JPH01224399A - Anti-idiotypic antibody - Google Patents

Abstract

NEW MATERIAL:A monoclonal anti-idiotypic antibody against a monoclonal antibody which specifically reacts with rat bladder cancer and human bladder cancer. USE:Preventive, therapeutic agent and diagnostic for bladder cancers. PREPARATION:For example, N-butyl-N-(4-hydroxybutyl)nitrosoamine is given to a rat to generate bladder cancer and the cancer tissues are a rat of the same system to cause immunization. After final immunization, spleen cells are collected. The spleen cells are fused with mouse myeloma cells and cultured in the HAT culture to prepare a hybridoma. The hybridoma is cloned according the limit dilution method to obtain monoclonal hybridoma. Then, the monoclonal hybridoma is given peritoneally to a mouse, dehydrated to give a monoclonal antibody solution. Further, the solution is given to a rat together with adjuvant to effect immunization and cell-fused with spleen cells in the similar manner to effect cloning. The cloned hybridoma is cultured to give anti-idiotypic type antibody.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a monoclonal anti-idiotype antibody, more specifically a monoclonal anti-idiotype antibody to a monoclonal antibody that specifically reacts with rat bladder cancer and human bladder cancer. The present invention relates to a hybridoma that produces the monoclonal anti-idiotype antibody and a method for producing the hybridoma.

BACKGROUND OF THE INVENTION The ultimate goals of conventional cancer research are the search for substances that exhibit anticancer and anticancer effects, and the early detection of cancer, that is, the establishment of early diagnostic methods. In recent years, a method has been developed to produce monoclonal antibodies that react with specific antigens by fusion of splenocytes and myeloma cells from animals that have been immunized against specific antigens. One point is to create monoclonal antibodies with high specificity for.

If an antibody that specifically recognizes a cancer-specific antigen is obtained, this antibody will not react with normal cells even from the same individual, making it possible to identify and react with only cancer cells. become able to.

That is, it is expected that the use of this antibody will enable cancer treatment and early diagnosis.

To realize this expectation, attempts are being made to create monoclonal antibodies against various cancer cells. -429 (1981))]
and 11 different antigens of human bladder cancer cells [Furado et al.
roc, Natl, Acad, Sci, , 8
1.224-228 (1984))] have been reported.

Further, Japanese Patent Application No. 62-064695 describes a monoclonal antibody that specifically reacts with rat bladder cancer and human bladder cancer by syngeneic immunization.

° In addition to these, monoclonal antibodies against various bladder cancers have been created at present, but bladder cancer can be diagnosed using these monoclonal antibodies because the antigen specific to bladder cancer is secreted. It was not possible to detect non-secreted antigens. Also,
When these monoclonal antibodies are administered to patients as therapeutic agents, anti-monoclonal antibodies are generated in the serum, making repeated administration impossible.

Purpose of the Invention Under these circumstances, the present inventors have conducted various studies to address the above-mentioned problems, and have developed a monoclonal anti-idiotypic antibody against a monoclonal antibody that specifically reacts with bladder cancer produced in syngeneic rats. The present inventors have found that it is useful to create such a system, and have completed the present invention.

That is, the present invention provides a monoclonal anti-idiotype antibody to a monoclonal antibody that specifically reacts with rat bladder cancer and human bladder cancer, a hybridoma that produces the monoclonal anti-idiotype antibody, and a method for producing the hybridoma. be.

Structure and Effects of the Invention The monoclonal anti-idiotype antibody and the hybridoma producing the monoclonal anti-idiotype antibody according to the present invention can be produced as follows: i) Infecting syngeneic rats with rat bladder cancer. immunization, fusing the splenocytes of the rat with mouse myeloma cells, and selecting a hybridoma that produces a rat monoclonal antibody that specifically reacts with rat bladder cancer and human bladder cancer; iii) Immunizing a rat monoclonal antibody produced by the hybridoma obtained in step ii) that specifically reacts with rat bladder cancer and human bladder cancer using the antigen as an antigen, and iv) immunizing the spleen cells of the dentist. and mouse myeloma cells to select a hybridoma that produces a monoclonal anti-idiotype antibody against a rat monoclonal antibody that specifically reacts with rat bladder cancer and human bladder cancer, and obtained in step V) step iv). The hybridoma is cultured under appropriate conditions, and the monoclonal anti-idiotype antibody is collected.

