JPS63254992A - Monoclonal antibody - Google Patents

Abstract

PURPOSE:To obtain plural Kinds of antiCEA monoclonal antibodies having different antigenic determinants, by forming a fused hybrid between an antibody- producing cell and a myelomatous cell and cloning the above-mentioned hybrid. CONSTITUTION:An antibody-producing cell, such as splenic cell, lymph node cell or B-lymphocyte, is separated and collected from an animal, such as mouse, immunized with established cancer cell, such as esophageal cancer, pulmonary cancer, gastric cancer or hepatic cancer, to carry out cell fusion to a myelomatous cell derived from a mouse, rat, human, etc., and select a clone capable of producing the aimed monoclonal antibody. Thereby, (i) a monoclonal antibody reactive with CEA, colonic cancer, intestinal mucosa, granulocyte and bile duct, (ii) a monoclonal antibody reactive with CEA, colonic cancer, intestinal mucosa and granulocyte but unreactive with bile duct, (iii) a monoclonal antibody reactive with CEA, colonic cancer and bile duct but unreactive with intestinal mucosa and granulocyte and (iv) a monoclonal antibody reactive with CEA, colonic cancer and intestinal mucosa but unreactive with granulocyte and bile duct are obtained from the afore-mentioned clone.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a monoclonal antibody against carcinoembryonic antigen (hereinafter referred to as CEA). In particular, the present invention relates to multiple types of anti-CEA monoclonal antibodies having different antigenic determinants.

[Prior art]

CEA is a glycoprotein with a molecular weight of approximately 180,000, and because it is present in the serum of patients with gastrointestinal cancer, it is widely used as a cancer marker in clinical diagnosis.

By the way, a substance other than CEA that exhibits cross-reactivity with anti-CEA antibodies (CEA-related antigen) has been found in normal tissues. That is, CEA-related antigens are considered to be substances that are partially similar in structure to CEA.

Known CEA-related antigens include non-specific cross-antigens (present in the lungs and lungs of normal people), normal fecal antigens (present in normal adult feces), and biliary glycoproteins (present in bile).

With the establishment of monoclonal antibody technology that recognizes only one antigen-determining site, the relationship between CEA or CEA-related antigens and anti-CEA monoclonal antibodies is also being studied (Japanese Patent Application Laid-Open No. 59-5120, 6
0-500427 and JP-A-60-18765).

[Problem that the invention seeks to solve]

Therefore, even though it is an anti-CEA monoclonal antibody, the antigen-determining site that it recognizes may be different, so it cannot necessarily be said that it is the same antibody, and its utility value is expected to vary.

In view of the above circumstances, the present inventors have conducted intensive research on anti-CEA monoclonal antibodies, and have found that monoclonal antibodies with different antigen-determining sites have been found to have different reactivity with CEA or CEA-related antigens derived from normal tissues. Furthermore, because some of them have specific reactivity, they have found that they are extremely promising clinically, leading to the completion of the present invention.

[Means for solving problems]

That is, the present invention can be obtained using carcinoembryonic antigen or carcinoembryonic antigen-producing cancer cells as an immunogen, and has reactivity to carcinoembryonic antigen and colorectal cancer as well as other characteristics. The present invention provides a monoclonal antibody having the following properties.

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 as the immunogen.

CEA, CEA-producing cancer cells, or an extract thereof is used as the immunogen.

As CEA, for example, one derived from ascites of a cancer patient can be used.

CEA-producing cancer cells include esophageal cancer (TE-2), lung cancer (PC-3), lung cancer (PC-9), and gastric cancer (MKN-28).
), gastric cancer (MKN-45), gastric cancer (KATO-III)
, liver cancer (HEK), and other established cancer cell lines are used.

In the case of an extract, the cancer cell line is disrupted by ultrasonication, etc., and then centrifuged (e.g., 10,000-20,000 G,
10 to 60 minutes) to obtain a cell extract, and this supernatant was filtered using a gel filtration carrier (e.g., Sephadex, Sephacryl, Sepharose, biogel, etc.) capable of separating substances with molecules of 1.1 to 2 million molecules. The polymer fraction (molecular weight approximately 700,000 to 1,500,000) is collected through molecular sieving and used. For immunization, the immunogen is used as it is or reacted with an appropriate anti-CEA antibody (immunoblock method).

The immunoblock method, for example, uses CEA and a suitable anti-CEA.
This is a method of mixing with a monoclonal antibody at a ratio of about 1:1 to 10.

