WO2004096244A1

WO2004096244A1 - Method of preparing tumor vaccine for the inducement of anti-tumor activity and a pharmaceutical composition containing the same

Info

Publication number: WO2004096244A1
Application number: PCT/KR2004/001025
Authority: WO
Inventors: Taehwan Kwak; Myunggyu Park; Taek Joon Yoon
Original assignee: Md Bioalpha Co. Ltd.
Priority date: 2003-05-02
Filing date: 2004-04-30
Publication date: 2004-11-11
Also published as: KR20040094635A

Abstract

The present invention relates to a method of preparing a tumor vaccine to induce anti-tumor activity, a pharmaceutical composition containing the same tumor vaccine, and a method of providing enhanced immunity against tumors using the tumor vaccine or composition to prevent and treat the tumor. The tumor vaccine of the present invention provides enhanced immunity against tumors to allow the tumor-specific CD4+ or CD8+ T cell to be activated, thereby inducing the humoral and cell-mediated immune response. Moreover, the tumor vaccine of the present invention has cross-reactivity with respect to tumors so that it can induce the immune response against a variety of tumors, thereby expressing a vaccine effect for preventing the proliferation and metastasis of tumors and also a therapeutic effect on existing tumors.

Description

METHOD OF PREPARING TUMOR VACCINE FOR THE

INDUCEMENT OF ANTI-TUMOR ACTIVITY AND A

PHARMACEUTICAL COMPOSITION CONTAINING THE SAME

FIELD OF THE INVENTION

The present invention relates to a method of preparing a tumor vaccine, a pharmaceutical composition containing the tumor vaccine, and a method of enhancing immunity against tumors using the vaccine or pharmaceutical composition to inhibit the tumors, thereby ultimately treating or preventing tumor growth. More specifically, the present invention provides (a) a method of heating tumor cells originated from tumors to prepare a tumor vaccine comprising inactivated tumor cells and/or tumor antigens obtained from the inactivated tumor cells, and conferring the enhanced immunity against tumors; (b) a pharmaceutical composition comprising the inactivated tumor cells and/or tumor antigens, capable of inducing the anti-tumor activity; and (c) a method of enhancing the immunity of a patient against tumor, using the tumor vaccine or pharmaceutical composition, to inhibit the tumor proliferation and metastasis, thereby ultimately preventing the generation of tumors and also treating the tumor.

For convenience of explanation, the term "inactivated tumor cell" or "tumor antigen" is sometimes used, instead of the term "tumor vaccine", in the present disclosure, and these terms are intended to have the same meaning. BACKGROUND OF THE INVENTION

Extensive research has been conducted to develop a tumor vaccine to ultimately induce anti-tumor activity, whereby the prevention and treatment methods of some tumors, based upon the induction of anti-tumor immunity, were found; however, clinically applicable method have not been developed yet.

The key reason for the failure of cancer immunotheraphy is the immune escape mechanism of tumors per se, that is, the poor immunogenicity of tumors. More specifically, expression of class I- and II-MHC is suppressed, or an antigen inducing immune response is not present, or even though an antigen is present, it is hidden by muco-polysaccharides containing sialic acid, thereby inhibiting the function of CTL and helper T cells. Moreover, since tumor cells do not make a co-stimulator signal stimulating T cells, T cells become anergic. Tumor cells also produce TGF-β having immunosuppressive function and do not express FASL so that effector cells such as CTL becomes inactive. Unfortunately, tumor-specific antigens of many tumor cells have not been identified so far. Since effective immunity is not induced by any known antigen alone, many researchers anticipate that a multivalent antigen, using simultaneously at least three antigens for immunization, may induce an effective anti-tumor immune response, hence the immune response generated by whole tumor vaccine is now being researched.

Generally, as in vivo effector cells involved in anti-tumor immunity, known are tumor-specific helper T cells, cytotoxic T lymphocytes, etc. and tumor-nonspecific macrophages, NK-cells, etc. Cytotoxic T lymphocytes ("CTL") involved in the cell-mediated immunity are best known as effector cells having anti-tumor activity. CTL activity against tumors is induced mainly by recognizing MHC-I presented tumor antigen. However, tumor cells are known to rarely express MHC-I, hence research is focused on a cross priming method mainly via APC. Accordingly, attention is focusing on an immunization method using a dendritic cell as a strong APC.

Meanwhile, anti-tumor NK-cells are known as cells inhibiting the metastasis or proliferation of tumors. In recent, it was ascertained that activated NK-cells, which react with specific ligands (Rael and H60) expressed in tumor cells, induce the perforϊn-mediated tumor killing mechanism, thus a possibility of using these ligands as a vaccine against tumors is suggested.

In the in vivo mechanism of conferring immunity by tumor vaccine for treatment or prevention of malignant tumors, induction of the antigen-presenting ability (hereinafter, referred to as "antigen presentation ability") by DC, as a professional APC, and macrophages is regarded as the most important factor. In previous experiments, several researchers co-cultured DC or macrophages with apoptotic or necrotic tumor cells, as a tumor vaccine, and inoculated them into a tumor-bearing host, thereby having been able to induce a significant inhibition of the tumor proliferation and metastasis. This result suggests that the immune escape mechanism of tumors, as mentioned previously, may be overcome by using a strong APC to present a tumor antigen.

For this reason, research using immune adjuvants to improve the cancer antigen presentation ability for induction of anti-cancer activity, is being carried out. This research intends to ultimately enhance the immunity against cancer antigens which are poorly expressed. Some researchers developed methods of creating cancer antigen-bearing tumor using molecular biological techniques, or creating tumor cells to which the cytokine gene directly involved in induction of the antigen presentation has been introduced. These methods are an important fundamental research to study the mechanism of tumor immunogenicity clinically.

The induction of cancer immunity using tumor vaccine proceeds in the following three stages: an antigen-presentation stage, a stage of priming T cells by APC, and an effector stage by the activated T cell. To present an antigen, a tumor antigen must have in vivo the function of differentiating the immature APC to the mature APC. The uptake of antigen by APC leads to the differentiation thereof to the mature form capable of presenting antigen to induce expression of TNF-α and IL-12. These cytokines are the most important factors in immunity induction. As these cytokines induce the maturation of T cell in response to antigen presentation, antigen presented T cells produce GM-CSF, which again induces the proliferation of APC. Therefore, production of GM-CSF from T cells is indispensable for the proliferation of professional APC. By such regulation of cytokines, the maturation of CTL, which has acquired the ability to kill a specific tumor, is induced. As CTL acquires the tumor-killing ability by contact with tumor cells, IFN-γ is produced and simultaneously the co-stimulatory molecule is expressed. As such, the production of IFN-γ from T cell is an important index of the tumor-killing activity of CTL. As a result, the tumor antigen presentation by APC and the production of Thl-type cytokine from T cells are regarded as highly important, thus a method of preparing a tumor vaccine capable of inducing the activity thereof is required.

In this connection, Gough MJ et al. conducted an animal experiment testing the inhibition of tumor proliferation by immunization with apoptotic cells and necrotic cells (Cancer Res. 2001, 61, 7240-7247). According to these authors, the necrotic cells activated macrophages to induce significant production of TNF-α, IL-1 and IL-6, thus tumor proliferation was significantly inhibited, and further prevented by immunization with the macrophages cultured along with necrotic cells. They also reported that immunization with apoptotic cells removed tumors by the phagocytosis of macrophage, whereas the immunization with necrotic cells provided the vaccine effect through induction of some phagocytosis and induction of immune activity by production of the above cytokines. Furthermore, they asserted that this result was attributed to the expression of hsp 70 mainly resulting from stress.

In connection with the antigen presentation of macrophages, Barker RN et al. supported the above result by finding that necrotic cells, having come in contact with macrophages, induce the expression of CD40 to activate T cells (Exp Immunol. 2002, 127, 220-225).

On the other hand, Schnurr M et al. reported the vaccine activity of apoptotic cells by illustrating that when TNF-α was produced in co-cultures of DC and apoptotic cells, DC expressed CD83 to differentiate into mature DC, thus the co-culture thereof induced a significant production of IFN-γ (Cancer Res, 2002, 62, 2347-2352). They also reported that the freeze/thawed necrotic cells failed to significantly induce production of IFN-γ so that anti-tumor immunity could not be induced. Meanwhile, Mass D et al. reported that since the co-culture of apoptotic cells and DC allowed the immature DC to differentiate into a mature form capable of expressing CD86⁺, DC maturation plays an important role in activation of T cells (Cancer Res. 2002, 62, 1050-1056). In contrast, Strome SE et al. reported that the maturation of DC by irradiated tumor cells induced a significant inhibitory effect on the cancer proliferation, whereas the freeze/thawed necrotic cells failed to achieve the same effect (Cancer Res 2002, 62, 1884-1889), which conflicts with the result as reported by Schnurr M et al. above. In other words, Strome SE et al. asserted that only irradiated cells can induce the production of IFN-γ, associated with CTL, from T cells, and that the above result can be obtained when the cell is maintained under the stress condition by radiation, which implies that hsp is involved in anti-tumor immunity.

