JPS61167699A - Monoclonal antibody - Google Patents

Abstract

PURPOSE:The titled antibody recognizing a cell surface antigen of IgG1 class showing positivity in a specific cancerous cell, produced by a hybridoma prepared by using a cell strain (MKN-45) derived from glandular carcinoma of stomach low differentiation type as an immune source. CONSTITUTION:An extracted solution of a cell of cell strain (MKN-45) derived from glandular carcinoma of stomach low differentiation type is subjected to gel filtration to give a high polymer fraction having about 700,000-1,500,000 mols.wt. which is blended with Freund Complete Adjuvant, and the blend is used as an immune source. For example, a mouse is immunized against the immune source, the spleen of the mouse is taken out, subjected to cell fusion with mouse myeloma p3U1 to give a hybridoma. Then, cloning is carried out several times by limiting dilution method, etc., and from the supernatant liquid of the culture solution is obtained the aimed antibody having the following properties. The Ig class (Light chain):IgG1(K), recognition antigen type:cell surface antigen, positive in the following cancerous cells, esophagus cancer (TE-3), lung cancer (MKN-45), (KATO-III), and liver cancer (HEK).

Description

[Detailed Description of the Invention] [Technical Field] The present invention relates to a cell line derived from gastric poorly differentiated adenocarcinoma (MKN=45).
relates to the production of hybridomas created as immunogens to produce monoclonal antibodies with specific properties.

[Prior art]

It can be said that the ultimate goals of cancer research are the search for substances that exhibit anticancer and anticancer effects and the early detection of cancer, that is, the establishment of early diagnostic methods. To date, various drugs, treatments, and reagents have been developed for cancer, but all of these affect not only cancer cells but also normal tissues and cells, and no matter how effective they are, they At present, its use is severely restricted due to side effects.

Immune reactions (antigen-antibody reactions) are highly specific, but with conventional polyclonal antibodies, no matter how much you manipulate the absorption procedure, they can only detect very minor antigens, such as a subset of lymphocytes. It was difficult to recognize what was distinguished by determinants. Milstei
The monoclonal antibody (Ki; h
ler, G. and Mtlstein, C.:
Nature, 256.495 (1975)) aims to break through this barrier by obtaining monoclonal antibodies that specifically recognize cancer-specific antigens on cancer cells or cancer-related antigens, thereby preventing damage to normal mm. It is expected that cancer cells can be specifically eliminated without causing damage. Furthermore, diagnostic agents or test reagents using monoclonal antibodies are thought to be capable of detecting cancer-related antigens and cancer-specific antigens with high sensitivity without cross-reactivity with normal serum components.

[Problem that the invention seeks to solve]

The present invention provides monoclonal antibodies that specifically react with specific cancer antigens.

Furthermore, the present invention provides a reagent for detecting an antigen against the above-mentioned specific cancer.

[Means for solving problems]

The present invention consists of a monoclonal antibody that specifically reacts with gastric cancer, particularly gastric cancer cell membrane antigens.

The monoclonal antibody of the present invention is produced by so-called cell fusion. That is, a fusion hybrid is formed between an antibody-producing cell and a myeloma cell, and the hybrid is cloned to produce an antibody specific to the cancer cell (i.e., a specific antigen having the above characteristics). Manufactured by selecting. The operation may be carried out in accordance with conventionally known methods, except for using the following cells as immunization cells.

Antibody-producing cells include, for example, lung cells, lymph node cells, and B cells from animals immunized with antigens obtained from established cancer cell lines.
-They are lymphocytes. An example of the cancer cell line is a cancer cell line derived from poorly differentiated adenocarcinoma (MKN-45).

Examples of animals to be immunized include mice, rats, horses, goats, and rabbits.

Antibody-producing cells are produced, for example, as follows. That is, the gastric cancer-derived cell line MKN-45 (poorly differentiated adenocarcinoma) was destroyed by ultrasonication, etc., and centrifuged (e.g., 10,000 to 2
0.000 G for 10 to 60 minutes) to obtain a cell extract, and transfer this supernatant to a gel filtration carrier capable of separating substances with a molecular weight of 100,000 to 2,000,000 (e.g., Sephadex, Sephacryl, Sephacose, Biogel). etc.) to separate into a high molecular fraction and a low molecular fraction. The polymer fraction with a molecular weight of about 700,000 to 1,500,000 obtained by
After mixing with T, it is used for animal immunization. For immunization, administer approximately 1.5 x 10' subcutaneously, intramuscularly, or intraperitoneally to the animal.
~10" cell equivalent/time 1-2 times a week, 3-7
It is carried out by weekly administration. Approximately 3 times more than the final immunization
~5 days later, antibody-producing cells are collected from the immunized animal.

