JPS63222700A - Monoclonal antibody - Google Patents

Abstract

PURPOSE:To obtain a readily producible monoclonal antibody, reactive with a specific antigen without sialic acid present in an antigenic determinant, having properties of reacting with human cancers in colon, stomach, bile duct, etc., useful for blood serum diagnosis of cancer and belonging to IgM class. CONSTITUTION:A mouse is immunized with an antigen (e.g. colonic cancerous cell) recognizable by a desired monoclonal antibody and antibody-producing cells are separated from the immunized mouse. The separated cells are then fused to myelomatous cells having self propagation ability to afford fused cells capable of producing a monoclonal antibody. The resultant fused cells are subsequently cultivated to provide an antibody, which is recovered to separate a monoclonal antibody, having properties of reacting with a glycoprotein antigen having >=1,000,000mol.wt. (measured by a gel filtration method) in ascitic fluid of a patient suffering from colonic cancer, human colonic cancer, human ovarian mucocele adenocarcinoma, part of human bronchial epithelium, cultivated cell strain of human colonic cancer, human pancreatic cancer and cultivated cell strain of human cholangiocarcinoma H-1, etc., and called NCC-CO-450, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to monoclonal antibodies useful for serodiagnosis of cancer.

In recent years, many monoclonal antibodies that recognize many SS-related antigens, including CEA and AFP, have been produced. or,
Cancer patients are also diagnosed by measuring the levels of the antigens contained in the serum or body fluids of cancer patients using these monoclonal antibodies.

Examples of the monoclonal antibodies currently used in serum diagnosis for screening for gastrointestinal cancer, pancreatic cancer, breast cancer, etc. include CA-19-9, DU-PAN-2, and F36.
/22 and NCC-ST-439. These monoclonal antibodies are used for clinical diagnosis and prediction of recurrence after surgery in cancer patients.

By the way, the conventionally known P! Immunohistological studies have revealed that tumor-associated antigens are expressed not only in various cancer tissues but also in various normal tissues. For example, CA-19-9 also reacts with some of the bronchial wires of the lungs and salivary glands. DU-PAN-2 also weakly reacts with normal pancreatic and gastrointestinal epithelium. On the other hand, these tumor-related antigens show high levels in the serum or body fluids of gastrointestinal cancer patients, but are absent or have low levels in the serum of normal people.

Most of the antigens in serum or body fluids of cancer patients that are detected by these monoclonal antibodies are high-molecular mucin-like glycoproteins, whereas these antigens in cancer tissues are not only mucin-like glycoproteins but also glycolipids. Some are.

It has become known that the sensitivity and specificity of cancer serodiagnosis can be increased by using several such monoclonal antibodies in combination.

Therefore, in order to obtain a monoclonal antibody that reacts with a polymeric signature component that can be isolated from the serum or body fluids of cancer patients, the present inventors conducted intensive research and developed a polymeric glycoprotein isolated from the ascites of colorectal cancer patients. The present invention was achieved by obtaining a monoclonal antibody that can react with

That is, the present invention reacts with a glycoprotein antigen with a molecular weight of 1 million or more (gel filtration method) in the ascites of a colorectal cancer patient, and recognizes an antigenic determinant that does not contain sialic acid and has the following properties: (a) Reacts with human colon cancer, human gastric cancer, human bile duct cancer, human lung adenocarcinoma, and human ovarian mucinous cystadenocarcinoma.

(b) Reacts with human bronchial epithelium, a portion of bronchial mucus glands, and a portion of human cervical glands.

(C) Human colon cancer cultured cell line, c-Co-4, S
Reacts with W1116 and CCK-81, as well as the human gastric cancer cultured cell line KATO-1.

It provides:

In addition, in the following description, all cells, tissues, etc. mean human cells, tissues, etc. unless otherwise specified.

The monoclonal antibody of the present invention is suitable for colon cancer and gastric cancer.

It responds to pancreatic cancer, bile duct muscle, and ovarian mucinous cystadenocarcinoma.

Furthermore, the monoclonal antibody of the present invention is applicable to lung adenocarcinoma.

For breast cancer, ovarian serous cystadenocarcinoma, pancreatic cancer and uterine adenocarcinoma,
It doesn't happen very often, but it does happen.

The antigen targeted by the monoclonal antibody of the invention is present in the cytoplasm of cancer cells and in the vaginal secretions.

The monoclonal antibodies NCC-C0-450 and NCC-CO-473 of the present invention are both cultured colon cancer cell lines, c
-Reacts strongly with Co-4, SW1116 and CCK-81.