In summary, the above results show that a tumor vaccine may induce anti-tumor activity depending upon experimental conditions, regardless of apoptotic cells or necrotic cells, and that the most important factor for prevention of malignant tumors is that the antigen presentation (the degree of maturation) of APC acts as a priming factor in induction of the anti-tumor immunity to contribute to induction of CTL as the cell-mediated immunity system, thereby resulting in the vaccine effect. They also show that hsp is directly or indirectly involved, as a type of tumor antigen, in induction of the tumor immunity, and that hsp is considered more in the cancer proliferation-inhibiting aspect than in the cancer metastasis-inhibiting aspect.

While the nature of cancer antigens capable of inducing an immune response effective to remove cancer is not well known, such antigens are believed to exist in that where some cancers, induced by carcinogens, were established in mouse, antigens generated during the tumor induction procedure induce the immune response against the cancers in a cancer-bearing host. For instance, it has been reported that some cancer antigens may induce an extensive, protective immunity against a variety of cancers in humans, and research for prevention or treatment therapy using the cancer antigen is being conducted, but has not yet achieved satisfactory results. As a result, the nature of cancer antigens has not been confirmed yet.

Generally, in order to induce an efficient immune response against tumors and ultimately to develop a method of effectively preventing or treating cancer proliferation and metastasis, the following is needed: (i) identification of malignant tumor antigens, (ii) a method of efficient induction of the immune response to the tumor antigen, and (iii) a method of overcoming the immune escape mechanism of tumors. The efficient induction of the immune response is known to be associated with use of an immune adjuvant. The identification of malignant cancer antigens and removal of the immune escape mechanism of tumors are now being researched. As a result, identifying a malignant tumor antigen, which induces anti-tumor activity against a variety of tumors, will be very important in view of the induction of anti-tumor immunity. However, it has been found that treatment methods using one kind of tumor antigen are limited due to appearance of variants thereof, thus many researchers are searching for a method for induction of the anti-tumor immunity using a whole tumor cell.

So far, as vaccines able to be used in the form of whole tumor cell known are the following: apoptotic cells, freeze/thawed necrotic cells, irradiated cells, formaldehyde-fixed cells, heat shock proteins, photoreactive or photosensitizer cells (photodynamic therapy: PDT), etc.; however, as mentioned previously, there are many different opinions with respect to practical applications thereof. Priming of a professional APC (dendritic cell) in those whole cell lysates gave good results in vivo, such that some entered the preclinical phase. But, no preparation method of tumor vaccines showing a satisfactory activity when applied clinically has been developed yet. SUMMARY OF THE INVENTION

The inventors of the present invention researched extensively to develop a preparation method of a vaccine capable of inducing an efficient anti-tumor immunity. Resultantly, we developed strong cancer vaccine having tumor metastasis-inhibiting ability to prevent death caused by remote metastasis, which is regarded as the most fatal factor in clinic, as well as tumor proliferation-inhibiting ability, and identified a material expressing the activity thereof, and also investigated the mechanism thereof.

Therefore, an object of the present invention is to provide a preparation method of a tumor vaccine capable of inducing anti-tumor activity. The tumor vaccine, prepared by the method according to the present invention, induces an excellent anti-tumor immune response for prevention of tumor proliferation and metastasis in a subject, and also expresses cross-reactivity against the proliferation and metastasis of other cancers.

Another object of the present invention is to provide a pharmaceutical composition comprising an active component expressing such an anti-tumor activity.

A further object of the present invention is to provide a method of enhancing the immunity against tumors, and a method of preventing and treating tumors by inhibition of tumor proliferation and metastasis.

In order to accomplish the above objects, the preparation method of a tumor vaccine according to the present invention comprises heating tumor cells to provide inactive tumor cells and/or tumor antigens obtained from the inactive tumor cells, capable of enhancing the immunity against tumors.

The tumor cells used in the preparation method of the present invention are not particularly limited and include, for example, colon 26-M3.1 carcinoma (Balb/c), CT-26 colon carcinoma (Balb/c), B16-BL6 melanoma (C57BL/6), L1210 leukemia (DBA2), as employed in Examples of the present disclosure. Further, the tumors may be primary tumors or malignant tumors metastasizing from the primary tumors. Induction of the anti-tumor activity by immunization with the tumor vaccine according to the present invention is effective with respect to the proliferation or metastasis of solid cancers and, particularly, malignant cancers acquiring metastatic ability, as elucidated in Examples below. The tumor vaccine of the present invention confers immunity against tumors of both syngeneic and allogeneic subjects. Thus, the tumor vaccine of the present invention may be based upon (i) autologous tumors in which a donor subject of tumor antigen is identical with a donee subject thereof, or (ii) allogeneic tumors in which a donor subject is different from a donee subject. Moreover, the tumor vaccine of the present invention has cross-reactivity enhancing for the immune response against other kinds of tumors. Accordingly, the term "tumor" used in the present disclosure includes all of the above concepts, without distinguishing among, for example, tumor tissue, tumor cell, etc.

The heat treatment can be performed by various ways which include, but are not limited to water bath heating, pressure sterilization, wet sterilization, etc.

The temperature and duration of heat treatments are in the range of more than 45°C, preferably 60 ~ 130°C, and more preferably 90 ~ 110°C, and at least 5 minutes, preferably 10 - 60 minutes, and more preferably 20 ~ 40 minutes. They are particularly preferably in the range of 90 ~ 100°C and 20 ~ 30 minutes.

In some embodiments, the method according to the present invention may further include one or more selected from the group consisting of (a) a step of culturing the tumor cells derived from tumors under appropriate conditions to proliferate the cells, (b) a step of sonicating the tumor cells during and/or after the heat treatment, and (c) a step of purifying tumor antigens from the inactivated tumor cells.

The culture of tumor cells and the sonication of tumor cells can easily be conducted by those skilled in the art, thus the detailed description thereof is omitted in the present disclosure. On the other hand, the purification of tumor antigens can be carried out on the basis of information regarding the tumor antigens to be illustrated herein later.

The sonication of tumor cells, whereby supersonic waves are applied to tumor cells during and/or after the heat treatment, can result in a vaccine in which more many tumor-specific antigens are exposed. The sonication time is not particularly limited and is, for example, approximately 10 ~ 40 minutes.

The inactivated tumor cells may be in an intact form, i.e., maintaining the original cell membrane, or in a lysate form. Furthermore, the tumor antigen which is an antigen, being included in the inactivated tumor cell and also inducing the immune response, may be the lysate per se or some thereof.

As will be ascertained in Examples later, the inactivated tumor cells obtained by the method according to the present invention includes monovalent or multivalent antigens, which are one or more tumor antigens selected from 45, 57, 62, 74 and 75 kDa proteins obtained after heat treatment, providing immunity against tumor and/or inhibiting tumor proliferation and metastasis to express treatment and prevention activity against tumors. These tumor antigens may be antigen proteins having been denaturated deformation by heat treatment, or antigen proteins having been newly exposed on the surface of cells by heat treatment. However, it must be noted that the tumor antigen, obtained by the method according to the present invention, is not a heat shock protein (hsp), and that the tumor protein serves to present an antigen through MHC of the antigen-presenting cell (APC), thereby inducing the cell-mediated immune response in the subject being immunized.

