JPS6256136B2 - - Google Patents

Description

Detailed Description of the Invention The present invention relates to a novel antitumor agent, and more specifically, the present invention relates to a novel antitumor agent, and more specifically, an antitumor alkylating agent or an amino group or a carboxyl group introduced into an antibody against a tumor antigen purified by affinity chromatography. The present invention relates to an antitumor agent containing as an active ingredient a substance obtained by bonding an antitumor alkylating agent with an amide bond. The present inventor previously described mitomycin C, doxorubicin hydrochloride, bleomycin, daunorubicin,
He invented an anti-tumor agent whose active ingredient is a substance made by binding antibiotic anti-tumor agents such as actinomycin and sarcomycin to tumor antibodies.
53-161388). However, the above antibiotic-based anti-tumor agents are mainly produced by microorganisms and have complex structures, so what is the best way to combine them with tumor antibodies to produce anti-tumor substances? I can not say. As a result of further research, the present inventor has found that alkylating agent-based substances such as chlorambutyl, melphalan, ACNU, uramustine, and cyclophosphamide are suitable for producing antitumor substances by combining with tumor antibodies. This discovery led to the present invention. The present invention uses as an active ingredient a novel substance in which the above-mentioned highly cytotoxic antitumor alkylating agent is bound to an antibody against a tumor antigen through an amide bond (-NHCO-) under extremely mild conditions. The purpose of the present invention is to provide an antitumor agent that has an excellent antitumor effect but has significantly lower cytotoxicity than the antitumor alkylating agent mentioned above, which is one of the starting materials. shall be. Furthermore, the present invention provides means for purifying the antibody itself against a tumor antigen, and the above-mentioned anti-tumor alkylating agent having an amino group or carboxyl group, and the above-mentioned anti-tumor alkylating agent having an amino group or carboxyl group introduced therein. An amide bond (-
The object of the present invention is to provide a method for producing an antitumor agent containing the above-mentioned substance as an active ingredient, which comprises bonding through NHCO-). In recent years, various antitumor agents have been widely used.
It is having some effect. These antitumor agents include chlorambutyl, melphalan, ACNU,
Alkylating agents such as uramustine and cyclophosphamide are also used, but these substances themselves have high cytotoxicity and can cause side effects such as leukopenia, hair loss, and gastrointestinal disorders when administered. It is known that there are limits to the use of these drugs. Furthermore, attempts have been made to produce or isolate antibodies against certain tumor antigens and use them for the treatment of tumors, but the desired antitumor effects have not been achieved. Furthermore, it has recently been proposed to expect antitumor effects from new substances obtained by chemically bonding antitumor agents to tumor antibodies, but the chemical reaction conditions to obtain the above substances are too harsh. sufficient results have not been obtained. Furthermore, since the antibodies used in these experiments have only been purified to the immunoglobulin fraction, they contain general immunoglobulin and are difficult to recognize as tumor antibodies in the true sense of the word. Since the antibody is a common immunoglobulin, anaphylactic shocks such as damage to normal tissues, generalized convulsions, or stiffness often occur in living organisms to which the above-mentioned substances are administered.
The present invention collects antiserum containing antibodies against tumor antigens inoculated into animals, separates the immunoglobulin fraction therein, and further purifies it by specific means. First, tumor antibodies are
By isolating it and combining it with an antitumor alkylating agent, it is possible to provide an antitumor agent that can be produced under mild reaction conditions and does not cause the above-mentioned anaphylactic shock. The present invention will be explained in detail below. The substance that is the active ingredient of the present invention reacts with the amino group or carboxyl group originally present in both the antibody and the antitumor alkylating agent under mild reaction conditions, or it reacts with the above alkylating agent. Furthermore, it can be obtained by reacting an antibody into which an amino group or a carboxyl group has been introduced. Antitumor alkylating agents used in the production of this substance include chlorambutyl, melphalan, ACNU,
Preferred are uramustine and cyclophosphamide. The names of these alkylating agents are common names, and the structural formulas are as follows. Chlorambutyl: Melhuaran: ACNU: Ulamustine: Cyclophosphamide: Furthermore, as the antibody which is one of the starting materials for this substance, an antibody of a tumor for the purpose of treatment is used.