As myeloma cells, those derived from mice, rats, humans, etc. are used. Preferably, the antibody-producing cells and bone marrow cells are derived from animals of the same species.

Cell fusion can be carried out using e.g. G, K5hler (Natur
e 256+495 (1975)) or a method analogous thereto. At this time, 30 to 5
0% polyethylene glycol (average molecular weight 1,000~
4,000) at a temperature of 30 to 40°C, about 1 to 3
This is done by allowing the reaction to take place for about a minute.

Cells obtained by cell fusion are subjected to cloning. That is, the cells are cultured in, for example, a microplate, and the antibody titer in the culture supernatant of wells in which proliferation is observed is subjected to cloning using, for example, an enzyme antibody method to obtain clones. This clone is transplanted intraperitoneally, for example, into a BALB/C mouse that has been previously administered pristane, and 10 to 14 days later, ascites containing a high concentration of monoclonal antibodies is collected and assayed. The obtained clones are then subjected to screening for clones that produce the desired monoclonal antibody. Screening is followed up by enzyme-linked immunosorbent assays and the like.

The monoclonal antibodies produced by the selected clones can be collected using ammonium sulfate fractionation and PE, which are conventionally known methods for purifying IgG.
This can be easily achieved by applying G fractionation, ethanol fractionation, and anion converters.

〔Effect of the invention〕

The monoclonal antibody obtained by the present invention is thought to be specific for adenocarcinoma, recognizes cell membrane superficial antigens, and does not react with cultured cells derived from normal tissues, red blood cells, white blood cells, or normal tissues, and does not react with cancer cells. It is presumed that it recognizes a specific antigen. That is, the monoclonal antibody according to the present invention can be used for tumor imaging and tumor imaging.
Clinical applications such as tartetins+theraov (targeting therapy) are expected.

Example (1) Preparation of cancer-related antigen for immunization: Gastric cancer cell line (MKN-45 strain) was disrupted by ultrasonication and centrifuged (15.0 OOG, 30 minutes) to obtain a cell extract. . This supernatant was gel-filtered using a Sepharose 4B column and separated into a high molecular fraction and a low molecular fraction.

The polymer fraction with a molecular weight of about 700,000 to 1,500,000 was
After mixing with nd Complete Adjuvant,
Mice were immunized once a week for 5 weeks.

Four days after the final immunization, mouse lungs were removed and used for the following cell fusion.

(2) Cell fusion and cloning: The above mouse tile cells and mouse myeloma P3U 1
(Curr, Top, Microbiol, I
mmunol, +81+1 (1970)) and 4
: 1 and followed the method of K15h fen et al. (Im
Munologcal Method (Acade
nic Press), New York, 39
1. (1979)) was partially modified, and cell fusion was performed by reacting for 2 minutes using 42.6% polyethylene glycol (average molecular weight 1.000).

These cells were implanted into a 96-well microplate, and HA
After culturing in T medium (Table 1) for 19 to 14 days, the cells were transferred to HT medium (Table 1), and when they could be cultured in flasks (25cIA), they were cultured in D-MEM medium (Table 1). The antibody titer in the culture supernatant of wells in which proliferation was observed was measured by enzyme antibody method, and the desired hybridoma was cloned from appropriate wells by limiting dilution method.

i.e. 2 per well in a microtiter plate.
5.000 mouse peritoneal exudate cells were implanted and then D
-MEM medium, 10.5.2.5.1 pieces 10.1ml
The hybridoma was diluted so that 0.1 ml of this was inoculated into a microquiter plate and cultured.

After 4 days, 0.11111 of D-MEM medium was added, and thereafter 4
Half of the medium was replaced once every 7 days. 10 after starting culture
Colonies visible to the naked eye were formed in ~20 days, and a clone strain was obtained.

Table 1 (3) Screening method The obtained hybridomas were screened for clones producing the desired monoclonal antibody as follows.

(b) Explanation of the method Enzyme antibody method was performed as follows.

Add the specimen to a microplate coated with antigen (various cancer cell lines or partially purified cancer-related antigens or normal cells,
After reacting at 37°C for 1 hour and washing, peroxidase-labeled anti-mouse immunoglobulin (IgG + I gA + I
gM) Rabbit antibody was added, and the mixture was further reacted at 37°C for 1 hour. After washing the unreacted labeled antibody, 0-phenylenediamine solution was added, and after reacting at room temperature for 300 minutes, 2M
The reaction was stopped by adding sulfuric acid, and the absorbance at 490n+11 was measured.

Using this method, the reactivity with various cells was investigated. Note that a B-Galactosidase-labeled antibody was used for cross-reactivity with leukocytes. In addition, cross-reactivity with human red blood cells was investigated using the PHA method using a mixture of human type A, B, and 0 red blood cells.