In order to elucidate the mechanism of anti-tumor vaccine (hereinafter, sometimes referred to as "MB-J vaccine"), many experiments were carried out, as will be described in Examples later, and the results are summarized in below.

i) From the electrophoresis of MB-J vaccine and the Western blotting analysis using anti-MB-J vaccine antibody, it was ascertained that MB-J vaccine contains a new protein of abut 75kDa, not found in live tumor cells, and that this protein is not hsp inducing the immune-stimulating activity.

ii) From the experiment in which T cells of a host which had been immunized with MB-J vaccine produced GM-CSF inducing maturation of APC, it was ascertained that MB-J vaccine induces the maturation of APC.

iii) From the experiment in which TNF-α and IL-12 involved in the effector stage are found in a culture supernatant after in vitro co-culture of MB-J vaccine and each APC, it was ascertained that MB-J vaccine enhances the antigen presentation ability of dendritic cells as APC and macrophages.

iv) From the experiment in which the anti-metastasis activity of MB-J vaccine was also induced in a mouse from which NK-cells had been removed using anti-asialo-GMl antibody, it was ascertained that the anti-tumor activity of MB-J vaccine-immunized mouse is not attributed to the activation of NK-cells as a tumor-nonspecific effector cell.

v) From the experiment in which T cells of a host, immunized with MB-J vaccine, produced IFN-γ being expressed in the effector stage of CTL, and in which the production significantly increased when re-stimulated with MB-J vaccine, it was ascertained that the activation of APC-dependant T cells by MB-J vaccine induces CTL to attack tumor cell.

vi) From the experiment in which cancer metastasis inhibitory activity was entirely extinguished in CD8⁺ T cell-knockout mice and partial anti-tumor activity was observed in CD4⁺ T cell-knockout mice, it was ascertained that the induction of CTL, having the anti-tumor activity, by immunization with MB-J vaccine, which is dependent upon CD4⁺ and CD8⁺ T cells, is attributed to the activation of CD8⁺ T cells via CD4⁺ T cell or the direct activation of CD8⁺ T cells.

vii) From the experiment in which the MB-J vaccine-specific antibody was produced by immunization with MB-J vaccine, it was ascertained that the MB-J vaccine is directly or indirectly involved in the anti-tumor immunity of a host.

viii) From the other experiments, it was ascertained that some of a portion of antigens inducing the anti-tumor activity are present in the soluble fraction of MB-J vaccine, but are present mainly in the cellular fraction thereof, which indicates that the specific antigens (probably, 75 kDa protein or other antigens), produced in the preparation procedure of MB-J vaccine, are partly suspended in PBS buffer during the preparation procedure but major components inducing the anti-tumor activity are mainly present in cells.

Accordingly, induction of tumor immunity by immunization with the tumor vaccine according to the present invention is attributed to the mechanism of inducing tumor-specific cell-mediated immunity by presentation of tumor antigen, produced during preparation of the vaccine, by APC; whereby the vaccine according to the present invention can confer immunity not only against syngeneic tumors but also against allogeneic tumors, which is deemed to be attributed to a 75 kDa protein or another antigens produced during the heat treatment procedure.

The present invention also provides a pharmaceutical composition for prevention and treatment of tumors, comprising (a) a therapeutically effective amount of the inactivated tumor cell and/or tumor antigen obtained therefrom, as defined previously, and (b) a physiologically acceptable carrier, diluent, or excipient, or a combination thereof.

The term "pharmaceutical composition" as used herein means a mixture of the inactivated tumor and/or tumor antigen (hereinafter, referred to as "active component") of the invention with other chemical components, such as diluents or carriers. The pharmaceutical composition facilitates administration of the active component to a patient. Multiple techniques of administering the active component in the art include, but are not limited to injection, oral, aerosol, parenteral, and topical administrations.

The term "therapeutically effective amount" means that amount of the active component which is sufficient for prevention and treatment of a tumor. The term "carrier" means a chemical compound that facilitates the incorporation of an active component into cells or tissues. For example, dimethyl sulfoxide (DMSO) is a commonly utilized carrier as it facilitates the uptake of many organic compounds into the cells or tissues of a patient.

The term "diluent" defines chemical compounds diluted in water that will dissolve the active component of interest as well as stabilize the biologically active form of the component. Salts dissolved in buffered solutions are utilized as diluents in the art. One commonly used buffered solution is phosphate buffered saline because it mimics the ionic strength conditions of human blood. Since buffer salts can control the pH of a solution at low concentrations, a buffered diluent rarely modifies the biological activity of active components.

The term "physiologically acceptable" defines a carrier or diluent that does not abrogate the desired biological activity and properties.

The term "prevention and/or treatment" includes enhancing the immunity of a subject against tumors, inducing the clinical effect based upon such enhancement of immunity, and inducing the clinical effect by the active component even though it is not attributed to the enhancement of immunity against tumors. The prevention or treatment of cancer can be achieved, for example, by inhibiting the proliferation and metastasis of cancer, but it is not limited thereto. Since the prevention includes immunizing a subject as a matter of course, the pharmaceutical composition according to the present invention, is also intended to be understood as a vaccine composition in the present disclosure.

Suitable routes of administration may, for example, include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intravenous, intramedullary injections, as well as intrathecal, direct intraventricular, intraperitoneal, intranasal, or intraocular injections. Preferably, intradermal, intermittent intradermal, intramuscular and intravenous administration can be employed for the pharmaceutical composition according to the present invention.

The pharmaceutical composition of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active component into preparations, which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. Any of the well-known techniques, carriers, and excipients may be used as suitable and as understood in the art; e.g., in Remington's "Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, 18th edition, 1990.

For injection, the composition of the present invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

For the active component used in the composition of the present invention, the therapeutically effective dose can be estimated initially from cell culture assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC_SQ as determined in cell culture. The dosage of the active component lies preferably within a range of circulating concentrations that include the ED₅₀ (the dose achievng the therapeutical effect in 50% of the population) with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the attending physician in view of the patient's condition (See e.g., Fingl et al. 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p. 1).

Other ingredients may be further added to the pharmaceutical composition of the present invention within the range where the effect of the present invention is not degraded.

The present invention also provides a method of enhancing the immunity of a subject being treated by administering a therapeutically effective amount of the inactivated tumor cell and/or tumor antigen, as defined previously, to the subject. Such enhancement of the immunity, for example, can inhibit the proliferation and/or metastasis of tumor in a subject being treated.

The above prevention and/or treatment method includes a method of obtaining tumor from a subject and preparing the inactivated tumor cell and/or tumor antigen from the tumor according to the preparation method of the present invention to administer them to a genetically syngeneic subject being treated; and a method of obtaining tumor from a subject and preparing the inactivated tumor cell and/or tumor antigen from the tumor according to the preparation method of the present invention to administer them to a genetically allogeneic subject being treated.

The subject is a vertebrate animal and preferably human.

In an embodiment, the method further includes administering APC stimulated by the active component alone, or together with the active component.

The enhancement of immunity to tumor according to the present invention can be achieved either by one administration of the active component or by repeated administration of the active component. Since the active component has cross-reactivity against different kinds of tumors besides the tumor used for preparation of the active component, as already mentioned, the tumor used for preparation of the active component need not be the same as the tumor to be treated.

The present invention also provides a method of administering a therapeutically effective amount of the inactivated tumor cell and/or tumor antigen, as defined previously, to a subject being treated to prevent and/or treat tumors. Various routes of administration can be employed and include, for example, preparing a pharmaceutical composition as defined previously and then administering it to a subject being treated by one or more of administration manners as described above.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A ~ 1C are graphs showing the effect of a vaccine on the inhibition of tumor proliferation, on the survival rate, and an the inhibition of tumor generation, respectively, in which the vaccine was prepared using colon 26-M3.1 carcinoma (Balb/c) according to the method of the present invention; FIGS. 2 A ~ 2C are graphs showing the effect of a vaccine on the survival rate, and on the inhibition of tumor generation, respectively, in which the vaccine was prepared using CT-26 colon carcinoma (Balb/c) according to the method of the present invention;

FIGS. 3 A and 3B are graphs showing the effect of a vaccine on the inhibition of tumor proliferation and on survival rate, respectively, in which the vaccine was prepared using B16-BL6 melanoma (C57BL/6) according to the method of the present invention;

FIG. 4 is a graph showing the survival rate of tumor-bearing mice inoculated with the vaccine prepared using L1210 leukemia (DBA2) according to the method of the present invention;

FIG. 5 discloses photographs showing the results of electrophoresis and Western blotting analysis of a vaccine, as obtained in an embodiment, prepared by the method of the present invention (hereinafter, sometimes referred to as "MB-J vaccine");

FIG. 6 is a photograph showing the result of Western blotting analysis of MB-J vaccine against anti-hsp70 antibody;

FIG. 7 is a graph showing the effect of MB-J vaccine on the production of IL-12 from DC;

FIGS. 8 A and 8B are graphs showing the amount of TNF-α produced by the pellet fraction and supernatant fraction of MB-J vaccine, respectively;

FIGS. 9 A and 9B are graphs showing the effect of endotoxin on production of

TNF-α by the pellet and supernatant fractions of MB-J vaccine, respectively; FIG. 10 is a graph showing the survival rate of mice, in which the mice were immunized with MB-J vaccine and control vaccines, respectively, and then inoculated with tumor cells;

FIG. 11 is a graph showing the effect of MB-J vaccine and control vaccines in NK-cell knockout mice;

FIG. 12 is a graph showing the activity of MB-J vaccine and control vaccines, respectively, concerning the induction of TNF-α from peritoneal exudative cells;

FIG. 13 is a graph showing the proliferation ability of peritoneal exudative cells by MB-J vaccine and control vaccines, respectively;

FIGS. 14A and 14B are graphs showing the production of cytokines following, the stimulation of macrophages by MB-J vaccine and control vaccines, respectively;

FIG. 15 is a graph showing the re-stimulating activity of spleen cells immunized with MB-J vaccine;

FIGS. 16A ~ 16D are graphs showing the induction pattern of Thl type cytokines (IFN-γ, GM-CSF) after immunization with MB-J vaccine and control vaccines, respectively, and then inoculation with cancer cells;

FIGS. 17A and 17B are graphs showing the induction pattern of Th2 type cytokines (IL-10) after immunization with MB-J vaccine and control vaccines, respectively, and then inoculation with cancer cells;

FIG. 18 is a graph showing the experimental result regarding the re-stimulation of spleen cells immunized with MB-J vaccine and control vaccines, respectively, and then inoculation of cancer cells;

FIGS. 19A and 19B are graphs showing the antibody levels and the subisotype analysis of antibodies in mice immunized with MB-J vaccine.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention will now be illustrated in more detail, including preparation of a tumor vaccine according to the present invention, identification of active components contained in the tumor vaccine, validation of the anti-tumor inhibition activity in the in vivo animal test by immunization with the tumor vaccine, elucidation of the mechanism of immunogenicity of tumor vaccine, and the like, by the following examples and experimental examples; however, it will be understood that the present invention is not limited to these specific examples and experimental examples, but is subject to various modifications that will be recognized by one skilled in the art to which the present invention pertains.

