JPH0653682B2 - Human immunoglobulin-binding antitumor agent - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to human immunoglobulin binding anti-tumor agents.

In recent years, with the development of immunochemistry, many tumor-associated antigens have been discovered, and tumor-specific antibodies that selectively bind to them have been developed. Furthermore, attempts have been made to bind antitumor substances to these tumor-specific antibodies and to centrally transfer the drug only to the tumor site. Here, the tumor-specific antibody is prepared by a method of immunizing a rabbit, a horse, a sheep or the like with tumor cells or a tumor-associated antigen, and is used by obtaining an immunoglobulin fraction from animal serum. Recently, after immunizing mice with tumor cells or tumor-associated antigens, the antibody-producing cells are taken out and fused with mouse myeloma cells such as NS-1 to obtain monoclonal antitumor antibody cells, from which anti-tumor antibody cells are obtained. Taking out the tumor antibody.

These attempts have been carried out in the form of an antitumor antibody alone or a form in which a certain cytotoxic substance is bound to the antitumor antibody, but it has not been put to practical use. The reason is that the above-mentioned antitumor antibody is a heterologous protein for humans because it is produced by immunizing a heterologous animal. In other words, when an antibody obtained from a heterologous animal is administered to humans, it cannot be used for a serum illness such as anaphylaxis after the second administration, and therefore it can be used only once. there were. To solve this, it is necessary to use alloantibodies, and although a monoclonal antibody using human lymphocytes is ideal, it is still under study.

Therefore, in order to improve these drawbacks and solve matters concerning practicality, it was necessary to search for antibodies that aggregate in tumor cells from among alloantibodies. Therefore, the present inventors diligently investigated the biodistribution of ¹²⁵ I-labeled substances of various antibodies. As a result, general natural antibodies reach the tumor site,
Moreover, they have found that they remain for a long time. The present invention has been completed, knowing that an antitumor substance is bound to these immunoglobulins, and if the substance is administered to a cancer-bearing individual, the drug stays at the tumor site for a long time and exhibits an antitumor effect. The human immunoglobulin-binding antitumor agent has the greatest feature and advantage in that it can be administered more frequently than the antitumor antibody derived from a xenogeneic animal and that it remains at the tumor site for a long time. Therefore, the present invention provides a new type of drug containing a highly practical human immunoglobulin-binding antitumor agent. The present invention relates to alkylating agents such as chlorambucil, melphalan, ACNU, cyclophosphamide, antibiotics such as mitomycin C, doxorubicin hydrochloride, daunorubicin hydrochloride, bleomycin, actinomycin D, neocarzinostatin, cytarabine, 8-azaguanine. , 5-
Antitumor based on a novel compound in which a highly cytotoxic antitumor substance belonging to the group consisting of antimetabolites such as fluorouracil, methotrexate, sodium aminopterin, and leucerine is bound to human immunoglobulin under extremely mild conditions It is a drug and has an excellent antitumor effect,
The purpose of the present invention is to provide an antitumor agent having significantly lower cytotoxicity than an antitumor substance which is one of the raw materials. The present invention will be described in detail below.

In recent years, various antitumor agents have been widely used and have been effective to some extent. As these antitumor agents, chlorambucil, melphalan, ACNU, cyclophosphamide, cytarabine, 8-azaguanine, 5-fluorouracil, methotrexate, aminopterin sodium,
Mitomycin C, doxorubicin hydrochloride, bleomycin, daunorubicin, actinomycin D, sarcomycin and the like have been used, but these substances have high cytotoxicity per se, and after administration, leukopenia, It is known that side effects such as hair loss and gastrointestinal disorders are exhibited, and for this reason, the actual use of these drugs is limited.

Further, although attempts have been made so far to produce or isolate antibodies against tumor cells or tumor-associated antigens and use them for the treatment of the tumor, a desired antitumor effect has not been obtained. Furthermore, recently, it has been proposed to expect an antitumor effect by a novel substance obtained by chemically binding an antitumor antibody to an antitumor antibody, but the chemical reaction conditions for obtaining the above substance are It's too harsh to get enough results. In addition, the antibodies used in these experiments were unable to avoid side effects such as serum sickness because the antibodies of different animals were used.

Therefore, the present inventors have invented a method of purifying antitumor antibodies obtained from xenogeneic animals by affinity chromatography (Japanese Patent Application Nos. 53-161388, 54-142152 and 54-14).
2153). Although it is possible to highly purify the antibody using this method, the problem remains in that frequent administration is performed.

