JPH11178593A - Monoclonal antibody specific to vegf 121 and measurement - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new monoclonal antibody specific to vascular endothelial cell growth factor(VEGF) 121 and useful for the immunological measurement, action mechanism clarification, etc., of the VEGF 121 having neovascular actions related to tissue construction, cancer proliferation, etc. SOLUTION: A new monoclonal antibody specific to vascular endothelial cell growth factor (VEGF) 121. The monoclonal antibody can widely be utilized for the immunological measurement, action mechanism clarification, etc., of the VEGF 121 having neovascular actions related to angioplasty and tissue construction in fetal periods, luteinization, endometrial hyperplasia, inflammation, wound cure, diabetic retinopathy, articular rheumatic diseases, solid cancer proliferation, etc. The monoclonal antibody is obtained by administering the VEGF 121 together with an adjuvant into the abdominal cavity of BALB/c mouse to immunize the mouse, fusing the splenic cell to the myeloma cell of a mouse to immunize the mouse, fusing the splenic cell of the finally immunized mouse to a myeloma cell of a mouse, culturing the hybridoma in HAT culture medium, selecting an antibody-producing strain, cloning the selected strain, administering the obtained monoclonal unicellular strain into the abdominal cavity of a mouse, separating the antibody from the abdominal dropsy of the mouse and subsequently purifying the antibody.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a monoclonal antibody specific to VEGF121, a hybridoma producing the monoclonal antibody, and a method for measuring VEGF using the monoclonal antibody.

[0002]

BACKGROUND OF THE INVENTION Angiogenesis normally involves fetal angiogenesis and tissue formation, luteal formation and endometrial proliferation, but in pathological conditions, inflammation, wound healing, diabetic retinopathy, joint disease, etc. Rheumatoid arthritis plays an important role in the growth of solid tumors (Experimental Medicine 15 998-1002 (1997)). Vascular endothelial cell growth factor (vascular end factor)
othelial cell growth factor (VEGF), vascular permeability factor (VPF), fibroblast growth factor (FG)
F), platelet-derived growth factor
factor, PDGF), transforming growth factor
α (transforming growth factor-α, TGF-α),
Tumor necrosis factor (TNF), interleukin-8 (IL-8) and the like are known, but VEGF plays the most central and important role among them. . Subsequently, it has been confirmed that VEGF and VPF are the same factor.

[0003] VEGF is a protein found as a vascular permeability factor secreted by tumor cells (Science, 219, 983-985 (1983)) and as an endothelial cell growth factor in pituitary follicle cell culture supernatant ( Biochem. Biophys. Res. Commu
n., 161, 851-858 (1989)), and the gene was isolated in 1989 (Science, 246, 1306-1309 (1989), Scien
ce, 246, 1309-1312 (1989)). Subsequent research has shown that V
EGF was found to have four isomers having different numbers of amino acid residues. 121 amino acid residues
(Hereinafter referred to as VEGF121 in the present specification) and 165 (hereinafter referred to as VEGF1 in the present specification).
65, 189 (hereinafter, described as VEGF189), and 206 (hereinafter, described as VEGF206) of the four isomers were identified as VEGF12 by gene level studies.
2 and VEGF165 were suggested to have strong effects on vascular endothelial cells (Endocrine Reviews, 13, 1).
8-32 (1992)). However, VEGF121 and VEGF1
It is not clear whether there is a qualitative or quantitative difference in the effect on vascular endothelial cells between the 65.

[0004] When VEGF is to be captured as a protein, it is convenient to use an immunoassay using an anti-VEGF-specific antibody. Monoclonal antibodies to VEGF have already been obtained (Growth Factor, 7, 53-64 (199
2), Hybridoma, 14, 475-480 (1995), Clinical Chemis
try, 42, 1777-1784 (1996), JP-A-8-16989
JP-A-8-53498, JP-A-7-330795
No., Japanese Patent Application Laid-Open No. Hei 9-124697), there is no antibody that discriminates and sorts between VEGF121 and VEGF165, and the role of each of VEGF121 and VEGF165 has not been elucidated.

