JPH06116163A - Tumor inhibitor - Google Patents

Abstract

PURPOSE:To obtain a tumor inhibitor for inhibiting the function of vascular permeability factor inducing neogenesis and wandering closely related to tumor and metastases of tumor and indirectly suppressing activity of tumor cell. CONSTITUTION:This function inhibitor of vascular permeability factor (VPF) has specific cell growth promoting activity and blood permeability promotion activity against vascular endothelial cell and about 20,000 molecular weight and exists as a dimer in vivo. The tumor inhibitor is effectively used in combination with various medicines, efficiently carries out chemotherapy of cancer, suppress metastases of tumor and has extremely light side effect such as influence on hematopoietic organs.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a drug which inhibits the function of a vascular permeability factor which induces neovascularization and migration of blood vessels closely related to tumor growth and metastasis. Since it effectively suppresses the activity of tumor cells, it is effective as a tumor suppressor and is used in the pharmaceutical industry and medical field.

[0002]

2. Description of the Related Art Most of the currently widely used tumor treatment methods target tumor cells per se, whether chemotherapy or radiation therapy. Selective attack on tumors by drug administration causes tumor cells to divide much more vigorously than other normal cells,
Much depends on the nature of repeated proliferation. That is, the objective is to kill the target cells by destroying or inhibiting the cell growth mechanism itself.

On the other hand, a method for suppressing the growth of solid tumors is
The idea of so-called military food blame, which cuts off the sources of nutrition and oxygen, has been proposed. In other words, without attacking the tumor cells themselves, they are deficient in nutrients and oxygen, resulting in the inhibition of tumor growth and the therapeutic effects of regression. Specific targets for this approach include blood vessels that reach the tumor.

That is, it is generally said that even if cells are transformed into malignant cells and cancer cells are generated, the growth thereof is very slow in the initial stage, and the tumor that has occurred has a diameter of 2 mm or more without reaching blood vessels. It is even said that it does not multiply (MA Gimbrone et al., J. Exp. Med. 13
6, 261, 1972). However, once a blood vessel reaches this lesion, the tumor, which has an inexhaustible supply of nutrients and oxygen, not only starts explosive growth, but also causes distant metastasis via the blood vessel. Therefore, it is said that angiogenesis is inseparable from tumor progression and metastasis.

Further, it has been observed that, when a cancer cell is generated, a new blood vessel newly branched from the surrounding blood vessel migrates toward the cancer cell. From this phenomenon, it has been considered that cancer cells may exert some factor that induces the formation and migration of blood vessels, and the existence of the cancer angiogenesis factor (Tumor Angiogenesis Factor) has been proposed (J. Folkman). , Cancer Res. 46, 467, 1986)).

[0006] In addition, as substances that induce neovascularization or promote the growth of vascular endothelial cells, which are the constituent cells of blood vessels, aFGF, bFGF, EGF, PD-ECGF, VPF (also known as vascular endothelial growth factor: VEGF) (Abbreviated), TGF-b, An
Many substances such as giogenin have been reported (R. Bicknell
and AL Harris, Eur. J. Cancer 27, 6, 781, 199
1).

[0007]

DISCLOSURE OF THE INVENTION The present inventors have found a factor that induces vascularization and migration in cancer cells, inhibits its function, and suppresses tumor growth to target conventional tumor itself. Therefore, we conducted a diligent study, considering that a new and effective cancer treatment method different from the above-mentioned cancer treatment method could be provided.

[0008]

[Means for Solving the Problems] The present inventors have found out by what mechanism and what mechanism of the above-mentioned substances that induce neovascularization or promote the growth of vascular endothelial cells, which are the constituent cells of blood vessels. Among the aforementioned factors, the present inventors have found that the vascular permeability factor (VPF) has a signal peptide involved in extracellular secretion, although it is not known whether it is responsible for the action of the aforementioned cancer angiogenic factor (TAF). , And that the expression was observed in various cancer cells, we hypothesized that vascular permeability factor (VPF) might have something to do with tumor angiogenesis. Sex factor
It was found that the action of (VPF) is specifically exerted on vascular endothelial cells, not on tumor cells themselves, and promotes neovascularization in vivo, and suppresses the action of this vascular permeability factor (VPF). It was found that tumor growth can be suppressed by the above, and the present invention was completed.

That is, the present invention has a specific cell growth promoting activity for vascular endothelial cells and a vascular permeability promoting activity, has a molecular weight of about 20,000, and exists as a dimer in vivo, a vascular permeability factor (VPF). And a vascular permeability factor (V
(PF) against a polyclonal antibody against PF).