Furthermore, NCC-CO-450 is a cultured gastric cancer cell line MK.
N-45 and KATO-1 and bile duct muscle culture cell line H
Reacts with -1. Also, N CC-CO-473 is also KAT
Although it reacts with O-1, the number of positive cells is small and the reactivity is weak compared to the reaction with Maki colorectal cancer cultured cell line.

Here, c-Co-4 is Co-4 from a nude mouse xenograft tumor, and c-Li-7 is 1i-7
These are cultured cell lines established in the inventors' laboratories from xenotransplantation.

The monoclonal antibody NCC-C0-450 of the present invention is
It reacts with large intestine epithelial cells, and also with parts of intestinal metaplasia of the gastric mucosa, bronchial epithelium, parts of bronchial mucus glands, and parts of cervical glands.

On the other hand, the monoclonal antibody NCC-GO-4 of the present invention
73 reacts with deep epithelial cells of the large intestine crypts, bronchial epithelium, bronchial mucus lines, and parts of cervical glands, but NCC-G
The reaction is somewhat weaker than that of O-450.

Monoclonal antibody ID3 (Gan0OI)adhya produced from mice immunized with human ovarian cystadenocarcinoma
V, A.

et al. Cancer Res, 45:'1744-17
52.1985. Immunohistological antigen distribution in normal tissues of the monoclonal antibody NCC-C of the present invention
Similar to O-450. However, the reactivity against gastric cancer and colorectal cancer was determined by the monoclonal antibody NC of the present invention.
C-C0-450 and NCC-Co-473t; T is not expensive. Furthermore, N CC-CO-450 of the present invention reacts with ovarian serous cystadenocarcinoma, but 1D3 does not react with it.

Monoclonal antibodies MOv1 and lyl Qv2 (Taaliabue) obtained by immunization of mucinous cystadenocarcinoma
, E.

et al. Cancer Res, 45:379-385.
1985. ref.) both react with high molecular weight glycoproteins extracted from ovarian mucinous f1 cystadenocarcinoma, and MOvl and MOv
The antigen recognized by No. 2 is present in ovarian mucinous cyst adenocarcinoma and normal colonic mucosa, and shows an immunohistological distribution similar to the antigen recognized by the monoclonal antibody of the present invention. however,
MOvl does not react with cancers in other organs, and
Both monoclonal antibodies are different from the monoclonal antibody of the present invention in that Ov2 also reacts with normal gastric mucosa and normal laryngeal mucosa.

The antigen in the ascites of a colon cancer patient with which the monoclonal antibodies of the present invention (NCC-CO-450 and NCC-G O-473) react is a high molecular weight glycoprotein with a molecular weight of 1 million or more (gel filtration method). Furthermore, by treating this antigen with alkali, its reactivity with the monoclonal antibody of the present invention disappears, indicating that this antigen is not a ganglioside but a mucin-type glycoprotein. Even if the antigen in patient ascites is treated with neuraminidase, which is a sialic acid degrading enzyme, the reactivity of the monoclonal antibody of the present invention to the antigen does not disappear. Therefore, the antigen site recognized by the monoclonal antibody of the present invention It can be seen that (antigenic determinant) does not contain sialic acid.

CO-4 reacting with N CC-G O-450 of the present invention
Antigens in xenograft tumors (purified by immunoaffinity chromatography and gel filtration) were analyzed by 5DS-PAGE,
Its molecular weight is 70,000 to 4 by Western blotting.
It is in the range of 00,000.

The antigen in the graft that reacts with the NCC-CO-473 monoclonal antibody of the present invention was also analyzed by 5O8-PAGE.
, shows a molecular weight distribution of 70,000 to 400,000 by Western blotting.

Also, NCC-CO-450 and NCC-C0-41
3 also mutually react with the antigen in the transplanted mai purified using the other monoclonal antibody. or,
The binding of NCC-C0-450 to this antigen is
Not inhibited by CC-G O-473. Also N
The binding of CC-473 is slightly inhibited by NCC-CO-450. Therefore, it is thought that the antigenic determinants on the above-mentioned antigens for these two types of monoclonal antibodies are different from each other or partially shared.

Incidentally, monoclonal antibodies N 319-9 and N CC-ST-439, which are useful for serodiagnosis of cancer, do not react with the antigen recognized by the monoclonal antibody of the present invention.

Furthermore, it reacts with c-Go-4, a cultured human colon cancer cell line established from a Co-4 xenograft It4 tumor, and the monoclonal antibody of the present invention purified from its culture supernatant by immunoaffinity chromatography. The antigen also exhibits a similar molecular weight distribution as the antigen in the transplanted I layer.

