JPH06157346A - Selective carcinostatic agent - Google Patents

Abstract

PURPOSE:To provide the carcinostatic medicine capable of selectively acting only on cancer cells. CONSTITUTION:The selective carcinostatic agent is characterized in that an amino group in a monoclonal antibody produced from a hybridoma originated from a human cancer antigen is bound to a carcinostatic substance containing one or more saccharide units as a part of its constituting components through a carbonnitrogen covalent bond in an aminomethyl bond. The hybridoma includes the fused cell of the splenic cell of an animal (BALB/mouse) sensitized with human malignant melanoma with the myeloma cell of an animal. The carcinostatic substance containing the saccharide unit as a part of the constituting components includes adriamycin, daunomycin, cytarabine, thionosine and FT-207 (tegafur). The bound product is bound to the cancer-specific antibody of a target cancer cell and is taken in the cancer cell by its phagocytosis and pinocytosis, but the bound product does not express a lethal action on other normal cells because it is not bound to the other normal cells not having the adoptive antigen.

Description

Detailed Description of the Invention

[0001]

[Object of the Invention]

FIELD OF THE INVENTION The present invention relates to a selective antitumor agent, in particular, a monoclonal antibody (hereinafter referred to as "MoAb") against a human cancer antigen produced by a hybridoma and an antitumor agent are bound to each other by an aminomethyl bond. By a carbon-nitrogen covalent bond, which has a specific affinity for human cancer cells.

[0002]

BACKGROUND OF THE INVENTION As a therapeutic drug for malignant tumors, that is, cancer, a large number of cytotoxic substances have already been clinically used or tested, but all of them are nonspecific to cancer cells and It is also toxic to cells and has the essential drawback that it is not possible to increase the dose until an effective concentration is reached. For this reason, even today, the reliability of chemotherapeutic agents for cancer is poor, and early detection and surgery are still the main roads for cancer treatment.

Therefore, in recent years, the idea of binding a substance having an antitumor effect or cytotoxicity to a special carry and selectively transmitting this to a cancer cell to act on it has been born,
Along this line, the use of antibodies against the antigens carried by cancer cells has been studied. Originally, the same or different antitumor antibody of the same individual does not always exert a damaging effect on cancer cells, but they have a common property that they have a high probability of binding to cancer cells. . Utilizing this property, for example, an antitumor immunoglobulin and an antitumor agent such as daunomycin
Example in which Fab 'dimer is covalently bound (JP-A-51-14)
4723 No.), the complex cross-linked with Diphtheria toxin and rabbit SV ₄₀ antibody glutaraldehyde (FL Moolten et al., J. Natl.
Cancer Inst., Vol.55, pp.473-477 (1975)), a conjugate in which diphtheria toxin and an anti-lymphocyte antibody are bound via chlorambucil (Nature, Vol.271, pp, 752-754).
(1978)), etc., and in addition to these, JP-A-55-136235, 55
Inventions such as -49321, 56-16418, and 56-16417 have also been published.

However, the antibodies used in these documents are not single antibodies, but also contain antibodies against antigens of normal cells, so that the selectivity for cancer cells is insufficient, and therefore , It is highly possible that the drug will have a damaging effect on normal cells.

[0005]

SUMMARY OF THE INVENTION Therefore, an object of the present invention is to provide a selective anticancer drug having a high purity of a cancer antibody carrying a drug and having a property of selectively acting on cancer cells. .

[0006]

However, as a result of efforts by the present inventors to create an antitumor drug that can selectively act only on cancer cells, fused cells (hybridomas) obtained by the cell fusion method. Succeeded in obtaining MoAb against human cancer cell antigen by purifying the protein produced by Saccharomyces cerevisiae. -By covalent bonding with nitrogen, it was possible to obtain a selective antitumor agent having specificity for cancer cells.

That is, the drug of the present invention comprises an active ingredient having an action of suppressing the growth of cancer cells or killing them, and a carrier ingredient carrying the active ingredient and inducing it into cancer cells. It The active ingredient preferably has a strong cytotoxic activity regardless of whether it is currently used as a carcinostatic agent, and examples thereof include alkylating agents such as endoxane, nitromine and sarcolicin; amethopterin, 8-
Antimetabolites such as azaguanine, 5-fluorourazil, cytosine arabinoside, 6-mercaptopurine; antibiotics such as mitomycin C, actinomycin D, adriamycin, daunomycin, zarcomycin;
Alkaloids such as vinblastine; basic polypeptides derived from wheat (SP ₁ , SP ₂ , SP-H, etc.), diphtheria toxin,
Examples include toxic proteins or peptides such as botulinum toxin, tetanus toxin, and ricin.

