JPH03103189A - Monoclonal antibody and hybridoma capable of secreting same antibody - Google Patents

Abstract

NEW MATERIAL:A monoclonal antibody inhibiting a biological activity of basic fibroblast growth factor(bFGF) and exhibiting no crossing-over reaction with bFGF inactivated by heating. USE:Useful for basic research of bFGF and diagnosis and treatment of malignant tumor by measuring bFGF which is malignant tumor maker in high precise. PREPARATION:A mouse is immunized with bFGF separated from cow brain, etc., and then a spleen cell of the above-mentioned mouse is fused with a mouse myeloma cell. Then cell capable of producing the aimed antibody from the resultant hybridoma is selected and the selected cell is proliferated in abdominal dropsy of mouse to provide the aimed antibody.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field 1] The present invention relates to two types of novel monoclonal antibodies and hybridomas that secrete each of these monoclonal antibodies. [Problem to be solved by the prior art and the invention 1 Fibroblast growth factor (FGF)
owthfactor) is a protein found in the cerebrum and pituitary gland extracts, and is a protein found in the cerebrum and pituitary gland extracts, and is a protein found in the cerebrum and pituitary gland extracts, and is similar to basic fibroblast growth factor (bFGF) and acidic fibroblast growth factor (aFGF).
It has been known. Fibroblast growth factors (FGFs) are
In addition to cultured fibroblasts, the proliferation of many cultured mesoderm cells including vascular endothelial cells was stimulated at low concentrations (several pg to several ng/m).
l) [Gospodarowicz et al., J.
．． Cell. Physiol. , Supplem
ent 5, 15-26 (1987)]. The actions of basic fibroblast growth factor (bFGF) and acidic fibroblast growth factor (aFGF) are almost similar, but basic fibroblast growth factor (bFGF) is more widely distributed in the body. The fact that I have a tumor, Ia! It is known that there are many cases of production in cells. The in vivo effects of basic fibroblast growth factor (bFGF) are known to include differentiation from ectoderm to mesoderm in embryos, promotion of nerve cell survival and differentiation, and especially angiogenesis. Its effects are attracting attention. Basic fibroblast growth factor (bFOF) produced by tumor cells is considered to be one of the tumor angiogenesis factors, and is thought to play a role in the formation of solid tumors. Therefore, basic fibroblast growth factor (bFGF)
has the potential to be a biologically active tumor marker;
There is still no accurate measurement of blood levels. However, basic fibroblast growth factor (bF) is found to be high in the urine of patients with urinary tract tumors.
There are reports that there is a release of GF) [Chodak et al., Cancer Res. , 48, 2083-20
88 (198 g)], polyclonal antibodies against basic fibroblast growth factor (bFGF) have been obtained using synthetic peptides or natural molecules as antigens [e.g.
er et al., Nature, 325, 257-259 (
1987)]. ! However, it has been very difficult to generate monoclonal antibodies against basic fibroblast growth factor (bFGF). Massoglia et al. first reported the production of four monoclonal antibodies against basic fibroblast growth factor (bFGF) (Massoglia et al.
soglia et al., J. Cell. Physiol.
, 132, 531-537 (1987)], but all of these have their basic fibroblast growth factor (bFGF)
did not inhibit the biological activity of Subsequently, Seno et al. also reported the generation of four monoclonal antibodies against basic fibroblast growth factor (bFGF) [Seno et al., Hy
bridoma, 8, 209-221 (1989)
], but these also have basic fibroblast growth factor (bFG)
F) did not inhibit the biological activity of F).

As a result of various studies, the present inventors have discovered that the biological activity of basic fibroblast growth factor (bFGF) can be inhibited, and that the active and inactive forms of basic fibroblast growth factor (bFGF) can be inhibited. We have discovered a monoclonal antibody that can distinguish between [Means for Solving the Problems 1] The present invention provides a monoclonal monoclonal that inhibits the biological activity of basic fibroblast growth factor and does not show cross-reactivity with basic fibroblast growth factor that has been inactivated by heating. Regarding antibodies. Additionally, the present invention relates to monoclonal antibodies that inhibit the biological activity of basic fibroblast growth factors and exhibit cross-reactivity with basic fibroblast growth factors that are inactivated by heat.

