JPS6356299A - Tumor-related antigen - Google Patents

Abstract

PURPOSE:To obtain a tumor-related antigen having a specific molecular weight and isoelectric point, present in a human tumor organism without existing in an erythrocyte in a adult peripheral blood, lymphocyte, etc., and useful as a remedy for tumor and a marker of diagnosis. CONSTITUTION:A culture medium containing a lymph node cell containing production cell of human immunoglobulin obtained from a perifocus of stomach cancer and B cell strain ATCC.CRL-8118 is incubated in a well of microtest plate to provided a culture supernatant, which is then added to a target cell strain KATO-111, etc. A washing liquid containing biotinated antihuman kappa, etc., and then a washing liquid containing vectastain ABC reagent are added to the resultant reaction liquid and reacted with said reaction liquid and the reaction product is subjected to washing treatment and cloning to provide an antibody-producing cell strain 418-59, from which an antibody is then obtained. The resulting antibody 59 is brought into contact with a gel membrane containing an antigen originated from a stomach cancer cell strain MKN-45, subject to antigen-antibody reaction and stained by an enzyme immnostaining method to provided tumorrelated antigen, having 45,000+ or -3,000 molecular weight and 4.7+ or -0.6 isoelectric point and combined with the antibody 59.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention relates to a tumor-associated antigen to which antibodies of cancer-bearing patients bind.

(Prior art) The analysis of cancer cell membrane antigens that mouse-derived monoclonal antibodies bind to originated from the hybridoma production technology disclosed by Keller and Milstein, and the antigen analysis of colon cancer by Kobrowaski et al. I was able to proceed.

However, many efforts have been made to discover the autoantigen system of cancer cells using human monoclonal antibodies, but they have not always been successful.

There are various reasons for this, but it is because there is currently no method for efficiently cultivating antibody-producing cell lines.

With human hybridomas, the probability of obtaining fused cells is too low, and chromosomes tend to fall off from the hybridoma. In addition, Epstein Perr virus (hereinafter referred to as G
There are two methods for transformation (abbreviated as BV), but the success rate is low and apart from melanoma antigens, there are no specific examples (abbreviated as BV).
= Scarce.

In addition, it is currently impossible to induce specific antibody-producing cells using a human lymphocyte culture system, and the probability is that the desired antibody-producing cells exist in the lymphocytes of cancer-bearing patients at a sufficient level that they can be utilized. I can't help but hope that.

However, the autoantigen system of cancer cells is one of the targets of the cancer elimination function carried out in the human body, and is considered to have great industrial utility value.

(Points of the Invention) The present inventor has succeeded in culturing and maintaining a cell line that produces a human monoclonal antibody that binds to tumor cells, and has demonstrated that the antigen to which the antibody binds exists in gastric cancer tissue. In addition to confirming the new molecular weight 4 from the gastric cancer cell line MKN-G!
tJ-cOO±3θ0θ,.

It was isolated as a tumor-associated antigen with an isoelectric point of 2±0.6.

(Means for carrying out the present invention) The present inventor has already invented a method for infinitely proliferating human immunoglobulin-producing cells using only human cell lines, which is different from the conventional technology, and has filed a patent application (patent application). Ganshojter/J
'? B).

In hopes that cells that produce antibodies that bind to gastric cancer cells exist around cancer lesions in gastric cancer patients, a patent application was filed in 1983.
-/♂ Zoro 22 (2) Repeat the described method,
The desired antibody-producing cells were established and a patent application was filed (patent application 1986).
−, 2/4t4t♂θ). Then, a monoclonal antibody was obtained, and the antigen that binds to the antibody was isolated from the gastric cancer cultured cell line MKN-
4tj.

Hereinafter, means for implementing the present invention will be specifically introduced.

(1) Obtaining cells that produce human immunoglobulin from lymph node cells of gastric cancer patients.
Cut into small pieces using a 10θ mesh stainless steel sieve. Cells are suspended in Hank's solution to obtain 30 million lymph node cells containing human immunoglobulin producing cells.

(Preparation λ of 21B cell line ATc-C-CRL-♂//♂
Using a medium RPMI-/4gθ containing fetal comb serum (hereinafter abbreviated as FC8) of θ, the cell line is grown with good survival rate in a carbon dioxide gas incubator.