Therefore, when the tumor reachability was studied diligently using various antibodies, it was found that the natural antibody was transferred to the tumor site at a high concentration and the retention time was longer than that of other organs. Based on this fact, chlorambucil, melphalan, ACN
Preferred antitumor when U, cyclophosphamide, cytarabine, 8-azaguanine, 5-fluorouracil, methotrexate, sodium aminopterin, mitomycin C, doxorubicin hydrochloride, bleomycin, daunorubicin, actinomycin D, and sarcomycin are bound to human immunoglobulin It turned out to be effective. Furthermore, among this combination, melphalan and its esters are antitumor agents that are easily obtained synthetically,
The conjugate of human immunoglobulin with melphalan and its ester is most preferable because of its high stability. Natural antibodies include human immunoglobulin (Ig) and small molecule antibodies (F
(Ab ') ₂ ).

The binding between human immunoglobulin and antitumor substance is produced by the following method.

The antitumor substance is dissolved in an aqueous solvent. The aqueous solvent is an acidic aqueous solution, an alkaline aqueous solution, a neutral aqueous solution, a phosphate buffer solution, sodium borate or the like. To which a binder such as carbodiimide, dextran, glutaraldehyde,
A substance selected from diethylmalonimidate, isocyanate, and polyglutamic acid is added, and further human immunoglobulin (including F (ab ′) ₂ ) is added and reacted. The reaction temperature is -30 ° C to 50 ° C, preferably 0 ° C to 30 ° C, and the reaction time is 1 minute to 48 hours, preferably 10 minutes to 25
It's time. The reaction product is purified by means such as salting out, precipitation, recrystallization, elution and column fractionation to obtain a conjugate.

The acute toxicity to mammals of the conjugate of the human immunoglobulin of the present invention and an antitumor substance (hereinafter, abbreviated as this substance) was investigated by intravenously injecting into mice at a dose of 4000 mg / kg. No death was observed in the observation.

In addition, human immunoglobulin is converted to pepsin (Nison
off Science 132 1770 (197)
0)), plasmin (Sgouris Vox San
g 18 71 (1967)), thermolysin (Japanese Patent Application No. Sho 50-19871), papain, trypsin, for the low molecular antibody obtained by enzymatic hydrolysis with chymotrypsin, was performed examined allowed coupling the anti-tumor agent. Even with these substances such as F (ab ') ₂ , the toxicity was 4000 mg / kg or more.

Therefore, this substance has extremely low toxicity, can be administered frequently, and is effective against various human cancers. For example,
It is effective for acute leukemia, malignant lymphoma, carcinoma, sarcoma, malignant ciliated epithelioma, acute myelogenous leukemia, melanoma, acute lymphocytic leukemia, bone marrow cancer and the like. As the formulation method and administration method when this substance is used as an antitumor agent, known methods for antitumor agents can be applied. The method of administration includes oral, parenteral, such as injection or rectal administration. The dosage form may be any of powder, granules, tablets, capsules, injections and suppositories. Particularly, administration by tablets or injection is preferable. Injectable formulations include physiological saline, sterile water, water-soluble solvents such as Ringer's solution, water-insoluble solvents, isotonic agents, soothing agents, stabilizers, preservatives, suspending agents, buffers. , An emulsifier, etc. can be optionally used.

As an example, 1 g of this substance and 5 g of mannitol are dissolved in distilled water to make 50 ml, and the bacteria are sterilized by a conventional method. Then, it is divided into a small bottle for injection or lyophilized as it is to obtain an injection. When used, this drug is diluted with physiological saline to prepare an injection solution. This substance is generally 0.01-90 during formulation.
%, Preferably 0.1 to 60%.

The dose of this substance mainly depends on symptoms, but it is 0.1 to 10 g, preferably 1 to 6 g per adult per day.

According to the present invention, the tumorigenicity of human immunoglobulins and enzyme-treated human immunoglobulins, and the antitumor properties of antitumor substances are retained in the above compounds without loss, so that when this substance is administered It efficiently reaches the target tumor site, remains for a long time, and exerts an antitumor effect.

It can be said that the present invention is industrially very advantageous because the antibody does not necessarily have to be selected from antitumor antibodies.

Hereinafter, the present invention will be described in more detail with reference to Examples.

Example 1 Distribution of Human Immunoglobulin In order to find out what kind of antibody is a practical antibody, in order to investigate the biodistribution of anti-S-180 rabbit immunoglobulin, normal ICR mouse immunoglobulin, and human immunoglobulin. Each substance was labeled with ¹²⁵ I-.