The present invention is also formulated by fusion of mouse myeloma cells with mouse spleen cells immunized with basic fibroblast growth factor, which inhibits the biological activity of basic fibroblast growth factor and is inactivated by heating. It also relates to hybridomas that secrete monoclonal antibodies that do not exhibit cross-reactivity with activated basic fibroblast growth factor.

Additionally, the present invention provides that basic fibroblast growth factor is formed by fusion of mouse spleen cells and mouse myeloma cells immunized with basic fibroblast growth factor, which inhibits the biological activity of basic fibroblast growth factor and is inactivated by heating. It also relates to hybridomas that secrete monoclonal antibodies that cross-react with modified basic fibroblast growth factor. The monoclonal antibody of the present invention is useful for basic research such as the bioimplantable properties of basic fibroblast growth factor (bFGF), and furthermore, the monoclonal antibody of basic fibroblast growth factor (bFGF), which is attracting attention as a malignant tumor marker, ) Diagnosis of malignant tumors by measuring with high precision and diagnostic agents used therein,
It is expected to be applied as a therapeutic agent for malignant tumors. The present invention will be explained in detail below. ] Mu [Basic fibroblast growth factor (hereinafter referred to as bF) as an immunogen
(abbreviated as GF), for example, is derived from the bovine brain and pituitary gland.
It can be separated and purified using known methods. Specifically, the immunogen was purified as follows. Namely, the method of Gospodarowicz et al. [G
ospodarowicz et al., Proc. Natl.
Acad. Sci. USA 81, 6963-6
967 (1984)] bFGF was purified from bovine brain. Eluted from the column, DNA synthesis stimulating activity [
Nishikawa et al., Methods Enzymol-14
6.11-22 (1987): Yoshitake et al., Cel
l StrucLFunct. 7, 229-243
(1982) collected fractions giving one protein peak with reference 1. A reduced sample of purified bFGF gave a single band of protein with a molecular weight of 18,000 on 19.5% SDS-PAGE. Mice are immunized by an in vivo immunization method using a bFGF immunogen solution purified from Yutan and Ichthyon [Nishikawa et al., Method.
ds Enzymol. 146. 1 1-22 (1
987): - Yoshitake et al., Arch. Biochem
．． Biophys- 263, 437-446 (
1988)]. Specifically, immunization was performed as follows. Briefly, the purified bFGF immunogen solution was mixed with an equal volume of Freund's complete or incomplete adjuvant until emulsified. This bFGF1
Immunization was performed by subcutaneously administering a mixture containing 0 μg to 6-week-old female BALB/c mice (first
immunization). Thereafter, the same procedure was performed at intervals of 2 to 4 weeks for a total of 5 immunizations. Quantification of antibody titer in mouse serum is
Massoglia et al., J. Cell Physio
l. 132, 531-537 (1987). Four days after the final immunization, the spleen was aseptically removed from the mouse and used in the following cell fusion step.