Tumors include Sarcoma 180, Sato lung cancer, Yoshida sarcoma, Ehritzch cancer, L-1210 leukemia, acute lymphocytic leukemia, bone marrow cancer, chronic lymphocytic leukemia, malignant lymphoma, ovarian cancer, breast cancer, sarcoma, leukemia, and carcinoma. These tumors include: The antibodies used in the present invention are produced by the method described in Japanese Society of Immunology General Meeting Records, Vol. 6, p. 198 (1976).
or the method of Dauphin, MJ et al. [J.Immunol. 113
948 (1974)]. The former is Freund's complete
In this method, antibodies are obtained by first injecting tumor cells subcutaneously into an animal to immunize the animal, and then boosting by injecting tumor cells intravenously. This is a method in which a tumor antigen is repeatedly administered intraperitoneally to an animal 3 to 4 times to immunize the animal, and antibodies are obtained from this. Note that the antibody used in the present invention may be either a homologous antibody or a heterologous antibody, but a homologous antibody is preferable. The antibody obtained in this way is first bound to the tumor antigen for treatment using bromcyan to the packing material of a column in Affinity chromatography, and then this is packed into the column, and the immunoglobulin image is packed in the column. Infuse a solution of antibody purified to minutes. After binding the antigen and antibody in the column, a special solvent is flowed into the column to break the antigen-antibody bond and elute only the antibody, which is then dialyzed to obtain a purified antibody aqueous solution. obtain. That is, the antibody thus obtained is an anti-tumor immunoglobulin with higher purity than conventional immunoglobulin fractions. Generally, to purify antibodies, a method is often used to obtain fractions from antiserum using ammonium sulfate salt precipitation and ion exchange chromatography using a DEAE cellulose column. In addition to this, in the present invention, a specific purification procedure was performed using affinity chromatography to selectively obtain only antibodies specific to tumor cells. Affinity chromatography is based on the principle of utilizing the specific affinity between biological substances such as enzymes and substrates, antibodies and antigens, and selectively separating one biological substance from the other. It is. The affinity chromatography used in the present invention involves (1) covalently bonding the antigen extracted from tumor cells to a carrier such as Shepharose (using bromcyanide), filling this in a column, and adding an antibody solution. After that, a sufficient amount of solvent is poured to wash away the unbound antibodies, and then a buffer solution with a lower pH is poured in to break up the antibody-antigen bond and release the separated antibodies. (2) Without using a column, mix the antigen-bound carrier with an antibody solution to bind the antibody to the antigen, wash the carrier particles to remove unbound antibodies, and then elute the antibody. and (3) a method of using tumor cells themselves instead of the carrier to which the antigen is bound. In the Examples of the present invention, methods (1) and (3) above were adopted. Therefore, antibodies purified by affinity chromatography are anti-tumor immunoglobulins, ie, anti-tumor antibodies, with much higher purity than conventional immunoglobulin fractions. In order to bind the antibody thus obtained to an anti-tumor alkylating agent, an anti-tumor alkylating agent having an amino group or a carboxyl group or an anti-tumor alkylating agent into which an amino group or an amino group has been introduced is used. The antibody is dissolved in a water-soluble solvent, carbodiimide is added thereto as a catalyst, and the mixture is heated at 0 to 50°C, preferably 10 to 40°C, for 10 minutes to 8 hours, preferably 30°C.
The reaction is allowed to proceed for 5 minutes to 5 hours, and the reaction is stopped by adding an acetic acid-sodium acetate buffer. Next, in order to remove excess antitumor alkylating agent, catalyst, components of the reaction termination solution, and salts from this reaction solution, either dialysis gel filtration or ultrafiltration is performed, or these operations are performed. Perform combined operations. The carbodiimides used as catalysts in the above reaction are 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, 1-cyclohexyl-3
-(2-morpholinoethyl)carbodiimide or dicyclohexylcarbodiimide. In order to introduce an amino group or a carboxyl group into an antitumor alkylating agent if necessary, the antitumor agent may be used as it is or in the form of a salt such as Na, K, or Ag to form a compound with the general _formula is Cl or
Br and n is an integer of 1, 2, or 3) or the general formula HOl・NH ₂ (CH ₂ )nCOX (in the formula
represents Cl or Br, and n represents an integer from 1 to 3) in an aqueous solvent such as methanol, water, ethanol, dimethyl sulfoxide, dioxane at 0 to 50°C, preferably at 10 to 40°C for 10 minutes. It is recommended to react for ~72 hours. The resulting reaction product is then recrystallized using a solvent such as water, alcohol, chloroform, dioxane, etc. to obtain an antitumor alkylating agent derivative into which an alkyl group or a carboxyl group has been introduced. Note that monochloroacetic acid is preferable as the above compound. This substance has 1 to 10 molecules of an antitumor alkylating agent bound to one molecule of antibody. The acute toxicity of this substance to mammals was investigated using mice by intravenous administration of 100 mg/Kg, but no mortality was observed after one week of observation. Therefore, the substance has low toxicity and is effective against various human cancers. For example, it is effective against chronic lymphocytic leukemia, malignant lymphoma, bone marrow cancer, ovarian cancer, breast cancer, sarcoma, leukemia, carcinoma, and the like. When this substance is used as an anti-tumor agent, known methods relating to anti-tumor agents can be applied as methods for formulation and administration. The administration method is oral;
Injection or rectal administration may be mentioned, and the dosage form may be powder, granules, tablets, injections, suppositories, or the like. Administration by injection is particularly preferred. Injectable drug formulations include physiological saline, water-soluble solvents such as sterile water Ringer's solution, non-aqueous solvents, isotonic agents, soothing agents, solubilizing agents, stabilizers, preservatives, suspending agents, and buffering agents. , emulsifiers, etc. may optionally be used. To give an example, 10 mg of the substance and 50 mg of mannitol
Dissolve mg in distilled water, make 10 ml, sterilize it using the usual method, and then dispense 2 ml into small injection bottles or freeze-dry it as it is to make an injection. Before use, this drug is diluted with physiological saline to form an injection solution. This substance is generally 0.01 to 90% preferably during formulation.
