JPH1149701A - Anticancer agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an anticancer agent having extremely minute degree of side effect, and capable of suppressing the growth of tumor more significantly than in the case of single use, as a chemotherapentic agent which can be used efficiently in combination with another agent by compounding a vascular endothelial cell growth factor (VEGF)/a vascular permeability factor(VPF) antagonist and a basic fibroblast growth factor (bFG) antagonist. SOLUTION: A human VEGF/VPF is collected in a purified form, for example, from culture liquid of a yeast which has been transformed with cDNA of a separated human VEGF/VPF, and its complex is formed by using KLH and glutaraldehyde. A mouse monoclonal antibody (especially, reactive to at least one selected from KPSCVPLMR, SFLQHNKCECRP and KCECRPKKDRAR, which are parts of the amino acid sequences of VEGF/VPF) is obtained according to a conventional method by using the obtained protein as an antigen. The objective agent is produced by compounding the mouse monoclonal antibody and optional an anti-bFG antibody.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a VEGF / VPF
(vascular endothelial growth factor: vascular permeability factor) antagonist and bFGF (basic fibroblast gro)
The present invention relates to an anticancer agent containing a wth factor (basic fibroblast growth factor) antagonist as an active ingredient.

[0002]

2. Description of the Related Art Generally, the growth of cancer cells is very slow at the initial stage of malignant conversion from normal cells. However, when a cancer cell is generated, it is observed that a new blood vessel newly branched from the surrounding blood vessel migrates toward the cancer cell. And once blood vessels reach this lesion, cancer cells are supplied with inexhaustible nutrients and oxygen, which not only explodes and starts to proliferate,
A distant metastasis will also occur via the blood vessel. From this, it is said that angiogenesis or proliferation of vascular endothelial cells, which are constituent cells of blood vessels, are closely related to tumor progression and metastasis.

[0003] Cancer cells are capable of inducing such angiogenesis and proliferation of vascular endothelial cells (Tumor Angiogene).
sis Factor), such factors as aFGF, bFGF, EGF, PD-ECGF, VEGF / VP
Many substances have been reported, including F, TGF-b, Angiogenin (R. Bicknell and AL Harris, Eur. J. Cancer 2
7, 6, 781, 1991).

With respect to VEGF / VPF, certain anti-VEGF / VPF
It has been reported that the use of neutralizing antibodies can suppress the growth and metastasis of cancer cells (M. Asano et al., CANCER
RESEARCH 55, 5296-5301, November 15, 1995). Similarly, for bFGF, it has been reported that tumor growth can be suppressed by using an anti-bFGF neutralizing antibody (AH
ori et al., CANCER RESEARCH 51, 6180-6184, November 15,
1991).

[0005]

The method of treating cancer using the antagonism of such an antibody is different from the current general treatment method targeting the tumor itself, indirectly by suppressing the formation of tumor blood vessels. It is based on a new mechanism of action that inhibits the growth of tumors, has very slight side effects, and is expected as an effective cancer chemotherapy that can be effectively used in combination with other drugs.

In view of the fact that the interrelationship between the above-mentioned cancer angiogenesis factors has not yet been studied, the present inventors provide a more effective method for treating cancer based on the antagonistic action against such cancer angiogenesis factors. As a result, the inventors of the present invention have conducted intensive studies focusing on a system in which the actions of a plurality of cancer angiogenic factors are simultaneously suppressed by respective neutralizing antibodies, and as a result, the present invention has been completed.

[0007]

That is, the present invention relates to
The present invention relates to an anticancer agent containing an EGF / VPF antagonist and a bFGF antagonist as active ingredients. According to the present invention, by using such two kinds of antagonists in combination, the growth of tumors can be significantly suppressed as compared with the case of using them alone.

[0008] VEGF / VPF acts directly on vascular endothelial cells to induce angiogenesis, a phenomenon of proliferation, migration and infiltration of capillary endothelial cells, fetal growth, wound healing, and cancer cell degeneration. It plays an important role in physiological or pathological phenomena such as proliferation. VEGF /
VPF is secreted by mouse, rat, guinea pig, bovine and human normal or tumor cell lines, and has been shown to be present in the brain, pituitary, kidney and ovary by tissue [Ferrara, N. ., Endocrine Reviews 13: 18 (1
992)].