The obtained immunogen may be prepared, for example, in Freund Complete Adjuvant.
After mixing with t), it is used for animal immunization. Examples of animals include mice, rats, horses, goats, and rabbits. Immunization is administered subcutaneously, intramuscularly, or intraperitoneally to the animal.
In the case of CEA, approximately 1 to 100 μg/dose is injected, and in the case of cancer cells, the amount equivalent to approximately 10' to L O' cells is injected per time.
Immunization is performed four times, followed by a final immunization approximately 1 to 4 weeks later. Approximately 3 to 5 days after the final immunization, antibody-producing cells are collected from the immunized animal.

Antibody-producing cells include lung cells, lymph node cells, and B-lymphocytes.

As myeloma cells, those derived from mice, rats, humans, etc. are used, for example. Examples include mouse myeloma P3U1, X 63-A g 8-6.5.3, and the like. Preferably, the antibody-producing cells and myeloma cells are derived from the same species of animal.

Cell fusion can be achieved, for example, by Gee, Galfer (G.

Ga1fre) (Neach#, -(Nature)
266, 550 (1977)) or a method analogous thereto. At this time, 30-50%
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.

The cells obtained by cell fusion are subjected to screening for clones that produce the desired monoclonal antibody. 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 measured by, for example, an enzyme antibody method, to obtain wells producing appropriate antibodies. . From such wells, cloning is further performed, for example, by the limiting dilution 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 fluid containing a high concentration of monoclonal antibodies is collected and assayed.

Alternatively, the cells can be grown in a dish-free medium (such as RITC55-9 medium) containing bovine serum albumin (0.1 to 1%), and the culture supernatant can be collected. Recovery of monoclonal antibodies produced by selected clones can be easily achieved by applying conventionally known immunoglobulin purification methods such as ammonium sulfate fractionation, PEG fractionation, ethanol fractionation, and anion converters. be done.

(i) Classification The obtained monoclonal antibodies are classified according to their reactivity with CEA or CEA-related antigens.

In the case of CEA, CEA itself or CEA-producing cancer cells (eg, colon cancer) are used.

As the CEA-related antigen, normal tissues such as intestinal mucosa, granulocytes, bile ducts, or their extracts are used.

Among these, intestinal mucosa includes mucosal epithelium of the large intestine or small intestine,
As the granulocytes, it is preferable to use peripheral blood leukocytes, lung tissue, etc.

As a reaction method, known immunoreaction means such as enzyme antibody method can be used.

(ii) Properties The monoclonal antibodies of the present invention can be classified into four groups having the following properties, but they all have in common that they react against CBA and colon cancer.

(alcEA, a monoclonal antibody reactive against colon cancer, granulocytes and bile ducts.

tblcEA, a monoclonal antibody that reacts with colon cancer and granulocytes, but not with bile ducts.

(C) CEA, which reacts against colon cancer and bile ducts,
A monoclonal antibody that does not react with granulocytes.

A monoclonal antibody that reacts against fdlcEA and colon cancer, but not against granulocytes and bile ducts.

More specifically, it can be classified into four groups having the following properties.

(alcBA, a monoclonal antibody reactive against colon cancer, intestinal mucosa, granulocytes and bile ducts.

fbl CEA, a monoclonal antibody that reacts with colon cancer, intestinal mucosa and granulocytes, but not with bile ducts.

(CICEA, which is reactive against colon cancer and bile ducts,
Monoclonal antibody that does not react with intestinal mucosa and granulocytes.

(dlcEA, a monoclonal antibody that reacts with colon cancer and intestinal mucosa, but not with granulocytes and bile ducts.

〔Effect of the invention〕

The monoclonal antibody obtained by the present invention is CEA
In order to recognize cancer, tumor imaging or targeting therapy (tar
It is expected that it will be used in clinical applications such as getting therapy, or as a diagnostic agent useful for early detection of cancer and follow-up of prognosis.

In particular, since the antigen-determining sites are different from each other, it is possible to use different antibodies of the present invention depending on the type of tumor, or to improve the sensitivity as a diagnostic reagent by using two or more kinds of antibodies of the present invention.

Furthermore, among the monoclonal antibodies of the present invention, those that do not react with antigens derived from normal tissues such as granulocytes and bile ducts are CE
This antibody is specific to A and is presumed to be a cancer-specific antibody. Therefore, such antibodies are considered to be extremely useful clinically.

[Example/Experiment example]

Example 1 +11 Preparation of immunogen: Poorly differentiated adenocarcinoma gastric cancer cell line MKN-45 is added to isotonic phosphate buffer (pH 7.2)! ! ! The mixture was immunized three times every two weeks by intraperitoneal administration of approximately 1.5×10' cells/time to BALB/C mice (Ca sex, 4 weeks old or older), and a final immunization was performed three weeks later.

Four days after immunization, the mice [intestines were removed].