It can be contained from 0.1 to 60%. The dosage of this substance mainly depends on the symptoms, but it is 0.01 to 3000 mg per adult, preferably 0.1 to 3000 mg per day.
It is 500mg. According to the present invention, the tumor-promoting and anti-tumor properties of the antibody and the anti-tumor properties of the anti-tumor agent are maintained in the above-mentioned substance without loss, so that the substance efficiently achieves its intended purpose when administered. It reaches the tumor site and exerts an antitumor effect. Therefore, if we consider the weight of the antitumor agent that this substance contains as a component as a standard, the amount of antitumor agent as an ingredient is equivalent to 1/10 to 1/20 of the dose of the same antitumor agent itself. The same degree of tumor growth inhibition can be obtained by administering this substance, and the side effects caused by the antitumor agent contained as an ingredient are 1/10 to 2/10 that of administration of the same antitumor agent itself. It is hoped that it will not be too much. This can be said to be the combined effect of the favorable properties of each of the components of this substance. The present invention will be explained in more detail below with reference to Examples. Example 1 1-1: Preparation and purification of antibodies using affinity chromatography (3) Yoshida sarcoma cells subcultured using DONRYU rats were suspended in physiological saline, and mitomycin C was added to the suspension. (50 μg/ml) and treated at 37° C. for 30 minutes, the supernatant was removed by centrifugation, and the cells were washed three times with 0.85% physiological saline. The Yoshida sarcoma cells, which had thus lost their proliferation ability, were mixed with Freund's Complete Ajuvant (abbreviated as FCA), and ¹⁰⁸ cells per rabbit were placed subcutaneously into the footpads of rabbits weighing 2.9 kg. A rabbit was immunized by injecting in the same manner two weeks later, and after another two weeks, 10 ⁸ cells of the above were injected into the vein of the rabbit. After one week, a cannula was inserted into the carotid artery to collect whole blood, from which antiserum was separated and purified as described below. That is, ammonium sulfate is added to 100 ml of antiserum at 20 to 30% of the saturation amount, the resulting salting-out fraction is collected, this is redissolved in 20 ml of water, and this solution is added to 1 mM phosphate buffer at pH 7.0. 4℃ for saline solution (abbreviated as PBS),
Desalination was performed by dialysis for 72 hours (during which time, the external dialysis fluid was replaced every 24 hours). Equal amounts of normal DONRYU rat blood cells, which had been washed three times with saline, were mixed with the dialysis fluid, and the mixture was left at 4°C for 30 minutes to allow absorption, and then centrifuged to obtain a supernatant. This absorption operation was repeated four more times for a total of five absorption operations. The antibody thus obtained is called a pre-purified antibody (commonly known as IgG). Further purification was then carried out using the method described below. That is, equal amounts of Yoshida sarcoma cells are mixed with the above-absorbed supernatant, left at 4°C for 30 minutes to allow antibodies against Yoshida sarcoma to bind to Yoshida sarcoma tumor cells, and then centrifuged to remove the supernatant. PH to precipitation
3.0 glycine-HCl buffer was added to liberate the antibody, and the mixture was centrifuged to collect the supernatant containing the antibody. After adjusting the pH of the supernatant to near neutrality with a 0.1 M aqueous sodium hydroxide solution, dialysis was performed against PBS at 4° C. for 24 hours (external dialysis solution was replaced every 8 hours). The result was an aqueous solution of rabbit anti-Yoshidada sarcoma tumor immune antibody. 1-2: Test for cytotoxicity against tumor cells and normal cells (Part 1) The rabbit anti-Yoshida sarcoma tumor immune antibody obtained above was subjected to a cytotoxicity test in the presence of complement (guinea pig serum). That is, the above antibody aqueous solution or diluted 10, 100, and 1000 times, and Yoshida sarcoma tumor cell suspension or normal
DONRYU rat splenocyte suspension (Eagle Minimum Essential Medium)
(hereinafter abbreviated as MEM) was used as a solvent, and the cell concentration was 5 x 10 ⁶ cells x ml) were mixed to 100 µm and left at room temperature for 15 minutes to allow the antibodies to bind to each cell. Then, dilute the guinea pig serum to 2 times with Eagle's MEM (this is called complement) and add 100μ of this to the above mixture.
