JPH05117165A - Anticancer medicine - Google Patents

Abstract

PURPOSE:To provide a system of widely alleviating side effects and improving safety and treating effects by utilizing an existing anticancer agent, especially an anticancer antibiotic and concentratedly transferring the anticancer agent to the target cancer cells. CONSTITUTION:An anticancer agent comprising an antibody-linked anticancer agent obtained by utilizing a partial peptide of an extracellular hydrophilic site in a protein amino acid sequence of cancer gene C-erbB-2 product as an antigen to give a monoclonal antibody, utilizing the monoclonal antibody as an antibody to recognize a membrane protein of adenocarcinoma cell and linking the antibody to adriamycin, as an active ingredient. This anticancer agent effectively acts on adenocarcinoma cell, especially cancer cells to yield product of cancer gene C-erbB-2.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to an anticancer agent, and more particularly to an adenocarcinoma cell, particularly an oncogene c-er.
The present invention relates to an anticancer agent effective against a cancer cell that produces a product of bB-2.

[0002]

2. Description of the Related Art Adriamycin
n) is a Streptomyces peucet actinomycete.
iusvar. Caesius) is known to have an antitumor activity (Translated by Seiji Miyano et al., "Medicinal Chemistry (II) -Clinical Pharmacy and Structure-Activity Relationship of Drugs") Hirokawa Shoten (Sho 55-9-20) p.
474-475), this adreamycin is mainly used for the treatment of adenocarcinoma.

However, not only adreamycin but conventionally used anticancer agents generally have strong side effects,
It also damages normal cells that are actively dividing and proliferating. Therefore, there is no choice but to limit the dose, and it is the current situation that effective treatment cannot be performed.

[0004]

The object of the present invention is to develop an excellent anti-cancer agent, but instead of newly developing a new anti-cancer substance, the side effect of the existing anti-cancer agent can be improved while utilizing the existing anti-cancer agent. The purpose is to develop a new system that can be widely reduced.

[0005]

Means for Solving the Problems The present invention has been made to achieve the above object, and even if a small amount of an anticancer agent is effective if the anticancer agent administered in the body is specifically concentrated in cancer cells. From the viewpoint that treatment is possible and side-effect problems are alleviated, it was decided to achieve the above-mentioned object by developing a system for specifically concentrating an anticancer agent on cancer cells.

As a result of various studies, the oncogene c-erb, which is overexpressed in adenocarcinomas, especially breast cancers.
Focusing on the expression product of B-2, that is, a protein that penetrates the cell membrane, an antibody that recognizes the extracellular hydrophilic site of this adenocarcinoma was prepared, and a conjugate of this and an anticancer agent was prepared. In particular, it was confirmed that the anticancer drug is extremely effective against adenocarcinoma cells, and the present invention has been completed.

[0007] That is, the present invention has as its basic technical idea an anticancer agent containing as an active ingredient a conjugate of an antibody-conjugated anticancer agent and an antibody that recognizes a membrane protein of adenocarcinoma cells.

In the present invention, an antibody that recognizes a membrane protein of adenocarcinoma cells is used. It uses the membrane protein and / or partial peptide as an antigen and, if necessary, binds with a carrier protein and then uses this as an antigen. The antibody may be prepared according to a conventional method.

As described above, the membrane protein and / or its partial peptide can be widely used as the antigen, and for example, the expression product of the oncogene c-erbB-2 can also be advantageously used as the antigen. The oncogene c-erbB-2 is amplified in adenocarcinoma tissues such as breast cancer and gastric cancer, and its product contains a transmembrane protein having a molecular weight of 185 kDa. Can be used as

The latter was obtained by synthesizing a peptide (hydrophilic residue number about 10 to 20) at the hydrophilic site corresponding to the extracellular portion in the protein amino acid sequence of the c-erbB-2 product. A polyclonal antibody can be obtained by treating a hydrophilic peptide as an antigen (by binding to a carrier protein) and treating the antigen according to a conventional method. This polyclonal antibody, as will be clear from the later section,
Since it can specifically recognize cancer cells, it can be used for producing an excellent anticancer agent according to the present invention by conjugating it with various anticancer agents. Further, in order to further enhance the specificity of the antibody to produce a more effective anticancer agent, a monoclonal antibody is prepared according to a conventional method using the above-mentioned antigen, and this is conjugated with various anticancer agents to produce the desired anticancer agent. It is also possible.

As the antigenic peptide, for example, among the peptides shown in the reference examples described later, 5 peptides can be most advantageously used, but not limited thereto, and peptides 1 to 4 are also advantageously used. be able to.