The carrier component is a single antibody against human cancer antigens and constitutes the main part of the invention. This is a fusion cell (hybridoma) with an appropriate experimental animal sensitized with human cancer cells, for example, a myeloma cell derived from a BALB / c mouse. By cloning this, a human cancer cell MoAb capable of binding to cancer-specific antigen of cell
It can be obtained by selecting and separating the fused cells that produce the antibody, and then proliferating the fused cells having the antibody-producing ability capable of binding to the selected cancer-specific antigen.

The fused cells (hybridomas) grown in the culture method or in the animal body are collected, and the antibody is separated therefrom by an appropriate means. For purposes of the invention, the cancer antibody should be as pure as possible, and ideally it is preferably electrophoretically purified to a single purity. In addition, the present antibody needs to contain an amino group in order to form an aminomethyl bond with an antitumor drug or a cytotoxic substance having a sugar as a component.

The purified antibody is then covalently bonded to the carbon-nitrogen bond by forming an aminomethyl bond with an antitumor drug or cytotoxic agent having the desired sugar as a component of its amino group. Specifically, for example, after cleaving between the adjacent hydroxyl groups (some of which may be amino groups) in the sugar moiety of the drug with sodium metaperiodate to generate an aldehyde group, the basicity of the antibody A Schiff base is formed between the amino group and the Schiff base is reduced with a selective reducing reagent such as sodium borohydride to give the corresponding aminomethyl derivative. This method is suitable when the drug contains sugar as a constituent component, and specific examples of the drug compound include, for example, daunomycin, adrimycin, FT-207 (tegaflu), cytarabine, thioinosine, etc. It is not limited to only the ones.

[0011]

The substances of the present invention will optimally be administered clinically in the form of intravascular or intramuscular injection or applied directly to cancerous tissue. As shown by the experimental results described below, the conjugate binds to the cancer-specific antigen of the target cancer cell, and is then taken up internally by the phagocytosis (pagocytosis) and pinocytosis of the cancer cell, resulting in lethality to the cell. It is presumed to be effective. At this time, since this conjugate does not bind to other normal cells that do not have the adaptive antigen, it can be considered that the lethal action does not appear.

[0012]

EXAMPLES The following will specifically describe the means for carrying out the invention with reference to the following examples, but the description is of course merely an example and does not limit the inclusion or extension of the invention.

Example 1 (Culture of hybridoma (225, 28S) and purification of MoAb) Dulbecco's modified Eagle medium (Dulbecco's Mod) was previously prepared.
ified Eagle Medium) (D-MEM), (containing 10% calf serum)
Hybridoma (225.28S) cultured in BALB / c
Mice (♀, 8 to 12 weeks old) are inoculated into the abdominal cavity 5 × 10 ⁶ each. In addition, 1 week before the inoculation, the mice were treated with pristine (2,6,10,14-tetramethylpentadecane) at 0.2.
Administer 3 ml each intraperitoneally to make hybridomas easy to grow.

Ascites fluid is collected from the second week after the hybridoma inoculation until the mice die. This ascites is 3000 r.p.
Centrifuge for 10 minutes to remove red blood cells and pristine. Add saturated ammonium sulfate solution to the supernatant with sufficient mixing so that the final concentration is 33.3%, and leave at 4 ° C for 1 hour or more.
Centrifuge the formed precipitate (10000r.pm, 10 minutes, 4
C). The collected precipitate was dissolved in a small amount of phosphate buffered saline (without Ca ⁺⁺ and Mg ⁺⁺ ) (PBS), and the dialysis tube (Cellophane Tubing-Seamless, Union Carb, USA) was used.
ide) and dialyze overnight against PBS. During this time, the external solution was exchanged at least 4 times, and finally dialyzed against M / 100 Tris-HCl buffer (pH 8.0), and then DE-52 column (2.5 x 16.5 cm) buffered with the same buffer in advance. ) Load. The column is eluted using the same buffer, but adding salt such that the salt concentration in the solution changes directly from 0 to 0.5M. MoAb activity in the extracted peak fraction (at 280 nm) is measured by the amount of binding to human malignant melanoma (Hu-man Mallignant Melanoma) using ¹²⁵ I-Protein A.