Regarding the human VEGF / VPF gene, its cDNA has already been isolated, its nucleotide sequence has been determined, and its amino acid sequence has been estimated. Four types of proteins having different numbers of amino acid residues (121 amino acid residues, 1
65, 189, and 206 types were produced, among them, those having 121 amino acid residues (VEGF
₁₂₁ ) and 165 amino acid residues (VEG
F ₁₆₅ ) is said to be a mature protein [Ferrara, N .;
Endocrine Reviews 13:18 (1992)]. Although VEGF ₁₂₁ are those 44 amino acids of the carboxyl-terminal of the VEGF ₁₆₅ are deleted, while the VEGF ₁₂₁ and VEGF _165, not been clarified whether there are differences in effects on vascular endothelial cells .

[0010] In the present invention, the VEGF / VPF antagonist refers to a substance having a function of inhibiting the action of VEGF / VPF, and any form having the function may be used. Has V
An antibody that acts on EGF / VPF, or a portion thereof, including but not limited to,
Inactive VEGF / V inhibiting the action of EGF / VPF
PF, or a portion thereof, impairs the function of the VEGF / VPF receptor, eg, an antibody against the vascular permeability factor receptor, or a portion thereof, or even VEGF / VPF
And the like, which suppress the production of the compound itself.

The antibody acting on VEGF / VPF may be an antibody against any of the four VEGF / VPF subtypes having 121, 165, 189 or 206 amino acid residues.

Representative VEGF / VPF antagonists include anti-VEGF / VPF monoclonal antibodies. In particular, the anti-VEGF / VPF monoclonal antibodies have the following sequences 1 to 3, which are part of the VEGF / VPF amino acid sequence: :

1. KPSCVPLMR 2. SFLQHNKCECRP 3. KCECRPKKDRAR

Preferably, the antibody reacts with at least one of the following.

[0015] bFGF is a protein found in cerebral and pituitary extracts, which reduces the proliferation of cultured cells derived from mesodermal cells including vascular endothelial cells in addition to cultured fibroblasts at low concentrations (several pg to several pg). ng / ml) [Gospodarowicz et al.
J. Cell Physiol., Supplement 5, 15-26 (1987)], known in vivo effects include the differentiation of ectoderm from mesoderm to the mesoderm in the embryo, the promotion of survival and differentiation of neurons, etc. In particular, attention has been paid to the angiogenic effect.

In the present invention, the bFGF antagonist refers to a substance having a function of inhibiting the above-mentioned action of bFGF, particularly an angiogenesis action, and may have any form as long as it has the function. Most commonly, antibodies that act on bFGF, or portions thereof, include, but are not limited to,
Inactive bFGF that inhibits the action of FGF, or a portion thereof, that impairs the function of the receptor for bFGF, eg, b
An antibody against the FGF receptor, or a part thereof, and a drug that suppresses the production of bFGF itself can be mentioned.

Typical bFGF antagonists include anti-bFGF monoclonal antibodies. Specifically, anti-bFGF monoclonal antibodies bFM-1 and bFM described in JP-A-3-103189 are mentioned.
FM-2 and A. Hori, CANCER RESEARCH 51, 6
180-6184, November 15, 1991, the anti-bFGF monoclonal antibody 3H3 and the like.

The above anti-VEGF / VPF antibody or anti-bF
Examples of the GF antibody include a mouse antibody, and a GF antibody that has been subjected to treatment for reducing side effects upon 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. Examples of the chimeric antibody or the humanized antibody include an IgG type or an IgA type, and the IgG isotype includes IgG1, IgG2,
IgG3 or IgG4.

When administering the anticancer agent of the present invention, the subject to be administered is not particularly limited. For example, it can be locally administered for the specific purpose of preventing or treating individual cancer types.
The method of administration is parenteral.