After culturing at 37°C for 30 minutes and centrifugation, the precipitate was washed 11 times with Eagle's MEM, to which trypan blue solution was added, and the degree of death of each of the cells was observed under a microscope. As shown in Table 1 below, the results show that the degree of cell death (cytotoxic activity) is +, +++, +++.
Yoshida's results are shown in three stages (non-killing is indicated by 1). The toxicity to sarcoma cells was not very different, but the toxicity to normal DONRYU rat splenocytes was extremely low and did not kill them. Thus, the purification of antisera is well shown to be fit for purpose. [Table] The cells used here are representative of normal cells.
After removing the spleen, DONRYU rat splenocytes were crushed into fine pieces in Eagle's MEM using tweezers, passed through a 200 mesh stainless steel mesh, washed once with MEM, and washed with tris-(hydroxylmethyl)aminomethane. Add 3 ml of buffered 0.75% ammonium chloride aqueous solution (PH7.4) to destroy the red blood cells in the sample, and then add 3 ml of buffered 0.75% ammonium chloride aqueous solution (PH7.4) to destroy the red blood cells in the sample.
It was obtained by washing twice. 1-3: Purification of antibodies by affinity chromatography (1) Purification was carried out as shown below using affinity chromatography using a column bound to a tumor antigen. First, the antigen itself was purified as follows. That is, Yoshida sarcoma tumor cells subcultured using DONRYU rats were freeze-dried, and 3M KCl solution (PH7.4) buffered with 5mM potassium phosphate buffer was added to 30g of this, and the antigen was extracted for 20 hours. Centrifuge the extract at 65,000G for 10 minutes to collect the supernatant, and then add this supernatant to
Centrifuge at 180,000G for 30 minutes and collect the supernatant.
Dialysis was performed at 4°C for 70 hours against distilled water (during this period
(Replace the external solution every 24 hours). After dialysis, the dialysate was further centrifuged at 65,000G to remove the precipitate, and ammonium sulfate was added to the supernatant to give a concentration of 2M.
The precipitate was collected by centrifugation at 65000G for 10 minutes. This precipitate was dissolved in distilled water, and this solution was dialyzed against distilled water for 72 hours (during which time, the external solution was exchanged every 24 hours). Using the thus obtained Yoshida sarcoma tumor antigen, a column for affinity chromatography was prepared as follows. First, agarose gel (Seharose4B;
Pharmacia Japan Co Ltd product) was swollen with water to 20 ml, 20 ml of 1 g/ml bromcyan aqueous solution was added to this, the two were allowed to react for 8 minutes while keeping the pH at 11.0, and then the reaction product was poured into a glass funnel. Distilled water, which was filtered and the precipitate cooled on ice,
Next, after washing with an ice-cooled 0.5M aqueous sodium bicarbonate solution, the suspension was immediately suspended in a 0.1M aqueous sodium bicarbonate solution, to which the purified antigen aqueous solution described above was added and allowed to react with stirring at room temperature overnight. Pass the product through a glass funnel and
0.1M sodium bicarbonate aqueous solution, then distilled water, and finally phosphate buffered saline solution (0.85%, PH
7.0). This washed reaction product was washed with an inner diameter of 13 mm and a height of 15 cm.