Example 1 : Preparation of tumor vaccines

A tumor cells used in this experiment were the colon 26-M3.1 carcinoma cell line. Colon 26-M3.1 carcinoma cell line was cultured in EMEM nutrient medium supplemented with 7% FBS at 37°C. Cells were grown on the surface of a culture flask until they covered about 60% of the surface, forming a monolayer of cells, then were collected for preparation of a tumor vaccine. For vaccination, the cells were subjected to the following treatments, respectively:

1) Heat-treated vaccine (MB-J vaccine) For preparation of a tumor vaccine according to the present invention, used was RPMI-1640 or EMEM medium supplemented with 7% FBS. In the case of adhesive cell lines such as colon 26-M3.1 carcinoma and B16-BL6 melanoma, cells were separated using trypsin-EDTA and then a culture medium were added and, in the case of floating cell lines such as L1210 leukemia, cells were to remove the medium. Then, the cells were suspended in PBS and centrifuged three times to remove fetal bovine serum (FBS) remaining in the medium. An appropriate amount of cells (lxlO⁷ cells/ml) were added to PBS to be subjected to water bath heating for 30 minutes. Thereafter, they were quenched in ice and then stored at 4°C for use as a vaccine (hereinafter, referred to as "MB-J vaccine") in the following experiments. For convenience of illustration, a vaccine according to the present invention is also sometimes referred to as "X-vaccine" in the below Examples and relevant drawings.

2 Formaldehyde-fixed vaccine (F- vaccine)

Tumor cells cultured in a medium were washed three times with PBS to remove FBS. 0.1% formaldehyde on the basis of PBS was added to fix the cells (lxlO⁷ cells/ml) for 1 day in an incubator. Then, the cells were washed with PBS and stored at 4°C for use as a control vaccine (hereinafter, referred to as "F-vaccine") in the following experiments.

3) Other control vaccines

(a) Tumor cells cultured in a medium were washed with PBS to remove FBS.

The tumor cells, which were cultured at 37°C in a CO₂ incubator for 5 days were centrifuged at 9000 rpm for 10 min to give a soluble surface antigen ("surface Ag"). Protein, contained in a supernatant, was quantified using a protein analysis kit (Bio-rad) and then stored at 4°C for use as a control vaccine (referred to as "Sur-vaccine") in the following experiments.

(b) Cells cultured in a medium were separated using trypsin-EDTA and washed once with a culture medium and then three times with PBS to remove all traces of FBS. The cells (l lO⁷ cells/ml) were suspended in PBS, and flash-frozen using liquid nitrogen, then thawed at room temperature. This procedure was repeated three times to make another control vaccine (hereinafter, referred to as "F/T- vaccine").

(c) Tumor cells were subjected to the cell lysis using a lysis buffer, then protein was quantified from the lysate and stored at 4°C. This lysate is called "cell lysate" hereinafter.

Example 2: Effect of vaccine immunization on the proliferation of solid cancer in animal model

In the present experiment, using colon 26-M3.1 carcinoma (Balb/c), CT-26 colon carcinoma (Balb/c), B16-BL6 melanoma (C57BL/6), and L1210 leukemia (DBA2), each vaccine was prepared in the same manner as in Example 1, and the effect of immunization with these vaccines was evaluated in view of their inhibitory effect against the proliferation of the above cell lines. Immunization was carried out three times at one-week intervals, and one week after the final immunization, cancer cells were subcutaneously or intraperitoneally (for L1210 leukemia) injected. The densities of vaccines were 1x10 cells, 1x10^s cells and lxlO⁶ cells, respectively, and live cancer cells were inoculated into the mice at a concentration of 1.5xl0⁴ cells. 15 days after appearance of cancer colonies, the cancer cell proliferation was checked at intervals of 1~3 days, and simultaneously the number of tumor-free mice and the survival rate of mice were checked. The results are disclosed in FIGS. 1 ~ 4. In these drawings, "X5" means the lxlO⁵ cell-density group of vaccines according to the present invention and "X6" means the 1 x 10⁶ cell-density group thereof.

As seen in FIGS 1A ~ 1C and 2 A ~ 2C, in the case of colon 26-M3.1 carcinoma and CT-26 colon carcinoma, the induction effect of vaccine on the anti-tumor activity was so good that the generation of cancers was inhibited by 80% and 60%, respectively, in immunization with lxlO⁶ cell-density vaccine. When the size of cancer became 25 mm³ after inoculation of colon 26-M3.1 cells it was inhibited for about one week by re-immunization with the vaccine, thereafter the cancer grew again. In the case of CT-26 colon carcinoma model, when the size of cancer became about 300 mm³, the vaccine immunization inhibited the cancer growth for about 4 days. The survival rate of mice and the growth rate of cancer in both cell lines were dependant on the concentration of immunization cells, and the optimal cell number for vaccination is deemed to be lxlO⁶ cells in mice models of the present experiment.

As seen in FIGS. 3 A and 3B, in the case of B16-BL6 melanoma model, the tumor proliferation and survival rate of mice were similar tendency to those of colon 26-M3.1 and CT-26 cell model, and the re-immunization of vaccine during growth of tumor colonies significantly inhibited the proliferation of tumor for about 7 days. In the case of control groups, all the mice died within 37 days after inoculation of tumor cells. On the other hand, the mice immunized with lxlO⁶ cell-density vaccine showed survival rate of 40% up to 50 days after tumor inoculation and 20% of the mice survived up to 100 days.

As seen in FIG. 4, also in the case of L1210 leukemia model, a significant inhibition effect versus the tumor proliferation was seen upon immunization with lxlO⁶ cell and lxlO⁵ cell-density vaccine, as compared to the control groups, and 40% and 20% of the mice survived up to 75 days after inoculation of tumor cells, respectively.

The above results suggest that the vaccine according to the present invention can be used to inhibit the proliferation of tumors, and it is anticipated that the continuous administration of the vaccine can further enhance the tumor proliferation inhibitory activity. Furthermore, it is deemed that the vaccine immunity according to the present invention induces significant immune response against tumor, of which the mechanism will be illustrated in more detail in later Examples.

Example 3: Anti-tumor activity dependence on the temperature of heat treatment in vaccine preparation

To find the optimal heating temperature for preparation of MB-J vaccine, cell lysates of colon 26-M3.1 carcinoma cell line were prepared at various heating temperatures and applied to the experimental metastasis model, in which the same colon 26-M3.1 carcinoma cell line was employed, to estimate the anti-metastasis activity thereof. The heat treatment was performed by water bath heating at 45 °C, 60°C, 80°C and 100°C, respectively, for 30 minutes. Immunization was conducted by using such heated cells at a concentration of 5xl0⁵ cells/mouse three times at 10-day intervals. For inoculation of tumor cells, colon 26-M3.1 carcinoma cells were intravenously injected at a concentration of 2.7x10⁴ cells/mouse 10 days after the final immunization. The results are summarized in Table 1 below.

[TABLE 1]

As seen in TABLE 1 above, it was ascertained that the anti-tumor activity is dependant upon the temperature of heating tumor cells and that 90°C ~ 100°C is an optimal temperature for such heat treatment.

Meanwhile, it is necessary to know whether the induction of anti-tumor immune response by heat treatment is associated with production of heat shock proteins ("HSPs"). HSPs are proteins expressed in various cells, bacteria, etc. when stresses such as heat treatment are applied thereto and these proteins are sometimes collectively called as "hsp family". Of them, hsp 60, hsp 70, hsp 90 and hsp 96 are well known as activating mainly the innate immune system of a host. Among the hsp proteins expressed in eukaryoic cells, particularly cancer cells, hsp 70 is most well known and this protein induces the anti-tumor activity by enhancing the antigen presentation ability of APC and also activates the innate immune system. Accordingly, it is required to confirm whether the results in TABLE 1 above were caused by the expression of HSP or not.