Parenteral administration includes injections such as subcutaneous, intramuscular, intravascular, intraperitoneal or intrathoracic injections, infusions, suppositories and the like. The dose and the ratio of the active ingredient vary depending on whether the animal or human, the age, the administration route, and the number of administrations, and can be widely varied. In this case, VEGF /
The effective amount of the VPF antagonist and the bFGF antagonist and the effective amount to be administered as a composition of a suitable diluent and a pharmaceutically usable carrier is 0.1 to 100 mg / kg body weight.
/ Day, divided from once a day to several times a day or several days
It is administered once or once every 1-2 weeks. VEGF / VPF
The antagonist and the bFGF antagonist can be used together in any ratio, and both can be used in a mixed state or in separate states.

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

[0022]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, preferred embodiments of the present invention will be described with reference to anti-VE as a VEGF / VPF antagonist.
Using a GF / VPF monoclonal antibody and bFG
This will be described in detail using an anti-bFGF monoclonal antibody as an F antagonist.

Production of Monoclonal Antibodies Each monoclonal antibody was prepared by culturing animals with VEGF / VPF or b
It can be produced by immunizing with FGF, 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) 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.
At least 2-3 immunizations are performed at intervals of 間隔 4 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.

(2) Preparation of myeloma cells As myeloma cells, mouse myeloma Sp2 / O-Ag14 (S
p2), P3 / NSI / 1-Ag4-1 (NS-1), P3 × 63-Ag.8.U · 1 (P3U1) and the like. Subculture of these cells is performed according to a conventional method.

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

(4) Selection of hybridoma Selection of fused cells (hybridoma) 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.

(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. In addition, the monoclonal antibody of the present invention can be accumulated in ascites by transplanting hybridoma cells into the peritoneal cavity of a mouse and growing the cells.

(6) Collection and Purification of Monoclonal Antibodies The monoclonal antibodies accumulated in the culture solution of the hybridoma cells or in the ascites fluid are prepared by the conventional ammonium sulfate fractionation, PEG fractionation, ion exchange chromatography and gel filtration. It is purified by a method using chromatography. In addition, a method using affinity chromatography such as protein A and protein G can also be used.

For the selection of monoclonal antibodies, an enzyme immunoassay, a Western blotting method or the like is used.
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.

[0031]

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

Reference Example 1 (Anti-VEGF / VPF Monochrome
Preparation of Null Antibody MV833) (1) Preparation of Hybridoma Producing Anti-VEGF / VPF Monoclonal Antibody Human VEGF / VPF was purified from a culture solution of yeast transformed with the isolated human VEGF / VPF cDNA.
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.

(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.

The dissociation constants for VEGF ₁₂₁ and VEGF ₁₆₅ measured by the following method are as follows, indicating 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 (VEGF ₁₂₁ ) ○ 1.10 × 10 ^-10 M ± 0.11 × 10 ^-10 M (VEGF
F ₁₆₅ )

Method for measuring dissociation constant The monoclonal antibody containing 0.1 M sodium chloride
It is adjusted to 2 μg / ml with 5 mM carbonate buffer (pH = 9.0), added to a detachable plate with 100 μl each, and left at 4 ° C. 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 in the above and ¹²⁵ I-labeled VEGF ( ¹²⁵ I-labeled V
EGF ₁₂₁ is labeled VEGF ₁₂₁ by the chloramine T method,
¹²⁵ I-labeled VEGF ₁₆₅ was purchased from Amersham.) Add 200 μl of the reaction mixture per well and leave overnight. VEGF concentration in the reaction mixture is VEGF ₁₂₁ is 0～1Ng / well,
0-10 ng / well of VEGF _165, 1 of ¹²⁵ I-labeled VEGF
× 10 ⁴ cpm / well ( ¹²⁵ I-labeled VEGF ₁₂₁ ; 66.7 pg / well,
^{1 25} I-labeled VEGF _165; 116pg / well) to. The reaction mixture was removed from the wells, and the wells were washed six times with 0.1% BSA-PBS. Then, the wells were cut off one by one, placed in an analysis tube, counted with a gamma counter, and the dissociation constant was calculated from the scatter diagram created from the results. Ask.