[0005]

SUMMARY OF THE INVENTION Accordingly, it is an object of the present invention to provide a monoclonal antibody capable of distinguishing between VEGF121 and VEGF165 and an immunoassay using the monoclonal antibody.

[0006]

Means for Solving the Problems In order to solve the conventional problems, the present inventors have repeated studies on VEGF isomers. As a result, a monoclonal antibody specific to VEGF121 was prepared, and immunological reaction using the monoclonal antibody was performed. The present invention was successfully completed by measuring VEGF121 by the measuring method.

That is, the present invention provides a monoclonal antibody specific to VEGF121, a hybridoma producing the monoclonal antibody, and a method for measuring VEGF using the monoclonal antibody.

[0008] By using the monoclonal antibody of the present invention,
Only VEGF121 can be directly detected,
It can be widely applied to immunoassay of F121, elucidation of the mechanism of action, and the like.

Hereinafter, the present invention will be described in detail.

In the present invention, VEGF121 is a protein having the amino acid sequence shown in SEQ ID NO: 1. VE
GF121 has the amino acid sequence of VEGF165
To 114, asparagine at position 115 has been replaced with lysine, and the C-terminal 6 amino acid residues have
Identical to the C-terminus of EGF165.

In order to obtain a monoclonal antibody specific to VEGF121 of the present invention, VEGF121 is used as an immunogen.
It is preferable to use a structural part unique to VEGF121 rather than to use, specifically, a three-dimensional structure specific to VEGF121, a peptide containing a continuous amino acid part unique to VEGF121 on the C-terminal side generated from the continuation of exon 5 and exon 8, and the like. Is mentioned. More preferably, VEGF1
It is desirable to use the amino acid residues 110 to 121 of 21 as an immunogen, but no matter what is used as the immunogen,
When establishing a monoclonal antibody, a monoclonal antibody specific to VEGF121 may be selected, and the immunogen is not limited to these.

[0012] The immunogen as described above is used for mouse, rat,
Immunize immunized animals such as hamsters. Immunization can be performed according to a conventional method, and the antigen is administered to an immunized animal alone or together with an adjuvant. After immunization, serum may be collected to confirm an increase in the antibody titer, and boosting may be performed as necessary. After confirming the increase in antibody titer, antibody-producing cells such as spleen cells and tumor cells such as myeloma cells are fused with a fusion agent such as polyethylene glycol to produce a hybridoma. Next, the hybridomas are selected using a selective medium such as a HAT medium, and are monoclonalized and cultured by an appropriate method such as a limiting dilution method. The culture supernatant is analyzed by a suitable immunoassay such as an enzyme immunoassay, and a clone producing a desired VEGF121-specific monoclonal antibody is selected. Upon selection, VEGF165, VEGF189, VEGF206
It is preferable to confirm the presence or absence of reactivity with other isomers. These monoclonal antibody preparation techniques are:
Known methods, for example, Koehler and Milstein (Natu
re 256 495 1975), Scherer (Nature 285 446 198)
0) and the like. Further, the monoclonal antibody prepared by the above-described method, ascites obtained by administering a hybridoma producing the monoclonal antibody to a pristane-treated mouse, or a culture supernatant of the hybridoma producing the monoclonal antibody, salt folding, Analysis such as ion exchange chromatography and affinity chromatography with immobilized protein A
It can be recovered by purification means.

The monoclonal antibody of the present invention can be used in a known immunoassay. For example, the monoclonal antibody of the present invention is immobilized, and a sandwich measurement method using a known anti-VEGF antibody labeled in a liquid phase or labeled V
VEGF121 by Competition Assay Using EGF121
Immunoassay. Examples of the labeling substance used for the immunoassay include an enzyme, a radioisotope, a fluorescent substance, and a luminescent substance, but the immunoassay of the present invention is not limited to these.

The measurement sample is not limited, and can be applied to the measurement of VEGF121 in various body fluids such as serum, plasma, whole blood, urine, and lymph, and cell tissue extract.

Further, the monoclonal antibody of the present invention can be used for tissue immunostaining based on a conventional method.
It is useful for elucidating the mechanism of action of 121.

