JPH10245347A - Agent for inhibiting restorage of body fluid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a therapeutic medicine having actin functions for inhibiting the actions of VEGF/VPF inducing the restorage of ascites, pleural effusion, pericardial effusion, etc., to treat the restorage of the fluids. SOLUTION: This agent for inhibiting the restorage of body fluids contains an antagonist against VEGF/VPF vascular permeability factor (VPF) [also called vascular endothelial cell growth factor (VEGF), and generically called as (VEGF/VPF)]} as an active ingredient. The VEGF/VPF antagonist includes a monoclonal antibody resistant to VEGF/VPF and an antibody reacting with at least one of the following sequences 1-3 which each is a part of the amino acid sequence of the VEGF/VPF. 1: KPSCVPLMR; 2: SFLQHNKCECRP; 3: KCECRPKKDRAR.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a drug useful for suppressing the recurrence of the accumulation of bodily fluids such as neoplastic ascites, pleural effusion, or pericardial effusion, which occur once. Sex factor (VPF) [Vascular endothelial cell growth factor (VEGF)
Also referred to as “VEGF / VP” in this specification.
F ”] to prevent the recurrence of stored water.

[0002]

2. Description of the Related Art It is known that body fluids are pathologically stored in the body due to malignant and benign tumors, inflammation of the peritoneum, cirrhosis and the like. In particular, pooled water (malignant pericardial effusion, pleural effusion, ascites, subarachnoid infiltration, etc.) due to malignant tumors is difficult to treat,
It also worsens the prognosis of patients and lowers the quality of life (QOL).

[0003] At present, as a treatment method for such stored water, administration of a diuretic can be cited, but it is necessary to keep the balance of electrolytes, and when excretion of urine is difficult, it may be exacerbated. There is a problem with the method. There is also a treatment that removes stored water by puncture, but a sudden change in body fluid circulation can cause hypotension, dizziness, nausea, vomiting, opacity or shock, and even death, such puncture In the removal by water, the cause of the stored water is not treated, so it only stops temporarily, causing re-storage, and as a result, repeated drainage by puncture,
This is a method that may further exacerbate the patient's symptoms due to the outflow of proteins and electrolytes. Further, in the case of a malignant tumor, an anticancer agent is also administered to a place where the stored water is stored, such as the abdominal cavity or the thoracic cavity.

[0004] In recent years, as a treatment for ascites due to malignant tumors of the digestive system, BRM (biological response modifier) therapy by local administration of OK-432, IL-2 and activated lymphocytes has been performed.
It has been reported that ascitic fluid retention was suppressed and QOL was improved (Yoshiyuki Yamaguchi et al., Biotherapy Today, 3
(2) 79-82 (1996)). However, this treatment is not perfect, and a better therapeutic agent is desired.

[0005] On the other hand, VEGF / VPF is known not only to stimulate vascular endothelial cell proliferation but also to induce vascular permeability and angiogenesis. In addition, human ovarian cancer (OVCAR-3) is found in the peritoneal cavity. It is also known that the accumulation of ascites in transplanted nude mice can be suppressed by anti-human VEGF / VPF165 rabbit polyclonal antibody (Olson,
TA, et al., Int.J. Oncology, 8, 505-511 (199
6)).

[0006]

The establishment of an effective treatment method for this pooled water, especially for re-storage after removal of the generated pooled water, would greatly contribute to improving the QOL of patients and prolonging the prognosis. . However, at present, there is no effective agent for suppressing storage and re-storage of stored water, and the present inventors have conducted intensive research to solve this problem, and as a result, have completed the present invention.

[0007]

That is, the present invention relates to
The present invention relates to a body fluid re-retention inhibitor containing an EGF / VPF antagonist as an active ingredient, and particularly to VEGF / VP.
The F antagonist is an anti-VEGF / VPF monoclonal antibody, and relates to a body fluid re-retention inhibitor. Furthermore, the anti-VEGF / VPF monoclonal antibody has the following sequences 1 to 3, which are part of the amino acid sequence of VEGF / VPF: KPSCVPLMR 2. SFLQHNKCECRP 3. The present invention relates to a body fluid re-retention inhibitor that is an antibody that reacts with at least one of KCECRPKKDRAR.