Example 4: Anti-tumor activity dependence upon the time of heat treatment in vaccine preparation

To find the optimal heating time for preparation of MB-J vaccine, an experiment was conducted in the same manner as in Example 3 except that the water bath heating was set to 100°C and the heating time was set to 10, 20, 30 and 60 minutes, respectively.

[TABLE 2]

As seen in TABLE 2 above, when the heating time went beyond 20 minutes at

100°C, no significant difference was recognized. For this reason, the heating time for preparation of vaccines in Examples below was set to 30 minutes of water bath heating. For convenience of illustration, tumor vaccines in Examples below were simply referred to as "MB-J vaccine" without specifically designating the names thereof.

EXAMPLE 5: Electrophoresis and Western Blotting Analysis of vaccine

In the present experiment, each vaccine and live tumor cells were suspended in a lysis buffer (a mixture of 10ml M-PER mammalian protein extraction reagent and complete mini EDTA-free protease inhibitor cocktail tablet), and the concentration of each protein was measured, then SDS-PAGE was run to ensure the existence of constitutional proteins. 3 μg of protein was loaded onto acrylamide gel and electrophoresis was performed at 20 V for about 2 hours. After completion of the electrophoresis, the gel was strained with Coomassie Blue and underwent decolorized, then dried for storage. For the Western blotting analysis of the protein analyzed by the electrophoresis, used was an antibody, which had been produced through immunization with each vaccine. The antibody used was prepared by the following method: only MB-J vaccine without conventional adjuvants was subcutaneously injected at 5xl0⁵ cells/mouse 3 times at 2-week intervals; 2 weeks after the final immunization, live colon 26-M3.1 carcinoma cells were intravenously injected at 2.7xl0⁴ cells/mouse as a booster immunization; and 14 days after the booster immunization, blood was collected from the immunized mice to separate the serum which was used as an antibody source. The second antibody used for Western blotting analysis was goat-anti-mouse IgG-HRP, which was examined using ECL kit.

The result is disclosed in FIG. 5 in which "(a)" shows the result of electrophoresis and "(b)" shows the result of Western blotting analysis. Also in FIG. 5, Lane M indicates a molecular marker, Lane 1 indicates a liver tumor, Lane 2 indicates

F/T-vaccine, Lane 3 indicates a vaccine heated at 45°C, and Lane 4 indicates MB-J vaccine.

As seen in (a) of FIG. 5, the lysate of live tumor expressed a similar pattern to the lysate of cells heated at 45°C in the electrophoresis analysis. Also, it was ascertained that F/T vaccine comprises many kinds of proteins. Although the differences between the live tumor lysate, 45°C heated-cell lysate, and F/T vaccine could not be exactly compared because they showed many protein bands, F/T vaccine showed relatively fewer protein bands at more than 10 kDa and less than 3 kDa, as compared to the live tumor lysate and 45 °C heated-cell lysate, in which there was no significant difference in view of the result of electrophoresis analysis. On the other hand, MB-J vaccine according to the present invention showed a very simple pattern as compared to these control groups; more specifically, MB-J vaccine expressed five plain bands corresponding to 102 kDa, 78 kDa, 34 kDa, 29 kDa and 28 kDa, which illustrates the difference between MB-J vaccine and other control groups in view of the result of electrophoresis analysis.

In order to identify the characteristics of proteins composing these vaccines,

Western blotting analysis was conducted using the antibody obtained from immunization with MB-J vaccine. The antibody of MB-J vaccine was prepared by collecting the serum from the mice immunized with MB-J vaccine and the antibody was diluted 200-fold for use in experiments.

The Western blot experiment using anti-MB-J vaccine antibody provided an interesting result. More specifically, very few proteins in each vaccine reacted with the anti-MB-J vaccine antibody and they were mainly 62 kDa, 74 kDa and 75 kDa proteins. Firstly, the protein of about 62 kDa, reacting with the antibody, tended to be dependant upon the temperature of heat treatment and expressed a plain protein band as compared to that of 45°C heated-cell lysate as a control group. This 62 kDa protein was not present in the live cell lysate and F/T-vaccine. Therefore, 62 kDa protein is thought to be expressed on heat treatment, and also, the higher the temperature of heat treatment is, the more it is produced. Secondly, the protein of about 74 kDa, reacting with the antibody, was found in three control groups, i.e., live tumor lysate, 45°C heated-cell lysate and F/T-vaccine, but not in MB-J vaccine according to the present invention. On the other hand, the protein of about 75 kDa was found only in MB-J vaccine. Accordingly, one of important proteins composing MB-J vaccine is deemed to be 75 kDa protein reacting with the antibody of MB-J vaccine. This 75 kDa protein was not detected by electrophoresis, but the Western blotting analysis showed that this protein is contained at a small amount in the lysate of MB-J vaccine. Owing to the biochemical characteristics of that protein, the Western blotting result under reducing conditions showed the same pattern under non-reducing conditions, indicating that the structure of the protein is a monomer.

As a result, proteins reacting with the antibody of MB-J vaccine are contained in the vaccine at a small amount and are thus not well detected by electrophoresis, and may be 62 kDa protein, which is increasingly produced as the temperature of heat treatment increases, and/or 75 kDa protein, which is specifically expressed in MB-J vaccine and also anticipated to act as an important material in induction of the anti-tumor immunity.

Example 6: Determination of which or not hsp is present in MB-J vaccine

HSP, expressed when cells undergo stresses such as heat, acts on macrophages and dendritic cells (DC) to produce IL-12, TNF-α, IL-lβ, IL-6, and the like, which are cytokines involved in the innate immune system, thereby ultimately activating APC and NK-cell. Such positive effect on APC induces the maturation of APC to induce the antigen-specific immunity that ultimately activates immune effector cells. For this reason, it is important to find whether or not HSP is expressed from tumor cells during the preparation procedure of tumor vaccine in the method according to the present invention, in view of identification of the tumor antigen. In an experiment to ascertain whether or not HSP is present in MB-J vaccine, the cell lysate of MB-J vaccine was centrifuged at 10,000 rpm for 10 minutes to separate only soluble materials, and after electrophoresis, Western blot was performed using anti-hsp 70 antibody. As control groups, normal cell lysate and 45°C heated-cell lysate were used, respectively.

Cell lines used in the experiment were colon 26-M3.1 carcinoma cell and U937 monocytes cell, and the result is disclosed in FIG. 6. Electrophoresis of both cell lines showed a similar result. As seen in FIG. 6, the normal cell lysate and 45°C heated-cell lysate showed many bands throughout the PAGE gel, whereas MB-J vaccine showed a specific band pattern in both cell lines. In the case of U937 cell, the Western blotting result using anti-hsp 70 showed that a hsp 70 band, meaning the specific reaction with antibody, is present in the normal cell lysate and 45°C heated treated cell lysate, respectively, but not in 100°C heated-cell lysate. In the meantime, in the case of colon 26-M3.1 carcinoma, the Western blotting result did not show any effective band, which is deemed to result from the characteristics of the antibody in this cell line. In summary, it was proved from these experiments that HSP is not expressed, at least when preparing MB-J vaccine with colon carcinomas used, in that even U937 cell did not express hsp 70 when heated at 100°C. Accordingly, the result of the present experiment strongly suggests that the induction of anti-tumor immunity by MB-J vaccine in the following Examples is not caused by HSP.

Example 7: Induction of IL-12 (p40) from DC depending upon heating time

It is well known that HSP stimulates DC to produce IL-12, thereby inducing the activation of NK cells and CD8⁺ T cells. In the present experiment, silica was intraperitoneally administered to balb/c mice to produce DC (DC like cell) and 1.5x10⁴ cells of MB-J vaccine were then administered (DC : vaccine = 1 : 50). After culturing for one day, the supernatant was separated, then IL-12 present in the supernatant was measured. Since IL-12 is produced from mature DC, the ability of DC to produce IL-2 by the co-culture with vaccine can be regarded as an index for the antigen presentation ability of DC and further as an important index for the following immune response.

The result of the experiment is disclosed in FIG. 7. As seen in FIG.7, the ability to produce IL-12 was dependant upon the heating time in co-cultivation of MB-J vaccine and DC, and the peak was achieved at the heating time of 60 minutes, which was not significantly different from the heating time of 30 minutes. In the meantime, although not illustrated in FIG. 7, when DC control group (cultured in media alone) was stimulated by F/T vaccine, 176.1±7.1 pg/ml of IL-12 was measured to be present in a supernatant.