Carcinoembryonic antigen
Cross-reactivity with (CE A) was determined by the PHA method using CEA-sensitized blood cells.

In order to investigate whether the monoclonal antibody recognizes the MKN-45 secreted antigen or the cell membrane antigen, the monoclonal antibody and MK
It was investigated whether reactivity with N-45 cells themselves was inhibited.

Inhibitior+ Te5 using enzyme antibody method
The specific method for determining t is as follows. That is, the supernatant of the hybridoma is subjected to Htration using an enzyme antibody method, and an appropriate dilution rate is determined based on the results. Next, M.K.
N-45 culture supernatant was concentrated 5 to 25 times and used as a stock solution, diluted with t's, t's''...1:5'' and added in equal amounts to appropriately diluted hybridoma supernatant. , incubation for 1 hour at 37°C.

Then, the usual enzyme antibody method (target: MKN-
The assay was performed using the system of 45).

(b) Screening flow Primary screening: Target cell
(MKN-45) and normal-derived cells (Plow 200
%1ell that was positive for MKN-45 and negative for Flow 2000 was selected by enzyme antibody method using 0 ).

Secondary screening: In addition, all assay systems using other normal cell lines, red blood cells, and white blood cells were negative.
Select ell.

Tertiary screening: 2 to 3 cell lines selected above
The culture supernatant was cloned twice and the culture supernatant was used to clone many cancer-derived cells.
In addition to examining the reactivity with ll 1ine, whether the antigen is recognized as a secreted type or a cell membrane type is identified using Inhibition Te5t using an enzyme antibody method.

(4) Collection and purification of monoclonal antibodies (a) The clone strain obtained by the above screening was preliminarily
+al/mouse BALB after 4 weeks of age when pristane was administered
/C into the abdominal cavity of a mouse (male) 2.0-3.0 x 10
' cells/mouse were transplanted, and 10 to 14 days later, ascites containing a high concentration of monoclonal antibodies was collected.

This ascites was diluted 5 to 10 times by adding 0.9% NaC# solution, then ammonium sulfate was added to a concentration of 40%, and the precipitate fraction was collected. This precipitated fraction was dissolved in as small a amount of 0.9% Na (1! solution as possible, then 0.9% NaC
The first solution was dialyzed as an external solution.

After dialysis, high performance liquid chromatography (TSK-G
ELL G-3000511) to obtain an IgG fraction, which was used as a purified monoclonal antibody.

(b) This Lorne strain can be grown in a BSA-containing serum-free medium. That is, the cells were grown in a serum-free medium containing 0.5% BSA (RITC55-9 medium), and the culture supernatant was collected. Add ammonium sulfate to this supernatant to a concentration of 40%, separate the precipitate fraction, and add 0.9%
After adding NaCl solution and dissolving it, ammonium sulfate was further added to a concentration of 40% and the precipitate fraction was collected. This precipitated fraction was dissolved in as small a volume of 0.9% NaCJ solution as possible, and then dialyzed against 0.02M physiological phosphate buffer as an external solution. After the dialysis, this solution was again passed through a 5epharose 4B column bound with an antibiotic fetal serum antibody (rabbit) and then transferred to a DEAE-Cellulofine column.The first peak of DEAE-Cellulofine chromatography was used as a purified monoclonal antibody.

(5) Properties of monoclonal antibodies The properties of the monoclonal antibodies produced by the clones thus screened are as shown in Tables 2 and 3. Immunoglobulin classes were assayed by the Ophthalony method.

Table 2 *The CD didn't cost 1nbition.

Table 3: RP) IA reagent and various human-derived cancer cell line culture supernatants and patent applicant Midori Juji Co., Ltd. Procedural amendment stamp button May 20, 1985 Patent Application No. 8129 2 of 1985, Invention Name Monoclonal Antibody 3, Relationship with the person making the amendment Patent applicant name Midori Juji Co., Ltd. 4, Agent ■541 Address New Life Hirano-cho 406, 4-53-3 Hirano-cho, Higashi-ku, Osaka Telephone (06) ) 227-1156 "Sodium pivalate" in Column 6 of "Detailed Description of the Invention" of the specification, contents of amendment (3) Table 1, page 9 of the same book
is corrected to "sodium pyruvate".

(4) Add "culture" before "supernatant" on page 11, line 10 of the same book.

(5) Add "culture" before "supernatant" on page 11, line 15 of the circular.

(6) On page 12 of the same book, line 8, after “consider”, “[
Measurement was performed using the CELISA method (Ani21 Dimensions in Biological Analysis, p. 376, Blenheim Press).

New York, London, 1980: ANewDi
n+ension in Biological An
lysis+ p, 376+Plenum Pres
s+ New York+ London+ 1980
) according to ]” is added.