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.

Measurement method of isoelectric point Isoelectric focusing of the monoclonal antibody was carried out using a commercially available agarose gel for isoelectric focusing (Washikamori) on the isoelectric focusing 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 reactive site of monoclonal antibody in VEGF / VPF (3-1) Preparation of peptide corresponding to part of amino acid sequence of VEGF / VPF 12 consecutive amino acids of amino acid sequence of human VEGF ₁₂₁ Was devised as one peptide, and 67 peptides covering the entire sequence were devised, and each peptide was synthesized using 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.

[0041]

[Table 1]

(3-2) Identification of Peptides Reacting with MV833 Antibody 67 kinds of peptides synthesized as described above are human VE
Which corresponds to the entire area of the GF _121. 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. In addition, for the measurement of the reactivity, in addition to the enzyme immunoassay described above, the Ouchterlony method, the Western blotting method, or the like may be used.

The MV833 antibody reacted strongly 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
FGF / VPF antibodies having a strong anticancer activity can be easily prepared by clarifying a portion that is important for inhibition of angiogenesis and the like among the sites exposed on the surface of F, and The method adopted in the present invention can also be applied as a method for clarifying a site exposed on the surface of a protein.

Reference Example 2 (Anti-bFGF monoclonal antibody
Preparation of bFM-1) The anti-bFGF monoclonal antibody bFM-1 can be prepared according to the following protocol.

(1) Purification of immunogen bFGF as an immunogen can be separated and purified from, for example, bovine brain or pituitary gland by a known method.

Specifically, the immunogen can be purified as follows. That is, methods such as ammonium sulfate precipitation, CM-Sephadex chromatography and methods such as Gospodarowicz including heparin-Sepharose chromatography [Go]
Natl. Acad. Sci. USA 81, 6963- spodarowicz et al.
6967 (1984)] to purify bFGF from bovine brain. One protein peak eluted from the column and having DNA synthesis stimulating activity [Nishikawa et al., Methods Enzymol. 146, 11-22 (1987); Yoshitake et al., Cell Struct. Funct. 7, 229-243 (1982)] Collect fractions that give The reduced sample of the purified bFGF contained 19.5%
On SDS-PAGE a single band of protein with a molecular weight of 18,000 is given.

(2) Immunization A mouse is immunized with a purified bFGF immunogen solution by in vivo immunization [Nishikawa et al., Methods Enz.
ymol. 146, 11-22 (1987); Yoshitake et al., Arch.Biochem.
Biophys. 263, 437-446 (1988)].

Specifically, the immunization can be performed as follows. That is, the purified bFGF immunogen solution is mixed with an equal volume of Freund's complete or incomplete adjuvant until emulsified. 10 μg of this bFGF
Is administered subcutaneously to a 6-week-old female BALB / c mouse (first immunization). Thereafter, the same operation is performed at intervals of 2 to 4 weeks, and immunization is performed 5 times in total. Quantification of antibody titers in mouse serum was performed by Massoglia
ELI described in J. Cell Physiol. 132, 531-537 (1987).
It can be performed by the SA method.

Four days after the final immunization, the spleen is aseptically removed from the mouse and used for the following cell fusion step.

(3) Preparation of Hybridoma Specifically, a hybridoma is prepared as follows. The spleen of the above (2) is placed in a DME medium and crushed on a stainless mesh to obtain a spleen cell suspension. The cells thus obtained are washed with a DME medium by a centrifugation method, and the number of living spleen cells is measured.