(2) Cell fusion and cloning: The above mouse tile cells and mouse myeloma X63-A
g 8-6.5.3 1:1 (cell number 6.2 x 10
') and mixed with 12.3% dimethyl sulfoxide and 42.6% polyethylene glycol (average molecular weight 10
Cell fusion was performed by reacting for 1 minute in 00).

7x10' treated cells 70.1 ml/well
After culturing in HAT medium (Table 1) for 9 to 14 days, transfer to HT medium (Table 1), and after culturing in a flask (25 cJ), D - Cultured in MEM medium (Table 1). The antibody titer in the culture supernatant of wells in which proliferation was observed was determined by enzyme antibody method, and the desired hybridoma was cloned from appropriate wells by limiting dilution method.

i.e. 25.0% per well in a microtiter plate.
000 mouse peritoneal exudate cells were implanted and then D-
M B M medium (Table 1), 10.5.2.5.1 pieces 1
The hybridoma was diluted to 0.1@7 and cultured by inoculating 0.1 mJ into a microtiter plate. After 4 days, 0.1 m7 of D-MEM medium was added, and thereafter, the medium was replaced once every 4 to 7 days. Colonies visible to the naked eye were formed 10 to 20 days after the start of culture, and a clone strain was obtained.

(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.

Samples were added to microplates coated with antigens (various cancer cell lines, partially purified cancer-related antigens, or normal cells), incubated at 37°C for 1 hour, washed, and peroxidase-labeled anti-mouse immunoglobulin (IgG + I gA +
r gM) rabbit antibody was added, and the mixture was further reacted at 37°C for 1 hour. After washing and removing unreacted labeled antibodies, 0-phenylenediamine solution was added, and the mixture was allowed to react at room temperature for 300 minutes. After that, 2M sulfuric acid was added to stop the reaction, and the absorbance at 490 nm was measured. Using this method, the reactivity with various cells was investigated.

Cross-reactivity with carcinoembryonic antigen (CEA) was performed using the CEA-sensitized sensitized blood cell-PHA method.

(b) Screening flow Primary screening: Target cell (MKN-45)
and enzyme-linked antibody assay using normal-derived cells (CODo3K), positive for MKN-45 and C0Das.
Select wells negative for SK.

Secondary screening: The selected cell line is cloned two to three times, and the culture supernatant is examined for reactivity with many cancer-derived cell lines.

(4) Collection and purification of monoclonal antibodies Prepare 0.5 ml of the clone strain obtained by the above screening.
2,0 to 3 days into the peritoneal cavity of BALB/C mice (male) after 4 weeks of age to which Bristan was administered. OX l O' c
ell/mouse was transplanted, and 10 to 14 days later, ascites containing a high concentration of monoclonal antibodies was collected.

0.9% NaC1 in this ascites? After adding fL and diluting the mixture 5 to 10 times, ammonium sulfate was added to a concentration of 40%, and the precipitate fraction was collected. After dissolving this precipitated fraction in as small a volume as possible of 0.9′AN a CIl solution,
．． Dialysis was performed using 9% NaCl solution as an external solution.

After dialysis, high performance liquid chromatography (TSK-G
ell G-3000S14) to obtain a globulin fraction, which was used as a purified monoclonal antibody.

Thus, the monoclonal antibody belonging to the bone [(al)
BD6) was obtained.

Example 2 Using the differentiated adenocarcinoma lung cancer cell line PC-9, all operations were performed in accordance with Example 1 except that the immunization schedule was 4 times per week and the final immunization was performed after 2 weeks. [(C1
A monoclonal antibody (AH3) belonging to this group was obtained.

Example 3 A monoclonal antibody (JA4) belonging to the above classification fbl was obtained by performing all operations according to Example 1 except for using the liver cancer-derived cell line HEK as an immunogen.

Example 4 Preparation of fil immunogen 30 pg of CEA derived from ascites of a cancer patient was mixed and treated with the purified monoclonal antibody (BD6) 12ON obtained in Example 1 to block some antigenic determinants of CEA.

After mixing 10.1 ml of this CEA30/Alr with an equal volume of complete soloin adjuvant, BAL B/C mice (sex A, 4 weeks old) were immunized by subcutaneous injection [3 times every 10 days], and A day later, final immunization was performed by intraperitoneal administration without adjuvant.

Three days after the final immunization, the mouse pI viscera was removed.

(2) Cell fusion The above lung cells and mouse myeloma P3U1 are combined for about 2
: 1 (3.3xlO' cells: 1.6x10' cells) and reacted for 2 minutes using 45% polyethylene glycol (average molecular weight [4000) to perform cell fusion. After centrifugation, discard the supernatant, leave for 5 minutes, suspend the cells in HAT culture solution, seed 0.1m7f in a 96-well flat-bottomed microplate, and culture in a 7.5% CO□ incubator. did.