The tumor suppressor of the present invention has specific cell growth promoting activity and vascular permeability promoting activity on vascular endothelial cells, has a molecular weight of about 20,000, and is a vascular permeability factor existing in vivo as a dimer ( VPF) [also referred to as vascular endothelial growth factor (VEGF)] may be in any form, and most commonly, as claimed in claim 2
The antibody or a part thereof that acts on the vascular permeability factor (VPF) described in the above, but the tumor suppressor of the present invention is not limited thereto, and for example, the vascular permeability factor can be used.
(VPF) inactive vascular permeability factor that inhibits the action, or a part thereof, impairs the function of the receptor of vascular permeability factor (VPF), for example, an antibody against the vascular permeability factor receptor,
Or part of the antibody, and further vascular permeability factor (VPF)
Examples include drugs that suppress the production itself of.

[0011]

According to the present invention, the action of vascular permeability factor (VPF), which is a growth factor for vascular endothelial cells, is suppressed, and migration of tumor blood vessels, which is a pathway for supplying nutrients and oxygen to the tumor, is prevented. , The effect of suppressing tumor growth is shown.

[0012]

【Example】

(a) Creation of VPF-producing cells Encode vascular permeability factor (hereinafter referred to as VPF only)
The cDNA was isolated from human promyelocytic leukemia cells HL-60. The isolated cDNA was incorporated into a plasmid DNA having a marker capable of expressing the human cytomegarovirus promoter and imparting resistance to the antibiotic geneticin (GIBCO, Co.). The plasmid DNA was introduced into human cervical cancer cell HeLa by the calcium phosphate method. After the introduction, the medium containing the antibiotic Geneticin (Dulbecco's Modified Eagle's Med
ium (DMEM) -10% Fetal BovineSerum (FBS) -0.4m
Culture was performed with g / ml Genetic in). When drug-resistant cells formed a colony, a single colony was taken out and cultured independently. From several groups of cells thus taken out, cells were cloned by the limiting dilution method.

VP of each cell line cloned in FIG.
It showed F-producing ability. The productivity of VPF was examined by the following means by the growth promoting activity on human umbilical cord-derived vascular endothelial cells HUVEC detected in the culture supernatant (conditioned medium). That is, 10 ⁴ human umbilical cord-derived vascular endothelial cells HUVEC were spread on a 12-well plate, and assay medium; RPMI1640
45%, DMEM 45%, FBS 10%, bovine insulin1
0mg / l, human transferrin 5mg / l, 2-mercaptoethano
l 0.01 mM, 2-amino-ethanol 0.01 mM, sodiun selen
Preparation medium prepared with each cell line in 1 ml of ite (10 nM)
0.2 ml was added and the cells were cultured at 37 ° C. and 5% CO ₂ for 5 days to compare the number of cells. The conditioned medium was prepared by culturing each cell line to confluence, and adding DMEM, bovineinsulin 10 mg / l, hu
Man transferrin was replaced with a medium of 5 mg / l, and the culture was continued for further 24 hours to obtain a conditioned medium. Among the cloned cells, Clone 5 showed the maximum growth promoting activity. this
HeLa / v5 (deposited at Institute of Microbial Science and Technology: Deposit No. "Micromachine Research Institute No. 13185") was used for the following experiments.

Next, in order to confirm that the obtained biological activity is specific to endothelial cells, HUVEC and HeLa were used as assay cells, and different amounts of HUVEC and HeLa were used in the same assay system as in the experiment of FIG. Conditioned medium was added and the effect was examined. As shown in FIGS. 2 and 3, the conditioned medium showed a growth-promoting effect only on HUVEC in a concentration-dependent manner and had no effect on HeLa. As a control, the original HeLa strain that had not been transformed, and the cell line He that was established by transforming only the VPF cDNA-free plasmid.
When the La / c conditioned medium was examined in the same manner, a remarkable growth-promoting activity on HUVEC was observed only in HeLa / v5, strongly suggesting that this physiological activity is due to VPF.

In order to investigate the effect of VPF in vivo, 10 ⁵
HeLa / c, HeLa / v5, and PBS (Phosphate Buffered Sa
line) was enclosed in a chamber made of a membrane with a pore size of 0.45 micrometer, and embedded subcutaneously in an ICR mouse. One week later, when I opened and examined the part where the chamber was embedded, He
Blood vessels were observed only in the skin in which La / v5 was encapsulated and embedded. From this, it was suggested that VPF not only promotes the proliferation of cultured vascular endothelial cells but also has the activity of inducing blood vessels in vivo.

