JPS6078998A - Monoclonal antibody of anti-carcinoembryonic antigen and its use - Google Patents

Abstract

NEW MATERIAL:A monoclonal antibody produced by a hybridoma system having specificity to carcinoembryonic antigen. USE:A cancer diagnozing reagent for immune determination of carcinoembryonic antigen. PREPARATION:For example, Hank's solution containing polyethylene glycol is added dropwise to a lymph node cell around focus of mammary cancer and an azaguanine resistant strain of human lymphoblast RPMI-1788, ATCC No. CRL8118, and they are subjected to cell fusion to give a hybridoma strain. The hybridoma strain is cultivated and multiplied in a medium containing 4X10<-7>M aminopterin, 10<-4>M hypoxanthine, and 1.5X10<-5>M thymidine, it is subjected to cloning by limiting dilution method. The cloned hybridoma strain is cultivated in a medium containing bovine fetal serum for 48hr, the supernatant liquid is adsorbed on Sepharose column with immobilized anti-human IgG antibody, and it is eluted with an eluting solution to give a monoclonal antibody.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a human monoclonal antibody produced in a hybrid cell line that is characterized by specificity for human carcinoembryonic antigen, and the use of the human monoclonal antibody. The present invention relates to a method for purifying human carcinoembryonic antigen and a method for immunostimulating human SIA fetal antigen.

In recent years, advances in theory and research techniques have led to significant progress in elucidating the immune response system of mammals.

In particular, since the discovery of a method for producing mouse monoclonal antibodies by cell fusion, it has become possible to obtain human monoclonal antibodies in less than 10 years.

The present inventors made full use of cell fusion technology to develop human carcinoembryonic anti-IIL (carcinoembryonic ant IIL).
With the aim of obtaining a miscellaneous a#l [I cell strain that produces human monoclonal antibodies specific for CEA (hereinafter abbreviated as CEA), the present invention has been completed after extensive research.

The human carcinoembryonic antigen referred to herein means a substance called CIA. CIA is Gold and Freedman
Blood C, E A
There is already a well-established reputation that the measurement of concentration is sufficiently useful in clinical practice, especially in follow-up after surgery and in determining the effectiveness of radiotherapy, chemotherapy, etc.

Since CEA is a protein with an extremely high sugar content, with a molecular weight of approximately 200,000 and a sugar content of approximately 50%, its structural Wf analysis has not progressed sufficiently, but the terminal amino acid residues of about 20 residues have not been elucidated. ing. (Refer to Cancer and Chemotherapy, August 1985, pp. 1754-1762) Research has already been carried out to create monoclonal antibodies against CEA in mice, but it has not been possible to give lymphocytes of cancer-bearing patients the ability to proliferate indefinitely. Although it was a desire to obtain an autologous monoclonal antibody against CEA, the desired autoantibody-producing lymphocytes were rare and could not be achieved using conventional techniques.

The present inventors have compared and studied various combinations of cell fusion methods, and have also conducted frequent cell fusion studies. As a result of the joint experiment, surprisingly, despite the fact that the existence of autoantibodies against CEA was not known, among the colonies of hybrid cells that had grown '#, we found that the hybrid cells produced anti-CEA monoclonal antibodies. I got the stock and got a 4+. Furthermore, by culturing the Mfl cell strain, it was possible to isolate an anti-CEA monoclonal antibody from the culture supernatant.

That is, the present invention provides human J+! /+ A human monoclonal antibody produced by a hybrid vession system characterized by % isomerism to fetal antigens.

To create a hybrid cell line, the cell line that should be used is:
It is preferred to use cell lines of mammals selected from the group of mice, rats, humans, guinea pigs, rabbits and monkeys. The reason for this is that it is possible to suppress the chromosome elimination phenomenon that occurs within the fused hybrid cell.
This is because.

Among these, the use of human cancer cell lines is effective because it is possible to create homogeneous hybrid cell lines, and the phenomenon of chromosome elimination within hybrid cells can be suppressed. Among human cancer cell lines, %n fc disclosed in Japanese Patent Application No. 57-97596
-H) It is preferable to use a cell line in which B#lil cells have been transformed into IJ+m, have immunoglobulin on the cell surface, and release the immunoglobulin into the culture medium. The reason for this is that even though the cell line is 11l'A, it retains traits that are extremely similar to those of human antibody-producing cells, so the traits of both types in the hybrid cell line that are formed may eliminate each other. This seems to be because it is relatively easy to balance the two.

Preferably, 7cATCC@CRL811B disclosed in Japanese Patent No. 57-97596 has a high 4411 cell fusion efficiency and is easy to use as a hybrid cell line.

There is no particular method for cell fusion, and any method such as using a polyethylene glycol solution or electrical stimulation may be used, but the method using a polyethylene glycol solution does not require any special equipment and is easy to use. .