This was packed into a glass tube to make a column for Affinity chromatography. In this column, the above 1-
Antibody solution (IgG) prepared by step 1 (however, the operation for binding with Yoshida sarcoma tumor cells is omitted)
3 ml, then 5 mM phosphate buffered saline solution (0.85%, PH 7.0) until no protein is detected in the eluate of the column, and then 50 m
A 0.5M saline solution to which M glycine-hydrochloric acid buffer aqueous solution (PH4.0) has been added is applied to the column, the eluted fraction is collected, this is immediately neutralized with sodium bicarbonate, and phosphate buffered saline aqueous solution (PH4.0) is added. 0.85%, PH7.0) for 72 hours (24
The dialysis fluid was changed every hour). In this way, an aqueous solution of a purified antibody against Yoshida sarcoma tumor was obtained using column-based affinity chromatography. 1-4: Cytotoxicity test on tumor cells and normal cells (Part 2) The anti-Yoshida sarcoma tumor immune antibody obtained in 1-3 above was subjected to a cytotoxicity test in the same manner as in 1-2. The results are shown in Table 2 below. [Table] These results indicate that purification of the antibody by Affinity chromatography significantly increased the activity of the antibody against Yoshida sarcoma cells and reduced the toxicity to splenocytes. It shows the excellence of refining. 1-5: Binding of anti-Yoshida sarcoma antibody and anti-tumor alkylating agent The rabbit anti-Yoshida sarcoma antibody prepared in 1-1 and 1-3 and purified by each method, chlorambutyl, melphalan (phenyl alanine mustard), ACNU, uramustine, and cyclophosphamide were reacted to synthesize each compound through an amide bond. An example of its synthesis will be described below. Synthesis example (Part 1) Binding reaction between antibody and chlorambutyl 1 ml of the purified rabbit anti-Yoshida sarcoma antibody obtained in 1-1
Add 40 mg of chlorambutyl to 10 ml of an aqueous solution containing 10.0 mg in water, and adjust the pH of the liquid with hydrochloric acid while stirring.
4.75, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride.
25.2 mg was added and the reaction was carried out for 40 minutes. Next, acetic acid-
The reaction was stopped by adding 20 ml of sodium acetate buffer (PH4.75). The reaction solution was then dialyzed against the distilled water from Step 5 at 4° C. for 72 hours (during which time, the external solution was exchanged three times).
After concentrating the dialyzed solution, it was passed through a column with a diameter of 3 cm and a height of 65 cm filled with a dextrin derivative (Sephadex G-200 manufactured by Pharmacia Japan) to completely separate high molecular weight substances and low molecular weight substances in the reaction liquid. The eluate was centrifuged at 40,000 g for 60 minutes using an ultracentrifuge. The supernatant was freeze-dried at -20°C to obtain the target substance.
The protein content in this substance was determined by the copper-phorin method using albumin as a standard, and the alkylation activity was determined by the Epstein method (Epstein J.Aval-Chem 27
1423 (1955), respectively. As a result, 10μ of chlorambutyl was used for 1mg of antibody.
It was found that it is g-bonded. Synthesis example (part 2) Binding reaction between antibody and melphalan 1 ml of the purified rabbit anti-Yoshida sarcoma antibody obtained in 1-1
Add 41.0 mg of melphalan to 10 ml of an aqueous solution containing 10.0 mg in water, and adjust the pH of the liquid with hydrochloric acid while stirring.
25.2 mg of 1-ethyl-3-
After adding (3-dimethylaminopropyl)-carbodiimide hydrochloride and reacting for 60 minutes, 20 ml of acetic acid-sodium acetate buffer (PH4.75) was added to stop the reaction. The reaction solution was then dialyzed against PBS at 4° C. for 72 hours (during which time the external solution was exchanged three times). Diameter: 3 cm, height: 65 cm, filled with dextrin derivative (Sephadex G-200, manufactured by Pharmacia Japan Co., Ltd.) after concentrating the dialysis fluid
High molecular weight substances and low molecular weight substances in the reaction solution were completely separated through the column. The eluate was centrifuged at 40,000 g for 60 minutes using an ultracentrifuge, and the supernatant was freeze-dried at -20°C to obtain the target substance. In this substance, 1 mg of antibody contains 10 melphalan.