(B) Preparation of Anti-VPF Polyclonal Antibody The isolated VPF cDNA was expressed in Escherichia coli as a fusion protein with glutathione S-transferase, and the obtained protein was used as an antigen to immunize a rabbit according to a conventional method. IgG was fractionated from the serum with increased antibody titer by DEAE chromatography.

In order to confirm that the obtained antibody reacts with activated VPF, the protein was taken out from the HeLa / v5 conditioned medium by ammonium sulfate salting out, developed by 12% SDS-PAGE, and the gel was applied to a nylon membrane. Western blotting was performed by reacting the antibody obtained by immunizing the protein with the obtained protein, and Western blotting was performed.Specific cross-reactivity was observed at the position corresponding to the molecular weight of VPF, and the prepared antibody recognizes the active VPF molecule. It has been shown.

It was examined whether or not the produced antibody affects VPF physiological activity in vitro. Different amounts of VPF antibody were previously added to 0.08 ml of HeLa / v5 conditioned medium and reacted overnight at 4 ° C. The VPF bioactivity of this solution was examined by the method using vascular endothelial cells HUVEC as described above. As shown in Figure 4, as the amount of antibody added was increased, HUVE
The C growth promoting effect was reduced. Anti-VPF created from this
The antibody was shown to inhibit the action of activated VPF on vascular endothelial cells.

(C) Test Using Living Body From the above-mentioned in vitro test results, VPF has no effect on HeLa, which is a tumor cell, but has growth-promoting activity on HUVEC, which is a vascular endothelial cell. And its activity was shown to be inhibited by anti-VPF polyclonal antibody. Transplantable tumors were prepared in order to investigate the effect of VPF on the solid tumors that actually grow in vivo in response to the observation of these in vitro experimental systems. One was transformed with HeLa / v5, which actively secretes and produces VPF, as a parent strain, and the other was transformed with only a plasmid vector as a control.
HeLa / c was used as a parent strain to create a transplantable solid tumor. Solid tumors were prepared by subcutaneously transplanting 10 ⁷ cells of each cultured cell to BALB / C nude mice, and substituting the established and growing tumors into the nude mice.

In order to examine whether or not cells that highly produce VPF and cells that do not produce VPF affect the growth in vivo, two transplantable tumors prepared by the above method were transplanted to nude mice and their growth was performed. I checked. Tumors cut into 2 mm square were subcutaneously transplanted into 6 nude mice per group, and their growth was recorded over time. As shown in Figure 5 where the average is plotted
It was shown that the VPF producing groups tended to be more active in growth.

Next, whether the promotion of the tumor growth rate, which is considered to be due to the effect of the action of VPF, observed in vivo, is affected by the anti-VPF polyclonal antibody whose VPF activity is suppressed in the in vitro experiment. I checked. HeLa / v5 tumors cut into 2 mm square were transplanted to 2 groups of rabbits in the same manner as in the above experiment, and one of them was treated with anti-VPF polyclonal antibody (IgG).
(0.1 mg / day, sc. 5 consecutive injections × 2), and compared with a normal rabbit as a control, the effect was examined. As shown in FIG. 6 in which the average of 3 animals per group was plotted, it was shown that tumor growth was suppressed in the VPF antibody-administered group as compared to the control group.

Human liver cancer cell line PLC / PRF / 5 which has not been genetically manipulated to investigate whether the effect shown in the artificially produced model experimental system with high expression of VPF is also applicable to normal tumors Similarly, transplanted to nude mice using anti-VPF
The effect of polyclonal antibody was investigated. As shown in Fig. 7, the tumor growth-inhibitory effect of anti-VPF polyclonal antibody was observed even using this system, and in addition to the fact that VPF is widely expressed in various tumors, angiogenesis by the action of VPF is observed. It was suggested that inhibiting the growth of tumors by suppressing the drug has a wide range of application.

[0023]

INDUSTRIAL APPLICABILITY As described above, the present invention provides a novel mechanism for indirectly inhibiting tumor growth by suppressing neovascularization of tumor cells without targeting the tumor cells themselves in treating tumors. Specifically, it was shown that tumor growth can be suppressed by using an anti-VPF polyclonal antibody. It is considered that the following effects can be expected from this.