That is, W. H. Hurter et al. Biochem.
J. According to the method of 89 114 (1963), ¹²⁵ I-labeling was carried out on the protein portion of immunoglobulin. Since the methods are the same, an example will be given. Mitomycin C-conjugated anti-S-180 antibody was added to a concentration of 0.
It was dissolved in 5M phosphate buffer (pH 7.4). The 0.
5 ml was put in a spic tube and 0.25 mCi of Na
Add ¹²⁵ I. Further 0.05M phosphate buffer 200μ
Add 0.7 mg of chloramine T dissolved in 1 and add at 0 ° C for 15
Let react for minutes. Then sodium pyrosulfite (1.75 mg) and KI (10 mg) dissolved in 0.05 M phosphate buffer
Was added to stop the reaction. The reaction solution is Sephadex
Unreacted radioactive iodine and reagents were removed using a G-25 (φ2.2 cm × 40 cm) column. In this way
¹²⁵ I-labeled mitomycin conjugated anti-S-180 rabbit immunoglobulin was obtained. In the same manner, ¹²⁵ I-labeled normal ICR mouse immunoglobulin and ¹²⁵ I-labeled human immunoglobulin were obtained. The biodistribution was investigated using these.

That is, S-180 tumor-bearing ICR mouse (2 weeks after transplantation)
Was administered intravenously, and the animals were sacrificed 24 hours and 144 hours later and dissected, and blood S-180 tumor site, liver,
Each organ such as the spleen, spleen and digestive organ was taken out and counted by a well type γ-counter, and the distribution in the form of the reached drug amount per tissue weight of the administered drug was displayed as follows (Table- 1).

Further, the ratio of the amount in each organ relative to the total amount remaining in each organ after 144 hours is expressed as a residual rate as shown in Table 2 below.

These results show that the heterologous antibody obtained by immunizing a heterologous animal with an antitumor antigen shows an excellent reaching rate.
However, it was found here that the natural antibody of the same species has a tumor arrival rate of 1/5 to 1/10 compared to the specific antibody, but the survival rate at the tumor site is higher than that of other organs. From this, it came to be known that the natural antibody is a highly practical antibody as a carrier.

Example 2 Preparation of Human Immunoglobulin 1000 ml of normal human serum is diluted with 1000 ml of 0.005 M phosphate buffered saline (hereinafter abbreviated as PBS). 2000 ml of saturated ammonium sulfate aqueous solution (pH 7.2) is gradually added to this diluted serum while stirring. When left at 4 ° C. for 60 minutes, salted-out substances start to precipitate, so centrifuge at 8000 rpm for 30 minutes to collect the precipitate. This precipitate was dissolved in PBS and the total amount was 10
Set to 00 ml. On the other hand, gradually add 250 ml of saturated ammonium sulfate to 20% saturation while stirring. If the solution becomes cloudy and precipitates, it is fibrinogen and is removed by centrifugation. Add 250 ml of saturated ammonium sulfate to the supernatant to make it 33% saturated. After standing for 60 minutes, centrifuge at 8000 rpm for 30 minutes to collect the precipitate. After dissolving this precipitate in 1000 ml of PBS, 500 ml of saturated ammonium sulfate is added. After stirring for 60 minutes, 30 at 8000 rpm
Centrifuge for minutes to collect the precipitate. The precipitate obtained is 30
It was dissolved in ml of PBS and dialyzed against PBS to remove ammonium sulfate. Further, after dialysis, using a DEAE-cellulose column (diameter 5 cm x 50 cm), 0.005M pH 8.0.
So I collected the passing fractions. The straight fractions were dialyzed against distilled water, desalted, and then freeze-dried to obtain 12.5 g of human immunoglobulin.

Example 3 A human immunoglobulin-binding antibiotic was synthesized by reacting a normal human-derived human immunoglobulin with mitomycin C, doxorubicin hydrochloride, daunorubicin hydrochloride, bleomycin, actinomycin, and sarcomycin. A synthesis example will be described below.

