JPH0611713B2 - 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 by binding a certain type of cytotoxic substance to the antitumor antibody, but have 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, the second and subsequent administrations cannot prevent sera diseases such as anaphylaxis and can be used only once. Met. 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 various antibodies. As a result, it was found that general natural antibody reached the tumor site and remained 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 an antitumor antibody obtained from a xenogeneic animal by affinity chromatography (Japanese Patent Application Nos. 53-161388, 54-142152 and 54-14215).
3). 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 was added to pepsin (Nison off Sci
ence 132 1770 (1970)), plasmin (Sgouris Vox Sang
18 71 (1967)), thermolysin (Japanese Patent Application No. 50-19871),
A low molecular weight antibody obtained by enzymatic hydrolysis with papain, trypsin, and chymotrypsin was also studied with an antitumor agent bound thereto. The toxicity of these substances such as F (ab ') ₂ was more than 4000 mg / kg.

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, sterilized water, water-soluble solvents such as Ringer's solution, non-water-soluble solvents, isotonic agents, soothing agents, stabilizers, preservatives, suspending agents, buffers, An emulsifier or the like may 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 sterilized by a conventional method, and then they are divided into small vials for injection or lyophilized as they are to give injections. 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 material was labeled with ¹²⁵ I-.

That is, ¹²⁵ I-labeling was carried out on the protein portion of immunoglobulin according to the method of WH Hurter et al., Biochem. J. 89 114 (1963). Since the methods are the same, an example will be given. Mitomycin C-conjugated anti-S-180 antibody was dissolved in 0.5 M phosphate buffer (pH 7.4) to a concentration of 5 mg / ml. Put 0.5 ml of it into a spic tube and add 0.25 mci of N
a ¹²⁵ I is added. Further 0.05M phosphate buffer 200μ
Melted into 0.7 mg of chloramine T was added and reacted at 0 ° C for 15 minutes. Then, sodium pyrosulfite (1.75 mg) and KI (10 mg) dissolved in 0.05 M phosphate buffer were added to stop the reaction. Add the reaction mixture to Sephadex g-25 (2.2 cm
X40 cm) column was used to remove unreacted radioactive iodine and reagents. Thus, 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 kidney, 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 each tissue weight of the administered drug was displayed as shown below (Table 1 ).

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

These results indicate that the xenoantibodies obtained by immunizing xenogeneic animals with tumor antigens show excellent arrival rates. However, compared to specific antibodies, natural antibodies of the same species have a higher tumor arrival rate.
Although it fell to 1/5 to 1/10, it was found here that 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 0.005 M phosphate buffered saline (hereinafter abbreviated as PBS) is added to 1000 ml of normal human serum to dilute it. To this diluted serum, 2000 ml of saturated ammonium sulfate aqueous solution (pH 7.2) is gradually added with 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, 250 ml of saturated ammonium sulfate is gradually added with stirring to 20% saturation. 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. The precipitate is dissolved in 1000 ml of PBS and then 500 ml of saturated ammonium sulfate is added. After stirring for 60 minutes, centrifuge at 8000 rpm for 30 minutes to collect the precipitate. 300m of the obtained precipitate
It was dissolved in l PBS and dialyzed against PBS to remove ammonium sulfate. After the dialysis was completed, a DEAE-cellulose column (diameter: 5 cm × 50 cm) was used to collect fractions that passed 0.005M pH 8.0. 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 mitomycin C 1.0 g of human immunoglobulin is dissolved in 100 ml of distilled water. 111.3 mg of mitomycin C is dissolved therein.
While adjusting the pH to 5.5 with 1.0N hydrochloric acid aqueous solution, 262.
6 mg of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride was added and reacted for the following time, and the reaction was stopped by adding 5 ml of acetic acid-sodium acetate buffer (pH 5.5). 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. The results of measuring the amount of mitomycin C bound to human immunoglobulin at each reaction time using ultraviolet absorption at 360 nm are shown in Table 3.

