JPH0585532B2 - - Google Patents

Description

[Detailed description of the invention]

TECHNICAL FIELD The present invention relates to antitumor agents. Alpha-phetoprotein (AFP) and embryonic antigen (CEA) are well known as cancer markers. CEA is said to exist on the membrane surface of cancer cells in the digestive system. On the other hand, AFP has been recognized to be produced in many liver cancers and cell lines, but it has only been confirmed that it exists in the cytoplasm, and it is not necessarily clear whether it exists on the membrane surface or not. . The present inventors have developed the so-called "missile therapy" in which anticancer drugs and toxins are attached to monoclonal antibodies.
In order to find a monoclonal antibody against human-derived AFP suitable for this purpose, various studies were conducted, and the present invention was achieved by discovering AFP present on the surface of human-derived membranes and a monoclonal antibody that recognizes it. That is, the gist of the present invention resides in an antitumor agent, which is obtained by encapsulating a toxin or anticancer agent for tumor cells in a liposome, and binding a monoclonal antibody that recognizes human-derived α-fetoprotein (AFP) to the surface of the liposome. The present invention will be explained in detail below. The monoclonal antibody used in the present invention may be any monoclonal antibody that recognizes human-derived AFP, but monoclonal antibodies that recognize human-derived AFP present on the surface of cell membranes are particularly preferably used. Such monoclonal antibodies can be obtained by the following method. That is, first, after immunizing, for example, BALB/C mice with AFP derived from human placenta, the spleen was removed, and P3 was inoculated using polyethylene glycol.
-Fuse with mouse myeloma cells such as U1 to obtain a hybridoma by a conventional method. Monoclonal antibodies are then obtained from the hybridoma supernatant. Next, using these antibodies obtained by conventional methods, liver cancer cell lines (e.g., PLC,
Immunohistochemically stain KN, NuE) and select antibodies that show positivity. This detection is based on abiocin:
Using a biotinylated horseradish peroxidase complex (ABC) kit, horseradish
The enzymatic activity of peroxidase is carried out using diaminobenzidine as a substrate. To obtain large quantities of antibodies that show positivity, hybridomas that produce the selected antibodies are
BALB/C mice were injected intraperitoneally and allowed to grow.
This can be done by collecting ascites fluid.
Alternatively, the above hybridoma may be cultured in large quantities in a culture tank. The monoclonal antibody thus obtained has the following properties. Using this i ¹⁴ C-labeled antibody, placenta-derived
Examining whether AFP inhibits the binding of the PLC liver cancer cell line to the membrane, it is found that AFP competitively inhibits the binding of antibodies to the cell line membrane. Label the PLC cell line with ¹⁴ C-leucine, solubilize its membrane components with "Triton X",
When looking at the binding with this antibody, clear binding is observed. Furthermore, binding to secreted proteins in the culture supernatant was confirmed. When two-dimensional electrophoresis was performed on solubilized membrane components of the PLC cell line and antibody reactants in the culture supernatant according to the method of O'Farrel et al., it was found that both membrane-derived and culture supernatant-derived bonds were detected. It can be seen that substances also have AFP properties (isoelectric point, molecular weight). When the membrane fraction of the PLC cell line is digested with proteolytic enzymes such as trypsin, protease, and thymotrypsin, the binding property with the present antibody decreases or tends to decrease. Furthermore, treatment with “Triton X” showed a decreasing tendency;
Lipase treatment increases its binding properties. The antitumor agent according to the present invention encapsulates a toxin for tumor cells or an anticancer agent in a liposome, and a monoclonal antibody that recognizes human-derived α-fetoprotein (AFP), preferably a human as described above, is attached to the surface of the liposome. and an antibody that recognizes AFP present on the derived membrane surface. Examples of the toxin include plant seed toxins such as ricin and abrin, and bacterial toxins such as diphtheria toxin. These toxins have a B chain that participates in cell binding,