C Hybridoma 1 Specifically, a hybridoma was created as follows. Jl! of (B) above in DME medium. ! ! 1 and crushed on a stainless steel mesh to obtain a spleen cell suspension. The cells thus obtained were washed with DME medium by centrifugation, and the number of living spleen cells was determined. On the other hand, pre-cultured mouse myeloma cells (myeloma cells) P3-X63-Ag8-Ul (P3U1)
(Dr. Oordon H. Sato, W. Alt.
on Jones Cell Science Center
ter, Inc. Add IXIO' of the above spleen cells to about 5
Centrifugation was performed (500 x g, 10 minutes).
Aspirate the supernatant, loosen the pellet well, and remove 50%
0.5 ml of (w/v) polyethylene glycol 4000 in DME solution (kept at 37°C) was added dropwise, and the polyethylene glycol solution and cell pellet were mixed by gently rotating the centrifuge tube by hand for 1 minute. Next, 24 ml of DME medium kept at 37"C was added in small portions, and the tube was gently rotated, followed by centrifugation (500Xg, 10 minutes). The cell pellet was mixed with 10% bovine After centrifugal washing with PRMI medium containing fetal serum, cells were incubated with HAT containing 10% fetal bovine serum.
Medium (RPMI medium with aminopterin 4 x 1σ 7M, thymidine 1.6 x 10'M, hypoxanthine I x 10
'M added) was suspended in 40 ml. Add 20 cells of this cell suspension to each well of a 96-well cell culture plate.
0 μl was dispensed and culture was started at 37'C in a carbon dioxide gas incubator containing 5% carbon dioxide gas. During culture, remove approximately 100 μl of the medium from each well at intervals of 2 to 3 days, and add freshly added H
Hybridomas growing in HAT medium were selected by adding 100 μl of AT medium. lO% from day 8
M' medium containing fetal bovine serum (thymisin 1 in DME medium)
．． 6 x 10"M, Hyboxanthin IX added to 10'M), observe the growth of hybridomas, and on the 10th day, perform the following ELIS
Hybridomas producing bFGF antibodies were screened by Method A. The presence or absence of produced antibodies in the culture supernatant of D hybridomas can be determined by ELIS.
Measured by method A. i.e. 96-well ELISA
Add the above b to each well of a plate for
Dispense 2 μg/ml of FGF immunogen solution and incubate at room temperature for 4 hours.
I left it for a while. After removing the supernatant, 1% BSA-PBS
was dispensed and blocked. Next, Q. 1%Twee
After washing four times with n20-PBS, 50 μl of the culture supernatant was added to each well and reacted at room temperature for 4 hours. after that,
Remove the supernatant and o. i%Tween20-4 on PBS
After washing twice, wash with Tween 20-PBS for 2,000
50 μl of peroxidase-conjugated rabbit anti-mouse antibody (Dako, Denmark) diluted 1:0 was added to each well. After the reaction, each well was washed 4 times with 0.1% Tween20-PBS, and 0.1M citrate-phosphate buffer (
pH 5.0), 0.4% 0-phenylenediamine and 0.4% 0-phenylenediamine.
Add 50 μl of a solution containing 0.005% hydrogen peroxide to each well, react for 15 minutes at room temperature, and
The absorbance at was measured. As a result, antibody production was observed in 5 out of 192 wells. After that, ELI again
After confirming the presence or absence of anti-bFGF antibody production using the SA method, cloning was performed three times using the limiting dilution method. Among these clones, clones with good growth, high antibody secretion ability, and stable clones were selected and re-cloned in the same manner as above to establish two types of anti-bFGF-specific antibody-producing hybridomas, each of which was bFM -1 and bFM-2.

Monoclonal construction: In “Vivo’Bristan” (
2, 6, 10. 14-tetramethylbentadecane) was administered intraperitoneally to a 7-week-old BALB/c mouse, and after 7 days, the in vitro-proliferated hybridoma bFM-1 and bFM
-2 was inoculated at IXIO' cells per mouse. Approximately 5-10 cells from one mouse per hybridoma
ml of ascites was obtained. The antibody concentration is 1-3 mg/
It was ml. Purification of monoclonal antibodies in ascites
It was done as follows. After the ascites was filtered through gauze and centrifuged (20,000 x g, 10 minutes), solid ammonium sulfate was added to give a 30% saturation concentration. After centrifugation (20,000 x g, 10 minutes), ammonium sulfate was further added to the supernatant to a saturation concentration of 50%, and a precipitate was obtained by centrifugation. The precipitate was dissolved in a small amount of 5mM Tris-HC1 [street solution (pH 7.5)] and dialyzed three times against 100 times the volume of the above buffer. The obtained dialysate was mixed with DE-5 equilibrated with the above buffer solution.
0 cellulose column and washed with the above buffer. The adsorbed monoclonal antibody was eluted by the concentration gradient method using the above buffer and a solution containing 0.15M NaCl. The homogeneous purity of the obtained monoclonal antibody was confirmed by SDS-electrophoresis. F Globulin class anti-bFGF specific monoclonal antibodies bFM-1 and bF
Identification of the M-2 immunoglobulin class and subclass was performed using the Mouse Monoclonal Antibody Isodiving Kit (
(manufactured by Amersham).