(8) Co-culture 70,000 lymph node cells obtained in step (1) and λ0,000 B cell line ATCC/CRL-J in each well of 30 9-well microtest plates. = θθμ selection medium containing ?//♂ (0.1mM hypoxanthine, θ, 4tAM aminopterin, /OμM thymidine, 0.7mM repamisole and λθ Fe2
Dispense RPMI −/64tO containing RPMI −/64tO. ) These microtest plates were cultured in a carbon dioxide gas incubator for 20 days, and colony formation was observed. The reactivity of the antibody in the culture supernatant of the well was examined.

(4) Target cell line KATO-III, MKN −/
and enzyme immunostaining of cell lines that produce antibodies that bind to either KATO-III, MKN-gj, or MKN-gj. MKN-4t
j for each! The culture supernatant obtained in step (2) (8) was added in 50 μm portions and allowed to react at room temperature for 7 hours with gentle stirring.

A U-bottomed microtest plate was placed in a phosphate-relaxed saline solution containing 0.2'11 bovine serum albumin (hereinafter referred to as
(abbreviated as washing solution) (: After washing 7 times, each !μf/
d concentration of piotinylated anti-human kappa and lambda (goat) antibodies were dissolved! Dispense θμl of washing solution into each well. After reacting again for 30 minutes at room temperature with gentle stirring, washing was performed seven times.

Step 3 Vector Laboratories, Bellingham, USA
Inc.

Pectastin ABC reagent consisting of a biotinylated peroxidase-avidin complex from Burlingame, [JSA] was added to the washing solution containing 2 parts, and after reacting gently at room temperature for 30 minutes with stirring, the mixture was incubated twice. Perform cleaning operations.

Diaminobentidine solution (diaminopentidine at 0, s iy/mA and θ,
0,! Tris (slow-tipping solution containing 70% hydrogen peroxide)
After adding 0μ aliquots and reacting at room temperature for 70 minutes, the staining properties of the target cell line were observed using an inverted microscope.

As a result,! A positive reaction was obtained in which the target cell line was stained brown in the well, but when the reaction was investigated again 5 days later,
<t/l! ? The results were reproduced only for /phenoν named -39.

(Cloning of 51g / / -jri cell line! In each well of a 96-well microtest plate, θ, 3 cells of the cell line in the well named G/♂-!ri and θ, / RPM with 20% FCs containing sheep red blood cells
I-/14tO medium/θ0μ■ was added and cultured for λ weeks. The reactivity of the culture supernatant of the emerging colony was examined again using the method described in (4) using the MKN-1'j target cell line, and the presence of antibodies that similarly bound to the target cell line was proved. The globulin class of this antibody (hereinafter abbreviated as ``antibody'') is IgM lambda type.

Cloned Gu/♂-! The antibody-producing cell line, named Wa, is currently housed at the European Collection Opportunity, Wiltshore, Chillispary, Bortondakun, UK.
Animal Cell Cultures° (formerly known as National
Collection Op°Animal Cell Cultures,
European Co11ection of An
imal Ce1l Cultures.

Porton Down, 8alisbury, W
illtshire, Llnited Kingdom
) (deposit number ♂j07)03ノ), and is ready to be disclosed as the basis of the invention at any time.

(6) Analysis of antigens present in gastric cancer cell lines! By using this antibody, the molecular weight of the antigen present in gastric cancer cell lines can be determined.

A sample of gastric cancer cell line MKN-1'j dissolved in 7 volumes of sodium lacryl sulfate (hereinafter abbreviated as SO8) and reduced with Komel Kabuto ethanol was prepared in the presence of 8D8!
Electrophoresis (hereinafter abbreviated as PAGE) using a polyacrylamide gel (hereinafter abbreviated as PAGE) (second electrophoresis) to separate the gel onto a nitrocellulose membrane (second transfer). Screening using cell bodies for the presence of J″99 antibodies that caused an antigen-antibody reaction upon contact
Detection is performed using the same enzyme immunostaining method used in 2.

The results using this method showed that the antigen to which the Polygonum antibody binds (
The molecular weight of the polygonum antigen (hereinafter abbreviated as Polygonum antigen) is 4tjθθθ±
It is 3000. This molecular weight is not changed by the reduction operation with 11-mercabutoethanol.