Antibodies obtained from such antigens are conjugated with various anti-cancer agents to produce the desired conjugated anti-cancer agents. The conjugation methods include diazo method, peptide method, alkylation method, cross-linking method and the like. In addition to the covalent bonding method, the ionic bonding method and other known bonding methods can all be used.

As the anticancer agent to be conjugated with the antibody recognizing the membrane protein of adenocarcinoma cells according to the present invention, all types of anticancer agents can be used in addition to the antibiotic anticancer agents such as adreamycin and its analog daunorubicin. Non-limiting examples include: actinomycin C, D, adreamycin, carcinostatin, carcinophylline, chromomycin, dactinomycin, daunorubicin, mitomycin, neocarzinostatin, misramycin, Bleomycin, streptomycin and others.

[0014]

The details of the mechanism of action of the anti-cancer system of the present invention must be covered in future studies, but at the present time it is presumed to be as follows. That is, since the antibody used in the present invention recognizes a gene product site that has penetrated the membrane but not the cell of a cancer cell and is out of the cell, it has extremely high specificity and accurately and rapidly detects a cancer cell. It has the characteristic of being recognizable. Therefore, in the conjugate according to the present invention, first, the antibody portion promptly and accurately identifies the cancer cell, and then forms a complex with the antigen (the gene product site penetrating to the outside of the cell) present on the cancer cell, and It is presumed that the complex invades into the cell, and at the same time, an anticancer agent such as adreamycin conjugated to the antibody also invades the cell, and the invaded anticancer agent inhibits the growth of cancer cells.

The pharmaceutical composition according to the present invention is a solid, semi-solid or liquid form in which a conjugate of an anti-cancer agent for conjugating an antibody and an antibody is added as an active ingredient to an inorganic or organic carrier. In addition to orally administered agents, it is also formulated into parenteral agents such as external preparations.

Preparations for oral administration include tablets, pills, granules, soft / extended capsules, powders, fine granules, powders,
Examples thereof include emulsions, suspensions, syrups, pellets and elixirs. Formulations for parenteral administration include injections, drip infusions, infusions, ointments, lotions, tonics, sprays, suspensions, oils, emulsions, suppositories and the like. In order to formulate the active ingredient of the present invention, conventional methods may be followed, including surfactants, excipients, coloring agents, flavoring agents, preservatives, stabilizers, buffers, suspending agents, isotonic agents. Other commonly used adjuvants should be used as appropriate.

The dose of the pharmaceutical composition of the present invention varies depending on the type, the type of cancer to be treated or prevented, the administration method, the age of the patient, the symptoms of the patient, the treatment time, etc. Is 0.01 to 1000 mg / kg, preferably 0. 0, of the active ingredient (this substance) per adult per day.
1 to 100 mg / kg is administered.

When the antibody used in the present invention was administered to mice, no change in body weight or life span was observed up to at least 100 mg / mouse. For adreamycin,
In general, in the case of an adult, 40 mg is administered 8 times, the maximum dose is 500 mg, and in the case of mouse, the maximum dose is 500 μg. Therefore, the acute toxicity of the conjugate according to the present invention is Since these values are above these values, the present conjugate is very safe. Hereinafter, the present invention will be described in more detail with reference to Examples and Examples.

[0019]

[Reference Example 1] The hydrophilic site of the product encoded by the c-erbB-2 oncogene was searched using the method of Chou-Fasman, and some of them were made into their synthetic peptides and bound to a carrier protein. Then, it was used as an antigen to immunize mice. The reactivity of the obtained antiserum to the breast cancer cell line SK-BR-III in which the expression of c-erbB-2 was recognized was examined by an ELISA method in which the cells were immobilized, and the reactivity shown in Table 1 below. The results shown in Table 1 were obtained.

[0020]

[Table 1]

As is clear from the above results, c-erb
It was confirmed that the partial peptides of the extracellular hydrophilic site in the protein amino acid sequence of the B-2 oncogene product are all excellent antigens, and the peptides of Table 1 are most preferable, but other peptides are also preferable. Was done.

[0022]

Example 1 Mouse splenocytes immunized with a peptide (a synthetic peptide shown in Table 1 above) of the N-terminal residues 495 to 509 of the product encoded by the c-erbB-2 oncogene as an antigen. And mouse myeloma cells (SP
A monoclonal antibody can be obtained by cell fusion of 2/0) to produce a hybridoma. The outline of the manufacturing procedure is as follows.