As a result, each was found to be IgG1/κ.

G monoclonal difference (a) Bovine pituitary bFcF(1-146) [Esch
et al., Proc. Natl. Acad. Sci. U
SA 82, 6507-6511 (1985) 1, with the sequence of 14-16 amino acids in the natural form and an additional Tyr, were prepared using a fully automated Biolynx machine.
4170 Peptide Synthesizer (
Pharmacia LKB Biotechnolo
gy, Sweden) or fully automatic Applied Bio
systems 430 A Peptide Synt
hesizer (Applied Biophysics
cs Inc. [Matsuo et al., In Vitro Cell-Dev.
Biol. 24, 477-480 (198g)]
, two of which are Try”-bFGF(1-15
) and Try”2-bFGF (133-146), which correspond to the amino-terminal and carboxy-terminal sequences of the natural molecule, respectively.
bFGF(36-50), which contained the major portion of the hydrophilic sequence of bFGF identified by whole sequence hydrophilicity analysis. Monoclonal antibodies bFM-1 and bFM-2 of the present invention
To determine the evitope on bFGF for b
The cross-reactivity between three types of linear fragments of the FGF molecule and monoclonal antibodies bFM-1 and bFM-2 was investigated. The results are shown in Figures 1 and 2. Figures 1 and 2 are
Monoclonal antibodies bFM-1 (Fig. 1) and bFM-2 (Fig. 2) of the present invention and bFGF fragment, bF
This figure shows the results of cross-reactivity with GF (described later) and heat-inactivated bFGF (described later) using a competitive binding assay. In Figures 1 and 2, the vertical axis B/Bo
is the 125■ label b in the presence of various concentrations of antigen.
It is the amount of specific binding (B) of FGF divided by the amount of specific binding (Bo) in the absence of unlabeled antigen. In the experiments shown in Figures 1 and 2, 6 ng of monoclonal antibody bFM-1 or monoclonal antibody b
75 ng of FM-2 was added to the reaction mixture (0.5 ml). When Bo is expressed as a percentage of the total tracer added, it was 56% for monoclonal antibody bFM-1 and 35% for monoclonal antibody bFM-2. In Figures 1 and 2, ◎ indicates active bFGF, ○ indicates heat-inactivated bFGF, and indicates Try+6-bFGF (1-15).
☆ is Ty? '-bFGF (36-50), ◇ is Tyr1
32-bFGF (133-146). As is clear from FIGS. 1 and 2, these peptides, namely T
ry"-bFGF (1-15), Tyr"-bFGF
(133-146) and Tyr"-bFGF (36-5
0) is the monoclonal antibody bFM-1 of the present invention
or bFM-2 at 100 μg/ml (this concentration is 10% higher than the molar reference concentration of natural bFGF, which shows clear cross-reactivity).
No cross-reactivity was observed even at 0-1000 times. This shows that these sequences, ie the amino terminus, the carboxyl terminus and the major hydrophilic region, are not recognized by the monoclonal antibodies of the invention as continuous evitopes.

(b) The biological activity of bFGF is thermolabile and acid labile. Therefore, the conformation of the bFGF molecule (which is not maintained by disulfide bonds) appears to be essential for its biological activity [Gos
Podarowicz et al., J. Cell-Phys
iol- 128, 475-484 (1986)]
, to determine whether the monoclonal antibody of the present invention recognized the conformation of the bFGF molecule.
The FGF solution was incubated in a boiling water bath for 5 minutes. Biological activity of heat-inactivated bFGF (BALB/c3
(measured by stimulation of DNA synthesis in T3-3K cells) was less than 0.2% of the biological activity of bFGF before heat treatment. Monoclonal antibody bFM-1 of the present invention (
The monoclonal antibody bFM-2 (Fig. 1) of the present invention did not show any cross-reactivity with heat-inactivated bFGF (Fig. 2).
Figure 〉 showed cross-reactivity with heat-inactivated bFGF. however,
Its reactivity was somewhat lower than that with untreated bFGF. This indicates that the monoclonal antibody bFM-
1 recognizes the conformation of the bFGF molecule, which is necessary for the biological activity of bFGF.