The immunization in step i) and the cell fusion and hybridoma selection in step ii) are carried out in accordance with the above-mentioned Japanese Patent Application No. 62-0646.
95, and its outline is as follows.

That is, for immunization, rat bladder cancer induced by N-butyl-N-(4-hydroxy-butyl)nitrosamine administration was subcutaneously administered to syngeneic rats, and 1 to 2
It can be carried out by repeated intraperitoneal and intravenous administration at weekly intervals.

Cell fusion can be carried out by a conventional method in an aqueous polyethylene glycol solution using the immunized rat pus cells and mouse myeloma cells, such as an 8-azaguanine resistant strain.

In addition, the obtained hybridoma was selected by collecting the culture supernatant of the hybridoma, reacting it with rat bladder cancer cells and human bladder cancer cells, and using the cell ELISA method using bar oxidase to induce both rat bladder cancer and human bladder cancer. This can be done by selecting hybridomas that produce specifically reactive antibodies.

The immunization in step iii) can be carried out as follows. That is, a rat monoclonal antibody that specifically reacts with rat bladder cancer and human bladder cancer was obtained from the hybridoma obtained in step ii), and a co(7) anti(F-
is added to complete Freund's adjuvant and injected subcutaneously into a suitable commercial animal such as a rat, mouse, hamster, guinea pig, rabbit, etc. This rodent 1
Immunize with further booster treatments at ~2 week intervals.

The cell fusion in step iv) is carried out in a conventional manner in an aqueous polyethylene glycol solution using the immunized rodent prey cells in step 111) and mouse myeloma cells, such as an 8-azaguanine resistant strain. Can be done.

Further, the obtained hybridomas can be selected as follows. That is, the rat monoclonal antibody that specifically reacts with both rat bladder cancer and human bladder cancer, which was used for immunization of M meat animals;
Two other types of antibodies were prepared, and these were reacted with the culture supernatant of the hybridoma using enzyme-linked immunosorbent assay (EL).
Hybridomas producing antibodies that react only with the rat monoclonal antibody used for immunization of rodents are first selected by ISA). Furthermore, hybridoma culture supernatants may be used to finally select hybridomas that produce antibodies that inhibit the reaction between the rat monoclonal antibody used for immunization and rat bladder cancer.

The anti-idiotypic antibody in step V) can be recovered by a conventional method from the culture supernatant of the hybridoma producing the antibody. Additionally, this antibody may be purified by an appropriate method.

Since the anti-idiotypic antibody of the present invention specifically reacts with a rat monoclonal antibody specific to rat bladder cancer and human bladder cancer, the anti-idiotypic antibody of the present invention has the same antigenic determinant as that common to rat bladder cancer and human bladder cancer. It is thought that it has. Therefore, when this anti-idiotypic antibody is administered in vivo, the same antibody that specifically reacts with bladder cancer is produced, making it possible to prevent and treat bladder cancer.

Furthermore, by preparing the anti-idiotype antibody of the present invention bound to biotin and using this as a secondary antibody, antibodies that react with bladder cancer should be produced when bladder cancer occurs in a living body. Therefore, it is possible to diagnose the presence or absence of bladder cancer by detecting this antibody.

Thus, the anti-idiotypic antibodies of the present invention enable the prevention, treatment, and diagnosis of bladder cancer.

The present invention will be explained in more detail with reference to Examples below, but these are not intended to limit the present invention.

Example 1 Preparation of rat monoclonal antibodies that specifically react with rat bladder cancer and human bladder cancer Immunization of syngeneic rats □ with rat bladder cancer, fusion of the rat's colon cells with mouse myeloma cells, and Hybridomas producing rat monoclonal antibodies specifically reactive with rat bladder cancer and human bladder cancer were selected as described in Japanese Patent Application No. 62-064695.

(a) Immunization of rats Wistar rats [Nippon Talea Co., Ltd.]
A 0.05% N-butyl-N-(4-hydroxy-butyl)nitrosamine (hereinafter abbreviated as BBN) drink was administered to the mice for 20 weeks, and an observation period of 20 weeks was added to induce bladder cancer. The obtained bladder cancer tissue was subcutaneously administered to syngeneic Wistar rats (7 weeks old), and subcutaneous, intraperitoneal, and intravenous administration were repeated at intervals of 1 to 2 weeks to induce immunization. I did it.