(3) Screening method Anti-CEA antibodies are produced in the hybridomas that have been cultured and proliferated in 96 large microplates - all
P using sheep red blood cells with CEA bonded to the surface
Screening was performed using the HA method.

Furthermore, among these, anti-CEA monoclonal antibody B
By the PHA method using sheep erythrocytes bound to CEA via D6, all antibodies producing antibodies that recognize antigenic determinants other than the antigen recognized by BD6 were screened.

From the obtained -all, four types of monoclonal antibodies A10 (belonging to the above classification +d+), B9 (belonging to the above classification (bl)), D6-1 (
belonging to the above classification fbl), D6-2 (belonging to the above classification +d+
) was obtained.

Experimental Example 1 Properties of Monoclonal Antibodies The properties of the monoclonal antibodies produced by the clones thus screened are shown in Tables 2 and 3. Immunoglobulin classes were assayed by the Ophthalony method.

The enzyme antibody method used in the present invention was developed by Kennet.
[Monoclonal Antibodies (Brenium Press) New York London, 3
76, (1980)), enzyme-linked immunosorbent assay (Enzyme-Linked Ia+munoso) using cells as is.
rbent＾5say) (ELISA)
It is the law.

Experimental Example 2 The reactivity of the monoclonal antibodies obtained in Examples 1 to 4 with CEA or CEA-related antigens was evaluated, and the results are shown in Table 4.
It was shown to.

Note that CEA used was obtained by purifying patient ascites fluid.

(Target tissues) Tissues and peripheral blood: Cryo-embedded human lung and liver fixed with 4% paraformaldehyde, and frozen sections of unfixed cryo-embedded human colon cancer, large intestine mucosa, and small intestine mucosa, and then fixed in acetone. Used for experiments. In addition, peripheral blood was used for experiments after smearing and fixing with acetone.

Immunohistochemical staining: Frozen sections of fixed tissues or blood smears were air-dried for 1 hour at room temperature and then washed with cold isotonic phosphate buffer.

Endogenous peroxidase was blocked by incubation in methanol containing 0.3% hydrogen peroxide at room temperature for 30 minutes, followed by washing with isotonic phosphate buffer. 3
% goat serum at room temperature for 30 minutes, and then reacted with a primary antibody (monoclonal antibody) at room temperature for 2 hours. The anti-CEA monoclonal antibody, which is the second antibody, was diluted to 2 μg/ml with 2% bovine blood serum albumin-strengthened phosphate buffer. After washing with isotonic phosphate buffer, incubate for 30 min in 3% human serum + 3% goat serum.
Incubate at room temperature for a minute. Thereafter, it was reacted with a secondary antibody at room temperature for 1 hour. The secondary antibody used was Tavo's sheep anti-mouse lgG+IgM peroxidase diluted 20 times with 2% bovine serum albumin, 3% goat serum, and 3% human blood serum in a cyclic phosphate buffer.

After washing with isotonic phosphate buffer, 0.05% hydrogen peroxide, 0
．． Tris containing 02% diaminobenzidine (Sigma)
Peroxidase reaction was carried out for 4 minutes in hydrochloric acid buffer, pl+7.6. After washing with isotonic phosphate buffer, tissue sections were nuclear stained with methyl green and blood smears were stained with hemadoxylin, followed by dehydration, clearing, mounting, and microscopy.

(Margin below) Table 1

Claims (1)

[Scope of Claims] A carcinoembryonic antigen monoclonal antibody selected from monoclonal antibodies having the following characteristics: (1) It can be obtained using carcinoembryonic antigen or carcinoembryonic antigen-producing cancer cells as an immunogen; , and a monoclonal antibody reactive against carcinoembryonic antigen, colon cancer, granulocytes and bile ducts. (2) can be obtained using carcinoembryonic antigen or carcinoembryonic antigen-producing cancer cells as an immunogen, and has reactivity to carcinoembryonic antigen, colorectal cancer, and granulocytes;
A monoclonal antibody that has no reactivity to bile ducts. (3) It can be obtained using carcinoembryonic antigen or carcinoembryonic antigen-producing cancer cells as an immunogen, and has reactivity against carcinoembryonic antigen, colorectal cancer, and bile ducts, but is reactive against granulocytes. A monoclonal antibody that has no reactivity against. (4) It can be obtained using carcinoembryonic antigen or carcinoembryonic antigen-producing cancer cells as an immunogen, and shows reactivity to carcinoembryonic antigen and colorectal cancer, but is reactive to granulocytes and bile ducts. A monoclonal antibody that has no reactivity against.