(C) bFGF and bovine-aF derived from various species.
The reactivity of GF with these monoclonal antibodies was determined by radioimmunoassay (RIA). The results are shown in Table 1. Both monoclonal antibodies showed cross-reactivity with mouse-bFGF, human-bFGF, and bovine bFGF, but not with bovine aFGF. Table 1: bFGF and bovine a of various species
Cross-reactivity between FGF and monoclonal antibodies Bovine bFGF Mouse bFGF Human bFGF Bovine aFGF (100) 130-170 70-100 0 (100) 100-110 90-140 0 The experimental conditions for radioimmunoassay (RIA) were as follows. 1
As described in relation to Figures and Figure 2. As mentioned above, numerical calculations were carried out using the FGF concentration estimated by radioimmunoassay (RIA) and FGF concentration.
This was done by dividing the concentration by the FGF concentration estimated from the DNA synthesis stimulating activity. Pure bovine bFGF was used as a standard in both cases.

The value of bovine bFGF was set as 100%. Radioimmunoassay of bFGF Purified bovine bFGF was purified by the chloramine-T method [Kan et al., J. et al. Biol. Chem. 263. 11306
-11313 (1988) 1 and labeled with 1251 as described in Heparin-Sepharose Affinity Chromatograph 4 [Neufeld et al., J. et al. Bio
l. Chem. 260. 13860-13868(
The method described in 1985) was purified using a slightly modified method. Briefly, 2.8 μg of bovine bFGF and 700 μCi of “'I (25.0 μg) were incubated with 2.8 μg of bovine bFGF for 2 min under room water in 110 μl of a reaction mixture containing 80 μg/ml of chloramine-TL.
9MBq) was incubated in the presence of 0.02% CHAPS [Matsuo et al., In Vitro Ce
ll. Dev. Biol. 24, 477-480
(198g)]. 0.02M dithiothreitol 100
The reaction was stopped by adding μl.

Next, using a salt solution containing 0.1% CHAPS,
Hebarin - 125 Free on Sepharose Power Ram
■Gara125■Isolated labeled bFGF +25
The specific activity of 1-labeled bFGF is 5.5 x 1 0'
cpm/ng. Labeled and unlabeled bFGF showed almost the same biological activity as judged by the same stimulation of DNA synthesis as described above, RI.
The reaction mixture for A (5 ml) was prepared using 0.1 M phosphate buffer (
pH7.4) /0.02% NaN30.35ml, P
BS/0.1%BSA/t). 02% NaN3 (PBS
-4 ng/ml 125I-bFGF (80
00-1 5000cpm) 0.05ml, PBS-B
- an appropriate concentration of unlabeled bFGF0.
Purified monoclonal antibodies (0.12-1.5 μg/ml) in 0.05 ml and PBS-B-A 0.0
5 ml was incubated overnight at 4°C in a test tube (Eiken).

1% normal mouse serum in PBS/0.02% NaN3
．． 1 ml and 0.77 m in PBS/0.02% NaN3
g/m 1 Goat anti-mouse immunoglobulin (Dako)
After adding 0.1 ml, the test tube was further incubated at 4°C for 4
Incubated for hours.

Radioactivity bound to the antibody was precipitated by adding 1 ml of 0.2% polyethylene glycol 6000 and centrifuging, followed by counting in an Aloka automated one-well gamma system (ARC-300). 0oetz etc.
In Vitro Cell Dev. Biol.
Bovine capillary endothelial cells were isolated and cultured from bovine brain cortex under conditions slightly modified from those described in 21, 172-180 (1985), and maintained. Cell culture was carried out using Tybu IV
In a plate coated with collagen (Sigma, USA),
Penicillin 100 units/ml, streptomycin 10
It was performed in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum supplemented with 0 μg/ml, 15 mM Hepes (pH 7.3) and bFGF 1 ng/ml. Cells were used for proliferation experiments at passages 5 to 9. cells,
■ Cultured at 37°C in a humid atmosphere in air containing 25%. However, 60mm Fal coated with Inuzaki type-IV collagen
In a con dish, add 2xl in 5ml of the same medium as the maintenance medium.
Bovine capillary endothelial cells were plated at a density of O'. bFGF and monoclonal antibodies were added only at the time of cell seeding. After 5 days, cells were harvested with tribucin and then plated in a Coulter counter. Cell numbers were counted. Proliferation in the absence of bFGF was analyzed in the same manner as described above, except that 10' cells were plated in bFGF-free medium and cell numbers were counted 4 days later. Values were averaged from two-dish experiments.