3-1 Binding of mitomacine C 1.0 g of human immunoglobulin is dissolved in 100 ml of distilled water. 111.3 mg of Might Machine C is dissolved therein.
While adjusting the pH to 5.5 with 1.0N hydrochloric acid aqueous solution, 4
At 26 ° C., 262.6 mg of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride was added and reacted for the following time, and 5 ml of acetic acid-sodium acetate buffer (pH 5.5) was added.
The reaction was stopped by the addition of. Then, the reaction solution was concentrated and desalted to 10 ml using an ultrafilter. 10 ml of the concentrated solution was passed through a column having a diameter of 5 cm and a height of 90 cm packed with Sephadex G-25 (Pharmacia Japan) to completely separate the high molecular weight substance and the low molecular weight substance in the reaction solution.
The eluate was centrifuged by ultracentrifugation at 40,000 g × 60 minutes, and the resulting supernatant was freeze-dried at 0 ° C. to obtain the substance as a product. Table 3 shows the results of measuring the amount of mitomaine C bound to human immunoglobulin at each reaction time using ultraviolet absorption at 360 nm.

3-2 According to the above procedure, 1.0 g of human immunoglobulin was reacted with each of doxorubicin hydrochloride, daunorubicin hydrochloride, bleomycin and actinomycin D to give about
800 mg of this substance was obtained. The binding amounts of doxorubicin hydrochloride per human immunoglobulin (mg) were 4.8 μg and 9.5 μg at reaction times of 60 minutes and 24 hours, respectively.

Example 4 A human immunoglobulin derived from a normal person was reacted with each of chlorambucil, melphalan (phenylalanine mustard) ACNU, uramustine, cyclophosphamide, and melphalan methyl ester to synthesize each compound by an amide bond. An example of the synthesis will be described below.

4-1 Melphalan binding 1.0 g of human immunoglobulin is dissolved in 100 ml of distilled water. Suspend 100 mg of melphalan there. 1.
While adjusting the pH to 5.5 with 0N aqueous hydrochloric acid,
And 1-ethyl-3- (3-dimethylaminopropyl)
-Adding carbodiimide hydrochloride and reacting for 24 hours, acetic acid-
The reaction was stopped by the addition of 5 ml of sodium acetate buffer (pH 5.5). Then, 10 ml of the reaction solution was filtered using an ultrafilter.
Was concentrated and desalted. Diameter 10 filled with Sephadex G-25 (Pharmacia Japan)
The high molecular weight substance and the low molecular weight substance in the reaction solution were completely separated through a column having a height of 90 cm and a height of 90 cm. The supernatant obtained by centrifuging the eluate by ultracentrifugation at 40,000 g x 60 minutes was
The product was obtained by freeze-drying at ℃. The protein content in this substance was determined by the copper-fluorine method using albumin as a standard, and the alkylation activity was determined by the Epstein method (Epstein method).
Stein J. Anal. Chem. 27 1423 (19
55)). As a result, it was found that 6 μg of melphalan was bound to 1 mg of human immunoglobulin.

4-2 According to the above operation, human immunoglobulin 1.0 g was reacted with each of chlorambucil, ACNU and uramustine to obtain about 900 mg of this substance. The binding amount of chlorambucil per human immunoglobulin (mg) is 60 minutes reaction time,
The values were 5.1 μg and 11.7 μg in 24 hours, respectively.

4-3 Coupling of melphalan methyl ester 1.0 g of human immunoglobulin is dissolved in 100 ml of distilled water. 100 mg of melphalan methyl ester hydrochloride is dissolved therein. The pH was adjusted to 5.5 with 1.0 N hydrochloric acid aqueous solution.
1-Ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride was added at 4 ° C. while adjusting the reaction temperature to react for 24 hours, and acetic acid-sodium acetate buffer solution (pH 5.5) was added.
The reaction was stopped by the addition of 5 ml. Then, the reaction solution was concentrated and desalted to 10 ml using an ultrafilter. 10 ml of concentrated solution is Sephadex G-25 (Pharmacia Japan)
A high-molecular weight substance and a low-molecular weight substance in the reaction solution were completely separated through a column having a diameter of 5 cm and a height of 90 cm packed with. The eluate was centrifuged by ultracentrifugation at 40,000 g × 60 minutes, and the obtained supernatant was freeze-dried at 0 ° C. to obtain a compound as a product. The binding amount per mg of human immunoglobulin was 10 μg.

Example 5 A human immunoglobulin from a normal person was reacted with each of cytarabine, 8-azaguanine, 5-fluorouracil, methotrexate and aminopterin sodium,
Each compound with an amide bond was synthesized. The synthesis example will be described below.