[0016]

EXAMPLES The present invention will be described in more detail with reference to Examples and Examples.

Reference Example 1 Recombinant human VE derived from Escherichia coli
Preparation of GF121 and human VEGF165 Human VEGF was prepared by PCR from a cDNA library of a cultured cell line HL-60 derived from human acute promyelocytic leukemia.
The cDNAs for 121 and human VEGF165 were isolated.
These were pGEMEX-1 (promega) and pGEX
PW6A-VEGF121 and pW6A-VEGF121 as vectors expressing human VEGF121 and human VEGF165 by incorporating into a 4.6 Kb pW6A vector (Japanese Patent Application No. 9-121803) prepared from -2T (manufactured by Pharmacia).
W6A-VEGF165 was produced. pW6A-VEG
After introducing F121 and pW6A-VEGF165 into the host E. coli, respectively, 1% bactotryptone, 0.5% yeast extract, 1% sodium chloride, 50 μg /
ml of ampicillin, pH 7.5 medium (hereinafter referred to as LB medium in the present specification) at 37 ° C.
M isopropylthiogalactopyranoside (hereinafter referred to as IPTG in this specification) was added, and the cells were cultured for 3 hours to induce expression. Escherichia coli transformed with pW6A-VEGF121 was sonicated in Tris buffer and then centrifuged. The precipitate was solubilized in Tris buffer containing 6M urea and then centrifuged. The centrifuged supernatant was purified by anion exchange chromatography using a QFF anion exchange column (manufactured by Pharmacia) and reverse phase chromatography using a resource RPC column (manufactured by Pharmacia), and was derived from Escherichia coli having a purity of 95% or more. Recombinant human VEG
F121 (hereinafter referred to as rVEGF121 in the present specification)
E). On the other hand, pW6A-VEGF16
Escherichia coli transformed in Example 5 was also prepared in the same manner as described above, and then Superdex 200 (manufactured by Pharmacia).
The purified product was purified by gel filtration using E. coli to obtain a recombinant human VEGF165 derived from Escherichia coli having a purity of 95% or more (hereinafter, referred to as rVEGF165E in the present specification).

Reference Example 2 Anti-VEGF121 / VEGF1
Preparation of Hybridoma Producing 65 Monoclonal Antibody rVEGF165E prepared in Reference Example 1 was used as an immunogen. rVEGF165E was mixed in equal volume with Freund's complete adjuvant, and injected intraperitoneally into BALB / c mice.
0 μl (about 40 μg / mouse) was administered. After about two weeks,
Similarly, rVEGF165E was mixed in an equal volume with incomplete Freund's adjuvant and administered intraperitoneally. After confirming the increase in antibody titer, about 40 rVEGF165E were used as final immunization.
μg was administered intravenously, and three days later the spleen was removed.
Isolated splenocytes and mouse myeloma cell line P3-x6
3-Ag8-U1 (purchased from Dainippon Pharmaceutical) (hereinafter referred to as P3U1 in the present specification) was mixed at a 3: 1 cell number, and cell fusion was performed using 50% polyethylene glycol 1500. Cells were HAT (1 × 10 ⁻⁴ M hypoxanthine, ⁴ × 10 ⁻⁷ M aminopterin, 1.6 ×
RPMI164 supplemented with 10 ^-5 M thymidine) and 10% fetal calf serum (hereinafter referred to as FCS).
0 medium (hereinafter, referred to as HAT medium in the present specification), dispensed into a 96-well microculture plate, and cultured. The culture supernatant of the wells in which the hybridomas had grown was examined by the following ELISA method,
F121 and VEGF165 A hybridoma producing a monoclonal antibody reacting with the above was selected.

