JPH09316000A - Vaccine for suppressing arterialization - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject vaccine containing a specific cell growth factor (segment), excellent in suppressing action on arterialization which participates in proliferation, infiltration and transfer of a solid cancer and useful for treatment of various kinds of diseases including cancer and suppression of onset thereof. SOLUTION: This vaccine contains a cell growth factor [e.g. an acidic fibroblast growth factor, a basic fibroblast growth factor, epidermal growth factor or a platelet-derived endothelial cell growth factor, especially a vascular endothelial cell growth factor (VEGF)] or its fragment [a peptide corresponding to epitope of VEGF (e.g. a peptide having an amino acid sequence of TryProAspGluIleGluTryIlePheLye)].

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a vaccine for suppressing angiogenesis, and more particularly to a cell growth factor having a specific cell growth promoting activity on vascular endothelial cells, particularly a vascular endothelial cell growth factor. It is related to immunotherapeutic agents that use as an antigen component, and treats and develops (including recurrence) various diseases caused by angiogenesis such as solid cancer
It is used for the purpose of deterrence.

[0002]

2. Description of the Related Art It is known that higher animals have the ability to produce substances called antibodies when substances called antigens enter the living body. The purpose of this is to protect the living body from substances or organisms, that is, antigens, which may harm themselves. This is to protect the living body by specifically recognizing the invading antigen and decomposing, neutralizing or inactivating the antigen.

Due to the remarkable progress of immunology in recent years, the so-called immune response, that is, how skillfully specific antibodies are generated against all kinds of invading antigens and various immunocompetent cells are mobilized to achieve their purpose, Mechanisms are being clarified one after another, and the sophistication and complexity of the mechanism is remarkable. These are described in detail in, for example, “Immunology” (edited by Jiro Koyama, Toshiaki Osawa, published by Nankodo).

One of the most astonishing points of the immune response mechanism is the property of memorizing an invading antigen once and inducing an immune response extremely rapidly and effectively when the same antigen is invaded again. Utilizing this property, the technology that has already contributed to human welfare is called vaccine or vaccination. Traditionally, it was empirically recognized that humans are not twice susceptible to the plague once infected, but Jenner's smallpox, which used this for medical practice, is a very famous event.

Since then, vaccination and vaccination have seen various applications, and they are still widely used as an extremely effective means in preventive medicine. This technique effectively inactivates the immune response memorized in the body at the time of real infection by inoculating a human in advance with a part of the inactivated infectious organism or a constituent substance of the infectious organism to establish a pseudo-infection. It is to develop resistance to drawer infections.

As described above, the vaccine technology has been developed as an extremely effective method in the field of infectious diseases.
Furthermore, as a result of recent research, it has been considered that the immune response mechanism may play an important role in the development of various neoplastic diseases occurring in the body, that is, cancer. That is, a certain type of immunocompetent cell has a function of constantly monitoring all cells and objects in the living body and excluding cells that are determined to be non-self, and the generation of transformed cells, that is, tumor cells is also monitored. It is constantly checked by the mechanism, and there is an idea that many of the tumor cells that have arisen may have been eliminated before becoming a malignant cancer.
It has become clear that this mechanism is mainly constituted by a mechanism called cell-mediated immunity, which is controlled by killer T cells that damage cancer cells, mononuclear phagocyte macrophages, neutrophils, NK cells and the like.

[0007] From these findings, the idea of cancer immunotherapy, in which cancer immunity is positively applied to therapy, has been proposed one step further. Its contents include active immunotherapy, which is basically common to the above-mentioned vaccines against infectious diseases, that is, inoculation of inactivated cancer cells and its components to elicit an anticancer effect, and immunity. Activation therapy, that is, non-specific activation of the immune response mechanism itself to enhance the anti-cancer immune effect can be mentioned.

Cancer immunotherapy applying the cancer antigen and the immune mechanism as described above has been studied as a new attempt for cancer treatment, but in many cases, the cancer antigen is unique to each cancer cell. It is not necessarily expressed in all cancer systems, in addition, it does not have a strong effect such as an immune response to infectious diseases, and the cancer cells themselves have a mechanism that escapes the establishment of cancer immunity. However, the usefulness as a general therapy cannot be concluded without waiting for future progress.