1. Since the point of action is a blood vessel extending to a tumor and is completely different from a conventional anti-cancer drug that targets tumor cells themselves, it can be effectively used in combination with various drugs, and cancer chemotherapy can be more effectively performed.

2. In order to inhibit the development of blood vessels that extend to tumors, in addition to restricting the supply of nutrients and oxygen and suppressing the growth of tumors, metastasis is required to inhibit the development of blood vessels themselves, which is the main pathway of cancer metastasis. Deter.

3. Since vascular endothelial cells are one of the cells in which proliferation is extremely inactive, this therapy targeting them has extremely slight side effects such as effects on hematopoietic organs, which are often observed with ordinary anticancer agents.

[Brief description of drawings]

FIG. 1 is a diagram showing the VPF producing ability of a cloned cell line.

FIG. 2 is a graph showing the effect of conditioned medium on HUVEC.

FIG. 3 is a diagram showing the effect of a conditioned medium on HeLa.

FIG. 4 is a diagram showing the effect of the prepared antibody on VPF.

[Fig. 5] Fig. 5 is a diagram showing the difference in the growth of VPF-producing cells and cells not producing VPF in vivo.

FIG. 6 is a diagram showing the effect of anti-VPF antibody on the proliferation of HeLa / v5 in vivo.

FIG. 7 is a diagram showing the effect of anti-VPF antibody on the growth of PLC / PRF / 5 in vivo.

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[Procedure amendment]

[Submission date] October 1, 1993

[Procedure Amendment 1]

[Document name to be corrected] Drawing

[Correction target item name] Figure 8

[Correction method] Added

[Correction content]

[Figure 8]

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] Full text

[Correction method] Change

[Correction content]

[Document name] Statement

Title of the invention Tumor suppressor

[Claims]

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a drug which inhibits the function of a vascular permeability factor which induces neovascularization and migration of blood vessels closely related to tumor growth and metastasis. Since it effectively suppresses the activity of tumor cells, it is effective as a tumor suppressor and is used in the pharmaceutical industry and medical field.

[0002]

2. Description of the Related Art Most of the currently widely used tumor treatment methods target tumor cells per se, whether chemotherapy or radiation therapy. Selective attack on tumors by drug administration causes tumor cells to divide much more vigorously than other normal cells,
Much depends on the nature of repeated proliferation. That is, the objective is to kill the target cells by destroying or inhibiting the cell growth mechanism itself.

On the other hand, a method for suppressing the growth of solid tumors is
The idea of so-called military food blame, which cuts off the sources of nutrition and oxygen, has been proposed. In other words, without attacking the tumor cells themselves, they are deficient in nutrients and oxygen, resulting in the inhibition of tumor growth and the therapeutic effects of regression. Specific targets for this approach include blood vessels that reach the tumor.

That is, it is generally said that even if cells are transformed into malignant cells and cancer cells are generated, the growth thereof is very slow in the initial stage, and the tumor that has occurred has a diameter of 2 mm or more without reaching blood vessels. It is even said that it does not multiply (MA Gimbrone et al., J. Exp. Med. 13
6, 261, 1972). However, once blood vessels reach this lesion site, the inexhaustible nutrients and oxygen supplied through the blood vessels not only cause the tumor to explode, but also cause distant metastasis via the blood vessels. It is said that angiogenesis is inseparable from tumor progression and metastasis.

Further, it has been observed that, when a cancer cell is generated, a new blood vessel newly branched from a blood vessel surrounding the cancer cell migrates toward the cancer cell. From this phenomenon, it has become possible that cancer cells may exert some factors that induce blood vessel formation and migration.
The existence of Tumor Angiogenesis Factor has been proposed (J. Folkman, Cancer Res. 46, 467, 198).
6)).

On the other hand, as substances that induce neovascularization or promote the growth of vascular endothelial cells, which are the constituent cells of blood vessels, aFGF, bFGF, EGF, PD-ECGF, VPF (also known as vascular endothelial growth factor: VEGF) (Abbreviated), TGF-b, Angiogen
Many substances such as in have been reported (R. Bicknell and A.
L. Harris, Eur. J. Cancer 27, 6, 781, 1991).

[0007]

DISCLOSURE OF THE INVENTION The present inventors have found a factor that induces vascularization and migration in cancer cells, inhibits its function, and suppresses tumor growth to target conventional tumor itself. Therefore, we conducted a diligent study, considering that a new and effective cancer treatment method different from the above-mentioned cancer treatment method could be provided.