The above result supports the in vivo experimental result concerning the anti-metastasis ability of vaccine depending upon the heating time, as illustrated in TABLE 1 of Example 4. It also suggests that the component which induces maturation of DC to induce the antigen presentation is not HSP but novel proteins, including 75 kDa protein, which newly appears during the preparation procedure of MB-J vaccine, because it was already ascertained in Example 6 (FIG. 6) that HSP is not present in MB-J vaccine. However, it should be noted that the antigen inducing such anti-tumor activity is not necessarily only 75 kDa, protein and there is also a possibility that another protein or a combination of two or more types of proteins, including 75 kDa protein, exert the effect. Example 8: Fraction containing active component in MB-J vaccine

The present experiment was conducted to identify the fraction containing an active component in MB-J vaccine by measuring TNF-α contained in a supernatant, with TNF-α being produced through the stimulation of macrophage by MB-J vaccine. The macrophage used in the present experiment was induced by intraperitoneal injection of 3% thioglycolate (TG) to C57BL/6 mice. Firstly, in order to identify whether the component in MB-J vaccine, inducing the anti-tumor immunity, is soluble or non-soluble, the vaccine was centrifuged at 1000 rpm for 5 minutes to be divided into a pellet part and supernatant part. Then, the precipitate part in which vaccine cells are mainly contained was suspended in an equal volume of PBS. The supernatant part was again centrifuged at 10,000 rpm for 10 minutes to separate a further purified supernatant which was then filtered by filter 0.2 μm. Then, protein was quantified using a protein kit and stored at 4°C for use later. As a result of the protein quantification, protein was contained at a concentration below the detection limit in the supernatant obtained after re-suspension of the pellet in PBS, whereas in the supernatant obtained directly from MB-J vaccine of 5x10⁵ cells/ml, the concentration of protein was 10 - 30 μg/ml (20 μg/ml).

In FIGS. 8 A and 8B, disclosed is an amount of TNF-α that was produced by stimulation of each fraction of MB-J vaccine, respectively. In addition to this experiment, in order to find whether the stimulation of macrophage by MB-J vaccine is caused by endotoxin or not, the amount of TNF-α was also measured after addition of

10 μg/ml polymicin, and the results are illustrated in FIGS. 9A and 9B.

Referring to FIG. 8A, a pellet part of MB-J vaccine showed a higher TNF-α production than MB-J vaccine per se, indicating that cancer cells, having been subjected to structural change during the preparation procedure of MB-J vaccine, were converted into an active component to stimulate the immune system.

The ability to produce TNF-α by a supernatant, containing a soluble antigen, was also estimated in which the supernatant had been obtained after removing a pellet part from MB-J vaccine. The supernatant was divided into a fraction of >50 kDa and a fraction of <50 kDa using an Amicon filter having pores corresponding to 50 kDa. The protein molecular weights of these fractions were measured using a kit and it was ascertained that the fraction containing protein of interest is the fraction of >50 kDa. Thus, the TNF-α production ability was estimated for the supernatant fraction, >50 kDa fraction and <50 kDa fraction, respectively. The result showed that the supernatant fraction and >50 kDa fraction have a significant TNF-α production ability and, in particular, the TNF-α production ability of >50 kDa faction is superior to that of supernatant faction. It is, therefore, inferred that a substance inducing the production of TNF-α has 50 kDa or more.

In the meantime, it was proven that Sur-vaccine also does not have the TNF-α production ability by stimulation of macrophage, although the relevant experiment is not illustrated herein. This result means that a soluble material, secreted from colon cells, has no macrophage-stimulating activity. On the other hand, the experiment result, that the supernatant fraction of MB-J vaccine has the TNF-α production ability, indicates that the material having the immune system-stimulating effect was derived from a tumor cell during the preparation procedure of MB-J vaccine. In other words, it can be inferred that the material having the immune system-stimulating effect was not originally in a supernatant fraction, but some of the material was secreted into the supernatant faction during the preparation procedure of tumor vaccine. In summary, it is inferred that a certain material, including cell wall components, was modified in preparation of MB-J vaccine to acquire the activity of stimulating macrophage and some of the material was dissolved in PBS and the other remained in the tumor cells.

In addition, in order to find whether the stimulation of macrophages by MB-J vaccine was caused by endotoxin or not, the above experiment was repeated after addition of 10 μg/ml polymicin (PM). The result showed that the macrophage-stimulating activity decreases by 70% in the control group ("LPS" in FIGS. 9 A and 9B) treated with PM, but not in the pellet fraction and supernatant fraction of MB-J vaccine, respectively, indicating that the macrophage-stimulating activity by MB-J vaccine was not caused by endotoxin.

Example 9: Anti-metastasis effect upon immunization with whole MB-J vaccine and each fraction thereof.

On the basis of the experimental result in Example 8, in the present experiment, the induction of anti-tumor immunity by entire MB-J vaccine and each fraction, i.e., supernatant fraction and pellet fraction, was estimated in a cancer metastasis model using colon 26-M3.1 cells, and the result is provided in TABLE 3 below.

[TABLE 3]

As seen in TABLE 3 above, the cancer metastasis inhibitory effect was about 73.3% for whole MB-J vaccine, 46.4% for the supernatant fraction, and 79.7% for the pellet fraction, respectively. MB-J whole vaccine and pellet fraction showed anti-metastasis activity coinciding with the result in Example 8 regarding the TNF-α production ability, whereas the supernatant fraction induced only weak anti-metastasis activity, similar to that of Sur-vaccine to be illustrated in Example 10 below. Therefore, it is inferred that a component inducing tumor immunity in MB-J vaccine is present mainly in tumor cells.

Example 10: Anti-metastasis activity of autologous cancer cell by vaccine immunization

Mice used in the present experiment were the autologous Balb/c mice from which the colon 26-M3.1 carcinoma cell line used in preparation of vaccines was derived. Sur-Ag (Surface Ag) as a soluble vaccine was inoculated at 20 μg/mouse, whereas MB-J vaccine, F-vaccine and F/T vaccine, as a whole vaccine containing tumor cells, were inoculated at 5x10⁵ cells/mouse 3 times at intervals of 2 weeks, respectively. Two weeks after the final immunization, the mice were challenged with colon 26-M3.1 tumor cells at 2.0x10⁴ cells/mouse (TABLE 4A) and 2.7 10⁴ cells/mouse (TABLE 4B), respectively, through a tail vein injection. Fourteen days after the tumor inoculation, the mice were sacrificed to separate the lung which was then put into Bouin's solution to stain tumor tissue colonies and measure the number of lung tumor colonies. The result is provided in TABLES 4A and 4B below.

[TABLE 4A]

[TABLE 4B]

As seen in TABLES 4A and 4B above, MB-J vaccine significantly inhibited the metastasis of tumors by more than 95% in both experiments. While F-vaccine, F/T-vaccine and Sur-vaccine as control groups showed some inhibitory effect, in the experiment described in TABLE 4A, they did not show a significant inhibitory effect in the experiment described in TABLE 4B. That is, like the prior art research showing that some anti-tumor activity is induced by immunization with cancer cells, the result of the present experiment showed that F-vaccine, F/T-vaccine and Sur-vaccine as control groups induce some anti-tumor activity in an animal model; more specifically, these control vaccines conferred detectable metastasis inhibition in the experiment that a weak metastasis was applied (TABLE 4A); however, they could not do so in the experiment that a strong cancer metastasis was applied (TABLE 4B). On the other hand, MB-J vaccine according to the present invention induced a high anti-metastasis activity of more than 90% under both experimental conditions, indicating that MB-J vaccine alone can induce an effective anti-tumor immunity.

Example 11: Anti-metastasis activity against heterogeneous cancer cells by vaccine immunization

In order to examine the induction of anti-tumor immune response in heterogeneous mice in an experimental metastasis model, MB-J vaccine of colon 26-M3.1 carcinoma cells (H-2d) was injected to C57BL/6 (H-2b) mice, which are allogenic mice of a different haplotype. Immunization was performed in the same manner as in Example 10. After completion of the immunization, the mice were challenged by tail- vein injection of B16-BL6 melanoma (4.5x10⁴ cells/mouse) derived from C57BL/6 mice, so that the cancer metastasis inhibitory effect could be estimated to find whether the cross-immune response can be induced in heterogeneous cancer cells. The result is presented in TABLE 5 below.

[TABLE 5]

As seen in TABLE 5 above, MB-J vaccine (H-2d) according to the present invention expressed a significant anti-metastasis effect even on B16-BL6 melanoma (H-2b), a tumor cell line derived from C57BL/6 mice which are allogenic mice of a different haplotype, whereas F-vaccine, Sur-vaccine and F/T-vaccine failed to induce such activity. From this result, it is noted that MB-J vaccine can induce an effective anti-tumor immune response even on heterogeneous cancer cell lines so that the vaccine according to the present invention can induce an effective response against diverse cancer cell lines, particularly, those having acquired metastatic ability. This also suggests strongly that the tumor vaccine prepared by the method according to the present invention can also induce an effective immune response with respect to other kinds of cancer lines, not used in Examples herein, particularly, cancers derived from humans.