(b) Fusion and selection The above rat splenocytes and mouse-derived 8-azaguanine-resistant strain (hyboxanthinguanine phosphoribosyltransferase-deficient strain) P3-X63Ag8 U 1 (
P 3 U 1 ) [Current Topics
in Microbiology and Immunology
ology, 81. l-7 (1978) and 4 =
Polyethylene glycol [PEG-
1540; Fusion was carried out in Hanka Kagakuyaku Co., Ltd. solution.

The fused cells were separated from this PEG solution, and RDM1164
Splenocytes were suspended at a concentration of 2 x 10/x (l) in 0.0 liters of water and dispensed into a 96-well microplate.The fused cells were cultured in a CO incubator at a temperature of 37°C, and after a few days. RPM11640 culture medium containing hyboxanthin (100 μM), aminopterin (0.4 μM), thymidine (16 μM) (i.e., hyboxanthin,
100 μρ of a medium containing aminopterin and thymidine (hereinafter abbreviated as HAT medium) was added to each well. 37°
Continue culturing in a C15% CO atmosphere, check the color of the medium every 1 to 2 days, and if the pH of the medium decreases and the color of the medium becomes yellowish, add 100% of HAT medium each time.
Added μe/well. About two weeks after the start of culture, it was confirmed that only hybridomas were forming colonies, so the replacement culture solution was replaced with HT medium, which was obtained by removing aminopterin from HAT medium.

(c) Detection and cloning of antibodies It was confirmed that the hybridoma had grown sufficiently, so the culture supernatant was collected and reacted with human bladder cancer culture line T-24 and rat bladder cancer cells, and peroxidase was used. Wells in which antibodies specifically reacting with both BBN-induced rat bladder cancer and human bladder cancer were produced were selected using the cell ELISA method.

The /1 hybridomas in the wells thus selected were cloned by the limiting dilution method. Dilute to a concentration of 0.5 to 1 hybridoma/well,
No feeder cells were used. The diluted hybridomas were incubated in RPMI 1640 medium at 37°C, 5% CO
Cell ELISA using NO-oxidase was performed on the wells that had grown sufficiently, and the cells were cultured under 1 atmosphere. selected.

(d) Preparation of antibody solution The hybridomas thus obtained were intraperitoneally administered to nude mice [6 years of age; Japan Yurea Co., Ltd.] to induce ascites. 14
Ascitic fluid was collected a day later. Phosphate buffered saline (8.1mM Na, HPO4, 1.5mM K) was added to the obtained ascites.
H*PO4, 150mM NaCC.

After adding 3.139 g of ammonium sulfate (for enzyme purification) to 50% saturation, salting out was carried out at 4°C for 2 hours.

Next, the mixture was centrifuged (10,000 rpm, 15 minutes) to obtain a precipitate, which was dissolved in PBS and dialyzed against the same buffer 2Q. Dialysis was performed for 20 hours, replacing the dialysate with fresh dialysate 2e every hour. After dialysis, fractionation was performed using a Sephacryl S-300 (manufactured by Pharmacia) column equilibrated with PBS at a flow rate of 1°2191 min. By collecting the globulin fraction, a rat monoclonal antibody that specifically reacts with rat bladder cancer and human bladder cancer was obtained, and this antibody was adjusted to Ln/x (1) with PBS.

Example 2 Preparation of rat monoclonal anti-idiotypic antibody and determination of subclasses (a) Immunization of rats The antibody solution obtained in Example 1 above was mixed with complete Freund's Aschwand in a ratio of 5:2 to make an emulsion. This was subcutaneously injected into Wistar rats [6 weeks old; Nippon Yurea Co., Ltd.]. Then, at 10-day intervals, an emulsion containing an antibody solution and incomplete Freund's adjuvant mixed at a ratio of 5:2 was intraperitoneally injected into the same rat twice, and then once again.
After 0 days, final immunization was performed by intravenously injecting 0.5 ml of the antibody solution into the same rat.

(b) Fusion Lungs were removed from the rats 3 days after the final immunization.