The molecular weight of the antigen is the same when the gastric cancer cell line NATO-111 is used as when MKN-Ej is used.

In addition, gastric cancer cell lines MKN-i and MKN-113 (cell lines established from gastric cancer tissue and subcultured by Mr. Haruto Hojo et al. of the First Department of Pathology, Niigata University School of Medicine) and N
ATO-Ill (a signet ring cell carcinoma-derived cell line established by Mr. Morimasa Sekiguchi of the Institute of Medical Science, the University of Tokyo, and subcultured) is not distributed by the creator of each cell line. It can also be purchased from Immunobiological Research Institute Co., Ltd., located in Nakaza Higashida, Fujioka City, Gunma Prefecture.

(7) Present in formalin-fixed paraffin-embedded human tissue sections! Detection of antigens present in formalin-fixed, paraffin-embedded tissue sections of well-differentiated and poorly differentiated gastric cancers! The distribution of the antigen was determined by deparaffinization using xylene and alcohol using a conventional method.
(It can be detected using the Vectastin ABC reagent and diaminobenzidine solution shown in 41.) From the results of m-tissue staining, the antigen is present in large amounts (= present) in cancer tissue in both well-differentiated and poorly differentiated gastric cancers. Particularly, in the case of well-differentiated adenocarcinoma, the membranous part of linear epithelial-like cells (: is localized. In addition, no warts are observed in the stromal tissue surrounding the cancer.
A small amount of polygonum antigen is observed in normal gastric mucosal epithelium.

(8) Reactivity of the Polygonum antibody to blood cells The reactivity of the Polygonum antibody to adult peripheral blood cells was determined using the method shown in +41 and the anti-Human IgM antibody conjugated with the fluorescent dye FITC. This can be investigated using the fluorescent antibody method. from this result,! Antigen that reacts with antibodies is not present in any appreciable extent in human red blood cells, lymphocytes, granulocytes, and monocytes.

(9) Reactivity of the !9 antibody against lung cancer cell lines (2) As a result of examining the reactivity with the lung cancer cell lines using the method shown in (4), the !9 antibody It also reacts with moderately differentiated squamous cell carcinoma (obtained from Immunobiological Laboratory Co., Ltd., Nakaza Higashida, Fujioka City, Gunma Prefecture).

This suggests that the antigen is also present in tumors other than gastric cancer.

αO gastric cancer cell line MKN-41! , from j! Isolation of antigen Gastric cancer cell line MKN-4tj was treated with nonionic surfactant N.
Solubilize with P-4tO, and the solubilized product! The antibody is immobilized on Sepharose. Subsequently, the antigen can be isolated by elution with an acidic glycine-hydrochloric acid solution.

The antigen was obtained using a similar method (from the second stage, the gastric cancer cell line KATO-1
11 was also isolated. The antigen is present in the presence of the above-mentioned 8DS and! It is a substance whose main component is protein, because it is electrophoresed by PAGg and stained well with Coomassie Brilliant Blue G-230.

al)! The isoelectric point of the protein is determined by LKB Co. (LKB-Productor A).
B, Promma, Sweden, LKB-Product
er AB, Bromna, Sweden) by isoelectric focusing using carrier amphorain (pH range 3.3-10). 0. The isoelectric point in the presence of the nonionic surfactant Tween θ (T W E W N , 2θ) of /4 is G,Z±0.6.

(Effects of the Invention) Antigens are antigens present in cancer cells to which humans can make an immune response, and are considered to be extremely valuable as markers for cancer treatment and diagnosis.

(Example) (1) j? Isolation of Antigen A single method for isolation of antigen using antigen-antibody reaction PMI-/
The cells were cultured and proliferated at Otag θ to obtain IO to the 70th power of cells.

2)! 0rnl of phosphate buffered saline (PBS)
(abbreviated as) (2. The cells obtained in step -1) were added to a solution containing NP-90 at a final concentration/mM, phenylmethylsulfonyl fluoride, orthophenylenediamine, and EDTA at a final concentration/mM. The cell components were dissolved by shaking at g°C for 2 hours. Subsequently, centrifugation was performed for 10 minutes at 1/2 θθ rpm, and the supernatant was collected.

・3) Amicon Corporation (Amicon Corporation, Lexington, Massachusetts, USA A+n1con
Corporation, Lexington, MA
O, 2/73.