Method of cell fusion using polyethylene glycol solution,
and the HAT selection method, which is a simple selection method for hybrid cells,
In addition to what is stated in the aforementioned Japanese Patent Application Laid-Open No. 57-97596,
Sokoki “Animal tissue culture method J Yukiaki Kuroda, Kyoritsu Shuppan (197
4) It is preferable to use this method in detail also for pages 313 to 329.

The cell line ATCC-1% CRL811B is a strain deficient in enzymes hypoxanthine, guanine, phosphoribosyl, and trannuferase, and is easy to use for selecting hybrid cell lines.

Cell colonies grown in HATt@ground are considered to be hybrid cells, and their reactivity to cEA is examined.

At this time, the colon adenocarcinoma cell line C0LO-205 (ATCC odd number C
CL・222) is used.

5 to prevent the strain from detaching from the polystyrene plate.
Cells are denatured and fixed in an aqueous solution containing 0% acetone.

The culture supernatant of the hybrid cell line was applied to target these cells, and after thorough washing, a peroxidase-conjugated anti-human immunoglobulin serum (made by rabbit) solution was added. An enzymatic reaction is performed to select anti-CEA antibody-positive hybrid cell lines.

By this operation, the positive hybrid cell line is diluted to about 6 cells/- and cultured. By this operation, it is possible to obtain a hybrid cell line in a monoclonal state.

Culture supernatant of the cell line purified to this condition and colon gland mucus '1M
The reactivity of the 1M perchloric acid-intoxicating fraction with CEA, which was separated and purified by the method of Gold et al., was examined by the Ophthalony method. IgG with anti-CEA activity used for this operation
The cell line could be identified as a type 1'4i cell line producing type 1'4i antibodies.

In addition, the culture supernatant of the hybrid h'1 cell strain was adsorbed with IgG using a column immobilized with anti-Human IgG antibodies, and then p
I (during elution with glycine-HCl buffer in step 3),
Anti-CEA antibodies can be obtained.

By this method, anti-CEA antibodies of any class can be obtained, but IgG% IgM and IgA type antibodies are easy to purify and are easy to use.

The present inventors analyzed lymph node cells surrounding breast cancer lesions 8
106 cells and the same number of ATCC code CRL 811B were fused in a polyethylene glycol solution, and the proliferated hybrid cell line was subjected to cloning operation. After fc, the reactivity of the antibody against CEA was assayed, and it was found that D25D2 and We were able to obtain a complete cloned cell line producing the named anti-CEA antibody. D25. B2 belonged to the subclass of IgG, and the 1L chain showed a kappa type. When this strain was cultured for 24 hours at a concentration of 106 antibodies/- in the medium RPMI+ 1640 (containing 10 grams of fetal bovine serum), it produced approximately 10 μg/- of antibodies.

An application was made for depositing this strain at the Institute of Microbial Technology of the Agency of Industrial Science and Technology, but the deposit was rejected on the grounds that it was outside the range of microorganisms that could be deposited. However, D25B2 is being cryopreserved in liquid nitrogen and is now ready for distribution.

025B2 culture supernatant 500- was treated with anti-human IgG antibody (
After immobilizing Lp (produced in rabbits) and adsorbing it on a 2 ml fc Sepharose column, 5g of p)13
, eluted with 0.1M glycine-hydrochloride buffer, dialysis and freeze-drying to obtain 11 ng of anti-CEA antibody.

This was again bound to CNBr-activated Sepharose 6MB to form an affinity column, and 1M peroxylic acid soluble fraction (200 ml) obtained from 100f colon tissue was passed through this column. 5-3 MK
Phosphorous buffered saline containing SCN (pH 7,4
), followed by dialysis and freeze-drying. This gold is made by Zymed Laboratory, Inc.
Laboratory Inc, 5outh 5an
Rabbit anti-CEA (I
gG7) Using phosphatase-conjugated anti-CEA, the content of CEA was 11%1.
When the voltage was constant for 1 time, it was 5 μV.

From the above results, the anti-CEA human monoclonal antibody
The scale used for adsorption purification of A was shown.

The anti-CEA antibody isolated from D25B2 by the method described above was treated with peroxidase using the two-step glutaraldehyde method (Naritai 1 acetic acid immunoassay method), edited by Eiji Ishikawa et al., Igakushoin 197.
(Refer to 8th edition pss).

Using this specimen, an attempt was made to determine the immunodeficiency of CEA. Ahot
Abbott Laboratories
esNorth Chicago USA) L j) CEA measurement using the enzyme immunoassay method obtained Using Kind, a standard curve for Abbott's standard CEA was created using a defective product as a substitute for the enzyme and antibody. Abb
The results were similar to those obtained with the enzyme-labeled antibody of ott (+-1).The above results did not indicate that the antibody could be used satisfactorily in the immunoassay of CEA.