μg was bound. Synthesis example (part 3) Binding reaction between antibody and uramustine 1 ml of the purified rabbit anti-Yoshida sarcoma antibody obtained in 1-1
Add 40 mg of uramustine to 10 ml of an aqueous solution containing 10 mg in water, and while stirring, adjust the pH of the liquid to 4.75 with hydrochloric acid and add 25.2 mg of 1-ethyl-3-(3dimethylaminopropyl)-carbodiimide hydrochloride. The reaction was carried out for 65 minutes. Next, the reaction product is acetic acid-
The reaction was stopped by adding 20 ml of sodium acetate buffer (PH4.75). The reaction solution was then dialyzed against the distilled water from Step 5 at 4° C. for 72 hours (during which time, the external solution was exchanged three times). After concentrating the dialysate, it was passed through a column with a diameter of 3.0 cm and a height of 65 cm filled with a dextrin derivative (Sephadex G-200 manufactured by Pharmacia Japan) to completely separate high molecular weight substances and low molecular weight substances in the reaction liquid. The eluate was centrifuged at 40,000 g for 60 minutes using an ultracentrifuge. The supernatant was lyophilized at -20°C. In the material thus obtained, almost no uramustine was bound to 1.0 mg of antibody. Therefore, we reacted uramustine with monochloroacetic acid to increase its binding properties. Next, an example will be described. 500 mg of uramustine and 139 mg of potassium methoxide were dissolved in 10 ml of methyl alcohol, and 188 mg of monochloroacetic acid was added thereto, followed by stirring at room temperature for 60 minutes. After the reaction was completed, the mixture was concentrated under reduced pressure, and the residue was recrystallized from methyl alcohol and chloroform to obtain 215 mg (yield: 35%) of crystals. Analysis of this crystal confirmed that it was a uramustin derivative having the following structure. [Formula] Results of decomposition elemental analysis near melting point 200° C% H% N% Calculated value 38.70 4.19 13.54 Experimental value 38.50 4.00 13.30 Next, the uramustine derivative obtained as described above was used as described earlier in this synthesis example. The target substance was obtained by reacting with the purified rabbit anti-Yoshida sarcoma antibody obtained in 1-1 using the same procedure as the method. This material contained 10 μg of uramustine derivative per 1 mg of antibody. Synthesis Example (Part 4) Reaction between the antibody and melphalan derivative according to 1-1 200 mg of melphalan-silver salt was dissolved and dispersed in 10 ml of dimethyl sulfoxide (DMSO). Next, 100 mg of monochloroacetic acid was added, and the mixture was stirred at room temperature while shielding from light for 64 hours. The precipitate was removed, and DMSO and monochloroacetic acid were removed under reduced pressure over hot water at 80°C. White crystals were precipitated by adding water and cooling to a low temperature. Next, the precipitate was dried under reduced pressure. The yield was 40%. Using the melphalan derivative, a substance binding to the antibody according to 1-1 was obtained by the same procedure as described in Synthesis Example (Part 2). Antibody 1 in the obtained substance
The amount of melphalan derivative bound was 16 μg per mg. Synthesis Example (Part 5) Next, using the purified rabbit anti-Yoshida sarcoma antibody obtained in 1-3, the antibody was mixed with chlorambutyl, melphalan, its derivatives (see Synthesis Example (Part 4)) and uramustin in the same manner as described above. Each was reacted with a derivative (see Synthesis Example (Part 3)). Each substance obtained was a binding substance almost similar to the substance obtained in the synthesis example described above. Example 2 2-1: Preparation and purification of antibodies against tumor cells ¹⁰⁷ ascites-type Yoshida sarcoma cells, which had been subcultured using DONRYU rats and had lost their proliferation ability with mitomycin C, were incubated four times every week. of animals
DONRYU rats were inoculated intraperitoneally, and on the 7th day after the fourth inoculation, the rats were anesthetized and opened, and blood was collected from the abdominal vena cava. Antiserum containing antibodies was prepared from this blood. Thus 70 out of 100 rats
ml of antiserum was obtained. Collection of antibody from antiserum and purification of antibody were performed according to 1-1, and the antibody was stopped until absorption by rat blood cells. 2-2: Purification of antibodies by affinity chromatography Yoshida sarcoma tumor cells subcultured using DONRYU rats were freeze-dried, and 30 g of
Antigen extraction was performed for 20 hours by adding a 3M KCl solution (PH7.4) buffered with mM potassium phosphate buffer. This extract was centrifuged at 65,000G for 10 minutes to collect the supernatant, and this supernatant was further centrifuged at 180,000G.
