JPH02113894A - Anti-human uterus cancer antibody, its production and detection of human uterus cancer cell - Google Patents

Abstract

NEW MATERIAL:A monoclonal antibody having specificity against human uterus cancer cell. USE:A diagnostic for uterus cancer. PREPARATION:The objective monoclonal antibody is produced e.g., by immunizing a Balb/c mouse with a human uterus cancer cell, extracting the spleen after the final immunization, collecting antibody-producing cell from the spleen, fusing the cell with a mouse myeloma cell, culturing the fused cell in HAT medium to obtain a hybridoma, selecting a clone capable of producing an antibody having specificity against human uterus cancer cell from the hybridoma, monoclonizing the clone by cloning with limited dilution analysis, etc., and culturing the obtained monoclonal hybridoma.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to an anti-human endometrial cancer antibody, a method for producing the antibody, and a method for detecting cancer cells using the antibody.

[Background technology] Uterine cancer has a high morbidity rate among cancers in women. 8
In terms of water, uterine body cancer still accounts for about 10% of uterine cancers, but in Europe and the United States, the ratio of uterine cervical cancer to uterine body cancer is approximately 1:1+, and even in Japan, the lifestyle It is increasing with the westernization of the world. Uterine corpus fr, (
If uterine endometrial cancer is detected in the first stage (early stage cancer) and treated with surgery, the five-year survival rate is as high as 87.3%, but it is 72.8% in the second stage and 44% in the third stage. .8%, 13 in the fourth period
．． This decreases to 5%. Therefore, early detection is important for endometrial cancer as well as for other cancers.

The current diagnostic methods for endometrial cancer include endometrial cystoscopy, endometrial scraping histology, and other auxiliary diagnoses. Since endometrial cytology can identify high-risk groups for endometrial cancer, it is possible to diagnose by adding cytology to women who have experienced abnormal bleeding within the past few months after menopause or over 50 years of age. It is possible to do 5
On the other hand, although intrauterine scraping histology is inferior to cytology in terms of detection rate and screening method, it is indispensable as a definitive diagnostic method for uterine cancer. However, these endometrial cytology or endometrial curettage histology
Because this method uses special instruments to collect cells or tissues from the uterine body, it involves pain and the risk of infection for the patient, and although it is not necessarily a simple and safe method, it do not have. Furthermore, it is said that diagnosis of uterine body cancer requires a significantly higher level of knowledge and training than cervical cancer.

[Purpose of the Invention] The purpose of the present invention is to improve staining methods in cytology or histology by obtaining antibodies specific to uterine cancer cells and using the antibodies to label cells using antigen-antibody reactions. The present invention aims to provide a means for specifically staining uterine body cancer cells by performing an antibody method, thereby making it possible to easily and quickly diagnose uterine body cancer at an early stage.

DISCLOSURE OF THE INVENTION 1 The present invention provides the antibodies described in (1) to (3) below, methods for producing the same, and methods for detecting human endometrial cancer cells using the antibodies.

(1) Monoclonal antibody with specificity for human endometrial cancer cells.

(2) Antibody-producing cells obtained by fusion of mouse myeloma cells and antibody-producing cells of mice immunized with human endometrial cancer cells.
A method for producing a monoclonal antibody against human endometrial cancer cells, which comprises culturing a hybridoma and producing a monoclonal antibody from a culture solution or mouse ascites.

(3) After contacting the tissue sample to be examined with a labeled antibody that has specificity for human endometrial cancer cells or with an unlabeled antibody, 1. A method for detecting human uterine cancer sacs, which comprises bringing the sacs into contact with two swabbing bodies which have been subjected to ``drift'', and detecting human uterine cancer cells by searching for the label in each case.

4: The invention will be explained in more detail below.

The above-mentioned monoclonal antibody can be obtained as follows.

First, a mouse is immunized with human endometrial cancer cells, and a hybridoma is formed from the mouse anti-human endometrial cancer antibody-producing cells and myeloma cells. Next, the hybridoma is cloned, and a clone producing an anti-uterine body cancer cell antibody that is reactive with endometrial cancer cells is selected to obtain a monoclonal hybridoma. This hybridoma is cultured in nutrient medium or B a
Inject into the cavity of lb/c mice to obtain culture solution or ascites fluid containing the monoclonal antibody. The monoclonal antibody is purified from the culture solution or ascites by salting out fractionation using ammonium sulfate, ion exchange chromatography using DEAE Sephace i, and specific purification of immunoglobulin using protein A cellulofine.