5-1 Binding of Methotrexate 1.0 g of human immunoglobulin is dissolved in 100 ml of distilled water. Dissolve 151.3 mg of methotrexate there. While adjusting the pH to 5.5 with 1.0 N hydrochloric acid aqueous solution, 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride was added at 4 ° C. and the reaction was carried out for the following time, and acetic acid-sodium acetate The reaction was stopped by the addition of 5 ml of buffer (pH 5.5). Then, the reaction solution was concentrated to 10 ml by using an ultrafilter and desalted. 10 ml of the concentrated solution was passed through a column having a diameter of 5 cm and a height of 90 cm packed with Sephadex G-25 (Pharmacia Japan) to completely separate the high molecular weight substance and the low molecular weight substance in the reaction solution. The eluate was centrifuged by ultracentrifugation at 40,000 g × 60 minutes, and the obtained supernatant was freeze-dried at 0 ° C. to obtain a compound as a product.
The amount of methotrexate bound to human immunoglobulin was measured using absorption at 305 nm, and the result was 8.3 μg per mg protein.

5-2 According to the above procedure, human immunoglobulin 1.0 g was reacted with each of cytarabine, 8-azaguanine, 5-fluorouracil, and aminopterin sodium to give about 91
0 mg of bound compound was obtained. The amount of bound cytarabine per mg of human immunoglobulin was 4.7 μm in the reaction time of 60 minutes and 24 hours, respectively.
g and 8.3 μg.

Example 6 Preparation of Human Immunoglobulin F (ab ′) ₂ 1 g of human immunoglobulin is dissolved in 100 ml of 0.1N sodium acetate buffer (pH 4.5). Pepsin is added at a ratio of enzyme to protein of 1/100 (weight / weight), and digestion is performed at 37 ° C for 16 hours. Solid tris hydrochloride is added to the solution to adjust the pH to 8.0 to stop the reaction. The reaction solution is concentrated to 10 ml with an ultrafiltration device. Diameter 5 cm
The column with a height of 90 cm is packed with Sephadex G-150, charged with 5 ml of the concentrated solution, and then pH 7 PBS.
Elute with. Separate into three peaks, but collect the first peak as F (ab ') ₂ . This fraction was put in a dialysis tube, desalted and freeze-dried to obtain human immunoglobulin F (ab ') ₂ .

Example 7 Binding of Human Immunoglobulin F (ab ′) ₂ to Antitumor Agent 7-1 500 mg of human immunoglobulin F (ab ′) ₂ is dissolved in 50 ml of distilled water. 55.6 mg of mitomycin C is dissolved therein. While adjusting the pH to 5.5 with a 1.0 N hydrochloric acid aqueous solution, 131.3 mg of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride was added at 4 ° C. and the reaction was carried out for the following time, followed by acetic acid- Sodium acetate buffer (pH
5.5) The reaction was stopped by the addition of 5 ml. Then, concentrate the reaction solution to 5 ml using an ultrafilter and Sephadex G.
-5c diameter filled with -25 (Pharmacia Japan)
High molecular weight substances and low molecular weight substances in the reaction solution were completely separated through a column of m and 90 cm in height. The eluate was centrifuged by ultracentrifugation for 40,000 × 60 minutes, and the resulting supernatant was freeze-dried at 0 ° C. to obtain a compound as a product. Table 4 shows the results of measurement of the amount of mitomycin bound to human immunoglobulin F (ab ') ₂ in each reaction when using ultraviolet absorption at 360 nm.

7-2 In accordance with the above operation, human immunoglobulin F (ab ') ₂
500 mg was reacted with each of doxorubicin hydrochloride, daunorubicin hydrochloride, bleomycin and actinomycin D to obtain about 800 mg of the bound compound. The binding amount of doxorubicin hydrochloride per ₂ mg of human immunoglobulin F (ab ′) was 8.5 μg and 19.6, respectively for a reaction time of 60 minutes and 24 hours.
It was μg.

7-3 Human immunoglobulin F (ab ') ₂ according to the above operation
500 mg was reacted with each of chlorambucil, melphalan, ACNU, uramustine, melphalan methyl ester and cyclophosphamide to obtain about 400 mg of a bound compound. Human immunoglobulin F (ab ') of melphalan
The binding amount per ₂ mg was 8.1 for a reaction time of 90 minutes and 24 hours, respectively.
μg and 17.6 μg.

