JPH02150293A - Monoclonal antibody against human cancer-causing gene product and hybridoma producing same antibody - Google Patents

Abstract

PURPOSE:To provide a monoclonal antibody corresponding to human cancer- causing gene erb B-2 product as an antigen and useful for the diagnosis and treatment of human glandular cancers, especially breast cancer, carcinoma, etc. CONSTITUTION:A vector containing a human c-erb B-2 gene is developed and a fibroblast originated from mouse embryo is transformed with the developed vector to prepare a cell expressing a human c-erb B-2 gene in a high yield. The antibody-producing cell of an animal immunized with the prepared cell as an antigen is fused with a myeloma cell to prepare a hybridoma producing a monoclonal antibody whose corresponding antigen is the product of the human c-erb B-2 gene.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] This invention relates to a novel monoclonal antibody and a hybridoma producing the same. The monoclonal antibody of this invention can be used as a diagnostic or therapeutic agent for human adenocarcinoma, particularly breast cancer, gastric cancer, etc.

[Prior Art] Research has been carried out from various angles to develop cancer diagnosis and treatment methods. It is becoming clear that canceration of cells begins with abnormalities such as damage to chromosomal DNA. Different types of DNA can be broadly divided into two types. One is an abnormality in which the function of a specific gene is lost due to the abnormality, and as a result, the cell becomes cancerous.The other is an abnormality in which the function of a specific gene is altered in a way that favors the canceration of the cell. This is an abnormality. The cause of the latter abnormality is called an oncogene. There are about 40 specific genes known so far.

A typical virus that infects chickens and causes cancer is the Rous sarcoma virus, which was discovered in 1911.

The oncogenic potential of this virus is found in the genome of the virus.
It has only recently been discovered that it is caused by the c gene.

Since then, dramatic progress has been made in the field of cancer research, one of which was the discovery in 1976 that a gene homologous to SrC existed on the chromosomes of normal chicken cells.
Found by Staelin et al. Many others (RN of
Yes from molecular biological analysis of A tumor virus
Oncogenes such as , erbB, fps, abl, and ras have been identified, but the oncogenes of these viruses are Sr.
Like C, all of the genes were shown to be derived from cell-derived proto-oncogenes (hereinafter referred to as c-onc).

The gene c-onc, which does not exhibit oncogenic activity as long as it normally exists in normal cells, exhibits properties as an oncogene by being incorporated into the genome of the RNA Ill tumor virus and expressed.

Normal cells have the function of maintaining living organisms through differentiation and proliferation, and it is known that these proto-oncogenes are genes that are closely linked to cell differentiation and proliferation. It is expected that this research will lead to the development of diagnostic and therapeutic methods for cancers directly related to cancer.

The present inventors have conducted research using molecular biological methods to explore the functions of the proto-oncogene (c-anal).
Following gene cloning of rbB, a new human proto-oncogene erbB-2 (hereinafter referred to as human c-erbB-2) (Semba et al., PNAS 82.6497 (1
985+, Yamamoto et al., Nature 3
19.230 (19861 was discovered.

Human c-erbB-2 is a human c-er that was later found to be derived from the epidermal growth factor (EGF) receptor gene.
This gene has extremely high homology to bB, and its gene product has tyrosine kinase activity that specifically phosphorylates tyrosine residues in proteins. human c-erbB
-2, like human c-erbB, is thought to be a type of receptor that exists on the cell surface, but its ligand has not been identified to date. However, overexpression of a growth factor receptor with tyrosine kinase activity It is quite possible that this plays some role in the development of cancer. From this point of view, the present inventors prepared DNA from cancerous tissue removed during surgery and conducted an experiment to confirm the prediction using a hybridization method with NA specific to the c-erbB-2 gene. As a result, the human c-erbB-2 gene was found to be amplified in about 20% of glandular epithelial cancers such as gastric cancer and breast cancer. This means that human c
This means that -erbB-2 contributes to the onset and progression of adenocarcinoma, and provides important information for the diagnosis of adenocarcinoma.

Regarding the expression function of erbB-2, Weinberg et al.'s rat c-erbB-2 gene (hereinafter referred to as neu)
649, (198611 was found to have acquired a point mutation, but neu was found to be a gene with the ability to cause cancer in cultured cells from tumors induced by administering the chemical carcinogen ethylnitrosourea to newborn rats. Served.

Diagnostic information regarding abnormal expression of erbB-2 in tumor tissues can be obtained using DNA detection methods, such as increase in copy number, but this method is not common as a cancer diagnostic method, and the information obtained is based on the DNA level. limited to things.