On the other hand, mouse myeloma cells (bone marrow cells) P3- × 63-Ag8-U1 (P3U1) (Dr. Gordo
n H. Sato, W. Alton Jones Cell Science Center, In
c.) Add about 1 × 10 ⁸ spleen cells to about 5 × 10 ⁷ cells, mix well in DME medium, and centrifuge (500 ×
g, 10 minutes). The supernatant is aspirated, the pellet is well disentangled, and 50% (w / v) polyethylene glycol 4000
0.5 ml of DME solution (incubated at 37 ° C.) is added dropwise, and the polyethylene glycol solution and the cell pellet are mixed by gently rotating the centrifuge tube by hand for 1 minute. Next, 24 ml of DME medium kept at 37 ° C. is added little by little, and the tube is gently rotated, followed by centrifugation (500 × g, 10 minutes). Cell pellet
After centrifugal washing with a PRMI medium containing 0% fetal bovine serum, the cells were washed with HAT medium containing 10% fetal bovine serum (4 × 10 ⁻⁷ M aminopterin in RPMI medium, 1.6 × 10 ⁷ thymidine).
^-5 M, hypoxanthine 1 × 10 ^-4 M added) 4
Suspend in 0 ml. This cell suspension was dispensed 200 μl into each well of a 96-well cell culture plate,
Then, culture is started in a carbon dioxide incubator containing 5% carbon dioxide. During the culture, the medium in each well was added to the medium for about 100 days at intervals of 2 to 3 days.
The hybridoma that grows in the HAT medium is selected by removing 100 μl and newly adding 100 μl of the above HAT medium. From day 8, HT medium containing 10% fetal bovine serum (DME
Thymidine 1.6 × 10 ^-5 M, hypoxanthine 1 × 1
^0-4 M), and the growth of the hybridoma was observed.
The bFGF antibody-producing hybridoma is screened by the ELISA method.

(4) Establishment of Hybridoma The presence or absence of the produced antibody in the hybridoma culture supernatant can be measured by ELISA. That is, 2 μg / ml of the above bF was added to each well of a 96-well ELISA plate (Falcon).
Aliquot 50 μl of the GF immunogen solution and leave at room temperature for 4 hours. After removing the supernatant, blocking is performed by dispensing 1% BSA-PBS. Next, after washing four times with 0.1% Tween20-PBS, 50 μl of the culture supernatant of each well is added and reacted at room temperature for 4 hours. Then, the supernatant was removed and 0.1% Tween20-PBS
Wash 4 times.

Next, 50 μl of peroxidase-conjugated rabbit anti-mouse antibody (Dako, Denmark) diluted 2,000-fold with Tween 20-PBS is added to each well. After the reaction,
Wash each well four times with 0.1% Tween 20-PBS and add 50 μl of a solution containing 0.1 M citrate-phosphate buffer (pH 5.0), 0.4% o-phenylenediamine and 0.005% hydrogen peroxide to each well. In addition, the reaction was carried out at room temperature for 15 minutes.
Measure the absorbance at 2 nm. As a result, the wells in which antibody production was observed were tested again by ELISA for anti-b
After confirming the presence or absence of FGF antibody production, cloning several times by the limiting dilution method allows hybridoma b
FM-1 can be established.

(5) Production of monoclonal antibody:
Vivo method pristane (2,6,10,14-tetramethylpentadecane)
0.5 ml was intraperitoneally administered to 7-week-old BALB / c mice.
After 1 day, 1 × 1 hybridoma bFM-1 grown in vitro was intraperitoneally injected into each mouse.
0 ⁷ inoculated so that the cell.

For hybridomas, usually about 5 to 10 ml of ascites are obtained from one mouse. The antibody concentration is 1-3 mg / ml. Purification of the monoclonal antibody in the ascites can be performed as follows. After centrifuging (20,000 × g, 10 minutes) the ascites filtered by gauze, solid ammonium sulfate is added to a 30% saturation concentration. After centrifugation (20,000 × g, 10 minutes), ammonium sulfate is further added to the supernatant to a 50% saturation concentration, and a precipitate is obtained by centrifugation. The precipitate is dissolved in a small volume of 5 mM Tris-HCl buffer (pH 7.5) and dialyzed three times against a 100-fold volume of the buffer. The dialysate obtained is applied to a column of DE-50 cellulose equilibrated with the buffer and washed with the buffer. The adsorbed monoclonal antibody is eluted by the above-mentioned buffer solution and a solution obtained by adding 0.15 M NaCl thereto by a concentration gradient method. The fact that the obtained monoclonal antibody has a uniform purity means that SDS-
Confirm by electrophoresis.