3-2 According to the above procedure, human immunoglobulin 1.0 g was reacted with doxorubicin hydrochloride, daunorubicin hydrochloride, bleomycin and actinomycin D to give about 80
0 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 Binding of melphalan 1.0 g of human immunoglobulin is dissolved in 100 ml of distilled water. Suspend 100 mg of melphalan 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 reacted for 24 hours to obtain an acetic acid-sodium acetate buffer solution ( The reaction was stopped by the addition of 5 ml of pH 5.5). Next, the reaction solution was concentrated and desalted to 10 ml using an ultrafilter. The 10 ml concentrated liquid 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 liquid. The eluate was ultracentrifuged at 40,000 g x 60
The supernatant liquid obtained by centrifugal separation was freeze-dried at 0 ° C. to obtain a compound as a product. The protein content in this substance was determined by the copper-Folin method using albumin as a standard, and the alkylation activity was determined by the Epstein method (Epstein J. Anal. Chem. 27 1423 (1
955)). 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 procedure, human immunoglobulin 1.0 g was reacted with each of chlorambucil, ACNU and raumustine to obtain about 900 mg of this substance. Human immunoglobulin (m
The amount of chlorambuyl bound per g) is 60 min.
The time was 5.1 μg and 11.7 μg, respectively.

4-3 Binding 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. While adjusting the pH to 5.5 with a 1.0 N aqueous hydrochloric acid solution, 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride was added at 4 ° C and the reaction was carried out for 24 hours, followed by acetic acid-sodium acetate buffer solution (pH 5. 5) The reaction was stopped by adding 5 ml. Next, the reaction solution was added to 10 m1 using an ultrafilter.
Was concentrated and desalted. A 10m1 concentrate filled with Sephadex G-25 (Pharmacia Japan) with a diameter of 5
A high molecular weight substance and a 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 eluate was centrifuged by ultracentrifugation at 40,000g x 60 minutes and the supernatant obtained was centrifuged at 0 ° C.
The product was obtained by freeze-drying at. 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.
1-at 4 ℃ while adjusting pH to 5.5 with 1.0N hydrochloric acid solution
Ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride was added and reacted for the following time, and the reaction was stopped by adding 5 ml of acetic acid-sodium acetate buffer (pH 5.5). Next, the reaction solution was concentrated to 10 ml using an ultrafiltration device and desalted. 10m1 of concentrated solution is 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 the high molecular weight substance and the low molecular weight substance in the reaction solution. Ultracentrifuge eluate 4
The supernatant obtained by centrifuging at 000 g × 60 minutes 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 1.0g was reacted with each of cytarabine, 8-azaguanine, 5-fluorouracil, and sodium aminopterin to obtain about 910m.
g of bound compound was obtained. The amount of cytarabine bound per mg of human immunoglobulin was 4.7 μg for 60 minutes and 24 hours of reaction, respectively.
It was 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). Add pepsin at a ratio of enzyme to protein of 1/100 (weight / weight), 37 ℃
Digest 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. A column having a diameter of 5 cm and a height of 90 cm is packed with Sephadex G-150, charged with 5 ml of the concentrated solution, and eluted with PBS of pH 7. Separated into three peaks, but the first peak was F (ab ') ₂
Collect as. This fraction was packed 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 1.0N hydrochloric acid, at 4 ℃
131.3 mg of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride was added and reacted for the following time, and the reaction was stopped by adding 5 ml of acetic acid-sodium acetate buffer (pH 5.5). Then, the reaction solution was made 5 ml using an ultrafiltration device, and the concentrated solution was passed through a column with a diameter of 5 cm and a height of 90 cm filled with Sephadex G-25 (Pharmacia Japan Co.). Was completely separated. 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. The amount of mitomycin bound to human immunoglobulin F (ab ') ₂ at each reaction time was 360n.
The result of measurement using ultraviolet absorption of m is as shown in Table-4.

7-2 In accordance with the above operation, human immunoglobulin F (ab ') ₂ 500
mg with doxorubicin hydrochloride, daunorubicin hydrochloride, bleomycin and actinomycin D respectively to give about 800 mg of the bound compound. The binding amounts of doxorubicin hydrochloride per ₂ mg of human immunoglobulin F (ab ') ₂ were 8.5 µg and 19.6 µg at a reaction time of 60 minutes and 24 hours, respectively.

7-3 Human immunoglobulin F (ab ') ₂ 500 according to the above operation
mg and chlorambucil, melphalan, ACNU, uramustine, melphalan methyl ester, and cyclophosphamide were reacted to obtain about 400 mg of the binding compound. Human immunoglobulin F (ab ′) ₂ mg of melphalan
The amount of binding per unit is 8.1 μg for reaction time of 90 minutes and 24 hours, 1
It was 7.6 μg.