Example 12: Survival rate of immunized mice

In the present experiment, estimated was the effect of various vaccines, including MB-J vaccine prepared from colon 26-M3.1 cell line, on the survival rate of mice which were first immunized with the vaccines, respectively, and then challenged with colon 26-M3.1 cell line. More specifically, the mice were immunized with each vaccine (5xl0⁵ cells/mouse) at intervals of 2 weeks, and 10 days after the final immunization, colon 26-M3.1 carcinoma cell (2.7x10⁵ cells/mouse) was intraveneously injected to measure the survival rate of mice. Referring to FIG. 10, all mice had died by 27 days after inoculation of tumor (24.5+1.6 days) in the control group and 31 days after inoculation of tumor (26.9±2.2 days) in Sur-vaccine-immunized group, respectively, whereby there was not a significant difference between them. However, in the case of F-vaccine-immunized group generally showing a similar anti-metastasis activity to that of Sur-vaccine-immunized group, some mice survived up to 66 days after inoculation of tumor, In other words, the Sur-vaccine-immunized group was ascertained to have a survival effect of only 4 days, statistically identical to the control group, but the F-vaccine-immunized group was ascertained to have a significant survival rate improvement of as compared to the control group. Such difference between Sur-vaccine and F-vaccine cannot be clearly explained but is anticipated to result from the activation of NK-cells, when considering the result of Example 14 below.

In addition, the following inference may be made in connection with the above result: although the soluble Sur-vaccine has immunogenic activity, the antigen thereof is immediately removed in vivo by macrophages; however, the antigen of F-vaccine can remain in vivo for a certain time. The lengthening of survival time by immunization with F-vaccine is deemed to be attributed not to the enhancement of the antigen-specific immunity but to the activation of NK-cells due to extension of the serum survival time, as suggested by the result of Example 14 below. A clearer examination regarding this theory should be carried out in the future, but if the above inference is correct, it would suggest that immunization with a whole vaccine may be more useful for induction of the anti-tumor immunity than immunization with a soluble antigen, and that the tumor vaccine of the present invention as a whole vaccine itself can serve to enhance the immunity against tumor.

Meanwhile, MB-J vaccine showed excellent immunogenicity, as compared to other vaccines including F-vaccine, so that 70% of mice survived even 100 days after inoculation of tumor cells, thus it can be seen that a strong anti-tumor activity was induced by MB-J vaccine. As will be illustrated in Example 14 below, it was ascertained that the tumor metastasis inhibitory effect is not associated with the activation of NK-cells, thus the lengthened life of tumor-bearing mice by immunization with MB-J vaccine is anticipated to result from induction of antigen-specific anti-tumor immunity associated with B cells and T cells, which will be discussed later.

Example 13: Duration of anti-tumor activity

In the present experiment, to the mice that survived by immunization with MB-J vaccine in Example 12, cancer was re-inoculated, and after 14 days, the number of cancer colonies generated was counted. If the tumor-specific anti-tumor immunity was induced in the mice immunized with MB-J vaccine, when the same antigen, colon26-M3.1 carcinoma is re-inoculated later, an effective protection effect will be induced. Accordingly, an experiment testing the induction ability of anti-tumor immunity was performed by applying the surviving mice of Example 12 and new mice, as a control group, of the same age as the MB-J vaccine-immunized mice, to an experimental metastasis model. The results are presented in TABLE 6 below.

[TABLE 6]

As seen in TABLE 6 above, the generation of cancer colonies was inhibited by 99.1% in the mice which were first immunized with MB-J vaccine and subjected to inoculation of cancer cells, and 100 days thereafter again subjected to inoculation of cancer cells. This result indicates that immunization with MB-J vaccine induces antigen-specific immunity against tumors in a host, and that the immunity can last at least for 100 days. Accordingly, it was clearly proven that immunization with MB-J vaccine could induce anti-tumor immunity in a host, and that once MB-J vaccine is administered the host acquires antigenic memory, which suggests a possibility for the application of MB-J vaccine in preventive inoculation. In summary, it is anticipated that immunization with MB-J vaccine allows some of immune cells to be converted to memory cells so that tumor immunity is induced upon re-exposure to tumor cells, thereby fighting the tumor.

Example 14: Metastasis inhibitory effect of tumor vaccine in NK-cell-knockout mice

As examples of effector cells expressing anti-tumor activity, there are NK-cells as tumor-nonspecific cells and cytotoxic T-cells as tumor-specific cells. The present experiment was conducted to find whether an effector cell induces the anti-tumor activity by each tumor vaccine by a mechanism involving a tumor-specific immune cell or other mechanism, for example, a tumor-nonspecific NK-cell, etc. The immunization schedule of each vaccine and kind of mice used were the same as those in Example 10. In order to remove NK-cells from mice, anti-asialo GM-1 antibody was used. More specifically, three days and one day before inoculation of tumor cells, respectively, 20-fold diluted anti-asialo GM1 antibody was administered to mice by tail vein injection and the anti-metastasis activity induction was estimated. The result is disclosed in FIG. 11.

Referring to FIG. 11, about 157 tumor colonies were generated in the lungs of the normal mice, which were not treated with anti-asialo GM1 antibody and to which only tumor was inoculated. To the contrary, in the mice treated with anti-asialo GM1 antibody, about 371 tumor colonies were generated, such that it was confirmed that removal of NK-cells by treatment of the antibody was successfully achieved. Furthermore, when the normal mice were immunized with MB-J vaccine, only about 4 tumor colonies were generated, thereby supporting that MB-J vaccine induces an excellent immune response. Also in the case of immunization with F-vaccine, about 21 tumor colonies were generated, indicating a significant anti-metastasis activity of F-vaccine, which was however less than that of MB-J vaccine. This result is similar to that of Example 9.

However, these vaccines showed a big difference in immunization of NK-cell-deleted mice; more specifically, 4 tumor colonies were generated in immunization with MB-J vaccine, whereas 171 tumor colonies were generated following immunization with F-vaccine. This means that the induction of anti-metastasis activity by immunization with MB-J vaccine and F-vaccine, respectively, is based upon different mechanisms, which is important in view of the following experiments. In other words, it is inferred that the immunity conferred by MB-J vaccine allows the tumor-specific immune system, not associated with the activation of NK-cells to be activated, while the induction of anti-metastasis activity by F-vaccine is associated with NK-cells reacting with anti-asialo GM1 antibody.

Example 15: Measurement of ability of macrophages to produce TNF-α and induce proliferation of PEC.

Experiments regarding the stimulation of peritoneal exudative cell (PEC) were conducted in two ways: (a) direct stimulation to the PEC of normal mice and (b) stimulation to the PEC by intraperitoneal injection of 3% thioglycolate FIG. 12 shows the amount of TNF-α in a supernatant, induced after stimulation of the PEC of normal mice by each vaccine. It is well known that macrophages, present in the peritoneal space of normal mice, are in a mature form or immature form. Therefore, the PEC were extracted from normal mice and plated in vitro to separate only cells, having the characteristic of macrophages, attached to a plate, followed by addition of MB-J vaccine, F-vaccine and F/T-vaccine, respectively, and cultured together with PEC for 3 days. After completion of the culture, the activation of PEC was estimated by measuring the amount of TNF- α and the proliferation thereof by MTT assay. TNF-α serves to convert pre-effector T cells into immunocompetent T cells and increase the production of IFN-γ from T cells so that the anti-tumor activity is induced. As such, the ability to produce TNF-α from PEC, including macrophages, conferred by MB-J vaccine, is believed to affect directly the induction of tumor antigen-specific immunity. As seen in FIG. 12, MB-J vaccine expressed the highest TNF-α production ability upon of PEC of normal mice, and F-vaccine and F/T-vaccine also expressed a TNF-α production ability to an extent less than 50% of that of MB-J vaccine. Also in the experiment regarding the proliferation of PEC upon treatment of vaccine, as seen in FIG. 13, the result similar to the TNF-α production ability was observed.