If a monoclonal antibody with the human c-erbB-2 gene product as the corresponding antigen can be obtained, it can be advantageously used in the diagnosis and treatment of adenocarcinomas such as breast cancer and gastric cancer.

[Problems to be Solved by the Invention] Therefore, an object of the present invention is to provide a monoclonal antibody that uses the human c-erbB-2 gene product as a corresponding antigen, and a hybridoma that produces the monoclonal antibody.

[Means for solving the problem] When an antigen has been isolated, it is comparatively difficult to use it as an antigen to produce a monoclonal antibody using that antigen as a corresponding antigen according to the conventional method of Köhler and Milstein. However, since the gene product of human c-erbB-2 has not yet been isolated, it is not easy to produce a monoclonal antibody using this as the corresponding antigen. As a result, we developed a vector containing the human c-erbB-2 gene, and transformed mouse fetal fibroblasts with this vector to generate human c-erbB.
The human c-erbB-2 gene product is present on the cell membrane). fused to human c-erbB-2
We succeeded in producing a hybridoma that produces a monoclonal antibody using the gene product as the corresponding antigen, and completed this invention.

That is, the present invention provides a monoclonal antibody that uses the human c-erbB-2a gene product as a corresponding antigen, and a hybridoma that produces the same.

[Effects of the Invention] The present invention provides a novel monoclonal antibody that uses the human c-erbB-2 gene product as a corresponding antigen, and a novel hybridoma that produces the same. The monoclonal antibody of this invention is directed against human c-erb, which is a proto-oncogene.
The product of the B-2 gene is used as the corresponding antigen, and as mentioned above, the human c-erbB-2 gene is significantly amplified in patients with adenocarcinoma. Human c-erbB-2 using antibodies
Adenocarcinoma can be diagnosed by checking gene expression. Furthermore, it is also possible to treat adenocarcinoma by inactivating the human c-erbB-2 gene product by binding the monoclonal antibody of this invention to the human c-erbB-2 gene product through an antigen-antibody reaction. be.

[Detailed Description of the Invention] As described above, the monoclonal antibody of the present invention uses the human c-erbB-2 gene product as the corresponding antigen, and specifically performs an antigen-antibody reaction with it. - The monoclonal antibody of this invention uses a substance that has not been isolated so far as the corresponding antigen.

That is, the monoclonal antibody of the present invention was first produced by using cells newly produced by the present inventors as an immunogen, and as will be described later, the monoclonal antibody of the present invention is a product of the human c-erbB-2 gene. specifically performs antigen-antibody reactions, therefore, pure human c-erbB-
Monoclonal antibodies produced using the two gene products as antigens are also within the scope of the invention.

The monoclonal antibody of this invention can be obtained as follows.

First, the present inventors prepared a recombinant DNA in which the human c-erbB-2 gene was integrated into a vector, which was capable of proliferating in animal cells and capable of transforming animal cells. used pBR322 as a vector.
4409 base pairs of human c-er into this vector.
Connect Hin d II + linker to the DNA of bB-2 (38th base to 4446th base),
Before and after this, the SV-40 transcription promoter (SV ori
SV-40 DNA (7) 17112-277 containing the base sequence of the region 270-5171) and the polyA signal part
1 and 4100-4711) pSV incorporating the base sequence
This was done by constructing a recombinant DNA called erbB2. The pSVerbB2 gene map is shown in FIG. 1.

The recombinant DNA thus obtained is then introduced into appropriate animal cells, such as fibroblasts derived from mouse fetuses, to transform the animal cells and express the human c-erbB-2 gene. By selecting the cells, it is possible to obtain cells that can be used as immunogens for producing the monoclonal antibodies of the present invention and that express the human c-erbB-2 gene on their cell surfaces. The present inventors used the above-mentioned pSVerbB2 in mouse fetal fibroblast cells NIH/3 using a calcium precipitation method according to a conventional method.
T3 (ATCC strain number CRL-16581 was introduced, and the SV-11 strain was established by screening. 5
The v-ti strain has been deposited with the Microbial Technology Research Institute of the Agency of Industrial Science and Technology, and its accession number is 10197.
This is the number.

Next, antibody-producing cells of an immunized animal using the cells thus prepared as an immunogen are fused with myeloma cells to produce a hybridoma, and human c-e
Hybridomas producing monoclonal antibodies with the rbB-2 gene product as the corresponding antigen are selected. This selection can be carried out, for example, by using a fluorescent antibody method according to a conventional method.
This can be carried out by selecting a hybridoma that is positive for V-11 and produces an antibody negative for NI)+3T3, which is a wild strain of SV-11.