(6) Properties of Monoclonal Antibody bFM-1 (6-1) Identification of Immunoglobulin Class The immunoglobulin class and subclass of the anti-bFGF-specific monoclonal antibody bFM-1 were determined using a mouse monoclonal antibody isotyping kit (Amersham). Has been identified to be IgG1 / κ.

(6-2) Cross-reactivity of monoclonal antibody (6-2-1) Bovine pituitary bFGF (1-146) [Esch et al., Proc.
Atl. Acad. Sci. USA 82, 6507-6511 (1985)], a fully automated Biolynx4170 Peptide Synthesizer with 14-16 amino acids and an additional Tyr sequence.
(Pharmacia LKB Biotechnology, Sweden) or fully automated Applied Biosystems 430 A Peptide Synthesizer (Ap
plied Biophysics Inc., USA) by a solid phase method [Matsuo et al., In Vitro Cell. Dev. Biol. 24, 4
77-480 (1988)]. Two of them are Tyr ¹⁶ -bFGF (1-15) and Tyr ¹⁶ -bFGF (1-15).
yr ¹³² -bFGF (133-146), which correspond to the amino and carboxyl terminal sequences of the native molecule, respectively. Another is Tyr ⁵¹ -bFGF (36-50), which contains the major portion of the hydrophilic sequence of bFGF identified by hydrophilic analysis of the entire sequence.

BF for monoclonal antibody bFM-1
To determine epitopes on GF, the cross-reactivity of three linear fragments of the bFGF molecule with the monoclonal antibody bFM-1 was examined in a competitive binding assay based on the following radioimmunoassay (RIA). As a result, these three
Species peptide fragment and monoclonal antibody bFM
-1 means 100 μg / ml (this concentration is 100-100 of the molar standard concentration of native bFGF showing a clear cross-reactivity.
0-fold) did not show any cross-reactivity. This confirms that these sequences, ie, the amino terminus, carboxyl terminus and major hydrophilic region, are not recognized by monoclonal antibody bFM-1 as a continuous epitope.

(6-2-2) The biological activities of bFGF are thermolabile and acidlabile. Thus, the conformation of the bFGF molecule, which is not maintained by disulfide bonds, is considered essential for its biological activity [Gospodarowicz et al., J. Cell Physiol. 128, 475-
484 (1986)]. Monoclonal antibody bFM-1 is bFGF
In order to determine whether the conformation has been recognized, the bFGF solution may be incubated for 5 minutes in a boiling water bath. The biological activity of heat-inactivated bFGF (measured by the activity of promoting DNA synthesis in BALB / c3T3-3K cells) was 0.1% of the biological activity of bFGF before heat treatment.
2%.

When the cross-reactivity of the monoclonal antibody bFM-1 with the heat-inactivated bFGF was examined in the same manner as in the above section 6-2-1, no cross-reactivity was observed. This indicates that monoclonal antibody bFM-1
This indicates that the recognition of the conformation of the bFGF molecule required for the biological activity of FGF is recognized.

(6-2-3) bFGF derived from various species
Table 2 shows the results of determination of the reactivity between bovine-aFGF and monoclonal antibody bFM-1 by radioimmunoassay (RIA). Monoclonal antibody bFM-
1 is mouse-bFGF, human-bFGF and bovine-b
It cross-reacted with FGF but not with bovine-aFGF.