The antigens purified by immunoaffinity chromatography from C0-4 xenograft tumors were WGA, PNΔ, 5
- Binds to WGA (succinyl WGA). Similarly,
Le Reacts with monoclonal antibodies against sugar chains. Furthermore, the monoclonal antibody of the present invention does not react with the glycolipid moiety extracted from the transplanted tumor. On the other hand, it reacts with the PBS extract after defatting.

When the antigen purified by immunoaffinity chromatography from the PBS extract of the C0-4 xenograft tumor was treated with alkali, the reactivity with the monoclonal antibody of the present invention was abolished. Furthermore, when the antigen was treated with neuraminidase, the reactivity with the monoclonal antibody of the present invention did not disappear.

From the above results, it is considered that the antigen contained in the cancer tissue that reacts with the monoclonal antibody of the present invention is mucin-like glycoprotein.

The monoclonal antibody of the present invention belongs to the IgM class, and its L! i is a chain.

The antigen that reacts with the monoclonal antibody of the present invention exists in large amounts not only in cancer tissues but also in the serum or body fluids of patients with gastric cancer, colon cancer, pancreatic cancer, and the like.

The monoclonal antibody of the present invention can be produced, for example, as follows. That is, an animal such as a mouse or a rat is immunized with an antigen recognized by the monoclonal antibody of the present invention (the antigen may be isolated and used, but cancer cells containing the antigen or a homogenate thereof may also be used), Obtain antibody-producing cells from the immunized animal, fuse these with myeloma cells, clone the resulting fused cells, select fused cells that produce the monoclonal antibody of the present invention, culture them, and collect the antibodies. do. Immunization methods, fusion methods, selection of fused cells, etc. can be performed by conventional methods.

More specifically, the monoclonal antibody of the present invention can be produced, for example, as follows.

First, mice are immunized with cancer cells such as colon cancer cells. The animal to be immunized is not limited to mice, and other murine animals such as rats may be used, but it is usually preferable to use mice. Also, homogenates or isolated antigens thereof may be used instead of cancer cells. For example, inject cancer cells or their homogenates or isolated antigens into B^LB/c mice for several days.
Vaccinations are given several times every few weeks. The inoculation layer is 1 G'' to 1 (preferably 18 1111 cells are used) per mouse at a time.Then, the spleen is removed from the mouse and centrifuged to obtain antibody-producing cells.These cells have the ability to proliferate. Therefore, it is fused with cells that have the ability to self-propagate. Bone marrow cells are particularly preferred as cells that have the ability to self-propagate. As myeloma cells, it is preferable to use cells from the same species of animal. It is preferable to select cells that do not produce antibodies.Antibody-producing cells and myeloma cells are mixed with a cell fusion agent such as polyethylene glycol to perform cell fusion.Antibody-producing cells and bone aIII
AI! 1 is preferably used in a cell number ratio of 2:1 to 10:1. The obtained fused mu was separated by limiting dilution method, the separated fused cells were grown, and then each well (
The antibodies produced in the wells are reacted with the desired antigen or various i-cells, etc. by a known method such as fluorescent antibody method or enzyme antibody method, and from the results, hybridomas producing the desired antibody are selected.

It is also possible to culture the selected hybridoma in an incubator and obtain antibodies from the supernatant, but it is also possible to obtain antibodies from the supernatant fluid in vivo, for example (nude).
It is also possible to inject the hybridoma into the peritoneal cavity of a mouse and collect the antibody from the (nude) mouse serum or ascites fluid.

Cancer cells such as colon cancer cells can be used as cells to be used as an immunogen to produce the monoclonal antibody of the present invention, but these cancer cells are not limited to specific strains.

The antibody-producing cells used to produce the monoclonal antibodies of the present invention are B cells, and B cells circulate in the body but accumulate in the spleen, etc., so it is preferable to remove the spleen and use it, but it is not always necessary to remove the spleen. It is not necessary to use the part where many B[l cells are present].

In addition to the above-mentioned method, the monoclonal antibody of the present invention can be prepared by
It can also be manufactured as follows.

That is, a cancer tissue, such as a colon cancer tissue, containing an antigen that reacts with the monoclonal antibody of the present invention is transferred to an athymic mouse, such as 8^
Transplant subcutaneously into LB/c nu/nu nude mice.