The specificity of the monoclonal antibody obtained as described above is examined as follows. Reactivity with cultured human fibroblasts and human uterine cancer tissue, which are non-cancerous cells, and reactivity with existing cancer-related antigens are determined by a known enzyme immunoassay.

Further, the immunoglobulin class and subclass of the above monoclonal antibody can be determined by enzyme immunoassay using anti-immunoglobulin class/subclass antibodies.

The above monoclonal antibody according to the present invention can be used, for example, in a method for detecting human endometrial cancer cells in histology. This detection method involves bringing a tissue sample to be examined into contact with the labeled antibody described above, and then using a label detection means to search for the label attached to the cells, or searching for the label attached to the cell using a label detection means, or searching for the label attached to the cell using the labeled antibody. Beaded a sign after making contact 2
This is carried out by bringing the cells into contact with a second antibody (an antibody capable of binding to the above-mentioned antibody) and searching for a label attached to the cell using a label detection means. Radioactive substances, enzymes, fluorescent compounds (eg, fluorescein isothio/anate), and the like are used as labels for applying the above-mentioned labels.

Production of the above secondary antibody and labeling of the antibody are performed by conventional methods. In carrying out the method for detecting human endometrial cancer cells according to the present invention, (a) the above-mentioned labeled antibody or label? (b) the above-mentioned secondary antibody which has been labeled as necessary;
c) It is convenient to create and use diagnostic kits containing other necessary reagents and equipment (eg, buffers, chamber slides, etc.). The above-mentioned antibody according to the present invention specifically reacts with human endometrial cancer cells, and therefore is extremely useful for speeding up and objectifying the diagnosis of endometrial cancer.

The present invention will be specifically explained below using examples. However, the present invention is not limited to these.

Example 1 Preparation of anti-human endometrial cancer monoclonal antibody (a) Anti-human endometrial cancer cells established in the Department of Obstetrics and Gynecology at Higashi-Toyama University of Medical and Pharmaceutical Sciences were used. Transfer these cells to EMEM medium)
10% calf fat blood! The cells were cultured in a medium supplemented with (FBS), and upon immunization, the cells were detached with 0.25% trypsin and fixed with O1% glutaldehyde, which was used as an immunogen.

(b) Preparation of antibody-producing cells: 1iIJ per 6-week-old Ba1b/c female mouse.
0.25 1x10'+'il of cells obtained in (a)
suspended in z+2 physiological saline, thoroughly mixed with O-25mQ 70 India complete adjuvant, and injected intraperitoneally;
The first immunization was performed. Obtained in 2 weeks with Gofuki (a)! : Cells IX1.0'1m were suspended in 045I+1 physiological saline and injected intraperitoneally for booster immunization. After another 30 days, 5 x 10' cells obtained in (a) were suspended in 0.25 mQ of physiological saline and injected intraperitoneally for final immunization. Immediately after that, add 2.5 x 10' pieces of the solution obtained in (a) above to 0.05 mQ of physiological saline.
The suspension was injected into the tail vein. On the third day after the final immunization, the spleen of the mouse was removed aseptically, cut into pieces with scissors, and then passed through a stainless mesh into RP'JI 16.
40 medium (described in detail below (CX1.)) to prepare a suspension of tile cells.

(c) Cell fusion (1) Use the following materials and methods.

RPMI 1640 medium: RPIJI No. 16
40 (Difco La-boracories)
Sodium bicarbonate (12mM), sodium pyruvate (1mM), L-glutamine (2mM), potassium peninline G (50U/week i2), streptomycin sulfate (50μg/mQ), BJ: amikacin bisulfate (100μg/mQ) mQ) and p with dry ice.
The H was set to 7.2, and the bacteria were filtered using a 0°2μ top Toyo membrane filter.

MS-1 medium 2 Add sterile-filtered FBS (M, A, Bioproducts) to the above RPMI 1640 medium at a concentration of 15% (v/v).

PEG 4,000 solution: RPI, II 1640 medium polyethylene glycol 4,000 (PEG
4.000. Merck & CO, Inc.) 5
Prepare a 0% (v/v) serum-free solution.

8-azaguanine-resistant myeloma cells MS-1 (P3-
Fusion with N5I-1) is 5elected Meth
odin Ce1lular Immunology
(ed, B, B, !+1ishelland S
, M, Shiigi) , W,)1. F
Reeman and Company (1980
), 351-372, with some modifications.

(2) Tile cells (viable cell rate 100%) and myeloma cells (
100% viable cell rate) at a ratio of 5=1. The suspension of tile cells prepared in the first step (b) and myeloma cells are washed once with Noboribetsu's RPMI1640 medium. Next, suspend in the same medium and mix at the above ratio for fusion.