In the present invention, VEGF / VPF antagonist refers to VEGF / V having a specific cell growth promoting activity on vascular endothelial cells or a vascular permeability promoting activity.
It refers to a substance that inhibits the function of PF, and may be in any form as long as it has the function. Most commonly, an antibody that acts on VEGF / VPF, or a part thereof, may be mentioned. Without limitation, for example, an inactive VEG that inhibits the action of VEGF / VPF
F / VPF, or a portion thereof, that impairs the function of the VEGF / VPF receptor, eg, an antibody to the vascular permeability factor receptor, or a portion thereof, or even VEGF /
Drugs that suppress the production of VPF itself can be mentioned.

VEGF / VPF contains 1 amino acid residue.
There are four subtypes of 21, 165, 189, and 206 (Masashi Shibuya, “Functional expression mechanism of angiogenic factor VEGF”), Clinical Immunity, 28 (6) 758-764.
(1996)), the antibody may be an antibody against any subtype. Examples of the antibody include a mouse antibody and the like, and those subjected to treatment for reducing side effects in administration to humans can also be used. For example, a mouse monoclonal antibody is chemically modified with a substance such as polyethylene glycol to reduce its antigenicity. A human chimeric antibody or a humanized antibody in which a CDR region, which is a binding region to an antigen of a mouse monoclonal antibody, is replaced with another region while retaining the binding force to the antigen by a human antibody can also be used. Furthermore, antibodies that are obtained by enzymatically cleaving them to reduce the molecular weight can also be used. The chimeric or humanized antibody may be an IgG.
Or IgG type, and the IgG isotype is IgG1, IgG2, IgG3 or IgG4.
Is raised.

According to the present invention, the action of the VEGF / VPF antagonist suppresses the re-accumulation of bodily fluids such as ascites, pleural effusion, and pericardial fluid that have once occurred.
It can improve the prognosis of cancer patients and improve the quality of life.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, preferred V
EGF / VPF antagonist anti-VEGF / VP
The present invention will be described in detail using an F monoclonal antibody as an example.

(1) Production of Anti-VEGF / VPF Monoclonal Antibody
It can be produced by immunizing with F / VPF, removing the spleen cells, and culturing the hybridoma cells obtained by fusing the spleen cells with myeloma cells. The production of this hybridoma is described, for example, by the method of Kohler and Milstein [Nature, 256:
495 (1975)].

(1-1) Preparation of antibody-producing cells As animals for immunization, rodents such as mice, rats and rabbits are used. Mouse or rat-derived cells are used as myeloma cells. Then, an amount of 10 to 100 μg of VEGF / VPF was used as an antigen for one immunized animal.
Immunize 2-3 times at least every 3 weeks. Animal breeding and spleen cell collection are performed according to conventional methods. In the case of immunization, a protein obtained by fusing, for example, glutathione-S-transferase or the like to an antigen, or a complex protein obtained by binding keyhole limpet hemocyanin or the like can be used as the antigen.

(1-2) Preparation of myeloma cells Myeloma cells include mouse myeloma Sp2 / 0-Ag
14 (Sp2), P3 / NSI / 1-Ag4-1 (NS-1), P3 × 63Ag8
U.1 and the like. Subculture of these cells is performed according to a conventional method.

(1-3) Cell fusion Cell fusion can be carried out by mixing spleen cells and myeloma cells at a ratio of 1: 1 to 1:10 and mixing with polyethylene glycol or subjecting to electric pulse treatment.

(1-4) Selection of hybridomas Selection of fused cells (hybridomas) is performed using hypoxanthine.
(10 ^-3 to 10 ^-5 M), aminopterin (10 ^-6 to 10 ^-7 M)
M) and thymidine (10 ⁻⁵ to 10 ⁻⁶ M) and used as a hybridoma.

(1-5) Culture of hybridomas Cloning of hybridomas is repeated at least twice by limiting dilution. If the hybridoma is cultured in the same manner as ordinary animal cells, the monoclonal antibody of the present invention is produced in the medium. The monoclonal antibody of the present invention can also be accumulated in ascites by transplanting the hybridoma cells into the peritoneal cavity of a mouse and proliferating the cells.