Most of the methods currently widely used for treating tumors are those targeting the tumor cells themselves, whether they are chemotherapy, radiation therapy or the above-mentioned cancer immunotherapy. Selective attack on tumors by drug administration is largely due to the fact that tumor cells divide and proliferate much more actively than other normal cells. That is, the objective is to kill the target cells by destroying or inhibiting the cell growth mechanism itself.

On the other hand, in a method for suppressing the growth of solid tumors,
Ideas of so-called blaming have been proposed to cut off their nutrient and oxygen sources. In other words, it does not attack the tumor cells themselves, but becomes deficient in nutrients and oxygen, resulting in the therapeutic effects of tumor growth inhibition and regression. A specific target for this approach is the blood vessels that reach the tumor.

It is generally said that, even if cells become malignant and cancer cells are generated, their growth is very slow in the initial stage. It has been reported that the tumors that have developed do not even grow to diameters greater than 2 mm without reaching the blood vessels (MA Gimbrone et al., J. Exp. M.
ed. 136, 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. This is why angiogenesis is said to be inseparable from tumor progression and metastasis. It has been observed that when a cancer cell occurs, a new blood vessel newly branched from the surrounding blood vessel migrates toward the cancer cell, and from this phenomenon, the cancer cell causes some factor that induces new blood vessel formation and migration. It has been thought that the cancer angiogenic factor (Tumor Angiogenesis Factor: TAF) is present (J. Folkman, Cancer Research 46,
467, 1986).

On the other hand, acidic fibroblast growth factor (acidic fibroblas growth factor) is used as a substance that induces neovascularization or promotes the growth of vascular endothelial cells which are constituent cells of blood vessels.
t growth factor (aFGF), basic fibroblast growth factor (ba
sic fibroblast growth factor (bFGF), epidermal growth factor (
Epidermal growth factor (EGF), platelet-driven endothelial cell growth fac
tor: PD-ECGF), vascular endothelial growth factor / vascular permeability factor
(vascular endothelial cell growth factor / vascular
permeability: VEGF / VPF), placenta-derived growth factor (Plaseuta
Although many substances such as growth factor (PIGF) have been reported,
(reviwed R. Bicknell and AL Harris, Eur. J. Cance
r 27, 6, 781, 1991), and it is not known in what mechanism these substances play the role of the above-mentioned TAF. The present inventors noted that VEGF / VPF has a signal peptide involved in extracellular secretion among these factors, and that expression was found in almost all cancer cells tested, and VEGF / VPF showed tumor angiogenesis. I made a hypothesis that there might be something to do with. As a result, it was found that the action of VEGF / VPF is exerted specifically on vascular endothelial cells, not on the tumor cells themselves, and promotes vascularization in vivo. Furthermore, it was found that tumor growth in vivo can be suppressed by suppressing this VEGF / VPF action with an anti-VEGF / VPF polyclonal antibody (S. Kondo et al., Biochemical and Biophy.
sical Research Communications 194, 1234, 1993).
In addition, Kim et al. In the US showed that anti-VEGF / VPF neutralizing monoclonal antibody could suppress tumor growth in vivo (KJ Kim et al., Na.
ture362, 841, 1993). These results show that tumor growth can be suppressed by administering in vivo an antibody that is a so-called humoral immune component that specifically binds to VEGF / VPF and interferes with its action.

Therefore, the present inventors, if antitumor properties can be obtained by administering the anti-VEGF / VPF antibody obtained by immunizing a rabbit or mouse to another individual, the individual who develops the tumor itself By stimulating the immune system of VEGF / VPF as an antigen, an anti-VEGF / VPF antibody derived from the individual can be generated, and as a result, antitumor properties can be imparted to the individual, which in turn suppresses angiogenesis to the individual. I thought that it could be given, and conducted research.

[0014]

That is, the present inventors
In order to broadly apply the immune response mechanism provided in the living body to the treatment of cancer, we will focus on the neovascularization mechanism that is deeply involved in the growth, invasion and metastasis of solid tumors and conduct research on a vaccine that can suppress angiogenesis. The present invention has been completed which can be applied to the treatment of various diseases including cancer and the suppression of onset.