The A chain, which enters cells and exhibits toxicity, is linked by a disulfide (SS) bond. This S
It is used by reducing the -S bond and separating the A chain having the -SH group. In addition, animal toxins such as cobra venom, which is known as a complement activator, can also be used. In addition, the above-mentioned anticancer drugs are not particularly limited, but include, for example, adriamycin, actinomycin, daunomycin, nogalamycin, bleomycin,
Examples include chromomycin and mithramycin. Binding to the monoclonal antibody can be carried out using diazotization, carbodiimidation, or other reactions depending on the type of toxin or anticancer drug, depending on its functional group.
A method using dextran, etc. is adopted. In the present invention, liposomes (lipid membrane vesicles)
It is preferable to encapsulate (incorporate) the liposome into a liposome and bind a monoclonal antibody to the surface of the liposome, from the viewpoint of drug uptake into cells, sustained release, and reduction of toxicity. As a liposome consisting of a double lipid layer mainly composed of phospholipids, any of () multilayer, () small unilamellar, and () large unilamellar can be used. As the lipid, phosphatidylcholine (lecithin), lysolecithin, phosphatidylethanolamine, cholesterol, etc. are used. The preparation is carried out according to a conventional method, and a particle size of about 20 nm to several μm is usually obtained. In this case, the encapsulation of toxins or anticancer agents is usually carried out by including the drug in solution during liposome preparation. Thereby, hydrophilic substances are encapsulated within the liposome and hydrophobic substances are incorporated into the liposome membrane. The amount to be encapsulated (incorporated) can be determined as appropriate depending on the type of drug, but the concentration can be increased compared to normal administration methods. When binding a monoclonal antibody onto the liposome surface, methods such as attaching a hydrophobic substance to the antibody and inserting it into the liposome, or crosslinking phosphatidylethanolamine and the antibody with glutar, etc. are used, but suitable methods include The following methods are used: (a) Synthesize phosphatidylethanolamine with a reagent that reacts with the -SH group covalently bonded to it, and incorporate it into liposomes. To this is bound an antibody that has been converted to SH, or an antibody that has been converted to SH by removing the S-S bond between the antibodies. SPDP, MBS, etc. are used for the above covalent bond. Alternatively, (b) a glycolipid is incorporated into the liposome, and the antibody is reacted with the aldehyde group generated by periodic acid treatment. Ganglioside or the like is used as the glycolipid and treated with periodate to aldehyde the liposome surface. The antibody is then bound to liposomes in the presence of NaBH ₃ CN. The antitumor agent according to the present invention can be administered by conventional methods. Since the antitumor agent according to the present invention can selectively transport toxins or anticancer agents to cancer cells, high therapeutic effects can be obtained. Hereinafter, the present invention will be explained in more detail with reference to Examples. Reference example (1) Production of mouse monoclonal antibody: Purity 99 purified from placenta as human AFP
% or more, immunochemically human albumin (HSA)
A material (manufactured by Morinaga Seikaken Co., Ltd.) that does not react with was used. 10 μg of this human AFP was administered to BALB/C mice.
The animals were sensitized once with complete Freund's adjuvant, and the final immunization was given by intravenous injection. After 3 days, the spleen was removed and polyethylene glycol #400 was used.
It was fused with P3-U1 mouse myeloma, and a hybridoma was produced by a conventional method. Cloning was carried out using the limiting dilution method, which was repeated four or more times. In addition,
A large amount of antibody was collected by BALB/C mouse ascites system. Hybridomas producing anti-AFP antibodies are 6
Approximately 400 clones were selected after multiple rounds of fusion.
Among them, the antibody titer IgG amount of the culture supernatant of 11 clones is shown in Table 1 (measured after concentrating the supernatant 10 times).