[0063]

[Table 2]

The conditions for the radioimmunoassay (RAI) were the same as those described in the above sections 6-2-1 and 6-2-2, according to the method described in the following section 6-2-4. The calculation of the numerical values in Table 2 is based on the FGF concentration estimated by radioimmunoassay (RIA).
And the FGF concentration estimated from the DNA synthesis stimulating activity. In all assays, pure bovine bF
GF was used as a standard. 100% bovine bFGF
And

(6-2-4) Radioimmunoassay of bFGF Purified bovine bFGF was purified by the chloramine-T method [Kan et al.
Biol. Chem. 263, 11306-11313 (1988)].
Labeled with ¹²⁵ I and heparin-Sepharose affinity chromatography [Neufeld et al., J. Biol. Chem. 260, 1386.
0-13868 (1985)] with a slight modification.
Briefly, bovine bF in 110 μl of reaction mixture containing 180 μg / ml chloramine-T for 2 minutes at room temperature.
2.8 μg of GF and 700 μCi of ¹²⁵ I (25.9 MBq) in 0.02% CHAPS
[Matsuo et al., In Vitr
o Cell. Dev. Biol. 24, 477-480 (1988)]. The reaction was stopped by adding 100 μl of 0.02 M dithiothreitol. Next, using a salt solution containing 0.1% CHAPS,
From free ¹²⁵ I on heparin -Sepharose column ^{12 5} I
Labeled bFGF was separated. BF with ¹²⁵ I label
The specific activity of GF was 5.5 × 10 ⁴ cpm / ng. The labeled bFGF and the unlabeled bFGF showed almost the same biological activity as judged from the same DNA synthesis promoting activity as described above. The reaction mixture for RIA (5 ml) was 0.1 M phosphate buffer (pH 7.4) /0.02% NaN ₃ 0.35 ml, PBS / 0.1% BSA /
4 ng / ml ¹²⁵ I-bFGF (8000-15000) in 0.02% NaN ₃ (PBS-BA)
cpm) of 0.05 ml, 0.05 ml of unlabeled bFGF at an appropriate concentration in PBS-BA and 0.05 ml of each purified monoclonal antibody (0.12-1.5 μg / ml) in PBS-BA. (Eiken) and incubated overnight at 4 ° C. 0.1 ml of 1% normal mouse serum in PBS / 0.02% NaN ₃ and PBS
0.77 mg / ml goat in /0.02PasentoNaN ₃ - Anti - after adding a mouse immunoglobulin (Dako) 0.1 ml, was incubated for 4 hours at further 4 ° C. The tubes. The 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 auto-well gamma system (ARC-300).

(6-3) Monoclonal antibody bFM-1 having the above properties inhibits the growth of bovine capillary endothelial cells in vitro in the presence as well as in the absence of exogenous bFGF.

Example 1 Anti-VEGF / VPF monoclonal antibody MV833 and
Tumor suppression test 1 by combined use of anti-bFGF antibody bFM-1

Human colon cancer cells (RPMI
4788), and the cell suspension was subcutaneously injected into BALB / c nude mice (male, 6 weeks old, n = 4 or 5) at a rate of 10 ⁶ cells by a conventional method. When transplanted at four sites, the growth of 16 to 20 tumor masses was observed.

On days 1, 3, 6, 9, 12, 15 and 19 after the transplantation, 0.2 ml of the following six groups of antibodies were intraperitoneally administered, respectively.

MV group: MV833 (200 μg / M) administration, n = 41 FMV group: MV833 (200 μg / M) + bFM-1 (200 μg / M) administration,
n = 51F group: bFM-1 (200 μg / M) administration, n = 44F group: bFM-1 (800 μg / M) administration, n = 5 HA group: HA (anti hEGF McAb) (800 μg / M) administration , N = 4 PBS group: PBS (0.2 ml / M) administration, n = 4

FIG. 1 shows the tumor growth over time in each group.
Further, on day 23, the tumor was excised and the tumor weight is shown in FIG. FIG. 2 shows the places where there was a significant difference between the two groups according to the Mann-Whitney method.

From FIG. 1 and FIG. 2, the tumor growth was measured using anti-VEGF / V
It can be seen that the PF antibody (MV group) is very well suppressed, and that the group administered simultaneously with the anti-VEFG / VPF antibody and the anti-bFGF antibody (1FMV group) is more effective than the group with anti-bFGF antibody alone (4F group). It turns out that it is suppressed significantly. Therefore, it was shown that the combined use of an anti-VEGF / VPF antibody and an anti-bFGF antibody was effective for inhibiting solid tumor formation.