[0008]

Means for Solving the Problems The inventors of the present invention have found out what kind of substances and what kind of mechanism of the above-mentioned substances that induce the formation of blood vessels or promote the growth of vascular endothelial cells which are constituent cells of blood vessels. In, it is not known whether it is responsible for the action of the above-mentioned cancer angiogenic factor (TAF), among which the vascular permeability factor (VPF) has a signal peptide involved in extracellular secretion, and various Focusing on the fact that expression was observed in cancer cells, we hypothesized that vascular permeability factor (VPF) might have something to do with tumor angiogenesis. ) Is specifically exerted on vascular endothelial cells, not on the tumor cells themselves, and promotes the formation of blood vessels in the living body, and by suppressing the action of this vascular permeability factor (VPF), To suppress the proliferation of The inventors have found that they can and have completed the present invention.

That is, the present invention has a specific cell growth promoting activity for vascular endothelial cells and a vascular permeability promoting activity, has a molecular weight of about 20,000, and exists as a dimer in vivo, a vascular permeability factor (VPF). And a vascular permeability factor (V
(PF) against a polyclonal antibody against PF).

The tumor suppressor of the present invention has specific cell growth promoting activity and vascular permeability promoting activity on vascular endothelial cells, has a molecular weight of about 20,000, and is a vascular permeability factor existing in vivo as a dimer ( VPF) [also referred to as vascular endothelial growth factor (VEGF)] may be in any form, and most commonly, as claimed in claim 2
An antibody that acts on vascular permeability factor (VPF) as described in,
Or a part thereof, but the tumor suppressor of the present invention is not limited thereto, for example, an inactive vascular permeability factor that inhibits the action of vascular permeability factor (VPF),
Or a part thereof, which impairs the function of the receptor of vascular permeability factor (VPF), for example, an antibody against the vascular permeability factor receptor, or a part of the antibody, and further vascular permeability factor (VPF).
Examples thereof include drugs that suppress the production of PF) itself.

[0011]

According to the present invention, the action of vascular permeability factor (VPF), which is a growth factor for vascular endothelial cells, is suppressed, and migration of tumor blood vessels, which is a pathway for supplying nutrients and oxygen to the tumor, is prevented. , The effect of suppressing tumor growth is shown.

[0012]

Examples Example 1 (a) Preparation of VPF-producing cells Encode a vascular permeability factor (hereinafter referred to as VPF only)
The cDNA was isolated from human promyelocytic leukemia cells HL-60. The isolated cDNA was incorporated into a plasmid DNA having a marker capable of expressing the human cytomegarovirus promoter and imparting resistance to the antibiotic geneticin (GIBCO, Co.). The plasmid DNA was introduced into human cervical cancer cell HeLa by the calcium phosphate method. After the introduction, the medium containing the antibiotic Geneticin (Dulbecco's Modified Eagle's Med
ium (DMEM) -10% Fetal Bovine Serum (FBS) -0.4 mg /
(ml Genetic in). When drug-resistant cells formed a colony, a single colony was taken out and cultured independently. From several groups of cells thus taken out, cells were cloned by the limiting dilution method.

VP of each cell line cloned in FIG.
It showed F-producing ability. The productivity of VPF was examined by the following means by the growth promoting activity on human umbilical cord-derived vascular endothelial cells HUVEC detected in the culture supernatant (conditioned medium). That is, 10 ⁴ human umbilical cord-derived vascular endothelial cells HUVEC were spread on a 12-well plate, and assay medium; RPMI1640
45%, DMEM 45%, FBS 10%, bovine insulin1
0mg / l, human transferrin 5mg / l, 2-mercaptoethano
l 0.01 mM, 2-amino-ethanol 0.01 mM, sodiun selen
Preparation medium prepared with each cell line in 1 ml of ite (10 nM)
0.2 ml was added and the cells were cultured at 37 ° C. and 5% CO ₂ for 5 days to compare the number of cells. The conditioned medium was prepared by culturing each cell line to confluence, and adding DMEM, bovineinsulin 10 mg / l, hu
Man transferrin was replaced with a medium of 5 mg / l, and the culture was continued for further 24 hours to obtain a conditioned medium. Among the cloned cells, Clone 5 showed the maximum growth promoting activity. this
HeLa / v5 (deposited at Institute of Microbial Science and Technology: Deposit No. "Micromachine Research Institute No. 13185") was used for the following experiments.