[Table] (2) Selection of antibody a Cell line was incubated with Eagle MEM for at least one week.
After culturing in a medium based on , the cells were washed four times with phosphate buffer, and immediately reacted with the hybridoma culture supernatant; and) After fixation with 4% paraformaldehyde, methanol, H ₂ O ₂
A method was used in which endogenous enzymes were inactivated in a solution and reacted with antibodies. Antibody detection was performed using Vectastain's ABC kit.
Due to the enzymatic activity of horseradish peroxidase, diaminobenzidine was used as the substrate. b. Cell lines and maintenance Cell lines for human liver cancer include KN,
PLC, also a cancer cell line derived from fetal liver cells
NuE was used as a control, and human gastric cancer culture lines KATO, MKN45, and colon cancer C-
1 of each cell line was used. In addition, cultured cells are 20
%37.0 in culture medium of RPMI1640 containing fetal bovine serum
The cells were maintained and grown at 5% CO ₂ and 95% Air. c The culture supernatant containing the anti-AFP monoclonal antibody in (1) above and its dilutions (up to 2 ^{to 8} times) were added to liver cancer cell lines PLC, KN and fetal liver cells NuE.
When immunohistochemically reacted with 19F12
A strong reaction was observed. This reaction was similar for unfixed samples, fixation, methanol, and H ₂ O ₂ treatment. In addition, a commercially available anti-AFP monoclonal antibody (manufactured by Hybritech) was used as a control.
Although the antibody titer was adjusted to ¹⁰³ , no positive reaction was observed as with other hybridoma clones. As shown in Table 1, 19F12 does not have a particularly strong antibody titer nor does it have a large amount of IgG, but as mentioned above, unlike the others, it showed positive. On the other hand, 19F12 did not react with cell lines MKN45, KATO, and C1, which do not produce AFP. Example 1 (1) Method and materials (a) Cell line: ((2) ^b ) PLC, NuE,
C-1, KATO and Lic (human liver cancer) were used. (b) Culture method: According to the method in the above reference example. (c) Monoclonal antibody: The antibody (19F12) obtained in the above reference example was used. (d) Incorporation of ³ H-Thymidine: Culture cells in a 96-well culture plate, and add 2μCi/well (for anticancer drugs) or 5μCi/ml (for toxins) into the culture medium. ^3H -thymidine was added, and after culturing, the cells were washed and the radioactivity in the cells was measured using a liquid scintillation counter. (e) Toxin: Using ricin A, its purification and binding to antibodies were carried out by the methods of Olsness et al. (Biochemistry Vol. 12, No. 16, 1973) and
Raso et al.'s method (Cancer Research 42, 457
-464, 1982). (f) Anticancer drug: Adriamycin was used. (g) Liposomes: Creation and binding of antibodies are performed by the following method. (i) Add 1 ml of anti-human AFP mouse monoclonal antibody (19F12) and SPDP (0.2 mM) at room temperature.
Incubate for 30 minutes, then add acetate buffer (PH
4.5) using “Sephadex G-50”
The protein fraction was eluted using 50mM dithiothreitol (DTT).
The reaction was carried out for 40 minutes at room temperature to cleave the S-S bond, and then further reacted with "Sephadex G-
50" to obtain 1 ml of protein fraction. (ii) On the other hand, as liposomes, 4 μmole of egg yolk phosphatidylcholine, cholesterol
Wash by adding adriamycin using 2 μmole of DTP-DPPE and 0.006 μmole of DTP-DPPE, and suspend in 1 ml of acetate buffer. (iii) Next, the products of () and () above are mixed, adjusted to pH 8.0, and incubated for 24 hours to obtain the desired adriamycin-encapsulated liposome-monoclonal antibody conjugate. (2) Ricin A-monoclonal antibody conjugate
Effect on PLC (in vitro) Although all ricin molecules showed strong cytotoxicity even at 10 ^-9 M, the toxicity of ricin A chain ( ³ H-
Inhibition of thymidine uptake) was hardly observed at 10 ^-8 M order. On the other hand, ricin A bound to a monoclonal antibody inhibited the incorporation of ³ H-thymidine even at a concentration of 10 ^-8 M, and the effect was concentration dependent. In addition, ricin A bound to the anti-HSA monoclonal antibody used as a control was 5 × 10 ^-7
Although ^3H -thymidine incorporation was inhibited by M,
At a lower concentration of 10 ^-8 M, there was no effect at all. (3) Effect of adriamycin-encapsulated liposome-monoclonal antibody conjugate (in vitro) After contacting the cells with the drug for 2 hours, they were washed.
Incorporation of ³ H-thymidine was carried out as described above. The results are shown in Figures 1-5. It is natural that the anticancer drug Adriamycin suppresses tumor growth and reduces ^3H -thymidine uptake in vitro, but when comparing adriamycin-encapsulated liposomes and liposomes further conjugated with monoclonal antibodies, PLC (Figure 1),
In AFP-producing tumor liver cancer cell lines of LiC (Fig. 2) and NuE (Fig. 3), strong antitumor effects were always exhibited by the monoclonal binding type, and especially with NuE, a stronger effect than adriamycin alone appeared. On the other hand, no difference was observed in tumors other than liver cancer, C-1 and KATO, regardless of the presence or absence of monoclonal antibody binding. (4) Nude mouse transplantable liver cancer cell line Li7
According to the Battel Columbus Laboratories protocol, the major axis (L) and minor axis (W) were measured in mm twice a week using a sliding caliper. Estimated tumor weight was calculated from (W ² ×L)/2, and treatment was started when the tumor weight was 100 to 300 mg and was repeated three times every 4 days. The results for adriamycin-encapsulated liposomes, adriamycin-encapsulated liposomes bound to monoclonal antibodies, and adriamycin alone are as shown in FIG. In other words, adriamycin alone was clearly more effective than the control group (no treatment), and even more so, the liposome conjugate was more effective, and the monoclonal antibody-conjugated liposome (adriamycin encapsulated) was the strongest, producing a therapeutic effect in vivo. .

[Brief explanation of drawings]

Figures 1 to 5 show in vitro results of the antitumor agent according to the present invention.
Figure 6 shows the in vivo test results.

Claims (1)

[Claims] 1. An antitumor agent comprising a liposome encapsulating a toxin or anticancer agent for tumor cells, and a monoclonal antibody that recognizes human-derived α-fetoprotein (AFP) bound to the surface of the liposome.