This was shredded, filtered through mesh, and suspended in Dulbecco's Minimum Essential Medium (hereinafter abbreviated as DME) to obtain a splenocyte suspension. This splenocyte and a mouse-derived 8-azaguanine resistant strain (hypoxanthine guanine phosphoribosyltransferase deficient + M strain) S P
2/O-A g14 [Nature, 276
, 269-270 (197 g)] at a ratio of 5:l and centrifuged (1,500 rpm, 5 minutes).

Add 50% polyethylene glycol 4000 to the resulting precipitate.
(manufactured by Merck)/DME solution 31Q was added to 37°C warm water over a period of 1 minute with stirring. This and DME15x
Q was added over 6 minutes with stirring to perform cell fusion. After fusion, add a large amount of DME and centrifuge (1,500
Rotate for 5 minutes) and remove the supernatant. Then, 1
Add 0% FC3-DME medium to reach a splenocyte concentration of 3X
It was adjusted to be 108 Uj/It (l).

The following operations (c) to (e), ie, selection of hybridomas, detection of antibodies, and cloning, were performed using an "automatic sorting device for monoclonal antibody-producing cells" (manufactured by Sumitomo Electric Industries, Ltd.).

(c) Selection of hybridomas The cell suspension prepared in (b) above was dispensed into 96-well microplates in 200 μρ portions. The fused cells were cultured in a COt incubator set at a temperature of 37°C and a carbon dioxide concentration of 5%. After 2 days, half of the medium was removed from each well, and then hyboxanthin (100 μM
), aminopterin (0.4 μM), thymidine (101
1M) containing 10% F C3-DME medium (HAT
100 μρ of culture medium) was added to each well. 37°C1
When culturing was continued in a 5% carbon dioxide atmosphere and half of the medium was replaced with HAT medium every 2 days, it was confirmed that only hybridomas were forming colonies after 7 to 10 days, so the medium was replaced with HAT medium. The medium was changed to HT medium without aminopterin.

(d) Detection of antibodies Since it was confirmed that the hybridomas were sufficiently proliferating, antibodies were detected using the culture supernatant by the enzyme immunoassay method described below. A rat monoclonal antibody solution (10 uLi/
The antigen was fixed in μρ portions (4°C 1-night).
. Next, the antigen solution was removed, and a protein solution [I% bovine serum albumin 7 (BSA); manufactured by Sigma] was added to the solution. 100μQ of the hybridoma culture supernatant was dispensed onto this assay plate and allowed to react at room temperature for 2 to 3 hours. After washing the plate with PBS, biotin-labeled M2, which recognizes only rat antibodies of the lJa type, was added.
1 antibody (0,1μ9/shoulder) was added and reacted for 1 hour at room temperature.Then, the plate was washed with PBS, and an enzyme (peroxidase) was bound using the Vectastin ABC kit (manufactured by Vector). It's already - degree P
After washing with BS, 0-phenylenediamine (4 m9/
112), 100 μQ of phosphate-citrate buffer (pH 5°0) containing hydrogen peroxide (0,012%) was added to this plate, antibodies were detected by color reaction, and used for rat immunization. Wells producing antibodies that specifically reacted with rat monoclonal antibodies that specifically reacted with rat bladder cancer and human bladder cancer were selected.

(e) Cloning Hybridomas growing in the wells selected in (d) above were cloned by limiting dilution method. IO% FC3-DM so that the cell concentration after dilution is 0.3 cells/well.
Dilute with E medium and add 200% to 96-well microplate.
It was dispensed in μa portions. Cultivate at 37°C in a 5% carbon dioxide atmosphere, count the number of colonies in each well from time to time, and add 10% FC3-DM to wells where the pH has decreased.
The E medium was replaced.

After the colony reached a certain size, it was again examined by enzyme immunoassay to see if antibodies against the rat monoclonal antibody were produced. The hybridomas in the wells that strongly reacted with the rat monoclonal antibody used for rat immunization were cloned by the limiting dilution method in the same manner as described above.

(r) Inhibition test Using the culture supernatant of the hybridoma obtained as described above, the degree of inhibition of the reaction of a rat monoclonal antibody that specifically reacts with rat bladder cancer, vesicle/throat bladder cancer, and human bladder cancer was determined. It was measured.