(7) On page 14 of the same book, in the second line from the bottom, "rRPHA reagent and culture supernatant of various human-derived cancer cell lines" is corrected to "various human-derived cancer cell lines."

(8) The entire text on page 11 of the same book is corrected as shown in the attached sheet.

Above (Attachment) Patent Applicant: Midori Juji Shisho Co., Ltd. Neho Official Book (Method) % Formula % ■, Indication of the Case Patent Application No. 8129 of 1985 2, Title of Invention Monoclonal Antibody 3, Person Making Amendment Case Relationship with Patent Applicant Name Midori Juji Co., Ltd. 4, Agent 541 Address 406 New Life Hirano-cho, 4-53-3 Hirano-cho, Higashi-ku, Osaka Telephone: (06) 227-1156 b, Tetsuto Egg Ren Day 1-6, "Detailed Description of the Invention" column 7 of the specification to be amended, Contents of the amendment (1) r Milstein on page 2, line 15 of the specification
Correct J to "Milstein j.

(2) Circular page 2, lines 16-17 r (K6hle
r, G.

and Mth 5tein + C,: Naturel as “[Kohler.

Gee and Milstein, C: Nature (K5hl
er, c, and Milstein, c:
Nature) Correct to J.

(3) r Freund Com on page 5, line 1 of the same book.
pleteadjuvantJ is called ``Freund Complete Adjuvant''.
vant) Corrected to J.

(4) r G, K5hler on page 5, line 10 of the same book
(Nature J is ``G, Köhler (G, 5h
ler) (Nature) Correct to J.

(5) Circular page 6, last line - page 7, first line r co
njugate targeting therapy
(targeting therapy)" to "conjugate, targeting therapy
erapy) Correct to J.

(6) r Freund Co, page 7, line 11 of the same book.
＋wplete＾djuvantJ worr complete Freund's adjuvant" is corrected.

(7) r (Curr, To
p, Microbiol, Immunol, +J
``Current Tobical Microbiological Immunology (Curr.

Top, Microbiol, Immunol,)
Correct to +J.

(8) On page 7, line 18 of the same book, rK5hlenJ is corrected to "K6hler J.

(9) r (Immun
ologtcal Method (Acadenic
Press) + New York, J.
(＾caden+ic Press), New Y
ork) + J.

(10) rB-Galact on page 10, line 17 of the same book
osidaseJ as “B-galactosidase (B-Ga
lactosidase) Corrected to J.

(11) Circular page 10, last line r Carcinoe
Carcinoembryonic antigen J
Correct to J.

(12) Circular page 11, line 8 rlnhibitto
n Te5tj as “inhibition test”
bition Te5t) Correct to j.

(13) rtitratio on page 11, line 10 of the same book.
n Correct J to "tight region".

(14) Circular page 11, lines 15-16 r 1ncu
Correct bationJ to "incubation".

(15) r targetJ on page 11, line 16 of the circular
is corrected to "target".

(16) In the same book, page 11, line 17, rassay J is corrected to [assay J].

(17) rTarget c on page 11, line 19 of the same book
Correct ell J to "target cell".

(18) r well J on page 12, line 2 of the same book as “
Corrected to "Well."

(19) Change rassay j on page 12, line 4 of the circular to “
Correct to "assay".

(20) Change r well J on page 12, line 5 of the circular to “
Uni (21) circular page 12, line 8 r cell l
Correct 1neJ to "cell line".

(22) Circular page 12, line 10 rlnhibiti
Correct on Te5tJ to "inhibition test".

(23) rSepharos on page 13, line 18 of the same book
e Correct J to "Sepharose".

(24) rDE^E-Cel on page 13, line 19 of the same book
lulofineJt-rDEAE-Cellulofine”
Correct.

(25) r DEAR-Cel on page 14, line 1 of the same book
lulofineJG rDEAE-Cellulofine”
Correct.

(26) On page 14 of the same book, in the third line from the bottom, “*CD” is 1
nbi tion did not apply. " should be corrected to "*: In the inhibition test, inhibition did not occur."

that's all 1 copy of the original text) June 20, 1985

Claims (1)

[Scope of Claims] A monoclonal antibody produced by a hybridoma prepared using a poorly differentiated gastric adenocarcinoma-derived cell line (MKN-45) as an immunogen and having the following characteristics: (1) Ig class (light chain): IgG_1 (K)
(2) Recognized antigen type: Cell surface antigen (3) Reactivity with cancer cells: Shows positivity for the following cancer cells. Esophageal cancer (TE-3), lung cancer (PC-3), gastric cancer (MKN)
-45), gastric cancer (KATO-III), and liver cancer (HEK).