Conical styrene resin test tube with a capacity of 501Q (iza
kiGIass), RP old 1640 of 40taQ
Centrifuge in medium at 400Xy for 10 minutes and aspirate the upper groove completely. PEG 4,000 heated at 37°C to precipitate cells
Add 2.4 mQ of the solution dropwise over 1 minute with gentle stirring, and stir for another 1 minute to resuspend and disperse the spores. Next, 2.41Q of RPMI 1640 medium heated at 37° C. is added dropwise for 1 minute. After repeating this operation once more, 16.8tnQ of the same medium is added dropwise for 2 to 3 minutes with constant stirring to disperse the cells.

This is centrifuged at 400Xy for 10 minutes, and the supernatant is completely removed by suction. Next, add 37'C21D to this precipitated cell.
Warm N5-1 medium 24. Immediately add 3+Q and carefully pipette to disperse large clumps of cells using a 10raQ pipette. Furthermore, the same medium 48.6I
Add Q and dilute to 6.0X per well in a polystyrene 96-well microwell (Ivaki Glass).
Add 10' 10.1 mQ cells.

In addition, the 96-well microwell used at this time is pretreated with 0.2y+Q MS-1 medium, kept warm in a carbon dioxide gas incubator (37°C) overnight, and the medium is removed by suction before use. . Add cells to the above microwells at 7%
The cells are cultured in carbon dioxide gas/93% air at a temperature of 37° C. and a humidity of 100%.

(d) Selective growth of hybridomas using selective media (1) The culture medium used is as follows.

) IAT medium: N5-1 medium I described in (C) above, hypoxanthine (100 μM), aminopterin (
0.4 μM), and thymidine (16 μ; l).

(Above except that HTT-based niaminopterin was removed) IA
It has the same composition as T medium.

(2) The next day (1st day) after starting the culture in (c) above, apply 2 drops of HAT medium (approximately 0, IhmQ) to the cells using a Pasteur pipette.
) is added. 2.3.5.8. On day m1, half of the medium (0,1 mQ) is replaced with fresh HAT medium, and on day 14, half of the medium is replaced with fresh HT medium. From then on 3
Replace half of the medium with fresh) IT medium every ~4 days. Sufficient hybridoma growth is usually observed within 2 to 3 weeks (100% melt). The solid phase-antibody binding test method described in the next section (c) (E
Check for positive wells by LISA). Next, as a feeder, 1 m of HT medium containing 10' mouse thymocytes was prepared using a polystyrene 24-well cell tube (1yak).
i Glass) to transfer the entire contents of each positive hybridoma detected above. This is cultured at 37° C. for about 1 week in the presence of 7% carbon dioxide as in (c) above. During that time, replace the supernatant (J, 5mQ) of each well with fresh HTT solution 0.5mα once or twice.

When the hybridomas have grown sufficiently, the positivity is reconfirmed by ELISA, and each is cloned by the limiting dilution method described in the next section (f). Note that the remaining liquid after being used for cloning is transferred to a 25 cm polystyrene tissue culture flask (Ivaki Giass) to prepare a sample for cryopreservation.

(e) Search for hybridomas producing anti-human endometrial cancer cell antibodies using solid phase-antibody binding test (ELISA) Made in Japan! + Journal of the Gynecological Society 38.53-58 (1986
) The following procedure was performed according to the method of Inoue et al.

The established endometrial cancer cell line used for immunization was treated with trypsin,
Make a cell suspension of 1X10' cells/7+Q5 degrees in E-MEM medium. The cells were aliquoted into 96-well Microtight 'V-Nyo 7 plates (Flow Laboratories) in 0.1 mQ increments and cultured for several days under 7% carbon dioxide gas and 37°C, and cells grew throughout the wells of the plate. After confirming that, the culture medium was removed, the wells were washed with phosphate buffered saline, and 0.1111 (!) of 4% neutral formalin was dispensed into each well.
It was left standing for 5 minutes. By this operation, the cells were fixed on the wall of the well, and after blocking the unfixed part of the 5° well wall with 1% bovine serum albumin, the cells were placed in the ibridoma growth well (35/752ffll) obtained in (d) above.
) was added and incubated at room temperature for about 1 hour. Horseradish peroxidase-labeled goat anti-mouse immunoglobulin (cappe tLa
b,) was added and further incubated at room temperature for about 1 hour. Next, add hydrogen peroxide and the substrate 〇-FPR-diamine, and qualitatively judge the degree of brown color produced with the naked eye, or use a microplate reader (1, fPR-
A4, Toyo Soda) to measure the absorbance at 492 nm. The serum of a non-immune mouse was used as a negative control, and the serum of an immunized mouse was used as a positive control, and when compared with the negative control, those that clearly developed color were determined to be positive (positive, x4.7%).