Next, in order to confirm that the obtained biological activity is specific to endothelial cells, HUVEC and HeLa were used as assay cells, and different amounts of HUVEC and HeLa were used in the same assay system as in the experiment of FIG. Conditioned medium was added and the effect was examined. As shown in FIGS. 2 and 3, the conditioned medium showed a concentration-dependent growth promoting effect only on HUVEC and no effect on HeLa. As a control, the original HeLa strain that had not been transformed, and the cell line He that was established by transforming only the VPF cDNA-free plasmid.
When the La / c conditioned medium was examined in the same manner, a remarkable growth-promoting activity on HUVEC was observed only in HeLa / v5, strongly suggesting that this physiological activity is due to VPF.

In order to investigate the effect of VPF in vivo, 10 ⁵
HeLa / c, HeLa / v5, and PBS (Phosphate Buffered Sa
line) was enclosed in a chamber made of a membrane with a pore size of 0.45 micrometer and embedded subcutaneously in an ICR mouse. One week later, when I opened the chamber and examined it,
Blood vessels were observed only in the skin in which HeLa / v5 was encapsulated and embedded. From this, it was suggested that VPF not only promotes the proliferation of cultured vascular endothelial cells but also has the activity of inducing blood vessels in vivo.

(B) Preparation of Anti-VPF Polyclonal Antibody The isolated VPF cDNA was expressed in Escherichia coli as a fusion protein with glutathione S-transferase, and the obtained protein was used as an antigen to immunize a rabbit according to a conventional method. IgG was fractionated from the serum with increased antibody titer by DEAE chromatography.

In order to confirm that the obtained antibody reacts with activated VPF, the protein was taken out from the HeLa / v5 conditioned medium by ammonium sulfate salting out, developed by 12% SDS-PAGE, and the gel was applied to a nylon membrane. Western blotting was performed by reacting the antibody obtained by immunizing the protein with the obtained protein, and Western blotting was performed.Specific cross-reactivity was observed at the position corresponding to the molecular weight of VPF, and the prepared antibody recognizes the active VPF molecule. It has been shown.

In order to examine the influence of the prepared antibody on the physiological activity of VPF in vitro, different amounts of VPF antibody were previously added to 0.08 ml of HeLa / v5 conditioned medium and reacted at 4 ° C. overnight. The VPF bioactivity of this solution was examined by the method using vascular endothelial cells HUVEC as described above. As shown in Fig. 4, as the amount of antibody added was increased, the HUVEC of the conditioned medium was increased.
The growth promoting effect was reduced. Anti-VPF created from this
The antibody was shown to inhibit the action of activated VPF on vascular endothelial cells.

(C) Test Using Living Body From the above-mentioned in vitro test results, VPF has no effect on HeLa, which is a tumor cell, but has growth-promoting activity on HUVEC, which is a vascular endothelial cell. And its activity was shown to be inhibited by anti-VPF polyclonal antibody. In response to the observation of these in vitro experimental systems, transplantable tumors were created in order to investigate what effect VPF exerts on solid tumors that actually grow in vivo. One is VP
HeLa / v5, which secretes and produces F actively, was used as a parent strain, and the other was HeLa / c transformed with only a plasmid vector as a control, which was used as a parent strain, to prepare transplantable solid tumors. Solid tumors were prepared by subcutaneously transplanting 10 ⁷ cells of each cultured cell to BALB / C nude mice, and substituting the established and growing tumors into the nude mice.

In order to examine whether or not cells that highly produce VPF and cells that do not produce VPF affect the growth in vivo, two transplantable tumors prepared by the above method were transplanted to nude mice and their growth was performed. I checked. Tumors cut into 2 mm square were subcutaneously transplanted into 6 nude mice per group, and their growth was recorded over time. As shown in Figure 5 where the average is plotted
It was shown that the VPF producing groups tended to be more active in growth.

Next, the effect of the anti-VPF polyclonal antibody with suppressed VPF activity was examined in an in vitro experiment on the promotion of tumor growth rate, which is considered to be due to the effect of VPF action, which was observed in vivo. As in the above experiment, 2 mm square HeLa / v5 tumors were transplanted into two groups of rabbits, one of which was anti-VPF.
Polyclonal antibody (IgG) was administered (0.1 mg / day sc.
The effect was investigated by comparing with a normal rabbit as a control. FIG. 6 in which the average of 3 animals per group is plotted.
As shown in, the tumor growth was shown to be suppressed in the group to which the VPF antibody was administered, as compared to the control group.