First, rat bladder cancer cells were grown at 5x1051/x in Minimum Essential Medium (MEM) containing 10% FC3.
(adjust to 100 μρ in a 96-well microplate.
Dispensed in portions. This was cultured for one day at 37° C. under 15% carbon dioxide gas concentration, and then the cells were fixed to the bottom of the well using a cell fixative solution. Next, after blocking with 1% BSA, 100 μa of the culture supernatant of the hybridoma and a rat monoclonal antibody (10μ9) that specifically reacts with rat bladder cancer and human bladder cancer was added to each well. The reaction was allowed to proceed at room temperature for 2 hours.Then, the degree of reactivity between the rat bladder cancer and the above rat monoclonal antibody was measured by enzyme immunoassay.

As a result, five hybridoma strains were selected that produced monoclonal antibodies that strongly inhibited the specific reaction between rat bladder cancer and the rat monoclonal antibody described above, and these monoclonal antibodies were confirmed to be anti-idiotypic antibodies.

(g) Determination of subclasses of anti-idiotypic antibodies anti-rat antibodies [anti-IgGI, Ig(ga, IgG1 type or I
gG2b, IgGxc, IgM, IgA (rat/Mo
no Ab-ID SP kit; manufactured by Zymet)]
5 obtained by an enzyme immunoassay method in which the anti-idiotype antibody in the culture supernatant of hybridomas is reacted with
The subclass of anti-idiotypic antibodies produced by the hybridomas of the strain was determined. As a result, three types of strains developed color only at the site where anti-1gG+ was added, and IgG1 type or IgG2b type.
Of the 5 hybridomas obtained in (f), 3 strains produced rgG-type anti-idiotype antibodies, and 2 strains produced I-type anti-idiotype antibodies.
gG! It was found that this strain produced type b anti-idiotype antibodies.

Example 3 Preparation of mouse monoclonal anti-idiotype antibody and determination of subclass (a) Mouse immunization The antibody solution obtained in Example 1 was mixed with complete Freund's adjuvant at a ratio of 5:2 and emulsified. , this is B A L
B/c mice [6 weeks old - Nippon Talea Co., Ltd.] were injected subcutaneously. Then, at 10-day intervals, an emulsion containing an antibody solution and incomplete Freund's adjuvant mixed at a ratio of 5:2 was intraperitoneally injected twice into the same mouse.
Furthermore, 10 days later, the final immunization was performed by intravenously injecting 0.51 of the antibody solution into the same mouse.

(b) Fusion and selection Lungs were removed from the mice 3 days after the final immunization.

After shredding this, it was filtered through a mesh and suspended in a DME medium to obtain a splenocyte suspension. These splenocytes and mouse-derived 8-azaguanine resistant strain SP 210-Ag14 were mixed at a ratio of 5:1 and centrifuged (1,500 rpm,
5 minutes). Add 3jl12 of 50% polyethylene glycol 4000/DME solution to the obtained precipitate in 37°C warm water.
The mixture was added over a period of 1 minute while stirring. This and DME15
JfC was added over 6 minutes with stirring to perform cell fusion.

Next, after adding a large amount of DME, the mixture was centrifuged (1,500 rpm, 5 minutes) and the supernatant was removed. 10% FC3 for precipitation
- Add DME medium to reach a splenocyte concentration of 3 xio” cells/xi
After adjusting the amount to be 2, 200 μσ was dispensed into a 96-well microplate.

CO set at a temperature of 37°C and a carbon dioxide concentration of 5%.

The cells after fusion were cultured in an incubator, and after 2 days, half of the medium was removed from each well.
100 μe of medium was added to each well. 37°C15
% carbon dioxide atmosphere, and half of the medium was replaced with HAT medium every 2 days. After 7 to 10 days, it was confirmed that only hybridomas were forming colonies, so the medium was replaced with HAT medium. The medium was changed to HT medium without aminopterin.

(c) Detection and cloning of antibodies Since it was confirmed that the hybridomas had grown sufficiently, the culture supernatant was taken and reacted with two types of rat monoclonal antibodies that specifically react with rat bladder cancer and human bladder cancer. Wells producing antibodies that specifically reacted only with the rat monoclonal antibody used for mouse immunization were selected by enzyme immunoassay.

Hybridomas growing in the wells thus selected were cloned by limiting dilution.