7-4 Human immunoglobulin F (ab ') ₂ 500 according to the above operation
mg and cytarabine, 8-azaguanine, 5-fluorouracil, methotrexate, and aminopterin sodium were respectively reacted to obtain about 400 mg of the bound compound.
Methotrexate human immunoglobulin F (ab ') ₂ mg
The binding amount is 7.5 μg for reaction time of 60 minutes and 24 hours, 1
It was 7.3 μg.

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 Aqueous solutions of F (ab ′) ₂ and the conjugates with various antitumor substances were injected into the abdominal cavity of each mouse 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) × 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 in Donryu rats
Intraperitoneal 1 × 10 ⁶ of each Donryu rat in a group of 10
Human immunoglobulin, human immunoglobulin F (ab ′) ₂ and various antitumor agents were transplanted 24 hours after transplantation
Aqueous solutions of human immunoglobulin and human immunoglobulin F (ab ′) ₂ and various anti-tumor substance conjugates were administered intraperitoneally to each rat 5 times every other day for a total of 5 times. The average survival days (T) of the sample-administered group after injection and the control group (C) were obtained, and the survival rate (T / C × 100) was calculated. The results are shown in Table-8 to Table-10.
Shown in.

Example 10 Antitumor Effect on Mouse Leukemia P-388 P-388 ascites cells subcultured using DBA / 2 mice were intraperitoneally 1 × 1 in each DBA / 2 mouse group consisting of 10 mice.
0 ⁶ cells / mouse were transplanted, and 24 hours after transplantation, various antitumor agents,
Aqueous solution of human immunoglobulin and human immunoglobulin F (ab ') ₂ and various anti-tumor substance conjugates were intraperitoneally injected once a day for 5 consecutive days, 5 times in total. Then, the average survival time (T) of the sample administration group and the average survival time (C) of the control group were obtained, and the survival rate (T / C × 100) was calculated. The results are shown in Table-11 to Table-13.
Shown in.

Example 11 11-1 500 mg of dextran is dissolved in 500 ml of distilled water, and pH is adjusted to 11 by adding 1N NaOH. At room temperature, an acetonitrile solution of BrCN adjusted to 250 mg / ml is added with rapid stirring.

Adjust pH to 10.8-11.0 by adding NaOH. BrCN
Keep the pH for 10 minutes after adding. Add 100 mg of hexamethylenediamine dissolved in 2.5 ml of water and add pH.
To 9.0 with 1N HCl. After stirring for 5 minutes, 250 mg of melphalan was added to adjust the pH to 6.5.
Hold for 15 minutes. After the reaction is completed, the reaction solution is kept at 4 ° C for 10
Concentrate to m1. 10m1 of concentrated solution is 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 the high molecular weight substance and the low molecular weight substance in the reaction solution. Ultracentrifuge eluate 4
The supernatant liquid obtained by centrifuging at 000 g × 60 minutes was frozen at 0 ° C. to obtain a compound as a product. This compound was a melphalan-dextran conjugate, and 30 to 50 molecules of melphalan were bound to one molecule of dextran. A conjugate was prepared from 100 mg of this conjugate and 100 mg of human immunoglobulin using glutaraldehyde. Similarly, a conjugate was obtained using human immunoglobulin F (ab ') ₂ .

11-2 500 mg of dextran is dissolved in 500 ml of distilled water, and pH is adjusted to 11 by adding 1N NaOH. A solution of BrCN in acetonitrile adjusted to a concentration of 250 mg / ml at room temperature is added with rapid stirring.

Adjust pH to 10.8-11.0 by adding NaOH. BrCN
Keep the pH for 10 minutes after adding. Add 100mg of hexamethylenediamine dissolved in 2.5m1 of water and p
Adjust H to 9.0 with 1N HCl. After stirring for 5 minutes, 250 mg of mitomycin C 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. A 10m1 concentrate filled with Sephadex G-25 (Pharmacia Japan) with a diameter of 5
The high-molecular weight and low-molecular weight substances in the reaction solution were completely separated through a column having a height of 90 cm and a height of 90 cm. Ultracentrifugation of eluate
The supernatant liquid obtained by centrifugation at 40,000 g × 60 minutes was freeze-dried at 0 ° C. to obtain a compound as a product. 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 to a final concentration of glutaraldehyde of 100 μg / m 1 and reacted at room temperature for 1 hour to obtain human immunoglobulin-bound dextran-mitomycin C.