[0015]

The present invention relates to an angiogenesis suppressing vaccine containing a cell growth factor or a fragment thereof having a specific cell growth promoting activity on vascular endothelial cells. Examples of the above cell growth factor include factors called vascular endothelial cell growth factor or vascular permeability factor. In the following, the vascular permeability factor (Vascular Permeab
ility factor) is VPF and vascular endothelial growth factor (Vasc
ular Endothelial Growth Factor) is abbreviated as VGEF.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION The angiogenesis-suppressing vaccine of the present invention can impart an angiogenesis-suppressing activity to an individual, and thus can treat and prevent diseases associated with angiogenesis, particularly in solid tumors. It is possible to impart an onset suppressing effect and an antitumor effect to an individual. Examples of the cell growth factor having a specific cell growth promoting activity for vascular endothelial cells in the present invention include, but are not limited to, the following ones described above.

That is, acidic fibroblast growth factor (acidic f
ibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), platelet-driven endothelial cell gr
owth factor: PD-ECGF), vascular endothelial cell growth factor / v
ascular permeability: VEGF / VPF), placenta-derived growth factor (P
laseuta growth factor (PIGF) etc.

The fragments thereof are those derived from the polypeptide of each factor, or synthetic peptides containing a part of the amino acid sequence of the factor prepared by chemical synthesis, and immunizing against the factor. The thing which can make an antibody is mentioned. Incidentally, a part of the peptide corresponding to the epitope of VEGF which is one of the cell growth factors having a specific cell growth promoting activity on vascular endothelial cells (for example, a peptide having the amino acid sequence of TryProAspGluIleGluTryIlePheLye) has already been prepared by the present inventors. It was found and is described in Japanese Patent Application No. 6-125569.

Of these, VEGF / VPF is a factor that plays a major role in tumor angiogenesis, and has been considered to be secreted by many tumor cells. A vaccine using / VPF as an antigen is an immunotherapeutic agent that can be expected to suppress the growth and metastasis of solid cancer in general. Further, the advantages of vaccination with VEGF / VPF as an antigen are as follows.

First of all, as mentioned above, it is considered that angiogenesis is essential for the development of tumors. Without angiogenesis, tumor growth is extremely slow, and the growth size is limited to the upper limit. There is a point. That is, tumor angiogenesis, which is an essential item for the development of solid tumors, can be delayed without completely stopping it by immunizing the individual with VEGF / VPF beforehand, and the resulting tumor cells do not die. In any case, it takes a huge amount of time to be recognized as the onset of cancer as a disease. For example, it takes several to several decades for a certain cell in the body to undergo transformation, continue to divide while mutating, and eventually progress to the disease of cancer accompanied by neovascularization. However, if this period can be extended several times, it will take a considerable period of time for human life from the occurrence of cell mutation to the establishment of cancer as a disease. Considering that it is concentrated in the middle-aged and elderly, the meaning of this extension of the period is extremely significant, and according to the present invention, it means that it does not require a perfect action to be effective against cancer as a disease. It is more tolerant than therapy.

Second, in addition to the above-described onset-suppressing effect, a cancer regression effect can be expected. Most of the effects obtained by experimentally administering anti-VEGF / VPF antibody to tumor-bearing animals are considered to be due to neutralization of VEGF / VPF activity by antibody administration, and such effects Depends on the abundance of the administered antibody in vivo, and in many cases, the in vivo half-life of proteinaceous macromolecules is short,
It is considered to be transient. Therefore, treatment by antibody administration is often repeated.

On the other hand, in the case of the present invention, since the antibody is theoretically supplied in vivo, its effect is considered to be persistent, and it is expected that the VEGF / VPF neutralizing effect is continuously exerted. It is possible. Furthermore, according to the present invention, it is expected that the vaccinated individual will be fully equipped with a series of immune responses to antigenic stimuli, and the antibody-induced VE
It is expected that an immune response including not only the neutralizing effect of GF / VPF activity but also the cellular immune mechanism to the complement activation mechanism will be activated to antigen presenting cells. As a cell that presents VEGF / VPF antigen in a situation where a tumor grows in vivo,
From the results of various immunohistochemical stainings, in addition to tumor cells that produce VEGF / VPF itself, endothelial cells of tumor blood vessels in which VEGF / VPF is bound to a cell surface receptor are known. That is, tumor cells themselves, or tumor vascular cells that supply oxygen and nutrients to the tumor can be considered as targets for tumor immunity. As a result of these, it is expected that, in addition to the effect of suppressing the neovascularization of tumor blood vessels, a tumor necrosis effect that directly targets the already formed tumor itself or the tumor blood vessels can be obtained.