As the cancer tissue to be transplanted, for example, several cancer tissues of 2 to 3 angles are used. M 1 to 1.5 months after transplanting cancer tissue
When the maximum diameter of the tumor has grown to 11th, lymphocytes (T cells and B cells) contained in the spleen of a mouse with a thymus gland, such as a BALB/c nul+ mouse, are transferred to the tumor-bearing nude mouse described above (
The drug is administered intraperitoneally to an athymic mouse (bearing a tumor approximately 1α in diameter). It is sufficient to use an administration sickle in an amount equivalent to the amount of lymphocytes contained in a sickle for one mouse. 1 in tumor-bearing mice after administration v & 1 month! The tumor shrinks or disappears. The spleen (antibody-producing cells) of mice whose tumors have shrunk or disappeared are removed and fused with osteoI tumor cells. Method of fusion and subsequent selection of fused cells 1 The cultivation of the selected fused cells and the method of collecting the monoclonal antibody of the present invention can be carried out in exactly the same manner as described above.

It can be seen that the antigen recognized by the monoclonal antibody of the present invention is mainly expressed in lung adenocarcinoma among lung cancers. What is even more interesting is that only goblet cell type lung adenocarcinoma, one of the subtypes of lung adenocarcinoma, reacts with NCC-CO-450; however, this goblet cell type lung adenocarcinoma produces mucin-like substances. known to occur. Therefore, it is presumed that the monoclonal antibody of the present invention is useful for classifying lung adenocarcinoma.

The antigen recognized by the monoclonal antibody NCC-CO-450 of the present invention is not expressed in the gastric mucosa, but is expressed in a part of the gastric mucosa with intestinal metaplasia and in gastric cancer.

Based on this fact, the monoclonal antibody of the present invention is considered to be useful for research on the origin of gastric cancer.

The antigen with which the monoclonal antibody of the present invention reacts is not detected in the body fluids of normal people, but appears in large amounts in the ascites of patients with gastric cancer and colorectal cancer, and in the serum of patients with pancreatic cancer. It can be used for serodiagnosis of patients. Particularly in the case of patients whose serum contains a large amount of the antigen recognized by the monoclonal antibody of the present invention, it can be used to determine therapeutic efficacy and predict recurrence. Furthermore, by adding a radioactive isotope to the monoclonal antibody of the present invention, it can be used for cancer localization diagnosis, or by combining an anticancer agent and the monoclonal antibody of the present invention, it can be used for missile therapy. Furthermore, by using the monoclonal antibody of the present invention for lesions that are histologically difficult to distinguish from cancer, it may be possible to differentiate the lesions.

Example 1 (1) Ascites from a patient with advanced colorectal cancer made by M, a high molecular weight component, was centrifuged at 12ρOOG for 30 minutes, and the supernatant was then transferred to a Superose 6 column (pre-equilibrated with PBS containing 0.1% sodium nitride). )? - Macia Fine Chemicals Co.) and fractionated. The fractions eluted when a solvent equal to the total volume of the void was flowed were collected and used as components with orders of magnitude higher molecular weight. Figure 1 shows the elution pattern. Here absorbance at 280 ns, O and Δ represent NCC-G O-450 and NCC-CO-473 antigens, respectively, measured by solid phase EIA.

■ A nude mouse transplant of monochrome human colon differentiated adenocarcinoma (Co-4) was cut into 2-3 #l pieces and BALB/c nu/nu (
The tumor was subcutaneously transplanted into athymic nude mice, and approximately one and a half months later, the maximum diameter of the tumor was approximately 10 m. On the other hand, B^LB/c
The spleen of one of the nu/← mice was cut into small pieces, passed through a stainless steel mesh, and made into a cell suspension using 0.5 d of physiological saline. It was administered intraperitoneally to a tumor-bearing BALB/c nu/nu).

The tumor disappeared 3 weeks after administration. Therefore, 0.2d of Co-4 homogenate was added to BALB/c nu/nu (7)
It was administered intraperitoneally. Three days later, the spleen was removed from the mouse.

The method of cell fusion is the method of Watanabe et al.
2963-2967, 1978).

That is, the removed spleen was cut into small pieces, passed through a stainless steel mesh, and centrifuged at 1500 rpm + 200 G. The resulting sediment was heated at 50°C. Mouse myeloma cells (P3-X63-
I'18-Ul) (hereinafter referred to as P3U1)
P3U1 2x obtained by washing twice with M I-1640
Mix 7 lO (5:1), 2000 rpl, 20
Centrifugation was performed at 0 G for 10 minutes. After thoroughly loosening the precipitated cells, 45% (w/v) polyethylene glycol 400 was added.
0 (Merck & Co.) at 37°C, I) R of H7,4
PM I-1640, lll1 was added and treated for 8 minutes.