From these results, the usefulness of this antibody is clear, and missile therapy using VC conjugated with a cytotoxic substance,
It is also possible to apply this method to imaging of %+ ir diary using the "Isotho" label.

Example 1 Culture medium RP containing azaguanine-resistant strain ATCC I11 CRL811B of human lymph bud ffRpMI-1788 and 10% beef tallow ff1l (hereinafter abbreviated as FC8)
C suspended in MI-1640 with a carbon dioxide concentration of 5%
Increase the culture to 8 x 1 in an O2 incubator.
A parent strain of 0' cells was collected.

Regional lymph nodes obtained during breast cancer removal are
i The cells were fragmented and separated using a 100-mesh stainless steel mesh, and 8x10% of lymphocytes were collected aseptically.

Both cells were washed with a Hakuras group, and the MII cells were pelleted under centrifugation conditions of 1600 rpm, and 4
Class 1 (p) containing 5% polyethylene glycol
H8,2) 2m7! It dripped slowly.

Subsequently, the yutsukushi and polyethylene glycol were diluted with 20% of the medium RPMI-1640, which does not contain white matter components. This 4-a solution was subjected to gentle centrifugation conditions,
A fused cell population was obtained.

Aliquot 1 cells into a 24-well tissue culture plate and add 4x10-7M aminopterin 10
Medium RP containing -'M hypoxanthin and 1.5 x 10-5 M thi, midine and 20% Fe2
1 d of MI-1640 was also dispensed into each well.

Half of the medium was replaced every 2 days, and after 3 weeks of incubation, observation using an inverted microscope revealed that human immunoglobulin could be strongly detected in the culture supernatant in 10 wells.

On the other hand, the human colon adenocarcinoma cell line C0LO-205 was cultured in a microtest plate with a total of 96 wells.
When cells grew to 4 cells/well, the cells were denatured and fixed by contacting with an aqueous solution containing 50% acetone for 10 minutes.

Using the fixed cell line as a target, 100 μt of each culture supernatant in which approximately one hybrid cell was growing was added to a 96-well microtest plate, and after standing for 6 W), the culture supernatant was added to a 96-well microtest plate. and washed 5 times.

peroxidase-conjugated rabbit anti-human) IgG, M, A antibodies diluted 1:1000 in this plate]
33 mM containing % Fe2! J acid buffer (pH 6
, 5) was added to the solution at 100μ, and the mixture was left to stand for 2 hours.

Subsequently, after washing six times with phosphate buffered saline, orthophenylenediamine'f8f1soμ was added.

added. After standing for 50 minutes, color development at 450 nm was measured to examine the reactivity of the culture supernatant to the fixed cells.

Here, reactivity was observed in the well named D2, and peroxidase m-conjugated IgG, anti-IgL, and anti-Ig
As for the immunoglobulin class analyzed using A, it was found that IgG is the main component of its reactivity.

After growing in culture hole D2Q, 3 cells/rn1. Dilute to
100 μt each was dispensed into a 96-well microtest plate and allowed to grow.

In this state, the hybrid cell line that grew uniformly as a colony was picked up using a microcapillary, transferred to a new microtest plate, allowed to grow, and a subline that was positive for C0LO-205 was again selected and named D25B2. . When we searched for the subclass of this substance, it was identified as IgG2, and L
The chain was copper-shaped.

When the reactivity of the culture supernatant of this strain with CIA, which was prepared by separating the 1M perchloric acid soluble fraction of colon adenocarcinoma tissue according to the method of Gold et al., was examined using the Ophthalony method, a sedimentation line was observed. was observed, and it was concluded that there was an antigen-antibody reaction between 025B2 and CIAO.

The strain was cultured for 48 hours in RPMI-1640 containing 10 g of Fe2 at a concentration of 106 cells/-, and 300 ml of supernatant was cultured.
je, anti-human) IgG antibody (engineered for rabbits) was immobilized on 2rnl of Sepharose Calano (・After adsorption treatment with a trowel, elution was performed with 5me of pH 3, 0, 1M glycine-hydrochloride buffer. , dialysis and maintenance MLII 'Ij:'
,,Dried. This dried product had a size of 1.0 m9 and was an antibody having anti-CEA activity.

Claims (1)

[Scope of Claims] A human monoclonal antibody produced by a hybrid cell line characterized by 11+% isomerism to a human fetal antigen. +21 A human monoclonal antibody tl- produced in a hybrid cell line having specificity for human carcinoembryonic antigen, which is characterized in that it is used for purification of human m-fetal antigen. Method. (31) A method for immunoassay of human carcinoembryonic antigen, characterized in that a human monoclonal antibody produced by a hybrid cell line having specificity for human carcinoembryonic antigen is used for immunoassay of human carcinoembryonic antigen.