7-4 In accordance with the above operation, human immunoglobulin F (ab ') ₂
500 mg was reacted with each of cytarabine, 8-azaguanine, 5-fluorouracil, methotrexate, and aminopterin sodium to obtain about 400 mg of a bound compound. Methotrexate human immunoglobulin F (a
b ′) The amount of binding per ₂ mg was 7.5 μg and 17.3 μg at the reaction time of 60 minutes and 24 hours, respectively.

Example 8 Antitumor Effect on Sarcoma 180 Solid Tumor Mouse Sarcoma 18 subcultured using ICR mouse
0 1 × 10 ⁶ tumor cells were subcutaneously transplanted into the axilla of each ICR mouse in a group of 10 mice, and 24 hours after transplantation, various antibodies, commercially available antitumor agents, human immunoglobulins, human immunoglobulins An aqueous solution of each of F (ab ') ₂ and a conjugate with various antitumor substances was injected intraperitoneally once every other day for a total of 10 times, and the mice were killed 5 days after the last injection. Tumors were excised and weighed to obtain an average value for 10 animals. By comparing this average tumor weight (T) with the average tumor weight (C) of 10 mice in the control group, which was administered 10 times physiological saline instead of the aqueous solution of the conjugate, inhibition of tumor growth of the conjugate of the present invention was confirmed. The rates are shown in Tables 5, 6 and 7 as (1-T / C) x 100 (%). Table-5 shows the results obtained by the conjugate synthesized with mitomycin C, Table-6 shows the results obtained by the compound produced by bleomycin, and Table-7 shows the results obtained by the compound produced by doxorubicin hydrochloride.

Example 9 Antitumor effect against Yoshida sarcoma Yoshida sarcoma ascites cells subcultured using Donryu rats were intraperitoneally transplanted at 1 × 10 ⁶ cells / animal in each Donryu rat group consisting of 10 rats, and 24 hours after transplantation. Later, human immunoglobulin, human immunoglobulin F (ab ') ₂ and various antitumor agents, alone and human immunoglobulin,
Aqueous solutions of the conjugates of human immunoglobulin F (ab ′) ₂ and various antitumor substances were injected into the abdominal cavity of each rat 5 times every other day for a total of 5 times. Mean survival time (T) and control group mean survival time (C)
Was calculated and the life extension rate (T / C × 100) was calculated. The results are shown in Table-8 to Table-10.

Example 10 Antitumor effect on mouse leukemia P-388 P-388 ascites cells subcultured using DBA / 2 mice were intraperitoneally 1 in each DBA / 2 mouse in a group of 10 mice.
X 10 ⁶ cells / animal transplanted, and 24 hours after transplantation, various antitumor agents, each alone or in the form of an aqueous solution of human immunoglobulin, or a combination of human immunoglobulin F (ab ′) ₂ and various antitumor substances. Once a day for 5 consecutive days, 5 in total
Each mouse was intraperitoneally injected once, and the average survival time (T) of the sample administration group and the average survival time (C) of the control group were determined, and the survival rate (T / C × 100) was calculated. The results are shown in Table-11 to Table-13.

Example 11 11-1 500 mg of dextran was dissolved in 500 ml of distilled water to obtain pH.
To 1 by addition of 1N NaOH. 250 mg at room temperature
BrCN / acenitrile solution adjusted to / ml is added with rapid stirring. Add NaOH to adjust pH to 10.8 ~
Adjust to 11.0. After adding BrCN, keep the pH for 10 minutes. To this, 100 mg of hexamethylenediamine dissolved in 2.5 ml of water was added, and the pH was adjusted to 9.0 with 1N HCl. After stirring for 5 minutes, 250 mg of melphalan is added to bring the pH to 6.5 and the pH is kept for 15 minutes. After completion of the reaction, the reaction solution is concentrated to 10 ml at 4 ° C. 10 ml of the concentrated solution was passed through a column having a diameter of 5 cm and a height of 90 cm packed with Sephadex G-25 (Pharmacia Japan) to completely separate the high molecular weight substance and the low molecular weight substance in the reaction solution. The eluate was ultracentrifuged at 40,000 g x 60
Minutes and centrifugation, the resulting supernatant was frozen at 0 ° C. to obtain the product compound. This conjugate is a melphalan-dextran conjugate, and 30 to 50 molecules per 1 molecule of dextran.
The molecule melphalan was bound. 100 mg of this conjugate
A conjugate was prepared by using glutaraldehyde and 100 mg of human immunoglobulin. Similarly, a conjugate was obtained using human immunoglobulin F (ab ′) ₂ .