10 so that the cell concentration after dilution is 0.3 cells/well.
%FC3-DME medium and dispensed into 96-well microplates in an amount of 200 μQ.

Culture was carried out at 37° C. in a 15% carbon dioxide atmosphere, and the number of colonies in each well was counted from time to time, and 10% FCS-DME medium was replaced in wells where the pH had decreased.

After the colony reached a certain size, it was again examined by enzyme immunoassay to see if antibodies against the rat monoclonal antibody were produced. The hybridomas in the wells that had strongly reacted with the above-mentioned cytoclonal antibody used for immunization of mice were prepared in the same manner as above.
Cloning using the limiting dilution method.

(d) Inhibition test f? of the hybridoma obtained as above? Using the i, fi supernatants, the degree of inhibition of rat bladder cancer and the reaction of a rat monoclonal antibody that specifically reacts with rat bladder cancer and human bladder cancer was measured.

First, rat bladder cancer cells were adjusted to 5×10 5 Ga/mQ with a minimum essential medium containing 10% FC3, and 100 μQ were dispensed into a 96-well microplate. This was cultured for one day at 37° C. under 15% carbon dioxide concentration, and then the medium was removed by suction, and the cells were fixed on the bottom of the well with a cell fixative solution of acetone:methanol=1:1. Next, after blocking with 1% BSA, the culture supernatant of the hybridoma and a rat monoclonal antibody (1
100 μρ of 0 μy/xυ was added to each well and allowed to react at room temperature for 2 hours. Next, the degree of reactivity between the rat bladder cancer and the above rat monoclonal antibody was measured by enzyme immunoassay.

As a result, four hybridoma strains were selected that produced monoclonal antibodies that strongly inhibited the specific reaction between rat bladder cancer and the above rat monoclonal antibodies, and these monoclonal antibodies were confirmed to be anti-idiotypic antibodies. .

(e) Determination of subclasses of anti-idiotype antibodies The subclasses of anti-idiotype antibodies produced by the four obtained hybridoma strains were determined by the Ophthaloniological immunodiffusion method. Into the center hole of an agarose gel that has a slightly large hole in the center and six small holes equally spaced on the circumference around this hole, add 1 blue 75 μσ culture medium for anti-idiotype antibody producing cells. anti-mouse I gM, IgG 111 gGza,
IgG type 1 or IgG2b, r gG3, IgA
(manufactured by Miles, USA) was added in an amount of 10 μQ each. When this agarose gel was observed after incubation for 24 hours at room temperature, IgG.

Since an inhibition line was formed between the culture supernatant and the culture supernatant, the obtained 4
The subclass of anti-idiotypic antibodies produced by hybridomas of this strain were all found to be Igc.

Patent applicant: Sumitomo Electric Industries, Ltd.

Claims (5)

[Claims] (1) Monoclonal anti-idiotype antibodies against monoclonal antibodies that specifically react with rat bladder cancer and human bladder cancer. (2) Anti-idiotype antibody is IgG_1 type or Ig
The monoclonal anti-idiotype antibody according to claim (1), which is a G_2b type rat antibody. (3) The monoclonal anti-idiotype antibody according to claim (1), wherein the anti-idiotype antibody is an IgG_1 type mouse antibody. (4) A hybridoma that produces a monoclonal anti-idiotypic antibody against a monoclonal antibody that specifically reacts with rat bladder cancer and human bladder cancer. (5) A method for producing a hybridoma that produces a monoclonal anti-idiotypic antibody against a monoclonal antibody that specifically reacts with rat bladder cancer and human bladder cancer, comprising: i) immunizing syngeneic rats with rat bladder cancer; ii) ) fusing the rat splenocytes with mouse myeloma cells and selecting a hybridoma that produces a rat monoclonal antibody that specifically reacts with rat bladder cancer and human bladder cancer; iii) the hybridoma obtained in step ii) is produce,
iv) immunizing a rodent with a rat monoclonal antibody that specifically reacts with rat bladder cancer and human bladder cancer, and iv) fusing splenocytes of the rodent with mouse myeloma cells; A method comprising selecting a hybridoma that produces a monoclonal anti-idiotypic antibody to a rat monoclonal antibody that specifically reacts with bladder cancer and human bladder cancer.