After centrifugation for 30 minutes, the supernatant was collected and dialyzed against distilled water at 4°C for 72 hours. (During this time, 24
The dialysis fluid was replaced every hour. ) Affinity chromatography using the thus obtained Yoshida sarcoma tumor antigen was performed as follows. First, agarose gel (Sepharose4B;
Pharmacia Japan Co, Ltd.) was swollen with water to 20 ml, 20 ml of 1 g/ml Promcyan aqueous solution was added thereto, and the two were allowed to react for 8 minutes while maintaining the pH at 11.0. The precipitate was washed with ice-cooled distilled water on a funnel, then with an ice-cold 0.5M aqueous sodium hydrogen carbonate solution, and immediately suspended in 0.1M sodium hydrogen carbonate, and the purified antigen aqueous solution described above was added to this. The mixture was added and allowed to react at room temperature overnight with stirring. Pass the product through a glass funnel and
0.1M sodium bicarbonate aqueous solution, then distilled water, and finally phosphate buffered saline (0.85% PH7.0)
I washed it with This washed reaction product was
It was packed into a glass tube with a height of 15 cm to form a column for affinitake chromatography. Said 2-
Antiserum (containing antibodies) solution prepared in step 1 3
ml of the above Affinity chromatography column, and then 5mM phosphate, buffered saline solution (0.85% PH7.0) was passed through the column until no protein was detected in the column eluate. Next 50m
A 0.5M saline solution to which M glycine-hydrochloric acid buffer aqueous solution (PH4.0) was added was applied to the column, and the eluted fraction was collected. This was immediately neutralized with sodium bicarbonate and dialyzed against a phosphate buffered saline solution (0.85% PH7.0) for 72 hours. The dialysis fluid was replaced every 24 hours. ) Thus, an aqueous solution of antibodies against Yoshida sarcoma tumor cells purified using Affinity chromatography was obtained. 2-3: Cytotoxicity test on tumor cells and normal cells The rat anti-Yoshida sarcoma tumor immune antibody obtained in 2-2 above was tested for cytotoxicity in the same manner as in Example 1. The results are shown in Table 3 below. [Table] As can be seen in Table 3, the activity against Yoshida sarcoma cells was significantly increased by affinity chromatography as described above. 2-4: Binding of rat anti-Yoshida sarcoma antibody and antitumor agent The rat anti-Yoshida sarcoma antibody obtained in 2-1 and 2-2 and chlorambutyl were combined in the same manner as described in 1-5 above. A compound in which both were bonded through an amide group was obtained. A similar method was used to obtain a substance in which each of melphalan, melphalan derivatives, and uramustine derivatives was bound to an antibody through an amide bond. All of these substances exhibited almost the same physical and chemical properties as the corresponding compounds obtained in 1-5 above. Example 3 Antitumor effect on Yoshida sarcoma Ascites-type Yoshida sarcoma cells subcultured using DONRYU rats were administered to each group of 10 rats.
1 x ¹⁰⁶ cells/animal were transplanted into the peritoneal cavity of DONRYU rats, and 24 hours after transplantation, various antibodies and various commercially available antitumor agents were used alone, and rabbit anti-Yoshida sarcoma antibodies and various commercially available antitumor agents were combined. Aqueous solutions of conjugates of rat anti-Yoshida sarcoma antibody and various commercially available antitumor drugs were intraperitoneally injected into each rat five times every other day, for a total of five times. (T) and the average survival days (C) of the control group were determined, and the survival rate (T/C×100) was calculated. The results are shown in Tables 4 to 8. [Table] [Table] [Table] [Table] Was.
[Table] [Table] Represents antibodies.
As can be seen from Tables 4 to 7 above, the survival rate of this compound for Yoshida sarcoma in rats was approximately 5 to 10 times higher than that of commercially available antitumor agents, and the suppression rate was almost the same as that of commercially available antitumor agents. equal. As can be seen from Table 8, this is not surprising since it shows that the antibody itself exhibits almost no tumor-suppressing power at doses of this level. The characteristics of the present compound are obvious when comparing the dose of the commercially available anti-tumor agent, which is a component of the present compound, and the dose of the commercially available anti-tumor drug component administered by administering the present compound, and the latter is 1/1/2 of the former.
Although it is only 10 to 1/20 times smaller, it shows almost the same tumor growth inhibition rate as the former. This fact shows how well the antibody, which is one of the components of this compound, allows the other component, a small amount of a commercially available anti-tumor agent, to reach the tumor site, and it is the idea behind the present invention. It is beautifully embodied. Through this action, this substance reduces the use of commercially available anti-tumor drugs, which originally have extremely high side effects, to 1/10 to 1/20 of conventional drugs, while still exhibiting the same level of tumor growth suppressive power as conventional drugs. It is. It should be noted that although the origin of the antibody does not matter in terms of tumor growth inhibition rate, when a substance prepared and purified using rabbits combined with a commercially available anti-tumor drug was administered to rats, 10 Approximately three of the rats exhibited anaphylactic shock, including generalized convulsions and rigidity. On the other hand, when the present substance, which was prepared using rabbits and synthesized using antibodies purified by affinity chromatography according to the present invention, was administered, the occurrence of such anaphylactic shock was greatly reduced. In the case of substances synthesized using antibodies created and purified using rats, the occurrence of anaphylactic shock was extremely rare; however, when the antibodies were purified by affinity chromatography, No occurrence of anaphylaxis was observed with this substance. Example 4 4-1: Preparation and purification of antibodies against tumor cells 50 ml of blood was collected from a rectal cancer patient (50 years old male), and 22 ml of antiserum containing antibodies was obtained from this blood. 4-2: Purification of antibodies by affinity chromatography The patient's tumor cells removed during surgery were freeze-dried, and 50 ml of 3M KCl solution (PH7.4) buffered with 5mM calcium phosphate buffer was added thereto.