(1-6) Collection and Purification of Monoclonal Antibodies Monoclonal antibodies accumulated in the culture medium or ascites fluid of hybridoma cells can be obtained by the conventional ammonium sulfate fractionation method, PEG fractionation method, ion exchange chromatography, and the like. Purified by a method using gel filtration chromatography. In addition, a method using affinity chromatography such as protein A and protein G can also be used. Enzyme-linked immunosorbent assay, Western blotting, and the like are used for selecting monoclonal antibodies. Further, the determination of the isotype of the monoclonal antibody can be carried out by the enzyme immunoassay of the monoclonal antibody or the Ouchterlony method.

(2) Identification of reaction site of monoclonal antibody (2-1) Preparation of peptide corresponding to part of amino acid sequence of VEGF / VPF Twelve consecutive amino acids in VEGF / VPF are treated as one peptide in VEGF / VPF Design multiple peptides covering the entire sequence of Each designed peptide can be prepared, for example, by a multipin peptide synthesis method [Maeji, NJ et a
l., J. Immunol. Method, 134: 23 (1990)] and the like. The synthesized peptide can be quantified by quantifying the amino group using orthophthalaldehyde.

(2-2) Identification of Peptide Reacting with Monoclonal Antibody A plurality of peptides synthesized as described above are VEGF /
This corresponds to the entire region of the VPF. Therefore, by investigating the reactivity of the monoclonal antibody with all of the plurality of peptides, the monoclonal antibody was identified as VEGF / VPF.
Which site is reacting to. For the measurement of the reactivity, an enzyme immunoassay, an octalony method, a western blotting method, or the like is used.

(3) Use of Monoclonal Antibody as Body Fluid Reretention Inhibitor In the present invention, when a monoclonal antibody, a chimeric antibody or a humanized antibody is administered as a body fluid reretention inhibitor, the subject to be administered is not particularly limited. For example, it is locally administered to a site for storing a bodily fluid such as neoplastic ascites, pleural effusion, or pericardial fluid. The method of administration may be oral or parenteral, and oral administration includes sublingual administration. Parenteral administration includes injections such as subcutaneous, intramuscular, intravascular or intraperitoneal, intrathoracic injections, infusions, suppositories and the like. The dose varies depending on whether the animal or human, the age, the route of administration, the number of administrations, and can be widely varied. In this case, the effective amount of the monoclonal antibody and the effective amount to be administered as a composition of a suitable diluent and a pharmaceutically usable carrier are 0.1 to 10 mg / kg body weight / day, which is reduced from once to several times a day. It is administered once daily or several days, or once every 1-2 weeks.

When the monoclonal antibody is parenterally administered in the present invention, it contains additives such as a stabilizer, a buffer, a preservative, and a swelling agent, and is usually in the form of a unit-dose ampoule or a multi-dose container or tube. Provided. The compositions described above may be in powder form for reconstitution with a suitable carrier, for example, a sterile pyrogen-free dissolving agent.

In the case of oral administration, tablets, granules, fine granules, powders, capsules, and the like applied thereto are usually used in such compositions as binders, inclusion agents, and excipients commonly used in pharmaceutical preparations. -Contains additives such as lubricants, disintegrants and wetting agents. In addition, the liquid preparation for oral use may be in the form of a liquid solution for internal use, suspension, emulsion, syrup, etc.
Further, it may be a dried product that is redissolved when used. Further, the composition may contain any of additives and preservatives.

[0024]

The present invention will be described more specifically with reference to the following examples. However, the present invention is not limited to these examples.

(1) Preparation of Hybridoma Producing VEGF / VPF Monoclonal Antibody Human VEGF / VPF was purified from a culture solution of yeast transformed with the isolated cDNA of human VEGF / VPF.
A complex was prepared using keyhole limpet hemocyanin (KLH) and glutaraldehyde, and a mouse monoclonal antibody was prepared using the obtained protein as an antigen according to a conventional method. That is, KLH-
Splenocytes of mice immunized with VEGF / VPF were fused with mouse myeloma cells (Sp2) in the presence of polyethylene glycol. The obtained hybridoma was cloned by the limiting dilution method. The reactivity of the culture supernatant of the hybridoma cloned with VEGF / VPF was examined by enzyme immunoassay, and a hybridoma producing a monoclonal antibody reactive with VEGF / VPF was selected. or,
The monoclonal antibody produced by this hybridoma
V833. The hybridoma producing the obtained monoclonal antibody has been deposited as FERM BP-5669 with the Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry.

