JPH0469998B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to monoclonal antibodies. α-fetoprotein (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 AFP that is suitable for
We discovered AFP and a monoclonal antibody that recognizes it, and arrived at the present invention. That is, the gist of the present invention is to produce a hybridoma obtained by cell fusion of mouse spleen cells immunized with α-fetoprotein (AFP) and mouse myeloma cells, and to produce α-fetoprotein (AFP). It consists in monoclonal antibodies that recognize epitopes present on the surface of cell lines. The present invention will be explained in detail below. The monoclonal antibody according to the present invention 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-
Fuse with mouse myeloma cells such as U1 to obtain hybridomas by standard methods. Monoclonal antibodies are then obtained from the hybridoma supernatant. Next, using these antibodies obtained by conventional methods, liver cancer cell lines (e.g., PLC,
KN, NuE) are immunohistochemically stained and antibodies showing positive are selected. This detection is based on abiocin:
Using a biotinylated horseradish peroxidase complex (ABC) kit, horseradish
The enzymatic activity of peroxidase is carried out using diaminobenzine 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. (i) Using this 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. (ii) labeling the PLC cell line with ¹⁴ C-leucine;
When the membrane component was solubilized with "Triton X" and its binding with the present antibody was observed, clear binding was observed. Furthermore, binding to secreted proteins in the culture supernatant was confirmed. (iii) 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 membrane-derived and culture supernatant-derived It can be seen that both bound substances have the properties (isoelectric point, molecular weight) of AFP. (iv) When the membrane fraction of the PLC cell line is digested with proteolytic enzymes such as tribucin, 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. Since such a monoclonal antibody according to the present invention recognizes AFP present on the cell membrane surface,
It is useful as an antibody for liver cancer treatment, especially missile therapy, and is also useful as a diagnostic/testing agent for cytology and the like. Hereinafter, the present invention will be explained in more detail with reference to Examples. Example 1 (1) Production of mouse monoclonal antibody: Purity purified from placenta as human AFP
99% or more and does not immunochemically react with human albumin (HSA) (manufactured by Morinaga Seikaken Co., Ltd.)
was used. 10 μg of this human AFP was administered to BALB/C mice.
Sensitization was carried out once with Freund's complete adjuvant, the final immunization was given by intravenous injection, and 3 days later the spleen was removed and polyethylene glycol #400 was administered.
was used to fuse with P3-U1 mouse myeloma, and a hybridoma was produced by a conventional method. The mouse spleen cells used here were 8.2 × 10 ⁷ cells,
P3-U1 mouse myeloma cells are 3.2 x ¹⁰⁷ . Cloning was carried out using the limiting dilution method, which was repeated four or more times. In addition, a large amount of antibodies
The ascites was collected from BALB/C mice. Hybridomas that produce anti-AFP antibodies are
Approximately 400 clones were selected after 6 rounds of fusion. Among them, the antibody titer of the culture supernatant of 11 clones
The amount of IgG is shown in Table 1 (after concentrating the supernatant 10 times,
measurement. ).

[Table] (2) Antibody selection: a) Cell lines should be incubated with Eagle for at least one week.
After culturing in MEM-based medium, washing four times with phosphate buffer, (i) immediately reacting with hybridoma culture supernatant, and (ii)
After fixation with 4% paraformaldehyde, endogenous enzymes were inactivated with methanol and _{H2O2 solution} .
A method of reacting with antibodies was used. Antibody detection was performed using Vectastain's
Using ABC kit, horseradish
Due to the enzymatic activity of peroxidase, diaminobenzidine was used as the substrate. b Cell lines and maintenance Cell lines for human liver cancer include KN,
PLC, the fetal liver cell-derived cancer cell line NuE, and human gastric cancer culture lines KATO, MKN45, and colon cancer 1 cell lines were used as controls. The cultured cells were maintained in a culture medium of RPMI1640 containing 20% fetal bovine serum at 37.0°C, 5% CO ₂ and 95% Air.
Proliferated. c) Culture supernatant containing the antibody AFP monoclonal antibody mentioned in (1) above and its dilution (up to 2 ^{to 8} times)
When immunohistochemically reacted with the liver cancer cell line PLC; KN and fetal liver cell NuE, a strong reaction was observed with 19F12. This reaction can be performed even with unfixed specimens, fixed, methanol,
The results were similar for all H ₂ O ₂ treatment methods. We also used commercially available anti-AFP as a control.
Monoclonal antibody (manufactured by Hybritech)
was reacted with an antibody titer of 10 ³ , but as with other hybridoma clones, no positive reaction was observed. 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, cell lines MKN45, KATO, and C1, in which AFP production is not observed, have this 19F12
did not react. The monoclonal antibody (19F12) according to the present invention obtained as described above was further investigated as follows. A Competitive reaction of this antibody with placental AFP and PLC membrane: a ^14C label of antibody: in RPMI1640 medium without leucine
Add u- ^14C -leucine at a concentration of 5 μCi/ml,
The cells were cultured at a concentration of 5×10 ⁶ cells/ml for 60 hours, and the culture solution was purified using "Protein A Sepharose" to obtain a ¹⁴ C-labeled monoclonal antibody. b) Labeling of liver cancer cell line PLC and solubilization of membrane fraction: Cultured under the same conditions as in a) above, and the culture medium and cells were collected and used. The membrane fraction was used after sonicating the cells, washing the cells twice at 100,000 x g for 60 minutes, and washing the cells twice. Membrane solubilization was performed with 1% “Triton
It was used after being diluted 10 times with phosphate-physiological saline buffer. c A competition experiment was performed with the PLC cell line membrane and placenta-derived AFP using a ¹⁴ C-labeled antibody that was desalted using a PD10 column (manufactured by Pharmacia) and equilibrated with a phosphate-physiological saline buffer.
The results are shown in Figure 1. As is clear from FIG. 1, when AFP was added to the reaction solution, the amount of antibody that bound to the PLC membrane decreased depending on its concentration. In addition, soluble radioactivity, which appears to be a combination of AFP and antibody, increased depending on the AFP concentration. B Detection of monoclonal antibody-bound substances by two-dimensional electrophoresis: Label the liver cancer cell line PLC with ¹⁴ C-leucine, collect the polymeric substances in the culture supernatant,
When reacting with this antibody and examining the presence or absence of protein A-binding radioactivity, a clear antibody-bound substance was observed. In addition, a membrane-soluble fraction derived from 10 ml of culture solution and 5 x 10 ⁶ cells was reacted with 2 μg of this antibody and purified with "Protein A" to obtain an antibody-bound substance. When these bound substances are detected by two-dimensional electrophoresis according to the method of Oferle et al., in addition to the H chain and L chain derived from this antibody, molecular weight
An AFP of 64K and an isoelectric point of 4.7 was observed. All these spots were the same in the PLC culture supernatant and the solubilized membrane. C Liver cancer cell line PLC membrane enzyme and drug treatment binding: PLC membrane fraction was treated with trypsin, protease,
When digested with a proteolytic enzyme such as thymotrypsin, the binding property of this antibody decreased or showed a tendency to decrease. Treatment with "Triton X100" also showed a tendency to decrease, but treatment with ligase conversely increased the binding.

[Brief explanation of the drawing]

FIG. 1 shows the results of testing whether AFP inhibits the binding of the antibody to the PLC cell line membrane using the monoclonal antibody according to the present invention.

Claims (1)

[Claims] 1 Hybridomas obtained by cell fusion of mouse spleen cells immunized with α-fetoprotein (AFP) and mouse myeloma cells are produced,
A monoclonal antibody that recognizes an epitope present on the surface of AFP-producing cell lines.