The vaccine of the present invention can be applied to the treatment and prevention of diseases associated with angiogenesis, including the above-mentioned solid tumors. For example, angiogenesis of atherosclerosis can be suppressed and treated by this vaccine. In addition, when administered to a person with hyperlipidemia, the development of atherosclerotic disease can be suppressed, and the onset of myocardial infarction or cerebral infarction can be prevented. Furthermore, rheumatoid arthritis is a disease that develops due to the formation of blood vessels in the joints, and this symptom is exacerbated by the progress of this angiogenesis, but it is necessary to administer this vaccine to patients with rheumatoid arthritis. Due to
It is expected that the exacerbation of symptoms can be suppressed, and that the cause of the development of rheumatoid arthritis is angiogenesis, which is expected to lead to treatment. In addition, it has already been reported that VPF is involved in diabetic retinopathy, and administration of this vaccine to diabetic patients is expected to suppress the development of diabetic retinopathy and nephropathy. It is possible.

Other diseases associated with angiogenesis include central retinal vein occlusion, posterior lens fibroplasia, glaucoma, senile discoid macular degeneration, eye tumor, trachoma, retinopathy of prematurity, and blood vessels associated with corneal transplantation. Examples include neoplasia, psoriasis, pyogenic granulomas, hemangiomas, hypertrophic scars, granulation and edematous sclerosis, and the vaccines of the present invention can be expected to treat and prevent these diseases. Further, since VEGF has a VPF activity, that is, a substance permeation promoting activity, the vaccine of the present invention can be expected to suppress the accumulation of ascites and pleural effusion.

[0025]

[Examples] Examples of human VEGF / VPF121 expressed in insect cells using a baculovirus vector as an antigen and Freund's complete adjuvant as an adjuvant will be described below. It is not limited to the case of using such a thing. For example, as VEGF / VPF antigen VEGF / VPF derived from animal species other than human, VEGF / VPF having a length other than 121 amino acid residues even if derived from human, various VEGF / prepared by a method other than the above method. VPF and VE created by chemical synthesis
Included are synthetic peptides containing a portion of the GF / VPF amino acid sequence that are capable of producing immune antibodies. Human VEGF / VPF12
1 is a polypeptide having the amino acid sequence of SEQ ID NO: 1.

As an adjuvant, a B. pertussis vaccine,
Examples include streptococcal preparations, endotoxin lipopolysaccharide, BCG, aluminum hydroxide and the like. In addition, VEGF / VPF denatured with heat or acid to enhance antigenicity in vivo,
Alternatively, a product in which VEGF / VPF is bound to another protein or a high molecular substance, and a chimeric protein fused with another protein are also used. Further, the vaccination method is not limited to this example. In order to enhance the immune response, for example, granulocyte-macrophage colony stimulating factor (granulocyte-macr
ophage colony-stimulate factor: GM-CSF) or interleukin-2 (interleukin-2), or by administering various cytokines, or by culturing the antigen-presenting cells of the recipient and incorporating the antigen. A method of administering to an administrator and the like can also be used.

Example 1 Cancer Growth Inhibition Experiment on Vaccinated Mice To see the effect of the vaccine of the present invention on tumor growth inhibition,
Mice (C57BL / 6) were vaccinated, and after confirming an increase in anti-VEGF / VPF antibody titer, mouse Lewis lung cancer (LLC) was transplanted, and LLC growth was compared between the vaccinated group and the non-vaccinated group.

Vaccination: Five 8-week-old male mice (C57BL / 6) were initially treated with 0.0
2 mg / mouse of VEGF / VPF was added to 0.1 ml of Freund's complete adjuvant (DIFCO
2) (2 weeks (1 week))
And the third time (2 weeks), 0.02 mg / mouse VEGF / VPF was added to 0.1 ml of Freund's incomplete adjuvant [Freund's
Incomplete adjuvant (manufactured by DIFCO)] was intraperitoneally administered in a total volume of 0.2 ml (vaccine vaccination group). As a control, Freund's incomplete adjuvant 0.1 ml was intraperitoneally administered with water in a total volume of 0.2 ml for the first time (week 0), and Freund's incomplete adjuvant was used with water for the second time (1 week) and the third time (2 weeks). A group (adjuvant control group) administered intraperitoneally in a total volume of 0.2 ml, and only 0.2 ml of phosphate buffered saline (PBS) for the first time (week 0),
A group (buffer control group) that was intraperitoneally administered at the second time (1 week) and the third time (2 weeks) was prepared.