After 1 minute of reaction, gradually add RPMI-1640 to a total amount of 4
Cell fusion was completed as 0111. 11000rp,
After centrifugation at 100G, contain 10% tallow serum/v re
Add RPHI-164040m to make a cell suspension 3
The cells were cultured at 7°C in 5% CO2-filled JIB medium. Incubate for 24 hours, change to HAT medium (hyboxanthin, aminopterin, thymidine 10% tallow serum), and use C03tar.
The cells were cultured in 0.2 d portions per well on a m1cro culture plate. The supernatant was taken out on the 10th day, and the following screening was performed. As mentioned above, the fraction eluted when a solvent equal to the total volume of the cavity was flowed was diluted 10 times in PBSt', and divided into a 96-well costara + 1 crotiter plate with 50 components in each well. I noted it. PB 350m as control was also injected into the wells. This is 4
It was left overnight at °C. Next, to minimize non-specific binding in the wells, 5% 8 SA (
w/v) containing PBS (blocking buffer) 200
J11 was dispensed and left at room temperature for 2 hours. Dispense 50 g of the culture supernatant of the above hybridoma into each well, and store at room temperature for 2 hours.
I left it for a while.

After washing three times with PBS, rabbit anti-mouse gG immunoglobulin (Dako) diluted 200 times with blocking buffer was added to each well. It was added and left at room temperature. Each C-well was then washed with PBS as described above, followed by protein A diluted 1000 times with blocking buffer.
-Peroxidase (E, Y.

5011Il of LABS) was added to each well and left for 30 minutes. Thereafter, each well was washed with 0-phenylenediamine in 0.1 M citrate buffer solution C11119/d;
I) H4, 5) 200/ld was added. As a result of this reaction, the hybridomas in the wells that showed positive results were cloned by limiting dilution method so that each well contained 0.6 hybridomas. The medium was initially 1-IT (hyboxanthin, demidine, 10% live fetal serum) and fed
As an layer, 5×10 5 cells/well of 11111 breast cells of B/c nu/+ mice were added. then 10
The medium was replaced with RPHI-1640 medium supplemented with % live fetal serum.

Cloning by limiting dilution method was performed twice.

In addition, for large-scale cultivation, increase the amount of hybridoma from 1 well to 5 wells, then to 24 wells (Falcon 3008), and finally to Falcon tissue cultivation.
Add 0.1 NaN3 to the supernatant obtained from ure flask culture.
% and stored at 4°C.

In addition, mouse myeloma cells (P3-X63-AQ8-Ul)
culture supernatant was used as a negative control.

Through the above screening procedure, two monoclonal antibodies that react with the components of the polymer obtained from the ascites of colorectal cancer patients were obtained, and these were used for NCC-CO-450 and NCC-CO-450, respectively.
It was named CC-CO-473.

FIG. 2 is a graph showing the reactivity of these two monoclonal antibodies to serially diluted antigens.

O is N CC-C0-450, ・ is NCc-G O-4
Each represents 73 reactivities.

All fractions obtained by gel filtration as described above were subjected to solid phase EI.
A was tested using A, but only the fractions eluted when a solvent equal to the total volume of the void was flowed were these two.
It reacted with two monoclonal antibodies (see Figure 1). Furthermore, these two monoclonal antibodies did not react with any fraction obtained by gel filtration of normal human serum.

Paraffin blocks of various cancers and normal tissues were prepared at the National Cancer Center Hospital and Keio University Department of Surgery.

Staining of various tissues and cultured human cancer cell lines with the monoclonal antibodies NCC-CO-450 and NCC-CO-473 of the present invention was carried out by the method of Hsu, S, H, et al. (J.

Histochem, Cytochem, 29.
577-580.1981) by formalin fixation and staining of paraffin sections using the avidin-biotin-peroxidase complex method. That is, after fixing with 10% formalin, which is widely used, embedding in paraffin, thinly sectioned human colon cancer tissues, other human cancer tissues, and normal human tissues were deparaffinized in xylene, and 0.0%
Treated with methanol containing 5% H2O2 for 30 minutes. Then Rin! ! After washing with buffered saline (PBS), 5
z Treated with PBS containing normal pig serum for 30 minutes. For each cultured cell line, the cells were made into pellets, fixed in formalin, embedded in paraffin, and immunostained using the same method.