(f) Cloning Since there is a possibility that two or more types of hybridomas are growing in each well width, cloning is performed by the limiting dilution method to obtain monoclonal antibody-producing hybridomas. A cloning medium was prepared containing 107 mouse thymus taocytes as feeders per 1ls-1 medium tnQ, 5 per well in 36 wells of 966 microwells, 36 unil 3 and 24 wells,
Add 1 and 0.5 hybridomas. On Day 5 and Day 122, add about 0.1 m4 of NS-1 medium to all wells. E L f S for groups in which sufficient hybridoma growth was observed 14 to 15 days after the start of cloning, and 50% or more of the wells were negative for colony formation.
, perform method A. If all wells tested are not positive,
Confirm the number of colonies in the antibody-positive wells and add 1 in the frog.
Select 4 to 6 wells in which colonies have been confirmed and re-clon them. Finally, 114 hybrid strains producing monoclonal antibodies against human endometrial cancer cells were obtained.

(&) In vitro proliferation of monoclonal antibodies @ and in vivo proliferation Monoclonal antibodies are propagated by conventional methods. That is, the monoclonal antibody targets each hybridoma obtained in NS.
It can be obtained from the culture supernatant by culturing (in vitro growth) in an appropriate culture medium such as L medium (monoclonal antibody protein AK is io-100 μg/m (l)). In order to obtain the blistane (2, 6, to, 1
4-tetramethylpentadecane, Aldrich Ch
0.5:yl□Q per four mice was administered, and 1 to 3 weeks later, each Vibe II Doma lXl
By intraperitoneally administering 0', ascites with a monoclonal antibody protein concentration of 4 to 7?1 g/*Q can be obtained in vivo after 1 to 2 weeks.

(h) Heavy chain, light chain, and isotype of monoclonal antibody. Each of the ascites obtained in (g) above is first bound to a microtight rayon plate on which endometrial cancer cells are immobilized according to the ELISA method described above.

After washing with phosphate-buffered saline (PBS), an isotype-specific rabbit anti-mouse Ig antibody (
Zyzed Laboratories). P
After washing with BS, horseradish peroxidase-drifted goat anti-horseradish IgG (H+L) antibody was added, and 2
．． Detection was performed using 2'-azinodi (3-ethylbenzothiazoline sulfate-6) and hydrogen peroxide. The results are summarized in Table 1 below.

Among the obtained monoclonal antibodies against human endometrial cancer cells, 8 clones had immunoglobulin chains γ, 4 and 4.
Clones are μ/<wo, one clone is γ2. ', and the l clone is γ3. I had a party.

(I) Purification of monoclonal antibodies Each of the ascites obtained in (g) above was subjected to ammonium sulfate fractionation (40%!2!
After that, the IgG class was determined using D
A non-adsorbed fraction of EAE-Sephacel (pharmacia) was collected, and calf fat serum and mouse-derived protein in the medium were separated and removed. Ig! , 40m containing 0.1M sodium chloride for i-class purification.
DEAE equilibrated with M phosphate buffer (pH 8,0)
-5ephace I column chromatography, eluting with a gradient of sodium chloride from 0.1 M to 1,000 liters.

moreover,! gG; 7 ras was adsorbed on a Protein A Cellulofine (Seikagaku Corporation) column equilibrated with 50ffIM Tris-HCl buffer (pH 8,6) containing 0.15M sodium chloride, and after removing the non-adsorbed fraction, 0.15M g4 was prepared by elution with 50 mM acetate buffer (pH 4,0) containing sodium chloride. In addition, the eluate is 1.5! Neutralized with J Tris-HCl buffer (p) 18.9).

Example 2 Characteristics of monoclonal antibodies (a) Reactivity to normal human cells and human endometrial cancer tissues (sections) The reactivity of the monoclonal antibody according to the present invention to normal human cells was investigated. Human normal cells include human fibroblasts (Gin-i Q; American Type C
Cultured cells of ulture collaboration) were used. , the operating method is the ELI described in Example 1 (e)
In accordance with SA law.