Transition of antibody titer Anti-VEGF / VPF in uncollected blood by vaccination
Partial blood sampling was performed at 0, 1, 2, 3, 5, 7, 9 weeks from each individual in each group for the purpose of investigating whether or not the antibody titer is increasing (antibody positive: namely, seroconversion by vaccine). The anti-VEGF / VPF antibody titer in blood was examined. That is, the collected uncollected blood was immediately centrifuged to obtain plasma, and the plasma was diluted with 0.1% BS.
Dilute 1000 times with A-PBS (0.1% bovine serum albumin-containing phosphate buffered saline), and dispense 0.1 ml each into a resin immunoassay plate with VEGF / VPF protein immobilized.
An anti-VEGF / VPF antibody was specifically bound to the immobilized VEGF / VPF protein. The amount of bound anti-VEGF / VPF antibody was detected using an enzyme-labeled anti-mouse IgG antibody (by a so-called widely used enzyme immunoassay method). The transition of the average value of blood anti-VEGF / VPF antibody titer of each mouse examined by such a method is shown in FIG. That is, Fig. 1 plots the transition of the average value of anti-VEGF / VPF antibody titers in mouse blood in vaccinated mice for each of the vaccination group (●), the adjuvant control group (○), and the buffer control group (△). FIG. From this figure, a marked increase in antibody titer was observed in the vaccinated group starting at week 7, while no change was observed in the buffer control group over the entire period. In the adjuvant control group, a gradual increase in antibody titer was observed at 7th and 9th weeks, which was considered to be due to the nonspecific activation of immune reaction which is a property of Freund's adjuvant.

Solid tumor transplantation and follow-up of proliferation Since the antibody titer in the peripheral blood was considered to have reached the peak at the 7th week, the mouse Lewis lung cancer (LLC) solid tumor was transplanted at the 9th week, and the subsequent cancer was performed. Growth was followed. LLC solid tumors that had been sufficiently grown in another mouse were cut into 3 × 3 × 3 mm size, and subcutaneously subcutaneously injected into mice of 1 group of 5 vaccination groups, adjuvant control group and buffer control group. Transplanted.

The growth of cancer was measured on days 6, 9 and 13 after transplantation, and was calculated by the calculation method of tumor volume = minor axis × minor axis × major axis / 2. The transition of the average value of cancer growth in each group is shown in FIG. That is, FIG. 2 plots the transition of the average value of cancer growth in the cancer transplantation experiment to the vaccinated mouse for each of the vaccination group (●), the adjuvant control group (◯), and the buffer control group (Δ). It is a graph. From this figure, remarkable suppression of growth was observed in the vaccinated group as compared with other groups.

Statistical Analysis Whether or not the observed growth inhibition in the vaccinated group was statistically significant was tested by Student's t-test. As a result 6,
The results showed that the growth of the vaccinated group was significantly suppressed in both the buffer control group and the adjuvant control group at any of the 9th and 13th days (p <0.05: significant). In the comparison between the buffer control group and the adjuvant control group, the adjuvant control group appeared to grow slightly slower, but no statistically significant difference was found. From the above-mentioned research results, it became clear that the present invention, that is, the cancer vaccine containing VEGF / VPF as a main component, shows its effectiveness in a model system for solid tumor transplantation in mice.

Example 2 Cancer Metastasis Inhibition Experiment in Vaccinated Mice In order to see the effect of the vaccine of the present invention on tumor metastasis inhibition,
Mice (C57BL / 6) were vaccinated, and after confirming an increase in anti-VEGF / VPF antibody titers, mouse solid tumors (B16F1) were vaccinated from the tail vein, and formed in the lung between the vaccinated and non-vaccinated groups. The number of metastatic foci was compared.