The amino acid sequence of the antigen peptide is HTAN
It was prepared by crosslinking RPEDECVGEGL synthetic peptide and keyhole limpet hemocyanin (KLH) as a carrier protein using glutaraldehyde. The manufacturing method is described in G. Walter's method (P
ro C. Natl, Acad. Sci ,. 77 . 61
97, 1980). Next, for immunization, Balb / c mice were used with the above as an antigen. In addition, cell fusion
Behzadian, M .; A. Etc. Method (Cell
Struct, Funct. , 10 , 219, 198
It carried out according to 5). The ELISA method was used for the screening.

The obtained monoclonal antibody is human ce
Recognize the rbB-2 product. Whether or not this monoclonal antibody is directed to the c-erbB-2 product was determined by comparing the protein of the breast cancer cell line SK-BR-III whose expression of c-erbB-2 was confirmed with ³⁵ S-methion.
in which radiolabeled in is solubilized and immunoprecipitated,
It was confirmed by analyzing the autoradiography after development by SDS electrophoresis.

[0025]

[I. Manufacture of monoclonal antibodies]

[0026]

[(1) Preparation of Antigen Protein] The peptide was synthesized by a solid phase method using an automatic peptide synthesizer. further,
1 mg KLH and 3 mg peptide were crosslinked in a 0.1 M phosphate buffer (pH 7.2) with 2.5% glutaraldehyde. Furthermore, in order to remove free glutaraldehyde, 0.01 M phosphate buffer (pH 7.
4) Dialysis was performed in + 0.15M NaCl.

[0027]

[(2) Immunization] 100 μg of antigen protein and Freund's incomplete adjuvant were subcutaneously administered to Balb / c mice three times at 14-day intervals. Whether or not antiserum against the peptide was obtained was determined by immobilizing the peptide on a 96-well plate.
It investigated by the LISA method. At this time, the enzyme P was added to the anti-mouse IgG.
The one to which OX was bonded was used. OPD is the substrate.

[0028]

[(3) Cell fusion] The spleen of the immunized Balb / c mouse is excised, and the splenocytes are extruded in RPMI 1640 medium. Mouse myeloma cells SP2 / 0 are 1/5 of the number of splenocytes
˜1 / 10 amount was added, RPMI was removed by centrifugation, and then cell fusion was performed with polyethylene glycol (molecular weight 4000, Sigma). In order to remove polyethylene glycol and obtain certainty of fusion, centrifugation was immediately performed and the cells were cultured in HAT medium. Approximately one week later, cloning was performed in the serum medium by the limiting dilution method. Screening is ELISA
Method.

[0029]

[(4) Preparation of Monoclonal Antibody] The obtained hybridomas were grown in RPMI 1640 medium containing 15% FCS, glutamic acid and insulin, and cells (10 cells) were intraperitoneally administered to Balb / c mice treated with X-rays and treated with pristane. ⁷ / animal). After about 3 weeks, ascites was collected and centrifuged, and the antibody was purified from the supernatant using a DEAE cellulose column.

[0030]

[II. Properties of monoclonal antibody against c-erbB-2 product]

[0031]

[(1) Identification of antibody subclass] Ouchterlo
It was carried out according to the ny method. Anti-mouse IgM or anti-mouse IgG was allowed to react with the obtained monoclonal antibody in 1% agar, and it was examined whether or not a precipitation line was generated. As a result, the monoclonal antibody was confirmed to be IgM.

[0032]

[(2) Molecular weight] Sephacryl S-300 superf
It was examined by column chromatography using ine.

[0033]

[(3) Immunological characteristics of antibody] SK-BR-III cells radiolabeled with ³⁵ S-methionin were solubilized, a monoclonal antibody was added thereto and reacted, and the precipitate was washed several times by centrifugation and then subjected to SDS electrophoresis. (7.5
%), And the gel was subjected to autoradiography. As a result, one band was observed at around 185 kDa. This molecular weight is the same as the human c-erbB-2 product and it is clear that this antibody recognizes the c-erbB-2 product.

[0034]

Example 2

[0035]

[(1) Preparation of adreamycin] Adreamycin and N-succinimidyl 3- (2-pyridyldithio) propionate (SPDP) were mixed in a methanol or dimethylsulfoxide (DMSD) solution at an equimolar ratio, and mixed in an amount of 3 to 6
The adreamycin was modified by reacting for a period of time.

[0036]

[(2) Preparation of conjugate of adreamycin and antibody] The monoclonal antibody prepared in Example 1 and SPDP were mixed at a molar ratio of 30 to 150: 1, and the mixture was mixed at 30 ° C for 30 minutes.
Let react for minutes. Here, the final concentration of SPDP is 5 to 10
Adjusted to mM and unreacted SPDP was PD-1
Removed on column 0.