(2) Preparation of anti-VEGF / VPF monoclonal antibody The selected hybridoma was implanted intraperitoneally into a nude mouse, and ascites containing a large amount of the monoclonal antibody was collected.
From this ascites fluid, a protein G affinity column (M
AbTrapGII (Pharmacia) was used to purify the monoclonal antibody. The class of the antibody was determined by enzyme immunoassay using an anti-mouse immunoglobulin subclass-specific antibody. As a result, the class of the MV833 antibody was IgG1.

Further, the dissociation constants for VEGF121 and VEGF165 measured by the following method are as follows, which indicates that the monoclonal antibody of the present invention has a strong affinity for VEGF / VPF. ○ 5.70 × 10 ^-11 M ± 0.35 × 10 ^-11 M (VEG
F121) ○ 1.10 × 10 ⁻¹⁰ M ± 0.11 × 10 ⁻¹⁰ M (VEG
F165)

Method for measuring dissociation constant Monoclonal antibody containing 0.1 M sodium chloride
Prepare 2 μg / ml with 5 mM carbonate buffer (pH = 9.0)
Add 100 μl each to a detachable plate
Leave overnight. Next, remove the solution from the hole and add 1% BSA-PB
S is added in an amount of 300 μl and left at 37 ° C. for 4 hours. 1
After removing% BSA-PBS, 0.1% BSA-PBS
VEGF prepared in¹²⁵I-labeled VEGF (¹²⁵I label V
EGF121 labels VEGF121 by chloramine T method,
¹²⁵I-labeled VEGF165 purchased from Amersham)
Add 200 μl of solution per well and leave overnight. This anti
The VEGF concentration in the reaction mixture was 0-1 ng / well for VEGF121,
VEGF165 is 0-10ng / well,¹²⁵I-labeled VEGF is 1
× 10^Fourcpm / hole (¹²⁵I-labeled VEGF121; 66.7 pg / well,
^{1 twenty five}I-labeled VEGF165; 116 pg / well). Anti-hole
The reaction mixture was removed and washed six times with 0.1% BSA-PBS.
After that, cut the holes one by one and put them into the tube for analysis.
Counted at the mar counter and created from the result
Find the dissociation constant from the scatter plot.

The isoelectric point of the monoclonal antibody of the present invention measured by the following method was pI = 5.2 to 5.5.
Other IgG1-type anti-VEGF / V reported at this time
The isoelectric point of the PF monoclonal antibody was MV101 reported by our company with a pI of 7.0 to 7.5, and Genentech's A4.6.1 with a pI of 4.2 to 5.2 [Kim, KJ et al. .al. Grow
th Factors, 7:53 (1992)], and the substance of the present invention is different from any substance.

Isoelectric Focusing Method The isoelectric focusing of the monoclonal antibody was carried out using a commercially available agarose gel for isoelectric focusing (Washamori Co., Ltd.) in the isoelectric focusing electrophoresis layer of the company. The electrophoresis was performed at 3 W for 30 minutes using a power supply (Bio-Rad) capable of outputting an equal power. After the electrophoresis, the gel was stained with a protein using a silver staining kit (Bio-Rad). For the isoelectric point of the monoclonal antibody, the isoelectric point of the antibody was determined from the electrophoretic mobility of the isoelectric marker protein that was simultaneously electrophoresed.

(3) Identification of Reaction Site of Monoclonal Antibody in VEGF / VPF (3-1) Preparation of Peptide Corresponding to Part of Amino Acid Sequence of VEGF / VPF Twelve consecutive amino acids of the amino acid sequence of human VEGF121 were synthesized. As one peptide, 67 kinds of peptides covering the entire sequence were devised, and each peptide was synthesized by a multipin peptide synthesis method [Maeji, N, J, et.al. J. Immunol.method, 134: 23 (199)
0)].