The monoclonal antibody of this invention can be recovered from the culture supernatant by culturing the hybridoma thus obtained.

[Example] Hereinafter, this invention will be explained in more detail based on Examples.

Note that this invention is not limited to the following examples.

and tm Preparation of monoclonal antibody-producing hybridomas (il
Preparation of SV-11 strain In order to obtain a cell line that highly expresses the human c-erhB-2 gene, the present inventors developed the animal cell expression vector pSV2.
was selected, and a DNA in which the c-erbB-2 gene was inserted into this vector was prepared. This vector contains the transcription promoter of the SV40 virus (SV40 ori region 270
~5171 base sequence) and polyhe signal site (SV40
DNA (7) 1782-2771 and 4100-47
10 (7) nucleotide sequence), and human c-erbB
11 in. was added to the c-erbB-2 gene pre-fragment excised from the cDNA of the -2 gene using Smal and Aha3.
It was constructed by incorporating the dII+linker into the old ndI11 site downstream of the transcription promoter. This vector is called pSVerbB-2.
The genetic map is shown in Figure 1.

Next, the obtained recombinant DNA psVerbB-2
The fibroblasts derived from mouse fetus NIH3T3 (ATC
C strain number CRL-1658) cells using the calcium phosphate method to obtain transformed cells. Transformation 1@N
The SV-11 strain (Feikoken Bibori No. 10197) was established by screening IH3T3 cells for a strain expressing the human c-erbB-2 gene by DNA hybridization.

(11) Sensitization of mice The above cell line SV-11 was treated with DME supplemented with 10% fetal bovine serum.
Cultured in medium, detached cells with 0.05% EDTA, washed 5 times with phosphate buffered saline, and centrifuged (1000
rpm x 5 minutes), and the cells were used after adjusting the number of cells.

Immunization was performed by injecting l into the tail vein of 4-week-old BALB/c male mice.
x 10' cells of the cell line SV-11 were injected intravenously, followed by lx injection into the tail vein or intraperitoneally 2 to 4 times at 2-week intervals.
10' cells of cell line 5v-ii were boosted. During the course of immunization, the increase in antibody titer against the immunogen was monitored.

(iii) Cell fusion 3 days after the final immunization, tile cells were aseptically removed from mice with high antibody titers, loosened into single cells with a stainless steel mesh, and the 8-azaguanine-resistant myeloma cell line X-63 (8-azaguanine-resistant myeloma cell line Kohler G, and Milstei
n C, Nat, ure 256. pp, 495-
497 (19751, obtained from ATCC), and after washing and centrifugation, 1 ml of 50% polyethylene glycol (average molecular weight 1500) was added to the cell pellet to carefully perform a cell fusion operation.

Then, the fused cells were placed in a 96-well microplate at 1 x 1
0.1 ml was dispensed at a rate of 0'. Each microplate was carefully incubated in a serum-free medium selection method in an old incubator with 5% CO and 37°C (100% R).
I got a hybridoma.

(ivl) The hybridomas selected in this way were immediately subjected to repeated cloning using the limiting dilution method.The culture supernatant of the hybridomas, which were stabilized after five clonings, was reacted with cells in normal human peripheral blood, and the culture supernatants were reacted with cells in normal human peripheral blood, such as lymphocytes, granulocytes, etc. , monocytes and selected negative clones of SV2-61.
and SV2-61γ. Monoclonal antibody S
Hybridomas producing V2-61 and SV2-61γ (hybridoma SV2-61 and hybridoma S
V2-61γ) is a hybridoma S produced by producing ascites using the conventional Bresten method using BALB/c mice.
V2-61 and hybridoma SV2-61γ have been deposited with FIKEN, and their accession numbers are numbered 101, respectively.
No. 62 and Microtechnical Research Institute No. 10777.

The immunoglobulin subclasses of monoclonal antibodies SV2-61 and SV2-61γ were determined to be mouse IgM and mouse IgG, respectively, by the Ophthalony method.