The active fraction was concentrated at 4 ° C. and passed through a Sephadex G-200 column (1.5 × 90 cm) to give 2
The absorption pattern at 80 nm purifies to a single peak. This MoAb is also electrophoretically single. It can be lyophilized or stored frozen.

The above culture and purification / isolation means are used for other MoAbs against the human malignant melanoma antigen produced by the hybridoma, for example, 376.74,376.96,465.14,473.54,653.
25 (The above values are for hybridoma and Mo produced by it.
The type of Ab is shown. ) Also applies to. Each antibody specifically binds to an antigen of malignant melanoma, but since the binding site is slightly different depending on the type of the antibody, a mixture of several MoAbs and a drug conjugate does not bind to each other. We suggest the possibility of cocktail therapy with several antibody / drug conjugates at different sites.

Example 2 (Production of Drug / MoAb Conjugate) Adriamycin 40
Dissolve mg in 1 ml PBS, add a slight excess of sodium metaperiodate (NaIO ₄ ) to this solution, and leave it in the dark for 1 hour. Glycerin water (1M) is added to the obtained reaction solution to a final concentration of 0.05M to decompose excess sodium periodate.

To this drug solution was added 20 mg of the antibody of Example 1 dissolved in 1 ml of 0.15 M potassium carbonate solution (pH 9.
5), react at room temperature for 1 hour. Sodium borohydride was added to this reaction solution in an amount of 0.3 mg per 1 mL of the reaction solution,
Adriamycin / MoAb conjugate is obtained by gel filtration after standing at 37 ° C. for 2 hours.

Example 3 (Cytotoxic effect of the substance of the present invention) The drug-antibody conjugate according to the present invention has apparently selectivity for antigen cells. Below, we present the experimental facts that support this conclusion.

(A) Experimental Material Cells: Colo38 cells (human malignant tumor cells) and Raji cells (human lymphoid cells derived from B-cells) Culture medium: Colo38 for Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum (described above) For Raji-10% Calf Serum Added RMPI 164
0 Medium Drug: Adriamycin / MoAb conjugate described in Example 2, dissolved in PBS and applied. Each medium solution is used for dilution.

(B) Experimental method Each cell was dispersed at 2 × 10 ⁵ cells per plate, and at the same time, each conjugate was added at 0-100 μl per 1 ml of the medium.
After culturing at ℃ for 48 hours, a part of the cells was collected and its life or death was determined by the presence or absence of the staining property for the final concentration of 0.05% trypan blue (unstained one is "live", stained one is "dead").
Judgment) as Turk-Buerker
Calculated using a calculator.

(C) Experimental Results and Discussion As shown in FIG. 1, the control antibody alone has no lethal effect on Colo38 cells. The control adriamycin and the agent of the present invention both have a strong lethal effect on the same cells, while the latter is more potent than the former.

[0023]

As described above, the drug according to the present invention
The MoAb conjugate exhibits a strong affinity only for a specific cancer cell having a compatible antigen and has an effect of killing the cancer cell or inhibiting its growth, but has almost no effect on normal cells. , It is hoped that it will contribute as a selective cancer therapeutic agent.

[Brief description of drawings]

Fig. 1 Graph showing cytotoxic effect of adriamycin / MoAb conjugate on Colo38 cells

[Explanation of symbols]

 ○ Adriamycin / MoAb conjugate ● Adriamycin alone △ MoAb alone

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Soldano Farone (Nationality United States of America) Scenic Drive North 8936, La Jolla, California, United States (Postal code 92307)

Claims (2)

[Claims] 1. An amino group in a monoclonal antibody produced by a hybridoma derived from a human cancer antigen is covalently bonded to a carcinostatic substance having a sugar as a component by a carbon-nitrogen bond by an aminomethyl bond. Selective anticancer drug. 2. The hybridoma is a fusion cell of spleen cells of an animal (BALB / mouse) sensitized with human malignant melanoma and myeloma cells (myeloma cells) of the animal.
Anti-cancer drug.