Figures 3 and 4 show the effect of monoclonal antibodies on the proliferation of bovine capillary endothelial cells in the presence and absence of exogenously added bFGF. In addition,
In Figures 3 and 4, ○ indicates exogenous bFGF 1
Results are shown in the presence of ng/ml, ● indicates exogenous bFG
Results are shown in the absence of F. Also, thick arrow (Ino)
indicates the number of inoculated cells. Proliferation of bovine capillary endothelial cells in medium containing 10% fetal bovine serum was stimulated by added bFGP. The number of cells 5 days after inoculating 2 x 104 cells was 1 ng/ml of bFGF.
6. in the presence and absence of each. OX 105 and 1.
It was IX105. On the other hand, when these cells were seeded at a higher density (10 5 cells), they were able to grow in the absence of exogenously added bFGF. However, the growth rate was slightly slower.
b Doubling time and b at high inoculum concentration in the absence of FGF
Doubling times at low inoculum concentrations in the presence of FGF were 40 and 24 hours, respectively. Monoclonal antibody bFM-1 of the present invention (Figure 3) and monoclonal antibody bFM-2 of the present invention (Figure 4) both contain exogenous bFGF.
Proliferation of bovine capillary endothelial cells was inhibited in the presence as well as in the absence of D. in a dose-dependent manner (in the range of 0.1-10 μ-ml). monoclonal antibody b
The inhibitory effect of FM-1 was greater than that of monoclonal antibody bFM-2. This is consistent with the difference in Kd values of these monoclonal antibodies.

These results indicate that these monoclonal antibodies
It has been shown that it inhibits the biological activity of GF in vitro, and also inhibits the biological activity of bFGF produced and secreted from bovine capillary endothelial cells, suppressing the autocrine action of bFGF in these cells.

[Brief explanation of drawings]

FIG. 1 shows the monoclonal antibody bFM-1 of the present invention,
1 is a graph showing cross-linking with basic fibroblast growth factor (bFGF), heat-inactivated basic fibroblast growth factor (bFGF), and fragments of basic fibroblast growth factor (bFGF). Figure 2 shows monoclonal antibody bFM-2 of the present invention, basic fibroblast growth factor (bFGF), heat-inactivated basic fibroblast growth factor (bFGF), and basic fibroblast growth factor (bFGF).
This is a graph showing the cross-linkage with fragments of GF. FIG. 3 shows the monoclonal anti-abortion bFM-
FIG. 1 is a graph showing the effect of No. 1 on bFGF-promoted proliferation of bovine capillary endothelial cells. FIG. 4 is a graph showing the effect of the monoclonal antibody bFM-2 of the present invention on bFGF-promoted proliferation of bovine capillary endothelial cells.

Claims (4)

[Claims] (1) inhibiting the biological activity of basic fibroblast growth factor;
and a monoclonal antibody that does not show cross-reactivity with basic fibroblast growth factor that is inactivated by heat. (2) inhibiting the biological activity of basic fibroblast growth factor;
and a monoclonal antibody that cross-reacts with basic fibroblast growth factor that is inactivated by heat. (3) formed by the fusion of mouse myeloma cells with spleen cells of mice immunized with basic fibroblast growth factor, inhibited the biological activity of basic fibroblast growth factor, and was inactivated by heating. A hybridoma that secretes monoclonal antibodies that do not show cross-reactivity with basic fibroblast growth factor. (4) formed by the fusion of mouse myeloma cells with mouse spleen cells immunized with basic fibroblast growth factor, inhibited the biological activity of basic fibroblast growth factor, and was inactivated by heating. A hybridoma that secretes monoclonal antibodies that cross-react with basic fibroblast growth factor.