Determination of fiil specificity Monoclonal antibody SV2- obtained as above
61 and SV2-61-r are immunogen SV-114,1m
The test results were positive for T. aerialis and negative for the wild type NIH3T3. Specifically, this was tested as follows. That is,
2 x 10” S pre-cultured in microplates
V-11 cells were attached to a microplate and fixed with formalin PBS. Then, 20μ of the culture supernatant was added to the cells.
In addition, a fluorescent antibody method was performed on a microplate according to a conventional method to screen for positive hybridomas. For positive cells, 106 cells were further subjected to flow cytometry measurement using a fluorescent antibody method using a conventional method. The results are shown in the flow cytograms of FIGS. 2 and 3. Figure 2 shows the results for monoclonal antibody SV2-61, and Figure 3 shows the results for monoclonal antibody SV2-6.
The results for 1γ are shown. In addition, in FIG. 2 and FIG. 3, N indicates a negative peak and P indicates a positive peak.

Similarly, the established cell line MKN-7 was established from gastric cancer tissue and is known to express approximately 30 copies of erb-82 (References: S, Fukushige et al., M
o1ecular and Ce1lular
Biology, Mar, 955 (19861
(obtained from the Department of Cancer Control, Institute of Medical Science, University of Tokyo) was also positive.

(Molecular weight of the iii antigen The molecular weight of the antigen was determined by a method combining immunoprecipitation and electrophoresis. The cells used were NIH3T3.SV
-11, SV22, a c-erbB-2 gene expression strain
7 and the above MKN-7 cells. Cells were labeled with 3SS-methionine by culturing confluent cells in a 6c glassware for 4 hours in methionine-free DMEM and 100 μL of 31S-methionine, which was solubilized with RIPA buffer.

To this, monoclonal antibody SV2-61 or SV2-6
After adding 5 μg of 1y and reacting under water cooling, the mixture was precipitated with Protein A-Sepharose (registered trademark). Electrophoresis was performed on an 8% polyacrylamide gel at 30 mA for about 2 hours, and after drying the gel, autoradiography was performed.

As a result, monoclonal antibody SV2-61 or SV2
-The protein that reacts with antigen and antibody with 61γ has a molecular weight of 185
A single band was confirmed at the kD position.

That is, a main band was observed at a position of 185 kD in MKN-7, SV227, and SV-11 cells. This was consistent with the molecular weight of the antigen predicted from the base sequence of the human c-erbB-2 gene. On the other hand, as a negative control, the same operation was performed using NRS normal rabbit serum, but no band was observed at 185 kD.

(ivl tyrosine kinase activity When SV-11 cells were immunoprecipitated with monoclonal antibody SV2-61, tyrosine kinase activity was confirmed at the position of the molecule IL185KD. This indicates that monoclonal antibody SV2-61 inhibits the c-erbB-2 gene product. It was shown that it reacted as an antigen.

Diagnosis of Ftl emetic adenocarcinoma Tumor tissues removed during surgical operations from patients with various cancers were stained using the tissue staining method (using formalin-fixed paraffin sections) using the monoclonal antibody SV2-61 of the present invention. The results are shown in the table. From the table, monoclonal antibody SV2-61
This monoclonal antibody specifically reacts with adenocarcinoma, indicating that it is a monoclonal antibody with excellent performance in diagnosing adenocarcinoma.

[Brief explanation of the drawing]

Figure 1 shows the genetic map of pSVerbB2, which is the recombinant DNA used to produce the monoclonal antibody of the present invention. Figure 2 shows the genetic map of the monoclonal antibody SV2-61 of the present invention.
FIG. 3 is a flow cytogram showing the reactivity of the monoclonal antibody SV2-61γ of the present invention. 7 Choga Hakuyama” 11 [R -↑ Yoi Asahi stock (3rd circle A T 9th group B 3rd concave C □ Kakumaru Kiyoshi flute 3 turisumi 9 strong, supporting solid E

Claims (9)

[Claims] (1) Monoclonal antibody whose corresponding antigen is the human proto-oncogene erbB-2 product. (2) The monoclonal antibody according to claim 1, which is produced by a hybridoma obtained by fusing myeloma cells with antibody-producing cells of an animal immunized with cells expressing the human proto-oncogene erbB-2 as an immunogen. (3) The monoclonal antibody according to claim 1 or 2, which belongs to the IgM subgroup and reacts with SV-11 cells but not with NIH3T3 cells. (4) The monoclonal antibody according to claim 1 or 2, which belongs to the IgG subgroup and reacts with SV-11 cells but not with NIH3T3 cells. (5) Claim 3 which is monoclonal antibody SV2-61
The monoclonal antibodies described. (6) The monoclonal antibody according to claim 4, which is monoclonal antibody SV2-61γ. (7) A hybridoma producing the monoclonal antibody according to any one of claims 1 to 6. (8) The hybridoma according to claim 7, which is hybridoma SV2-61. (9) The hybridoma according to claim 7, which is the hybridoma SV2-61γ.