Human liver cancer cell line PLC / PRF / 5 which has not been genetically manipulated to investigate whether the effect shown in the artificially produced model experimental system with high expression of VPF is also applicable to normal tumors Similarly, transplanted to nude mice using anti-VPF
The effect of polyclonal antibody was investigated. As shown in Fig. 7, the tumor growth-inhibitory effect of anti-VPF polyclonal antibody was observed even using this system, and in addition to the fact that VPF is widely expressed in various tumors, angiogenesis by the action of VPF is observed. It was suggested that inhibiting the growth of tumors by suppressing the drug has a wide range of application.

Example 2 (a) Preparation of anti-VPF monoclonal antibody The isolated VPF cDNA was expressed in Escherichia coli as a fusion protein with glutathione S transferase, and the obtained protein was used as an antigen according to a conventional method to prepare a mouse (BALB / C) was immunized.
Spleen cells were taken out from the mouse with increased antibody titer and subjected to cell fusion with mouse myeloma cells (SP2) by the polyethylene glycol method (monoclonal antibody, Tatsuo Iwasaki et al., Kodansha Scientific).

On the other hand, the isolated VPF cDNA was isolated from yeast Sacchar.
It was linked downstream of the Gal1 transcription promoter from omyces cerevisiae. This DNA fragment was ligated to circular DNA containing a replication origin sequence derived from the yeast 2μ plasmid, and the yeast Saccharomyc
es cerevisiae to the lithium acetate method (Lundblad, V. Current
protocols: 13.7.1.-13.7.2., 1989).

The yeast into which the VPF expression plasmid was introduced was cultured under the following conditions. Yeast Nitrogen Bas
e (YNB))-ammonium sulfate 2% -casamino acid 0.5% -histidine 8
Raffinose 2 was used in pre-culture with 0 mg / l as the basic medium.
% Was used. The yeast was cultivated with shaking in 10 ml of the above preculture medium at 30 ° C. for 48 hours. Next, 6 ml of the culture
Was inoculated into 600 ml of the same medium and shake-cultured at 30 ° C. for 48 hours. Yeast cells were recovered from the culture solution by centrifugation at room temperature, and the recovered cells were re-inoculated into 12 l of a medium containing YNB-ammonium sulfate 2% -casamino acid 0.5% -histidine 80 mg / l-galactose 5%. This medium contains galactose and induces the expression of the human vascular permeability factor gene introduced downstream of the GAL1 transcription promoter. The 12 liter medium was divided into four 3 liter Erlenmeyer flasks and agitated at 30 ° C.

From the supernatant of the medium in which yeast was cultured for 4 days, VPF
Was purified by the following method. 12 liters of cultivated culture solution
Yeast was removed from the sample and the sample was finally concentrated from 12 liters to 50 ml using an ultrafiltration membrane having a molecular weight cut off of 10,000. The concentrated sample was dialyzed against 10 mM acetate buffer (pH 5.2) -0.1 M sodium chloride. The dialyzed sample was subjected to cation exchange column chromatography to obtain a sodium chloride concentration of 0.
The sample was fractionated with a linear gradient of 1M-2.1M. Ammonium sulfate was added to the fraction containing VPF and ammonium sulfate precipitation was performed under saturated conditions to collect the sample. The recovered sample was subjected to gel filtration column chromatography to purify VPF. In this way, it was possible to obtain a bioactive VPF (that is, to retain the correct higher-order structure).

The obtained VPF was adsorbed on a 96-well immunoassay plate, and 2% bovine serumalubumin (BSA) -ph was used.
It was coated with osphate buffered saline (PBS) and used as a plate for screening a monoclonal antibody. The culture supernatant of the fused cells is placed in each well of the plate, left at room temperature for 2 hours, and then the well is washed 4 times with PBS. An anti-mouse immunoglobulin antibody labeled with peroxidase is added thereto, and the mixture is allowed to stand at room temperature for 1 hour, and then the hole is washed 8 times with PBS. After that, an enzyme substrate was added to each well to cause reaction, and the color development was measured to detect the anti-VPF antibody. In this way, a fused cell line (hybridoma) producing the anti-VPF antibody was cloned.

The cloned anti-VPF monoclonal antibody-producing cell line was transplanted into the abdominal cavity of nude mice and proliferated,
Anti-VPF monoclonal antibody was purified from the accumulated ascites by affinity column chromatography. The purity of the purified antibody was assayed by SDS polyacrylamide gel electrophoresis and then dialyzed against PBS.