Next, it was reacted with a solution containing a monoclonal antibody at 1 m for 2 hours. After washing with PBS for 15 minutes, the cells were treated with biotinylated anti-mouse immune clopurin (7,5II!l/d) for 30 minutes. After washing this with PBS for 15 minutes, it was treated with an avidin-biotin peroxidase complex for 30 minutes at room temperature. After washing this with PBS for 15 minutes,
0.02% diaminobenzidine solution (0.02% H20
2. To! J Suhay 7 F 1187.6) at 8
Allowed to react for minutes. After staining the cell nucleus with hematoxylin,
It was sealed and examined using a microscope in the usual manner.

Table 1 shows the reaction results for various cancer tissues, Table 2 shows the reaction results for various normal tissues, and Table 3 shows the reactivity for various cancer cell culture cell lines.

Table 1 Esophageal cancer 015 015 Thyroid cancer
0/2 0/2 Lung squamous cell carcinoma 1/81/8 Lung adenocarcinoma
5/27 2/27 Lung large cell carcinoma 01
5 015 Small cell lung cancer 0/4
0/4 milk v! 11/12 0/12
Stomach cancer 17/20
11/21 Liver cancer 0/15
0/15 Cholangiocarcinoma 3/6
1/2 pancreatic cancer 1B726
0/26 Colorectal cancer 39/
40 33/38 Prostate cancer 0/4
0/4 Ovarian serous cystadenocarcinoma 3/4 0
/4 Ovarian mucinous cystadenocarcinoma 3/9
1/9 Ovarian cell carcinoma 0/3
0/3 Uterine adenocarcinoma 115 015 Uterine squamous cell carcinoma 0/3 0
/3 Kidney cancer 0/6
0/6 Malignant malignant layer 0/1 0/1 Second surface brain -- Peripheral nerve -- Salivary gland -- Thyroid -- Thymus -- Heart -- Mammary gland -- Lung Bronchial epithelium, bronchial mucus gland Bronchial epithelium, Bronchial mucus lineage - Stomach Intestinal metaplasia gastric mucosa - Small
Intestines -- Large intestine Large intestine epithelial cells Crypt deep epithelial cells Liver -- Pancreas -- Gallbladder -- Viscerum -- Al Kidney -- Kidney -- Ovary -- Uterus Cervical glands Cervical canal Adening circle -- Prostate -- Bladder -- Smooth muscle -- Transverse constrictor muscle -- Table 3 PC-3-- PC-9-- PC-13-- PC-14-- 0KADA -- WAMAKI -- Lung squamous cell carcinoma PC-10-- Lung large cell carcinoma Lu-65-- PC-13-- Lung small cell layer H-69-- N-231-- N-857-- Lu-134-- 8EKI -- TABATA -- Gastric cancer MKN-45+ -- MKN-74+ -- KATO-411+ + Table 3 continued 81exander --c-Li-
7-- +1tll -7-- Biliary muscle H-1, +- Pancreatic cancer PaCa-2-- Colorectal cancer c-CO-4+ + + 15W1116
++ + or CCK-81++
+Leukemia IL-60--Ni-562--Other NB-1--Ewing--1: Negative+: Approximately 50% of all cells are positive++: Almost all cells are positive (4) SDS-PAGE and immunoblotting human colon cancer cultured cell line c-Co-4, colon cancer patient ascites and affinity purified c-Co-4
Antigen derived from the culture supernatant of 5% (w/v) SDS-
Laeuli separated by polyacrylamide gel electrophoresis (under reducing conditions).

U, Ni, Nature (Lond,), 2
27; see G30-685.1970), Dura-p
ore filter (Millipore) by electrophoresis.
n, T.

and Gordon, J., Proc. Natl.
Acad, Sci, USA.

76:4350-4354.1979). Transfer I
1 m, 5X (w
The filter was immersed in PBS (blocking solution) containing 0.01% Antiform A (Sigma), 1% BSA, and 0.02% sodium nitride at room temperature for 2 hours (Johnson ,V,
G. et al., CanCerRcs, 46: 850-
857.1986 with some modifications). Next, the monoclonal antibody of the present invention was incubated at room temperature for 2 hours.
3 hours in PBS containing 0.1% Hitene 20.
After washing twice with PBS and then twice with PBS, the filter was left for 30 minutes at room temperature with biotinylated rabbit anti-mouse IQG (Vector) diluted 200 times with the blocking solution. After washing the filter as described above, it was left for 30 minutes with an avidin-biodin-peroxidase complex (Vector) diluted 200 times with the blocking solution.