11-2 Dissolve 500mg dextran in 500ml distilled water,
To 1 by addition of 1N NaOH. 250 mg at room temperature
A solution of BrCN in acenitrile adjusted to a concentration of / ml is added with rapid stirring. Add pH to 1 with NaOH
Adjust to 0.8-11.0. After adding BrCN, keep the pH for 10 minutes. There, 10 dissolved in 2.5 ml of water
Add 0 mg of hexamethylenediamine to adjust pH to 1N H
Adjust to 9.0 with Cl. 250 mg after stirring for 5 minutes
Might Machine C was added to adjust the pH to 6.5
Hold for 15 minutes. After the reaction was completed, the reaction mixture was heated at 4 ℃ for 10
Concentrate to ml. Add 10 ml of concentrate to Sephadex G-25.
(Pharmacia Japan Co.) was passed through a column having a diameter of 5 cm and a height of 90 cm to completely separate high-molecular weight and low-molecular weight substances in the reaction solution. Ultracentrifugation of eluate 40,000
The supernatant liquid obtained by centrifuging g × 60 minutes was lyophilized at 0 ° C. to obtain a product compound. This conjugate was a mitomycin C-dextran conjugate in which 30 to 50 molecules of mitomycin C were bound per one molecule of dextran.

100 mg of this conjugate and 100 mg of human immunoglobulin were added at a concentration of 100 μg / ml final glutaraldehyde, and the mixture was reacted at room temperature for 1 hour to obtain human immunoglobulin-bound dextran-mitomycin C.

11-3 Dissolve 500 mg dextran in 500 ml distilled water,
To 1 by addition of 1N NaOH. 250 mg at room temperature
A solution of BrCN in acetonitrile adjusted to a concentration of / ml is added with rapid stirring. Add NaOH to adjust pH
Adjust to 10.8-11.0. After adding BrCN, keep the pH for 10 minutes. 100m dissolved in 2.5ml water there
Hexamethylenediamine (g) was added to adjust the pH to 1N HC.
Adjust to 9.0 with l. 250 mg after stirring for 5 minutes
Add methotrexate to bring the pH to 6.5 and p
Hold H for 15 minutes. After completion of the reaction, the reaction solution is concentrated to 10 ml at 4 ° C. Add 10 ml of concentrated solution to Sephadex G-
5 cm diameter filled with 25 (Pharmacia Japan),
High-molecular weight and low-molecular weight substances in the reaction solution were completely separated through a 90 cm high column. The eluate was ultracentrifuged at 40,0
The supernatant liquid obtained by centrifuging at 00 g × 60 minutes was lyophilized at 0 ° C. to obtain a compound as a product. This conjugate was a methotrexate-dextran conjugate in which 30 to 50 molecules of methotrexate were bound per one molecule of dextran.

100 mg of this conjugate and human immunoglobulin F (ab ′) ₂ 1
20 mg of glutaraldehyde was added at a final concentration of 100 μg / ml to prepare a conjugate.

Similarly, a conjugate was obtained using human immunoglobulin.

Example 12 12-1 11.3 mg of mitomycin C was mixed with 0.01M phosphate buffer solution (pH
6.8) Dissolve in 1 ml, add 20 μl of 1% glutaraldehyde aqueous solution and stir at room temperature for 8 hours. A solution prepared by dissolving 100 mg of human immunoglobulin in 20 ml of a phosphate buffer (pH 6.8) is added to the mixture, and the mixture is further reacted at room temperature for 2 hours. After the reaction was completed, Sephadex G-25 (Pharmacia Japan Co.) was filled, and the diameter was 5 cm and the height was 90.
High-molecular weight and low-molecular weight substances in the reaction solution were completely separated through a cm column. Ultracentrifugation of eluate 40,000 g ×
The supernatant liquid obtained by centrifugation for 60 minutes was freeze-dried at 0 ° C. to obtain a compound as a product. The amount of Mitmachine C bound to human immunoglobulin was 9.6 μg per mg of protein.

12-2 Adriamycin 196 mg in 0.01 M phosphate buffer (pH
Dissolve 1 ml in 6.8). 20 μl of 1% glutaraldehyde aqueous solution is added thereto, and the mixture is stirred at room temperature for 8 hours.
A solution prepared by dissolving 100 mg of human immunoglobulin in 20 ml of a phosphate buffer (pH 6.8) is added thereto, and the mixture is further reacted at room temperature for 2 hours. After the reaction, Sephadex G-25
(Pharmacia Japan Co.) was passed through a column having a diameter of 5 cm and a height of 90 cm to completely separate high-molecular weight and low-molecular weight substances in the reaction solution. Ultracentrifugation of eluate 40,000
The supernatant liquid obtained by centrifuging g × 60 minutes was lyophilized at 0 ° C. to obtain a product compound. The amount of adriamycin bound to human immunoglobulin is 5.6μ / mg protein.
It was g.