(B) Test of VPF Physiological Activity Inhibitory Effect of Monoclonal Antibody It was tested whether or not the prepared monoclonal antibody exhibits the VPF physiological activity inhibitory effect as observed with the above-mentioned polyclonal antibody. Different amounts of anti-VPF monoclonal antibody were added to 0.4 ml of HeLa / v5 conditioned medium, and the mixture was allowed to stand at room temperature for 30 minutes. The VPF physiological activity of this solution was examined by the method using the aforementioned vascular endothelial cell HUVEC. That is, human umbilical cord-derived vascular endothelial cells HU
Spread 104 VECs, assay medium; RPMI1640 45%, DMEM
45%, FBS 10%, bovine insulin10 mg / l, human trans
ferrin 5 mg / l, 2-mercaptoethanol 0.01 mM, 2-aminoe
The mixture of each monoclonal antibody-HeLa / v5 preparation medium was mixed so as to be thanol 0.01 mM, sodiun selenite 10 nM, 1 ml, and cultured at 37 ° C. 5% CO ₂ for 5 days to compare the cell number. .

In 0.4 ml of the HeLa / v5 conditioned medium used, about 1.
Contains 5 ng VPF. The number of VPF molecules in the assay medium was calculated based on this value, and the antibody molecules added to VPF were set to 0, 1, 6, 36, 216 times (the number of molecules), and no antibody was detected. 10 cells without addition
The number of cells was set to 0, and the number of cells at the above-mentioned amount of antibody added was shown in FIG. In the figure, MV201 and MV302 are anti-VPF monoclonal antibodies, and Anti-alphapheto is an anti-alphafetoprotein monoclonal antibody.

As shown in FIG. 8, among the tested monoclonal antibodies, MV201 was observed to have an effect of suppressing the proliferation of HUVEC activated by VPF depending on the amount added. No effect was observed with MV302. A monoclonal antibody against alpha-fetoprotein, which is unrelated to the VPF protein, was also used as a control, and this antibody also produced HUVE activated by VPF as observed in MV201.
No effect of suppressing the growth of C was observed.

[0032]

INDUSTRIAL APPLICABILITY As described above, the present invention provides a novel mechanism for indirectly inhibiting tumor growth by suppressing neovascularization of tumor cells without targeting the tumor cells themselves in treating tumors. Specifically, it is shown that tumor growth can be suppressed by using an anti-VPF antibody that specifically inhibits the physiological activity of VPF. It is considered that the following effects can be expected from this.

1. Since the point of action is a blood vessel extending to a tumor and is completely different from a conventional anti-cancer drug that targets tumor cells themselves, it can be effectively used in combination with various drugs, and cancer chemotherapy can be more effectively performed.

2. In order to inhibit the development of blood vessels that extend to tumors, in addition to restricting the supply of nutrients and oxygen and suppressing the growth of tumors, metastasis is required to inhibit the development of blood vessels themselves, which is the main pathway of cancer metastasis. Deter.

3. Since vascular endothelial cells are one of the cells in which proliferation is extremely inactive, this therapy targeting them has extremely slight side effects such as effects on hematopoietic organs, which are often observed with ordinary anticancer agents.

[Brief description of drawings]

FIG. 1 is a diagram showing the VPF producing ability of a cloned cell line.

FIG. 2 is a graph showing the effect of conditioned medium on HUVEC.

FIG. 3 is a diagram showing the effect of a conditioned medium on HeLa.

[Fig. 4] Fig. 4 is a diagram showing the effect of the prepared polyclonal antibody on VPF.

[Fig. 5] Fig. 5 is a diagram showing the difference in the growth of VPF-producing cells and cells not producing VPF in vivo.

FIG. 6: HeLa / v5 in vivo with anti-VPF polyclonal antibody
It is the figure which investigated the influence which it has on the proliferation of.

FIG. 7: PLC / PRF in vivo with anti-VPF polyclonal antibody
It is the figure which investigated the influence which it has on the proliferation of / 5.

FIG. 8 is a graph showing the effect of addition of various monoclonal antibodies on the growth of VPF-activated HUVEC.

Claims (2)

[Claims] 1. A vascular endothelial cell having a specific cell growth promoting activity and vascular permeability promoting activity, and a molecular weight of about 20,000.
So, the vascular permeability factor (V
A tumor suppressor characterized by comprising a function inhibitor of PF). 2. It has a specific cell growth promoting activity for vascular endothelial cells and a vascular permeability promoting activity, and has a molecular weight of about 20,000.
So, the vascular permeability factor (V
Polyclonal antibody against PF).