The filter was washed once again as above and diaminobenzidine-HC! ! (20IIg) and hydrogen peroxide (
Tris buffer (1 d) containing 20 d of 30% solution
00rd'). In addition, at this time, P3-X63-
The culture supernatant of AC18-LJ1 cultured cell line was used as a negative control. Through the above operations, the above-mentioned molecular distribution results were obtained.

■ Anti-NC by affinity chromatography
C-G O-450 and N CC-CO-473 [0M
Sepharose CL-68 (
It was purified by gel chromatography using Pharmacia Fine Chemical Co., Ltd.). The purified antibody was added to coupling buffer (0, IHN a HCO3, ON 8.5
) k:, vs. TR analysis L/, L, t'Le It was added to the e-activated affinity matrix (Di-Get 10° Bio-Rad) and reacted overnight at 4°C with gentle stirring. Next, this affinity matrix was packed into a minicolumn and washed with PBS until the absorbance of the column washing solution at 28 on- became O. 0.2M glycine containing o
, H4NaHcO3 (pH 8,0) for 2 hours at room temperature. Add this column to the elution buffer (
1,0HNH40H, pH 11,5 and 10% ethylene glycol) and equilibrated with PBS.

C0-41 used as an immunogen for nude mice!
The tumor was cut into small pieces and treated with 0.1n+H phenylmethylphosphonyl fluoride, 1.0IHε-aminocaproic acid and 1
% aprotinin (P8S containing trypsin inhibitor 0.2 units/at (3x 1fiw/v)
t' homogenized. 1oooo of this homogenate
Centrifuge for 20 minutes at
-4737 affinity column. Unbound material was washed thoroughly with PBS. The bound substance is eluted with the elution solution (1,
0HN H40H, pH11.5 and U1096IchiLi
＞'j+) :I-ru) 30ad! was eluted by flowing at a speed of 0.5 m/min.

This eluate was mixed with 1. The mixture was immediately neutralized with OH vinegar 1111 Gae, dialyzed against Zenomizo water, and then freeze-dried. The lyophilized sample was dissolved in PBSlm and applied to a Superose 6 column (Pharmacia) equilibrated with PBS containing sodium nitride, and each fraction was measured by solid-phase EIA to obtain results that conformed to the molecular weight distribution described above. .

The elution pattern of this gel filtration is shown in FIG.

Figure 3A shows the gel filtration elution pattern of the product purified using the NCC-CO-450 affinity column, and Figure 3B shows the gel filtration elution pattern of the product purified using the NCC-Co-473 affinity column. show. O here
and are NCC-CO-450 and N in solid phase ETA, respectively.
It was measured using CC-CO-473.

Example 2 (1) Alkaline treatment and neuraminidase treatment of antigen The high molecular weight antigen isolated from the ascites of a colon cancer patient by gel filtration was treated with 0.1 N NaOH at 37° C. for 5 hours. Next, when this antigenic activity was measured by ELISA, it no longer reacted with the monoclonal antibody of the present invention (fourth
(See Figure A). The antigen II prepared from the PBS extract of the Co-4 xenograft tumor in the procedure (2) of Example 1 was also subjected to alkali treatment in the same manner (see FIG. 4B). In addition, in Fig. 4, the circles indicate the reactivity with NCC-G O-450.
Triangles indicate reactivity with NCC-G O-473, white blanks indicate reactivity before alkali treatment, and black solids indicate reactivity after the treatment.

This revealed that these antigens were not gangliosides.

Next, the high molecular weight antigen derived from Go-4 xenograft tumor was 96
Aliquot into a microtiter plate with 0.10/ad! neuraminidase (Boehringer). was diluted stepwise with acetate buffer (pH 5,5) and diluted with 10
0IJi were added to 96 wells and left at 37°C for 2 hours. Thereafter, the plate was washed with PBS, and the reaction of the monoclonal antibody of the present invention was measured by EIA in the same manner as in the above example, but the antigen activity was not eliminated by this treatment.

When the antigen in the ascites of a patient with colorectal cancer was similarly treated with neuraminidase, the antigen activity did not disappear.

■ Extraction TLC Lipids were extracted from Co-4 tumors grown in infected nude mice with chloroform/methanol/water (Hag
nani, J.L., et al., 5science, 212:
55-56° 1981). On the other hand, this lipid extract was developed by thin layer chromatography (TLC), and the reactivity with the monoclonal antibody of the present invention was measured by immunostaining. The defatted protein pellet was resuspended in 3 volumes of prechannel water and lyophilized. The obtained defatted powder is PBS
It was smelt inside and homogenized.