Similarly, a conjugate was obtained using human immunoglobulin F (ab ′) ₂ .

12-3 Methotrexate 200mg was added to 0.01M phosphate buffer (pH
6.8) Dissolve in 1 ml. 20 μl of 1% glutaraldehyde aqueous solution is added thereto, and the mixture is stirred at room temperature for 8 hours. 20 ml of 100 mg of human immunoglobulin F (ab ′) ₂
Add the solution dissolved in the phosphate buffer (pH 6.8) of
React for an additional 2 hours at room temperature. After the completion of the reaction, the high molecular weight and low molecular weight substances in the reaction solution were completely separated through a column having a diameter of 5 cm and a height of 90 cm packed with Sephadex G-25 (Pharmacia Japan). The eluate was centrifuged by ultracentrifugation at 40,000 g × 60 minutes, and the obtained supernatant was freeze-dried at 0 ° C. to obtain a compound as a product. The amount of methotrexate bound to human immunoglobulin F (ab ′) ₂ was 7.8 μg per mg of protein.

Example 13 13-1 Mitomycin C 11.3 mg and human immunoglobulin 100 mg
Is dissolved in 10 ml of 0.2 M sodium borate (pH 9.3). 5 mg of diethyl malonimidate was added thereto, and the mixture was stirred at room temperature for 1 hour while maintaining the pH at 8.6. 2.5 mg of diethyl malonimidate was added and stirred for 1 hour. After the reaction was completed, the pH was returned to neutral and 45% saturated ammonium sulfate was added to precipitate the human immunoglobulin-mitomycin C conjugate. Let 1 at 7000 rpm
The precipitate was collected by centrifugation for 5 minutes. The precipitate was dissolved in 5 ml of 5 mM phosphate buffer and dialyzed against distilled water until no ammonium sulfate was detected (72 hr). After completion of dialysis, low molecular weight substances in the reaction solution were completely removed through a column having a diameter of 5 cm and a height of 90 cm packed with Sephadex G-25 (Pharmacia Japan). The eluate was freeze-dried at −20 ° C. to obtain a compound as a product. The amount bound per mg of human immunoglobulin was 6.3 μg.

Similarly, a conjugate was obtained using human immunoglobulin F (ab ′) ₂ .

13-2 React 100 mg of human immunoglobulin with melphalan and sodium aminopterin according to the above procedure 85
Obtained mg of bound compound.

Example 14 The results of investigating the effects of the compounds synthesized in Example 11, Example 12, and Example 13 by using the antitumor test of Examples 8, 9, and 10 are shown in Tables 14 to 16.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Yoshiharu Oguchi 3-10-13 Nerima, Nerima-ku, Tokyo No.22 Daiichi Kurehaso (72) Inventor Chikao Yoshipu 2-19-46 East, Kunitachi, Tokyo

Claims (12)

[Claims] 1. An antitumor agent comprising as an active ingredient a compound obtained by binding an alkylating agent selected from the group consisting of chlorambucil, ACNU and cyclophosphamide to immunoglobulin derived from a normal person. 2. The antitumor agent according to claim 1, wherein the immunoglobulin is F (ab ′) ₂ . 3. The antitumor agent according to claim 1, wherein the alkylating agent is chlorambucil. 4. The antitumor agent according to any one of claims 1 to 3, which is bound using a water-soluble carbodiimide. 5. The antitumor according to any one of claims 1 to 3, which is bound by using isocyanate, diethylmalonimidate, glutaraldehyde, polyglutamic acid or dextran. Agent. 6. The antitumor agent according to any one of claims 1 to 5, which is in an oral dosage form. 7. The antitumor agent according to claim 6, wherein the oral dosage form is granules. 8. The antitumor agent according to claim 6, wherein the oral dosage form is a tablet. 9. The antitumor agent according to claim 6, wherein the oral dosage form is a capsule. 10. The antitumor agent according to any one of claims 1 to 5, which is a parenteral dosage form. 11. The antitumor agent according to claim 10, wherein the parenteral dosage form is a suppository. 12. The antitumor agent according to claim 10, wherein the parenteral dosage form is an injection.