That is, the rVEGF prepared in Reference Example 1
Each of 121E and rVEGF165E was diluted with a phosphate buffer (hereinafter, referred to as PBS in the present specification), dispensed into a 96-well ELISA plate, and allowed to stand at 4 ° C overnight to bind to the solid phase. Next, 0.05% Tween 2
After washing with PBS containing 0% (hereinafter referred to as PBST in the present specification), PBS containing 1% skim milk
Was dispensed and left at 37 ° C. for 1 hour to perform blocking. After washing with PBST, the hybridoma culture supernatant was
VEGF121E binding plate and rVEGF165E
Dispense into each of the coupled ELISA plates,
It was left at 7 ° C. Subsequently, after washing in the same manner, a 1000-fold diluted peroxidase (hereinafter, referred to as POD) -labeled anti-mouse immunoglobulin antibody (manufactured by Dako) was dispensed and left at 37 ° C. for 1 hour. . After the same washing, a POD substrate (ABTS-hydrogen peroxide) was added, and the mixture was allowed to stand at room temperature for 10 minutes.
The absorption at 5 nm was measured. Cells in positive wells were cloned by limiting dilution. The antibody activity of the cell supernatant of the well containing the single colony is examined by the above-described method, selected, cultured, and hybridoma VGF3-4 producing a monoclonal antibody reactive with VEGF121 and VEGF165.
Was established. The hybridoma VGF3-4 was cultured in large amounts and administered intraperitoneally to mice, and ascites was collected. further,
Antibodies were purified from ascites using Affi-prep Protein A Maps II Kit (Bio-Rad) to obtain monoclonal antibodies. This antibody is used for monoclonal antibody VG
It was named F3-4.

REFERENCE EXAMPLE 3 Recombinant human VEGF121 and human VEGF16 derived from baculovirus-insect cells
Preparation of 5 Cultured cell line HL-60 derived from human acute promyelocytic leukemia
CDNA of human VEGF121 and human VEGF165 isolated from the cDNA library by PCR was incorporated into a pBacPAK8 transfer vector (manufactured by Clontech) to prepare pBac-VEGF121 and pBac-VEGF165. These were co-transfected with Sac21 cells (manufactured by Clonetech) together with BacPAK6 DNA (manufactured by Clonetech) by the lipofectin method to construct recombinant baculoviruses AcVEGF121 and AcVEGF165. The recombinant baculovirus was purified by the plaque method, and then transfected into Sf21 cells to express recombinant human VEGF121 and human VEGF165 and purified. That is, AcVEGF121 or AcVEGF165 was added to Sf21 cells cultured in SF900-II serum-free medium (manufactured by GIBCO BRL) to infect the cells, and the cells were cultured at 27 ° C. for 3 days.
The culture supernatant was collected. Further, the monoclonal antibody VGF3-4 prepared in Reference Example 2 was replaced with HiTrap NH.
Using an affinity column bound to S-activated (Pharmacia), a recombinant human VEGF1 derived from a baculovirus-insect cell from the culture supernatant was used.
21 (hereinafter referred to as rVEGF121B) and human VEGF165 (hereinafter referred to as rVEGF165B) were purified.

Example 1 Preparation of Anti-VEGF121 Monoclonal Antibody-Producing Hybridoma A peptide represented by SEQ ID NO: 2 (hereinafter, referred to as P2 in the present specification) was synthesized, and was synthesized with KLH (Keyhol).
e Limpet Hemocyanin) to form a complex (hereinafter referred to as P2-KLH in the present specification), which was used as an immunogen.

Immunization was performed in the same manner as in Reference Example 2, and a hybridoma was prepared in the same manner.

Using the ELISA plate bound to rVEGF121B prepared in Reference Example 3, a hybridoma producing a monoclonal antibody reactive with VEGF121 was selected by the same ELISA method as in Reference Example 2. The culture supernatant of the well containing a single colony was examined in the same manner as in Reference Example 2 using the rVEGF121B and rVEGF165B-bound ELISA plates prepared in Reference Example 3 to produce a monoclonal antibody specifically reacting with VEGF121. Hybridoma VG2P3-
5 was established. A monoclonal antibody was obtained from the hybridoma in the same manner as in Reference Example 2. This antibody was named monoclonal antibody VG2P3-5.

The hybridoma was VG2P3-5
Deposited with the Biotechnology Research Institute, and its accession number is F
ERM P-16553.