Antigen extraction was performed for 20 hours. This extract
After centrifugation at 65,000 G for 10 minutes, the supernatant was collected and dialyzed against distilled water at 4° C. for 72 hours (during which time, the extra dialysis solution was replaced every 24 hours).
Affinity chromatography using the patient tumor antigen thus obtained was performed as follows. First, agarose gel (Sepharose4B;
Pharmacia JaPan Co, Ltd.) was swollen with water to 20 ml, 20 ml of 1 g/ml bromcyan aqueous solution was added thereto, the two were allowed to react for 8 minutes while keeping the pH at 11.0, and then the reaction product was poured into a glass tube. The precipitate was washed with ice-cold distilled water on a funnel, and then with an ice-cold 0.5M aqueous sodium bicarbonate solution, and then immediately diluted with 0.1M sodium bicarbonate.
The suspension was suspended in sodium hydrogen carbonate, the purified antigen aqueous solution mentioned above was added thereto, and the mixture was allowed to react at room temperature overnight with stirring. The resulting reaction product was filtered through a glass funnel and washed first with 0.1 M aqueous sodium bicarbonate solution, then with distilled water, and finally with phosphate buffered saline (0.85% PH 7.0). This washed reaction product was washed with an inner diameter of 13 mm and a height of 15 mm.
It was packed into a cm glass tube and used as a column for Affinity chromatography. Next, the above 4-
3 ml of the antiserum (containing antibody) solution obtained in step 1 was poured into the above-mentioned affinity chromatography column, and then 5 mM phosphate buffered saline solution (0.85% PH7.0) was added to the eluate of the column to ensure that the protein was not present. It was run until it was no longer detected. Next, 0.5M glycine-hydrochloric acid buffer aqueous solution (PH4.0) was added.
A saline solution was passed through the column and the eluted fraction was collected. Immediately neutralize this with sodium bicarbonate and use a phosphate buffered saline solution (0.85% PH).
7.0) for 72 hours. (The external dialysis solution was replaced every 24 hours.) Thus, an aqueous solution of antibodies against the patient's tumor purified using Affinity chromatography was obtained. The antibodies thus obtained were soluble in water and insoluble in organic solvents such as methanol, ethanol, acetone, and benzene. This purified antibody shows infrared absorption spectra and ultraviolet absorption spectra as shown in Figures 1 and 2 of the attached drawings, and its molecular weight is approximately 150,000, and disc electrophoresis shows that it exists between Rf0 and 0.1. It was a substance.

[Brief explanation of the drawing]

Figure 1 shows the infrared absorption spectrum of an antibody obtained by purifying the antiserum from a cancer patient prepared by the procedure described in Example 4 by affinity chromatography, and Figure 2 shows the same ultraviolet absorption spectrum. shows.

Claims (1)

[Scope of Claims] 1. An alloantibody against a tumor antigen obtained by purification by affinity chromatography bound to a tumor antigen, and an antitumor alkylating agent having at least one amino group or carboxyl group. An antitumor agent whose active ingredient is a substance formed by an amide bond. 2 Antibodies are Sarcoma 180, Sato lung cancer, L-
1210 Patent for immunoglobulin fractions derived from antigens of leukemia, Ehritzch cancer, Yoshida sarcoma, acute lymphocytic leukemia, bone marrow cancer, or other human cancers purified by affinity chromatography coupled with tumor antigens The antitumor agent according to claim 1. 3. The antitumor agent according to claim 1, wherein the antitumor alkylating agent is selected from the group consisting of chlorambutyl, melphalan, uramustine, ACNU, and cyclophosphamide. 4. The antitumor agent according to claim 1 or 3, wherein the amino group or carboxyl group of the antitumor alkylating agent is introduced. 5. The introduction of the amino group or carboxyl group is
X(CH ₂ ) _o COOH (in the formula, X represents Cl or Br, n
represents an integer of 1, 2 or 3).
The antitumor agent according to item 4 or item 4. 6. The antitumor agent according to claim 1, wherein the substance has 1 to 10 molecules of the antitumor alkylating agent bound per molecule of the alloantibody. 7. The antitumor agent according to any one of claims 1 to 6, which is in the form of an injection.