First, Fm was obtained from 9-fluorenylmethoxycarbonyl (Fmoc) -β-alanine introduced at the tip of a pin of a 96-well assay plate pin block by piperidine.
After removal of the oc group, the Fmoc-amino acid was condensed in the presence of dicyclohexcarbodiimide and hydroxybenzotriazole. After washing with N, N-dimethylformamide, the Fmoc-amino acid was again condensed in the presence of dicyclohexylcarbodiimide and hydroxybenzotriazole, and this operation was repeated to synthesize the desired peptide. After the completion of the condensation reaction, acetylation was performed with acetic anhydride, and the side chain protecting group was removed with trifluoroacetic acid. The peptide synthesized on the pin was excised by immersing the pin in a neutral solution. Quantification of the synthesized peptide was performed by quantifying amino groups using orthophthalaldehyde. Table 1 shows the amino acid sequences of the 67 peptides synthesized.
Numbers indicate peptide identification numbers.

[0033]

[Table 1]

(3-2) Identification of Peptide Reacting with MV833 Antibody 67 kinds of peptides synthesized as described above are human VE
This corresponds to the entire area of GF121. Therefore 6
By examining the reactivity between the seven peptides and the MV833 antibody, it is possible to clarify where in the VEGF / VPF the MV833 antibody reacts. Therefore, the reactivity of 67 kinds of peptides with the MV833 antibody was examined by enzyme immunoassay. 67 kinds of 20 μM peptide solutions were put in a 96-well NOS plate (manufactured by Coaster) and allowed to stand at room temperature for 2 hours. Plate 3 wells with 0.1% BSA-PBS
After washing twice, 2% BSA-PBS was added and left at room temperature for 1 hour. After removing 2% BSA-PBS, MV833 (1
% BSA-PBS solution) and left at room temperature for 1 hour.
After washing 6 times with 0.1% BSA-PBS, peroxidase-labeled sheep anti-mouse IgG (Amersham) (0.1% B
(SA-PBS solution) and left at room temperature for 1 hour. 0.1
After washing with 6% BSA-PBS six times, 0.2 M Tris-citrate buffer (pH = 5.2) containing 8.3 mg / ml orthophenylenediamine dihydrochloride and 0.01% hydrogen peroxide was added to develop color. I let it. After stopping the reaction by adding 2N sulfuric acid,
The absorbance (OD490 / 650) was measured.

The MV833 antibody strongly reacted with five peptides of peptide identification numbers 31, 32, 33, 60 and 63 among 67 kinds of peptides. Peptide identification numbers 31 to 3
Since peptide 3 contains the sequence KPSCVPLMR in common, it is considered that the MV833 antibody reacts with the amino acid sequence KPSCVPLMR in this region. Therefore, the MV833 antibody is
KPSCVPLMR array of F / VPF and SFLQHNK
It is expected that it reacts with the CECRP sequence and the KCECRPKKDRAR sequence. Since an antibody is considered to recognize a portion exposed on the surface of a protein, it can be said that these two amino acid sequence portions are portions exposed on the surface of VEGF / VPF. Monoclonal antibodies are said to have a single antigenic determinant, but when a macromolecular substance such as a protein having a higher-order structure is an antigen, the antibody recognizes the antigen three-dimensionally, and When the reactivity of an antibody is examined at the primary structure level, it may react to two or more discontinuous amino acid sequences. MV833 antibody is VE
Based on the reaction with two amino acid sequence portions in GF / VPF, it is considered that this antibody recognizes two amino acid sequence portions three-dimensionally and simultaneously.

When conducting research on trace proteins and viruses, the gene is currently cloned, and the amino acid sequence of the protein can be predicted from its base sequence. Search for a highly hydrophilic site based on this amino acid sequence,
Polyclonal and monoclonal antibodies against the synthetic peptide at that site have been prepared and used for immunological analysis. Hoop & Woods et al.'S method to search for highly hydrophilic sites
Analysis is performed using [Proc. Natl. Acad. Sci. USA 78: 3824 (1981)] or the like, but it does not always apply to all proteins. Therefore, according to the present invention, VEGF / VP
A portion of the site exposed on the surface of F that is important for the inhibition of storage of pooled water, such as ascites, has been clarified so that a VEGF / VPF antibody having a strong storage inhibitory activity can be easily prepared. In addition, the method adopted in the present invention is also applied as a method for clarifying a site exposed on the surface of a protein.

(4) Inhibition Test of Anti-VEGF / VPF Monoclonal Antibody on Carcinomatous Ascites Retention Human ovarian cancer K2 cells (obtained from Nagoya University School of Medicine) cultured in DMEM medium containing 10% fetal bovine serum were treated with an EDTA solution (50 mM HEPES). , 1mM EDTA, 125mM NaCl, 5mM KCl, 5mM
Glucose / pH 7.0) Peel from the flask and collect.
A cell suspension was prepared using BS.