The mixture was centrifuged at 10,0OOrp+a for 30 minutes, and the antigen activity of the supernatant was measured by solid-phase EIA. These measurements showed that only the defatted PBS extract contained a substance that reacted with the monoclonal antibody of the present invention.

■ Binding with lectin Example 1 (ω-purified antigen and ConA, DBA,
PNA, RCA120, SBΔ, UEA-T.

5-WGA and WGA (Vector Laboratories, B
The reaction with lectins such as urlingame, C^94010) was measured as follows.

Affinity-purified antigen was serially diluted to 50% each.
Four portions were dispensed into each well of a 96-well microtiter plate and left overnight at 4°C. Each well was incubated with PB3200 containing 5% BSA (w,/v) for 2 hours at room temperature.
I11 to block non-specific adsorption.

Next, various types of biotinylated lectins (100-fold diluted with blocking buffer) were added at 50/llj! The mixture was added in portions and left at room temperature for 2 hours. Washed three times with PBS, reacted with ABC peroxidavi (Vector Laboratories) diluted 200 times with blocking buffer for 30 minutes at room temperature, and then incubated with 0.0% of 0-phenylenediamine.
200 III of 1H citrate buffer (pH 4, 5) was added to each well to observe the reaction. As a result, it was found that it reacted relatively strongly with WGA, PNA and 5-WGA (5th
See diagram. In FIG. 8, Δ represents WGA, O represents PNA, and 5-WGA).

0) Competitive Fluorescence Inhibition Assay One of the monoclonal antibodies of the present invention obtained from nude mouse ascites in Example 1 was treated with N-hydroxysuccinimide biotin (Pierce Chemical Co., Rockfo
rd, IL, USA) was biotinylated (
Guesdon, J.L., et at., J.
Histochem.

Cytoches, 27:1131-1139.1
979). This labeled monoclonal antibody 25
/d was mixed with serially diluted unlabeled monoclonal antibodies.

A 96-well microtiter plate was coated with the affinity-purified antigen and left in the blocking buffer described above for 2 hours at room temperature. The monoclonal antibody mixture was added to each well and left at room temperature for 2 hours. After washing three times with PBS, the aforementioned blocking ffl diluted ABC peroxidase was added and allowed to react at room temperature for 30 minutes. After washing,
0-phenylenediamine in 0.1 M citrate buffer (1
)H4,5) 200111 was added to each well and reacted. As a result, it was found that the antigen recognition sites of the two types of monoclonal antibodies of the present invention are different or partially shared.

The results are shown in FIG. A and B are each NCC-Co
-450 and UNCC-Co-473 mono/regional antibody, where 0 is unlabeled N CC-C
It shows the reactivity when diluted and mixed with O-473, and * shows the reactivity when diluted and mixed with unlabeled NCC-G O-450.

[Brief explanation of drawings]

FIG. 1 shows the gel P'A elution pattern of a high molecular weight antigen from ascites of a patient with advanced colorectal cancer. FIG. 2 shows the reactivity of the monoclonal antibody of the invention. FIG. 3 shows the gel e-overelution pattern of Co-4 xenograft tumor-derived antigen. FIG. 4 shows changes in antigen reactivity due to alkali treatment. FIG. 5 shows the reactivity between antigen and lectin. FIG. 6 shows the pattern of competitive binding inhibition assay using monoclonal antibodies of the invention. Daughter u! , 450/7/F) 111 R'

Claims (3)

[Claims] (1) A monoclonal monoclonal that belongs to the IgM class and has the following properties, with no sialic acid present in the antigenic determinant that reacts with and recognizes glycoprotein antigens with a molecular weight of 1 million or more (gel filtration method) in ascites of patients with colorectal cancer. antibody. (a) Reacts with human colon cancer, human gastric cancer, human bile duct cancer, human lung adenocarcinoma, and human ovarian mucinous cyst adenocarcinoma. (b) Reacts with human bronchial epithelium, a portion of bronchial mucus glands and a portion of human cervical glands. (c) Human colon cancer cultured cell line, c-Co-4, SW11
16 and CCK-81, and human gastric cancer cultured cell line KA
Reacts with TO-III. (2) Furthermore, human pancreatic cancer, human ovarian serous/cystocarcinoma, human uterine adenocarcinoma, human breast cancer, part of intestinal metaplasia of human gastric mucosa, human colonic epithelial cells, and human gastric cancer cultured cell line MKN- 45 and human cholangiocarcinoma cultured cell line H-1, and is designated as NCC-CO-450. (3) Furthermore, it reacts with deep epithelial cells of the human large intestine, and
Monoclonal antibody according to claim 1 designated as C-CO-473.