The supernatant was concentrated using a PM-10 ultrafiltration membrane (LISA), filtered and washed with PBS 1: and 3-closed. (Estimated NP-4tO concentration at this time is 0.07%) 4) 59 antibody of /η subjected to affinity purification, /
7! Pharmacia Fine Chemicals, Uppsala, Sweden, Pharmacia
Fine Chemicals AB, Uppsa
la.

CNB r-activated Sepharose 4t from Sweden)
B/- (Two conventional methods (two-way binding) to Sepharose immobilized with !de antibody.

5) Add the entire amount of antibody-immobilized Sepharose obtained from the antigen solution obtained in 3) (2.1.4) and heat at 9°C! Shake for hours to allow antigen to bind. Next, transfer the j9 antibody-immobilized Sepharose to the column, and! Wash well with 0-PBS,
Elution was carried out with two volumes of 0.2M glycine-hydrochloride buffer.

6) The eluate was diluted to 0. / PBS containing NP-4tO (dialyzed twice!) and used as an antigen preparation.

7) In the presence of the standard SDB, male and female PAGB (secondary electrophoresis was performed and its purity and a box were assayed.
Approximately 2
It was found that 0 micrograms of 59 antigen was obtained.
Ta.

8) Using a column for isoelectric focusing with a total volume of 10 micrograms and a carrier amphorain (pH range 3.3-/0), transfer the equivalent of 10 micrograms of the standard to a non-ionic solution of θ, / Surfactant (TVI/EEN,
Isoelectric focusing was performed in the presence of 2(7) at 3,jtOV for 72 hours. After completion, 30 fractions (double fractions) were measured, and the pH of the fractions was measured, and the presence of J9 antigen was detected using the same method used in the analysis of the antigen present in (6) Gastric cancer cell line C. As a result, !The isoelectric point of the antigen was 7±θ, 7.

(2)! Reaction of anti-antibodies to blood cells Out of the adult peripheral blood of children whose coagulation was inhibited with heparin, 10-/θ
Centrifugation was performed at θQ rpm for θ minutes to obtain a precipitated red blood cell layer and a white deposited granulocyte layer (referred to as a buffy coat). After washing each cell fraction twice with PBS, , was used in the following experiments.

In addition, after diluting the remaining /θ- with twice the amount of PBS,
/! - Ficoll Metrishade high specific gravity liquid (specific gravity /, 077)
It was quiet (double 11 shi) on the top.

Subsequently, the container was centrifuged for 30 minutes at an acceleration of 1,000 to obtain a white cloudy interface cell fraction consisting of lymphocytes and monocytes. This cell fraction was centrifugally washed twice with PBS,
It was used in the following experiments.

Place 1 million cells of each fraction into a centrifuge tube (== volume), centrifuge the supernatant, and then transfer to a 70μ2
Culture supernatant/- containing antibodies was added, and antigen-antibody reaction was carried out at room temperature for 7 hours.

The culture supernatant was removed by centrifugation, and centrifugal washing was performed twice with washing solution, followed by fluorescent secondary antibody (goat anti-human IgM conjugated with fluorescent dye fluorescein isothiocyanate) diluted 20 and 50 times. Antibody) solution/- was added and allowed to react at room temperature for 30 minutes.

After removing the antibody solution by centrifugation, the cells were washed once and the degree of staining of the cells was observed using a fluorescence microscope.

As a result, the antibody No. 9 showed no significant reaction with any of the cell fractions of red blood cells, lymphocytes, granulocytes, or monocytes obtained from nine different adult peripheral blood samples.

Patent applicant: Asahi Kasei Kogyo Co., Ltd. Procedural amendment (method) % formula % 1. Indication of case 1986 Patent application No. -2000♂
No. 0 No. 2, Name of the invention Tumor-related antigen 3, Relationship with the person making the amendment Patent applicant No. 1-2-6 Dojimahama, Kita-ku, Osaka-shi, Osaka Prefecture゛＼, -No. 4, Full text of the specification subject to the amendment 5. Details of the amendment as shown in the attached sheet.

Claims (1)

[Claims] Molecular weight 4, present in human tumor tissues but not present in red blood cells, lymphocytes, granulocytes, or monocytes in adult peripheral blood.
5000±3000, tumor-associated antigen with isoelectric point 4.7±0.6