Example 2 Reaction specificity of anti-VEGF monoclonal antibody rVEGF121B and rVEGF prepared in Reference Example 3
165B was adjusted to a concentration of 0.1 μg / ml with PBS, and rVEGF121B binding ELISA was performed in the same manner as in Reference Example 2.
Plates and rVEGF165B binding ELISA plates were prepared. To this were added monoclonal antibodies VG2P3-5 and VGF3-4, each diluted to 2 μg / ml with PBS containing 1% bovine albumin, and the same measurement as in the ELISA method of Reference Example 2 was performed.
Table 1 shows the results. Monoclonal antibody VG2P3-5
Is a monoclonal antibody VG specifically for VEGF121.
F3-4 reacted with both VEGF121 and VEGF165.

[0026]

[Table 1]

Example 4 Measurement of VEGF121 10 μg of monoclonal antibody VG2P3-5 in PBS
/ Ml, and a monoclonal antibody VG2P3-5 binding ELISA plate was prepared in the same manner as in Reference Example 2. In addition, rVEGF121B and rVEGF
165B, recombinant human PDGF-BB (R & D System) and recombinant human P / GF (R & D System) were added and reacted at 37 ° C for 1 hour. Subsequently, after washing similarly, the POD-labeled monoclonal antibody VGF3-
4 was added and reacted at 37 ° C. for 1 hour. After a similar wash,
Add a POD substrate (OPD-hydrogen peroxide system) and add
After allowing the mixture to stand for 5 minutes, a reaction stop solution was added, and the absorption at 492 nm was measured. The results are shown in FIG. The sandwich ELISA method between the immobilized monoclonal antibody VG2P3-5 and the POD-labeled monoclonal antibody VGF3-4 is based on VEG.
F121 can be measured, but isoform V
EGF165 and PDGF- with homology in amino acid sequence
BB, PlGF could not be measured and VEGF12
It was confirmed to be 1 specific.

[0028]

Industrial Applicability According to the present invention, a monoclonal antibody specific to VEGF121, a hybridoma producing the monoclonal antibody, and a V-cell using the monoclonal antibody
A method for measuring EGF is provided, and VEGF121 can be specifically measured.

[0029]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 121 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence Ala Pro Met Ala Glu Gly Gly Gly Gln Asn His His Glu Val Val Lys 1 5 10 15 Phe Met Asp Val Tyr Gln Arg Ser Tyr Cys His Pro Ile Glu Thr Leu 20 25 30 Val Asp Ile Phe Gln Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys 35 40 45 Pro Ser Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn Asp Glu 50 55 60 Gly Leu Glu Cys Val Pro Thr Glu Glu Ser Asn Ile Thr Met Gln Ile 65 70 75 80 Met Arg Ile Lys Pro His Gln Gly Gln His Ile Gly Glu Met Ser Phe 85 90 95 Leu Gln His Asn Lys Cys Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg 100 105 110 Gln Glu Lys Cys Asp Lys Pro Arg Arg 115 120

SEQ ID NO: 2 Sequence length: 12 Sequence type: amino acid Topology: linear Sequence type: peptide sequence Arg Ala Arg Gln Glu Lys Cys Asp Lys Pro Arg Arg 1 5 10

[Brief description of the drawings]

FIG. 1 is a view showing the results of measurement of a sandwich ELISA method using an anti-VEGF121 monoclonal antibody.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI G01N 33/53 C12N 5/00 B 33/577 15/00 C 33/68 ZNAA // (C12P 21/08 C12R 1:91) (C12N 5/10 C12R 1:91) (72) Inventor Tetsu Ito 2-62-5 Nihonbashi Hamacho, Chuo-ku, Tokyo Inside Fujirebio Corporation

Claims (5)

[Claims] 1. A monoclonal antibody specific to VEGF121. 2. The monoclonal antibody according to claim 1, wherein the monoclonal antibody is a monoclonal antibody VG2P3-5. A hybridoma that produces the monoclonal antibody according to claim 1 or 2. 4. The hybridoma according to claim 3, wherein the hybridoma is VG2P3-5. 5. A method for measuring VEGF using the monoclonal antibody according to claim 1 or 2.