Example 2 Anti-VEGF / VPF monoclonal antibody MV833 and
Tumor suppression test 2 by combined use of anti-bFGF antibody bFM-1

A cell suspension was prepared using human squamous cell carcinoma cells (A431) cultured by a conventional method, and this cell suspension was used in BALB / c nude mice (male, 8 or 11 weeks old, n = 4). Or, in step 5), 10 ⁶ cells were transplanted into the subcutaneous back at four sites by a conventional method, and the growth of 16 to 20 tumor masses was observed.

Since the cancer cells (A431) rapidly grow the tumor, 0.2 days each of the following 6 groups of antibodies were intraperitoneally administered on days 1, 3, 5, 7, 9 and 11 after transplantation.

MV group: MV833 (200 μg / M) administration, n = 54 FMV group: MV833 (200 μg / M) + bFM-1 (800 μg / M) administration,
n = 51F group: bFM-1 (200 μg / M) administration, n = 44F group: bFM-1 (800 μg / M) administration, n = 5 HA group: HA (anti hEGF McAb) (1000 μg / M) administration , N = 5 PBS group: PBS (0.2 ml / M) administration, n = 4

FIG. 3 shows the time course of tumor growth in each group.
On the 14th day, the tumor was excised and the tumor weight is shown in FIG. FIG. 4 shows the places where there was a significant difference between the two groups according to the Mann-Whitney method. Incidentally, since there was one abnormal animal in the PBS group, the group excluding it was shown as the PBS 'group in FIG.

From FIGS. 3 and 4, tumor growth was not significantly suppressed by the anti-bFGF antibody (groups 1F and 4F).
It can be seen that it is well suppressed by the group of the anti-VEGF / VPF antibody alone (MV group),
In the group to which the antibody was simultaneously administered (4FMV group), anti-VEGF / V
It can be seen that the PF antibody is significantly suppressed compared to the group alone (MV group). Therefore, it was shown that the combined use of an anti-VEGF / VPF antibody and an anti-bFGF antibody was extremely effective in inhibiting solid tumor formation.

[0079]

According to the present invention, tumor growth can be effectively suppressed by using a VEGF / VPF antagonist and a bFGF antagonist together. Therefore, the present invention is effectively used for treatment of various cancers.

[0080]

[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 on the growth of human colon cancer cells over time in each antibody administration group.

FIG. 2 is a diagram showing the tumor weight on day 23 in FIG. 1 for each antibody administration group.

FIG. 3 is a graph showing the inhibitory effect on the growth of human squamous cell carcinoma cells over time in each antibody administration group.

FIG. 4 is a diagram showing the tumor weight on day 14 in FIG. 3 measured for each antibody administration group.

 ────────────────────────────────────────────────── ─── Continuing on the front page (72) Inventor Mitsuru Osawa 1-11-4 Higashikanda, Chiyoda-ku, Tokyo Inside Yatron Co., Ltd. (72) Inventor Makoto Asano 2nd Okubo Tsukuba, Ibaraki Pref. Inside the research institute (72) Inventor Ayako Koda No. 2 Okubo Tsukuba, Ibaraki Pref.Toagosei Co., Ltd. Within Tsukuba Research Co., Ltd. (72) Inventor Hideo Suzuki No. 2 Okubo Tsukuba, Ibaraki Pref.

Claims (4)

[Claims] 1. An anticancer agent comprising a VEGF / VPF antagonist and a bFGF antagonist as active ingredients. 2. The anticancer agent according to claim 1, wherein the VEGF / VPF antagonist is an anti-VEGF / VPF monoclonal antibody. 3. The anticancer agent 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. 1. KPSCVPLMR 2. SFLQHNKCECRP 3. KCECRPKKDRAR 4. The anticancer agent according to claim 1, wherein the bFGF antagonist is an anti-bFGF antibody.