[0038] 16.4g female nude mice The cell suspension 5-week-old (10 ⁷ 1.5 ×) 0.2 ml intraperitoneally (Balb / c-nu / nu )
Transplanted.

On days 4, 8, 12, 16 and 20 after transplantation, 100 μg / 0.2 ml / mouse of MV833 was administered intraperitoneally in a single dose (n = 25). As a control group, a group to which mouse IgG was similarly administered instead of MV833 (n = 25) was used. On the 22nd day, 4 mice died in the control group, but ascites could be collected, but ascites was collected from all individuals including 3 dead mice under Nembutal anesthesia, and the amount of ascites was measured. Is shown in FIG.

As is clear from FIG. 1, a remarkable inhibitory effect on ascites retention was observed in the MV833 administration group (p <0.00).
1).

(5) Inhibition Test of Anti-VEGF / VPF Monoclonal Antibody Against Carcinomatous Ascites Retention Among the mice of the control group used in the test of (4) above, the ascites of the mouse showing ascites retention was removed and then randomly removed. The test group was divided into two groups, a MV833 administration group (6 animals) and a control group (7 animals).

On day 0 and day 4 after removal of ascites, MV833 or mouse IgG was administered intraperitoneally to each mouse in each group in the same manner as in (4) above. On the 5th day, ascites was collected from all the animals except for one dead animal in the control group and the ascites volume was measured in the same manner as in (4), and the body weight and hematocrit value were measured. , 3 and 4.

As is clear from FIG. 2, a remarkable inhibitory effect on ascites re-accumulation was observed in the MV833 administration group (p <
0.01). As is clear from FIGS. 3 and 4, there was no significant difference in the weight of the mice in both groups, and the hematocrit value of the MV833-administered group was lower than that of the control group, but no significant difference was observed.

[0044]

Industrial Applicability According to the present invention, re-retention of stored water such as cancerous ascites is suppressed, and the therapeutic agent of the present invention is effectively used for treatment of re-retention of stored water.

[0045]

[Sequence list]

SEQ ID NO: 1 Sequence length: 9 Sequence type: amino acid Topology: Linear Sequence type: Protein Origin: Cell line: SEQ ID NO: 2 Sequence length: 12 Sequence type: amino acid Topology: Linear Sequence type: Protein Origin: Cell line: SEQ ID NO: 3 Sequence length: 12 Sequence type: amino acid Topology: Linear Sequence type: Protein Origin: Cell line:

[Brief description of the drawings]

FIG. 1 is a graph showing the inhibitory effect of anti-VEGF / VPF monoclonal antibodies on retention of cancerous ascites.

FIG. 2 is a graph showing the inhibitory effect of an anti-VEGF / VPF monoclonal antibody on cancerous ascites re-retention.

FIG. 3 is a graph showing changes in body weight associated with administration of an anti-VEGF / VPF monoclonal antibody for re-retention of cancerous ascites.

FIG. 4 is a graph showing a change in hematocrit associated with the administration of an anti-VEGF / VPF monoclonal antibody to re-reserve cancerous ascites.

Continuing from the front page (72) Inventor Ayako Koda 2nd Okubo Tsukuba, Ibaraki Prefecture Toagosei Co., Ltd. in Tsukuba Research Laboratories (72) Inventor Katsuhiko Matsuo 2nd Okubo Tsukuba City, Ibaraki Pref. Toshiaki Segawa 2nd Okubo Tsukuba City, Ibaraki Prefecture Toagosei Co., Ltd.

Claims (3)

[Claims] 1. A body fluid re-retention inhibitor comprising a VEGF / VPF antagonist as an active ingredient. 2. The method according to claim 1, wherein the VEGF / VPF antagonist is an anti-VEGF / VPF monoclonal antibody. 3. The method according to claim 2, wherein the anti-VEGF / VPF monoclonal antibody is an antibody that reacts with at least one of the following sequences 1 to 3, which is a part of the amino acid sequence of VEGF / VPF. Agent. 1. KPSCVPLMR 2. SFLQHNKCECRP 3. KCECRPKKDRAR