11-3 500 mg of dextran is dissolved in 500 ml of distilled water, and pH is adjusted to 11 by adding 1N NaOH. A solution of BrCN in acetonitrile prepared at a concentration of 250 mg / ml at room temperature is added with rapid stirring.

Adjust pH to 10.8-11.0 by adding NaOH. BrCN
Keep the pH for 10 minutes after adding. Add 100mg of hexamethylenediamine dissolved in 2.5m1 of water and p
Adjust H to 9.0 with 1N HCl. After stirring for 5 minutes, 250 mg of methotrexate are added to bring the pH to 6.5 and held for 15 minutes. After completion of the reaction, the reaction solution is concentrated to 10 ml at 4 ° C. Sephadex G with 10m1 concentrate
-5c diameter filled with -25 (Pharmacia Japan)
High-molecular weight and low-molecular weight substances in the reaction solution were completely separated through a m, 90 cm high column. Ultracentrifugation of eluate
The supernatant liquid obtained by centrifugation at 40,000 g × 60 minutes was freeze-dried 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 120 mg of human immunoglobulin F (ab ′) ₂
Was added at a concentration of 100 μg / m1 of the final glutaraldehyde to prepare a conjugate.

Similarly, a conjugate was obtained using human immunoglobulin.

Example 12 12-1 Mitomycin C 11.7 mg was added to 0.01 M phosphate buffer (pH 6.
8) Dissolve in 1 ml, add 1% aqueous 1% glutaraldehyde solution, and stir at room temperature for 8 hours. There 10
0 mg human immunoglobulin was added to 20 ml phosphate buffer (pH 6.
Add the solution dissolved in 8) and react at room temperature for 2 hours. After the reaction was completed, 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 mitomycin 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 6.
Dissolve 1 ml in 8). 20 μ of 1% glutaraldehyde aqueous solution is added thereto and stirred at room temperature for 8 hours. there
100 mg human immunoglobulin in 20 ml phosphate buffer (pH
Add the solution dissolved in 6.8) and react at room temperature for 2 hours. 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 (Falmachine 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 adriamycin bound to human immunoglobulin was 5.6 μg per mg of protein.

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

12-3 Methotrexate 200mg was added to 0.01M phosphate buffer (pH 6.
Dissolve 1 ml in 8). 20 μ of 1% glutaraldehyde aqueous solution is added thereto and stirred at room temperature for 8 hours. there
A solution prepared by dissolving 100 mg of human immunoglobulin F (ab ') ₂ in 20 ml of phosphate buffer (pH 6.8) is added, and the mixture is further reacted for 2 hours at room temperature. After the reaction, Sephadex G-25
(Pharmacia Japan) 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. 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. Human immunoglobulin F (a
The amount of methotrexate bound to b ′) 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. Furthermore, 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. The precipitate was collected by centrifugation at 7000 rpm for 15 minutes. The precipitate was dissolved in 5 ml of a 5 mM phosphate buffer solution and dialyzed against distilled water so that ammonium sulfate could not be 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. Human immunoglobulin mg
The binding amount per unit 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.
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 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 Yoshikumi 2-19-46, Higashi, Kunitachi, Tokyo

Claims (12)

[Claims] 1. An antitumor agent comprising a compound obtained by binding an antibiotic to an immunoglobulin derived from a normal person as an active ingredient. 2. The antitumor agent according to claim 1, wherein the immunoglobulin is F (ab ′) ₂ . 3. The antibacterial agent according to claim 1 or 2, wherein the antibiotic is selected from the group consisting of mitomycin C, doxorubicin hydrochloride, daunorubicin hydrochloride, bleomycin and actinomycin D. Tumor drug. 4. The antitumor agent according to any one of claims 1 to 3, which is bound using a water-soluble carbodiimide. 5. The anticoagulant according to any one of claims 1 to 3, which is bound using isocyanate, diethylmalonimidate, glutaraldehyde, polyglutamic acid or dextran. Tumor drug. 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.