Vaccination: Ten 8-week-old male mice (C57BL / 6) were vaccinated in the same manner as in Example 1, and as controls, an adjuvant control group and a buffer control group were also prepared.

Transition of antibody titer In order to confirm whether the transition of antibody titer of the vaccinated mouse is the same as in Example 1, 5 weeks (at the time when a marked increase in antibody titer was observed) and 9 weeks (antibody titer). (At the time when the decrease was observed), partial blood collection was performed, and the anti-VEGF / VPF antibody titer in blood was examined by the method described in Example 1. Anti-VEGF / V in blood of each mouse
The transition of average PF antibody titer is shown in FIG. That is, FIG.
Is anti-VEGF / V in mouse blood in vaccinated mice
It is the graph which plotted the transition of the average value of a PF antibody titer with respect to each of a vaccination group (■), an adjuvant control group (●), and a buffer control group (Δ). From this figure, it was confirmed that the antibody titer of the vaccinated group markedly increased at the 5th week and decreased at the 9th week, and the transition of the antibody titer of the vaccinated mouse was similar to that of Example 1. .

Inoculation of solid cancer cells and number of metastatic foci At 9 weeks, 5 B16F1 cells cultured with DMEM 10% FBS were cultured.
X 10 ⁵ cells / ml, add 0.2 ml each (1 x 10
⁽⁵ / mouse), 1 group of 10 vaccinated groups, an adjuvant control group, and a buffer control group were intravascularly administered through the tail vein, and the number of colonies colonized in the lungs was measured at 11th week.
The average number of engrafted colonies in each group is shown in FIG. That is, this figure is a graph in which the average value of the number of cancer colonization colonies in the cancer metastasis experiment to the vaccinated mouse was plotted for each of the vaccination group, the adjuvant control group, and the buffer control group. From this figure, a remarkable metastasis-suppressing effect was observed in the vaccinated group as compared to the other groups.

Statistical Analysis Student's T was used to determine whether the observed cancer metastasis-suppressing effect in the vaccinated group was statistically significant.
-Verified in the test. As a result, it was found that the vaccination group had significantly suppressed metastasis in both the adjuvant control group and the buffer control group (adjuvant control group p <0.05; significant, buffer control group p <0.001; significant). . In the comparison between the adjuvant control group and the buffer control group, the adjuvant control group seemed to have slightly less metastatic foci, but no statistically significant difference was found.

Example 3 Tumor Angiogenesis Induction Inhibition Experiment on Vaccinated Mice In order to see the suppressive effect of this vaccine on tumor angiogenesis, mice (C57BL / 6) were vaccinated and mouse Lewis lung cancer ( An LLC-enclosed chamber was inserted subcutaneously in the back of the mouse (subcutaneous dorsal method), and angiogenesis induced subcutaneously in contact with the chamber was observed.

Vaccination: 8-week-old male mice (C57BL / 6) were vaccinated in the same manner as in Example 1, and as controls, an adjuvant control group and a buffer control group were also prepared.

Preparation of Chamber A 0.45 μm filter (# HAWPO400 Millipore) was attached to both sides of Millipore (# PR0001401 Millipore) with an adhesive (# XX7000000 Millipore) and air-dried. When the chamber implantation, the LLC cells that had been cultured using DMEM 10% FBS was prepared in 6.6 × 10 ⁷ cells / ml, 0.15 ml each (1X10 ⁷ cells / chamber) chambers that had been air dried - placed in a nylon I plugged it with a stick. As a control, a chamber containing a phosphate buffer solution (PBS) was also prepared.

Transplantation of chambers Nine weeks after vaccination, mice were anesthetized with Nembutal and kept in the prone position, and the skin about 1 cm from the ridge to the head side was incised to the extent that the chamber can be inserted (about 1.5 cm). Then
A space was created using a ring tweezers so that the chamber could be inserted subcutaneously, and the chamber was inserted to the flank. The incision was closed with a suture instrument and disinfected with iodine tincture. A chamber containing LLC cells was inserted into the left back of each mouse, and a chamber containing PBS was inserted into the right back.