The monoclonal antibody thus modified with SPDP and the SPDP-modified adreamycin obtained in the above item (1) were treated with 4 mM in 50 mM triethylamine, 50 mM sodium chloride, 1 mM ethylenediaminetetraacetic acid solution at pH 8.0. The reaction was carried out at 90 ° C for 90 minutes. Unreacted SPDP or adreamycin was removed by a PD-10 column to obtain a conjugate.

[0038]

Example 3 The cell killing effect of the conjugate of adreamycin prepared in Example 2 and the antibody was confirmed by the following method. The cells used in this example were the following three types of breast cancer cells,
The expression levels of K-BR-III, BT-474, MCF-7, and oncogene c-erbB-2 in the cells were also decreased in this order.

[0039]

[(1) Method] The cell killing effect was determined by administering the sterilized main conjugate and adreamycin to the culture medium of the test cells in the logarithmic growth phase, and measuring the number of viable cells 72 hours after the administration. It was judged and evaluated.

The cell growth inhibition rate was calculated from the number of viable cells measured as described above, and the cell growth inhibition rate = (C ₀ -C ₁ ) / C ₀
It was calculated according to the formula of × 100. In the above formula, C
₀ represents the number of viable cells when saline was administered to the test cancer cells, and C ₁ represents the number of measured viable cells.

[0041]

[(2) Results] The relationship between the adreamycin concentration for SK-BR-III and the cell growth inhibitory rate is shown in FIG. In the figure, a represents this conjugate, and b represents adreamycin alone administration (control).

In the test cancer cells, 50% of the anticancer drug
As a result of obtaining the anticancer agent concentration IC ₅₀ showing the cell growth inhibition rate,
The results shown in Table 2 below were obtained.

[0043]

[Table 2]

[0044]

[(3) Conclusion] As is clear from the above results, the cytocidal effects of the anticancer agents a and b used in this example are as follows:
It is dependent on the expression level of rbB-2, suggesting that this mechanism of action is mediated by its expression product.

Further, from the item of b / a, the conjugate according to the present invention, in SK-BR-III cells, can obtain an equivalent IC ₅₀ value in a small amount of about 1/20 compared to the administration of adreamycin alone. Be understood. This indicates that the conjugate according to the present invention has a cell-killing effect about 20 times higher than that of adreamycin alone.

[0046] Further, in BT-474 cells, although the adriamycin administered alone not confirmed cytocidal effect at concentrations below 10- ⁷ M, it showed a sufficient effect in bonding member according to the present invention. This suggests that even if adrenamycin alone is not effective, it may be possible to expect a sufficient effect by using the conjugate according to the present invention.

[0047]

Example 4 Production of Injectable Formula (1) to (3) shown in Table 3 below.
After dissolving all the components of (4) in 1000 ml of distilled water, 1 ml each was dispensed into ampoules to produce 1000 injections.

[0048]

[Table 3]

[0049]

INDUSTRIAL APPLICABILITY As described above, the present invention exhibits a very effective cell killing effect on cancer cells producing the product of the oncogene c-erbB-2, as compared with anticancer agents such as adreamycin alone. It is possible to provide a safe and highly effective new anticancer agent with reduced side effects.

[Brief description of drawings]

FIG. 1 is a graph showing the relationship between the cell growth inhibition rate and the anticancer drug concentration in breast cancer cells SK-BR-III.

[Explanation of symbols]

 a: conjugate according to the present invention b: single administration of adreamycin (control)

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical indication location A61K 45/00 ADU 8415-4C C12N 5/18 C12P 21/08 8214-4B

Claims (5)

[Claims] 1. An anticancer agent comprising a conjugate of an antibody-conjugated anticancer agent and an antibody that recognizes an adenocarcinoma cell membrane protein as an active ingredient. 2. The anticancer agent according to claim 1, wherein the anticancer agent to which the antibody is conjugated is an anticancer antibiotic. 3. The anticancer agent according to claim 2, wherein the anticancer agent to which the antibody is conjugated is adreamycin. 4. Use of a monoclonal antibody obtained by using as an antigen a partial peptide of an extracellular hydrophilic region in a protein amino acid sequence of an oncogene c-erbB-2 product, as an antibody recognizing a membrane protein of adenocarcinoma cells. The anticancer agent according to any one of claims 1 to 3, which is characterized. 5. The anticancer agent according to claim 4, wherein the antigen is a peptide having an amino acid sequence represented by the following chemical formula 1. [Chemical 1]