In addition, a search for the specificity of the monoclonal antibody of the present invention for human uterine cancer tissues (sections) was carried out using the enzyme antibody method, according to the method of Nakaki et al. described in Interdisciplinary Project 72-88 (!985). Ta. That is, Rose L was immersed in the neck of 95% ethyl alcohol, 80% ethyl alcohol, and 70% ethyl alcohol, further washed with water, and treated with 0.3% hydrogen peroxide solution at room temperature for 30 minutes. The cells were then washed once with PBS and treated with 10% goat serum for 30 minutes at room temperature. 1
As a wipe, each monoclonal antibody was reacted for 300 minutes at room temperature, and after washing three times with PBS, horseradish peroxidase-labeled goat anti-mouse immunoglobulin (Cappel Lab,) was reacted for 300 minutes at room temperature as a second wipe. Ta.

After washing 3 times with PBS, 0025% diaminobenzidine.
0.01% hydrogen peroxide solution containing tetrahydrochloride at room temperature
The reaction was allowed to proceed for 5 minutes. The intensity of color development was observed using an optical microscope. The results are summarized in Table 2 below. None of the seven clonal antibodies reacted with normal human cells, and it was confirmed that these antibodies were specific to human endometrial cancer cells.

The reactivity to human uterine cancer tissues (sections) differs depending on each clone, with 2 clones showing strong reactivity, 6 clones showing reactivity, and 6 clones showing no reactivity. ;. In addition, the surface of the cancer cells and the luminal surface of the luminal structure were strongly stained. Clone number 16 - IFll and l5
-18 old antibody was considered suitable for tissue staining.

(b) Cross-reactivity with known cancer-related antigens It was searched whether the monoclonal antibody according to the present invention reacts with known cancer-related antigens. As known FIE-related antigens, CEA (Cosmo Bio), AFP (Fusmo Bio), and CA-125C Centocor) were investigated. Western blotting was performed using the pressure section method described in Cell Engineering 1&2.1061-+068 (1983). That is, 3.2 μ9 of CEA, 2.5 units of CA-+258 and 1 μ9 of AFP are pre-electrophoresed and then transferred to a nitrocellulose membrane. Monoclonal antibody (clone number 16-
18H1) is reacted and washed. Next, the cells were reacted with horseradish peroxidase-labeled goat anti-mouse immunoglobulin, washed, and stained with diaminobenzidine.

As a result, no staining was observed for any of CEA, C, 8-125, and AFP, indicating that the monoclonal antibody according to the present invention does not cross with these known cancer-related antigens.

Example 3 Histological Diagnosis The positive rate of tissue staining for human uterine cancer using the monoclonal antibody according to the present invention was investigated.

Human endometrial cancers include Adenocarcinoma 8 and Adenoacanthoma C%I n
Twenty-one patients with Fj5) were studied.

The monoclonal antibody used was the aforementioned clone number 16.
- IFll and 1.6-L, S old. Tissue sections were prepared by fixing the surgically removed cancer-bearing uterus in formalin.
Using paraffin-embedded samples, staining was performed according to the method described in Example 2 (a).

The results are shown in Tables 3, 4 and 5 below.
Table 3 (in the table, Ia, Ib, n, and ■ indicate the advanced stages of the International Federation of Obstetrics and Gynecology (FIGO)) shows the results of each of the more clonal antibodies for each of 21 cases of human uterine bodies. This shows reactivity.

Table 4 shows the reactivity of each monoclonal antibody as a positive rate for each tissue type of human endometrial cancer. Table 5 classifies human endometrial cancer by stage of early cancer (stage Ia and stage Ib) and advanced cancer (stage ■ and stage ■), and expresses the reactivity of each monoclonal antibody as a positive rate. .

The monoclonal antibody of Clone No. 16-IFII showed a tendency to react more with adenocarcinoma than with vA tumor cancer. Furthermore, it showed a tendency to be more responsive to early cancer than to advanced cancer. The monoclonal antibody of clone number 16-18) II showed a high positive rate (71.4%) and was thought to be more specific for adenocarcinoma than for adenocarcinoma. Similarly, high reactivity for early muscle (positive rate 68
．． 8%).

These results showed that it is possible to diagnose human endometrial cancer using these monoclonal antibodies.

No. table

Claims (1)

[Scope of Claims] 1) A monoclonal antibody having specificity for human endometrial cancer cells. 2) Human, characterized by culturing a hybridoma obtained by fusion of mouse myeloma cells and antibody-producing cells of a mouse immunized with human endometrial cancer cells, and purifying monoclonal antibodies from the culture solution or mouse ascites. A method for producing a monoclonal antibody specific for uterine cancer cells. 3) After contacting the tissue sample to be examined with a labeled antibody having specificity for human endometrial cancer cells or with an unlabeled antibody,
A method for detecting human endometrial cancer cells, which comprises further contacting the cells with a labeled secondary antibody and searching for the label in each case to detect human endometrial cancer cells.