Observation of Tumor Angiogenesis On the 4th day after the chamber transplantation, angiogenesis induced subcutaneously in contact with the chamber was observed. This result is shown in FIG.
Shown in In FIG. 5, (a) is subcutaneously in contact with a PBS (phosphate buffer) -containing chamber in the buffer control group, (b) is subcutaneously in contact with a PBS-containing chamber in the adjuvant control group, and (c) is a vaccine. Subcutaneous contact with PBS containing chamber in inoculation group, (d) Subcutaneous contact with chamber containing Lewis lung cancer cells in buffer control group, (e) Subcutaneous contact with chamber containing Lewis lung cancer cells in adjuvant control group ,
(F) shows subcutaneously in contact with the chamber containing Lewis lung cancer cells in the vaccinated group.
From this figure, in any group of mice, angiogenesis was not observed subcutaneously in contact with the PBS-containing chamber [see FIGS. 5a, 5b and 5c]. Significant angiogenesis was observed under the skin of the buffer control mouse and the adjuvant control mouse in contact with the LLC cell-containing chamber (see FIGS. 5d and e), whereas in the subcutaneous contact with the LLC cell-containing chamber of the vaccinated mouse, angiogenesis was observed. Was not observed [Fig. 5f
reference〕. In FIG. 5, PBS indicates phosphate buffered saline, LLC indicates mouse Lewis lung cancer, FCA indicates Freund's complete adjuvant administration group, and bVEGF indicates VEGF ₁₂₁ produced by baculovirus.

[0043]

INDUSTRIAL APPLICABILITY It is considered that by inoculating a living body with the vaccine of the present invention, particularly a vaccine containing VEGF / VPF, the immune response of the living body itself is elicited, and as a result, VEGF / VPF plays an important role. It suppresses angiogenesis during tumor growth and attacks tumors and tumor blood vessels to obtain an antitumor effect. Further, the following effects can be expected regarding the prevention and treatment of tumors.

1. Pre-vaccination of individuals with a vaccine containing VEGF / VPF is expected to have a preventive effect on the onset of cancer as a disease that does not increase the size of spontaneously-generated tumors and significantly delays the onset of tumors. it can. 2. Cancer metastasis is established when cancer cells derived from a primary cancer proliferate in different places. Even in this case, the transfer is approved,
That is, the key to ectopic cancer cell proliferation, is the key to the thought to be angiogenesis, before and after surgically resecting the cancer, by vaccination containing VEGF / VPF, Similar to the case of the primary cancer, the preventive effect of cancer metastasis is expected, that is, the carcinoma that is generated in a different place is not enlarged and the manifestation of the carcinoma is significantly delayed.

3. It is known that many cancer cells produce VEGF / VPF, and that accumulation of VEGF / VPF that is thought to be secreted from nearby cancers is observed on endothelial cells of tumor blood vessels. Vascular endothelial cells can be the target of a cytotoxic immune response. Therefore, it can be expected that the present vaccination will be effective as one of the cancer immunotherapies for the treatment of solid cancer accompanied by neovascularization of tumor.

[0046]

[Sequence list]

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

[Brief description of drawings]

FIG. 1 is a graph showing changes in the average value of anti-VEGF / VPF antibody titers in mouse blood.

FIG. 2 is a diagram showing changes in the average value of cancer growth.

FIG. 3 is a graph showing changes in the average value of anti-VEGF / VPF antibody titers in mouse blood.

FIG. 4 is a diagram showing an average value of the number of cancer colonies.

FIG. 5 is a photograph of subcutaneous (form of organism) in contact with a chamber in a tumor angiogenesis induction experiment in a vaccinated mouse.

[Procedure amendment]

[Submission date] June 6, 1996

[Procedure amendment 1]

[Document name to be amended] Drawing

[Correction target item name] Fig. 5

[Correction method] Change

[Correction contents]

[Figure 5]

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Ayako Koda No. 2 Okubo, Tsukuba City, Ibaraki Prefecture, Toagosei Research Co., Ltd. (72) Inventor Toshiaki Segawa No. 2 Okubo, Tsukuba City, Ibaraki Toagosei Co., Ltd.

Claims (2)

[Claims] 1. A vaccine for suppressing angiogenesis, which comprises a cell growth factor or a fragment thereof having a specific cell growth promoting activity on vascular endothelial cells. 2. The angiogenesis-suppressing vaccine according to claim 1, wherein the cell growth factor is a vascular endothelial cell growth factor.