor cells, the monoclonal antibody of interest, and a factor that binds to the receptor of the malignant tumor cells, a test solution containing complement components or not is added to the malignant tumor cells. Complement could be detected by measuring cytotoxicity resulting from lysis or growth inhibition.

As described above, this monoclonal antibody is used to react with malignant tumor cells in the presence of factors that bind to complement and receptors of malignant tumor cells, and by detecting malignant tumor cells damaged by the reaction. To detect one component of monoclonal antibodies involved in the reaction, complement, malignant tumor cells, and factors that bind to the receptors of malignant tumor cells, other necessary components involved in the reaction are used as reagents. Accordingly, the present invention can be implemented as a method for detecting malignant tumor cells, a method for detecting a factor that binds to a receptor on a malignant tumor cell, a method for detecting complement, and a method for detecting a receptor on a malignant tumor cell.

[Malignant tumor cell damage in animal experiments]

The tumor growth inhibitory effect of the monoclonal antibody TAGI-1 on tumor cells that binds the growth factor TAG was demonstrated by transplantation of human colon cancer BM314 cells into nude mice.
This was revealed by observing the subsequent growth.

That is, human amniotic fluid proliferation stamp T with a concentration of 5 μg/m (l)
AG and TAG sensitive human colon cancer cells BM314 10
bcells in vitro in Eagle's MEM medium containing 2% tallow serum at 37°C for 20 minutes at 5% coz.
After treatment in an incubator with 95% air, cells were washed with serum-free Eagle's MEM medium. Cells were then transferred back into serum-free Eagle's MEM medium at 10 bcel.
ls/Ill, and reacted with a 1/100 dilution of mouse ascites containing anti-TAG monoclonal antibody TAG■-1 at 37°C for 30 minutes in an incubator with 5% COz and 95% air. After that, the cells were collected by centrifugation and injected subcutaneously into the flank of a nude mouse.
Cells/mouse were injected and transplanted, and the proliferation of tumor cells was observed. As a result, it was confirmed that the growth of transplanted tumor cells was significantly delayed compared to a control group that was not treated with growth factors and monoclonal antibody TAG I-1. This tumor growth suppressive effect was further enhanced by administering rabbit complement, and it is believed that the expression of this antitumor effect in vivo is mainly due to the complement-mediated anti-11ffi tumor effect. Seem.

The present invention provides a malignant tumor cytotoxic agent containing as an active ingredient a monoclonal antibody against a factor that binds to a receptor on malignant tumor cells, and a monoclonal antibody against a factor that binds to a receptor on malignant tumor cells. Method of damaging malignant tumor cells using antibodies, method of detecting malignant tumor cells, malignant M, l! The present invention provides methods for detecting factors that bind to receptors on tumor cells, methods for detecting complement, and methods for detecting receptors on malignant tumor cells.

[Brief explanation of the drawing]

Figure 1 shows the growth promoting effect of malignant tumor cells by human amniotic fluid at 14 weeks of pregnancy, Figure 2 shows the growth promoting effect of malignant tumor cells by human amniotic fluid at 32 weeks of pregnancy, and Figure 3 shows the growth promoting effect of malignant tumor cells by human amniotic fluid at 32 weeks of pregnancy.
Figure 4 shows the growth curve of human lung adenocarcinoma A349 cells in 4-week human amniotic fluid, Figure 4 shows the cell proliferation activity of each fraction in Sephadex 075 column chromatography, and Figure 5 shows the cell growth activity of each fraction in heparin-Sepharose column chromatography. The cell proliferation activity of the elution fraction is shown, and Figure 6 shows the results of silver staining of the growth factor TAG by 5DS-PAGE. Lane 1 in the figure shows the growth factor TAG.
Lane 2 shows the blank, FIG. 7 shows the tight region curve of monoclonal antibody TAGI-1, and FIGS. 8 and 9 show the tight region curve of monoclonal antibody TAGI-1.
The cytolytic effect of I-1 on malignant tumor cells is shown.

Claims (9)

[Claims] (1) A malignant tumor cytotoxic agent whose active ingredient is a monoclonal antibody against a factor that binds to a receptor on malignant tumor cells. (2) The agent for damaging malignant tumor cells according to claim 1, wherein the factor is a growth factor or an activation factor for malignant tumor cells. (3) The malignant tumor cytotoxic agent according to claim 2, wherein the growth factor or activation factor for malignant tumor cells is the growth factor TAG, insulin, insulin-like growth factor, or EGF. (4) The monoclonal antibody is TAGI-1 strain (FERM
The malignant tumor cytotoxic agent according to claim 1, which is a monoclonal antibody against the growth factor TAG obtained from P-9851). (5) reacting the monoclonal antibody against the factor that binds to the receptor of malignant tumor cells according to claim 1 with the malignant tumor cells in the presence of complement and the factor that binds to the receptor of malignant tumor cells; A method of damaging characteristic malignant tumor cells. (6) The monoclonal antibody against the factor that binds to the receptor of malignant tumor cells according to claim 1 is reacted with the malignant tumor cells in the presence of complement and the factor that binds to the receptor of malignant tumor cells. A method for detecting malignant tumor cells, comprising detecting malignant tumor cells damaged by or existing malignant tumor cells. (7) The monoclonal antibody against the factor that binds to the receptor of malignant tumor cells according to claim 1 is reacted with the malignant tumor cells in the presence of complement and the factor that binds to the receptor of malignant tumor cells. A method for detecting a factor that binds to a receptor of a malignant tumor cell, the method comprising detecting a malignant tumor cell damaged by or existing malignant tumor cell. (8) The monoclonal antibody against the factor that binds to the receptor of malignant tumor cells according to claim 1 is reacted with the malignant tumor cells in the presence of complement and the factor that binds to the receptor of malignant tumor cells. A method for detecting complement characterized by detecting malignant tumor cells damaged by or existing malignant tumor cells. (9) The monoclonal antibody against the factor that binds to the receptor of malignant tumor cells according to claim 1 is reacted with the malignant tumor cells in the presence of complement and the factor that binds to the receptor of malignant tumor cells. A method for detecting a receptor for a malignant tumor cell, the method comprising detecting a malignant tumor cell damaged by or existing malignant tumor cell.