FIG. 14A shows the result of an experiment involving the stimulation of macrophages induced by intraperitoneal injection of 3% thioglycolate (TG). For the experiment, 1 ml of TG was intraperitoneally injected to mice, and after 3 days, PEC was extracted and plated on a 24-well plate at a concentration of 1.5xl0⁶ cells/ml per well, and after culturing for 2 hours, the adhered cells were washed with a culture medium. Then, each vaccine, including MB-J vaccine, was added at a concentration of 5xl0⁵ cells/ml per well and cultured for 24 hours. Then, a supernatant was separated to measure the amount of cytokines produced using ELISA kit. As seen in FIG. 4A, in case of co-stimulation by MB-J vaccine and live colon cell, a high level TNF-α production of 2767.9 and 2322.6 pg/ml was obtained, whereas F-vaccine and F/T-vaccine as control vaccines showed 686.1 and 791.3 pg/ml, respectively. Accordingly, the TNF-α production following the TG stimulation of macrophage was three times higher in the case of MB-J vaccine than other vaccines, which shows the same tendency as the normal macrophage not treated with TG. In the meantime, Sur-vaccine showed TNF-α production of less than 10 pg/ml, similar to that of a control group that was cultured with only culture medium. The above result means that MB-J vaccine has a strong ability to stimulate macrophages, as compared to control vaccines.

In addition, FIG. 14B shows the production levels of IL-12, similar to that of TNF-α. IL-12 is a cytokine produced mainly from activated macrophages and dendritic cells (DC) and acts in activation of NK-cells and Thl type cells, thus playing an important role in activation of the natural and acquired immune response. The fact that MB-J vaccine induces a high level of IL-12 production is important in illustrating the mechanism of antigen-specific anti-tumor immunity induction, including the activation stage of macrophages as an initial stage of immune response.

Example 16: Re-stimulation of spleen cell and induction pattern of cytokine by vaccine in vaccine-immunized mice

For experiments regarding re-stimulation of spleen cells, MB-J vaccine and F-vaccine were used at a concentration of 5x10⁵ and 5xl0³ cells/mouse, respectively, and intraderminally injected 3 times at intervals of 2 weeks. 10 days after the final immunization, MB-J vaccine was added at a density of lxlO⁵ cells/well to spleen cells (5xl0⁵ cells/well) extracted from each immunized mouse, and after re-stimulation for 3 days, the induction of cell proliferation was estimated by MTT assay. As a control group, Con- A (final concentration: 5 μg/ml) as a mitogen of T cells was used. The result is disclosed in FIG. 15.

In a Con-A-treated group, the spleen cells extracted from the normal mice and vaccine-immunized mice expressed similar proliferation rates, indicating that this re-stimulation experiment progressed normally. According to the experimental result of MB-J vaccine re-stimulation, proliferation of spleen cells was recognized in the mice immunized with 5xl0⁵ and 5xl0⁴ cells of MB-J vaccine, respectively, but not in the mice immunized with 5x10 cells of MB-J vaccine, so that the proliferation activity was dependent upon the concentration of vaccine used for immunization. On the other hand, a control group immunized with F-vaccine expressed a similar result to that of the normal mice, whereby the re-stimulating activity was not induced.

In that the re-stimulation activity is induced in only the MB-J vaccine-immunized group and not in the F-vaccine-immunized group, the induction of cell-mediated immune response by MB-J vaccine can be explained and the in vivo results of Examples 13 and 14 can also be supported. In addition, since the proliferation activity of spleen cells by re-stimulation of antigen was not induced in the case of F-vaccine, which was ascertained to express anti-tumor activity mainly due to the activation of NK-cells in the previous experimental metastasis model, the induction pattern of cytokine in a supernatant, produced through the immunity enhancement mechanism of MB-J vaccine, was estimated, as described below. The induction pattern of cytokines was measured after re-stimulating the spleen cells of MB-J vaccine and F-vaccine-immunized mice, respectively, with MB-J vaccine for 3 days. Such an antigen-specific (vaccine-specific) cytokines are classified into Thl type cytokine and Th2 type cytokine depending upon T cell. Thl type cytokines include IFN-γ, GM-CSF, TNF-α, etc. and Th2 type cytokines include IL-4, IL-10, etc. Thl type cytokines are known to induce cell-mediated immunity, including activation of CD8⁺ CTL cell, and Th2 type cytokines are known to enhance mainly the humoral immunity involved in the production of antibody. According to the present experiment, comparing (i) the induction pattern of cytokines after culturing spleen cells in a culture medium without re-stimulation by MB-J vaccine and (ii) the induction pattern thereof after re-stimulation by MB-J vaccine, the production of cytokines was increased by re-stimulation of MB-J vaccine and the tendency to increase applies to all types of cytokine. More specifically, in both Thl type cytokines (IFN-γ, GM-CSF and IL-2) and Th2 type cytokines (IL-4, IL-10 and IL-6), the MB-J vaccine-immunization showed a good induction pattern, as compared to the F-vaccine-immunization, and the induction activity was dependent upon the concentration of MB-J vaccine used for immunization (refer to FIGS. 16A ~ 16D and TABLE 7, the restimulation experiment results being illustrated in FIGS. 16B and 16D). Particularly, among diverse cytokines, the production of IFN-γ, directly involved in the activation of CTL, and the production of GM-CSF, affecting directly the antigen presentation by DC as an antigen-presenting cell, were greater in the spleen cells of the MB-J vaccine-immunized mice. For this reason, it is deemed that the anti-tumor activity of MB-J vaccine is attributed mainly to the direct activation of CTL or the activation of CTL by cross-priming via helper T cells. Meanwhile, it is inferred that production of Th2 type cytokines by immunization with MB-J vaccine will be connected with the production of a tumor-specific antibody later, and that this antibody will induce the anti-rumor activity mainly by antibody dependent cellular cytotoxicity (ADCC). For reference, some researchers have reported that Th2 type cytokine also participates in enhancement of cell-mediated immunity, thus it should not be excluded that Th2 type cytokine induced by the vaccine of the present invention may act as a complement for the induction of CTL.

[TABLE 7]

Example 17: Induction pattern of cytokines in co-culture of spleen cells and cancer cells, with the spleen cells separated from mice subjected to immunization and cancer inoculation

In the present experiment, live tumor cells (colon 26-M3.1 cells) were inoculated into mice immunized with each vaccine, and spleen cells were separated from the mice, then the spleen cells and live tumor cells (colon 26-M3.1 cells; lxlO³ cells/well) were co-cultured so that the induction pattern of cytokines could be estimated. MB-J vaccine used in the present experiment was an inactivated cancer cell of colon 26-M3.1 carcinoma. It was already ascertained in previous experiments that immunization with MB-J vaccine induces proliferation of spleen cells, and that the mechanism thereof is based upon the production of Thl type and Th2 type cytokines. However, MB-J vaccine should ultimately induce the activation of effector cells against cancer cells to induce protection against the metastasis and proliferation of tumors in vivo. For this reason, in order to ascertain that the immune response to a live cancer cell, colon 26-M3.1 cell, is induced by immunization with MB-J vaccine, the reactivity of the spleen cells of MB-J vaccine-immunized mice against live tumor cells was estimated in view of the induction pattern of cytokines as an immunity-mediated material in the present experiment.

According to the result of the experiment, re-stimulation by live tumor cells induced a large amount of INF-γ to be produced from the spleen cells of the MB-J vaccine-immunized mice and the F-vaccine or F/T-vaccine-immunized mice. More specifically, the spleen cells as a control group, not re-stimulated, expressed the same tendency as in Example 16, and the production of IFN-γ was in order of MB-J vaccine-immunized mice, F-vaccine or F/T-vaccine-immunized mice, and normal mice. The stimulation of live tumor cells to the MB-J vaccine-immunized mice induced about 10 times the amount of INF-γ than without stimulation, thus it was ascertained that a high level of immune response, particularly to live tumors, can be induced by immunization with MB-J vaccine. For instance, it was confirmed that the MB-J vaccine immunization induces the anti-tumor immune response sufficient to inhibit the metastasis of tumors by more than 90%, as illustrated previously in the in vivo experimental result.

In the meantime, F-vaccine and F/T-vaccine immunization also increased the production of IFN-γ by re-stimulation of live tumor cells, but the amount was less than 1/2 of that by MB-J vaccine immunization. This result shows that, as in the experimental result regarding survival rates of mice, F-vaccine immunization induces less anti-tumor immune response than MB-J vaccine immunization, but confers partial anti-tumor protection as compared to Sur-vaccine immunized and non-immunized control groups, and that as mentioned by other researchers, an inactivated whole tumor vaccine (apoptotic or necrotic cell) induces partial anti-tumor immune response.

This partial immune protection also appeared, as seen in FIG. 16C, in the production pattern of GM-CSF, another Thl type cytokine enhancing the antigen presentation ability of APC. It should, however, be noted that even in the case of spleen cells in only culture medium without re-stimulation of live cells, MB-J vaccine caused higher production of GM-CSF than F-vaccine and F/T-vaccine. As a result, it is inferred that MB-J vaccine-immunized mice undergo continuous antigen presentation against live tumors, to induce the immunity so long as the tumors are present in vivo.

In addition, it is known that IL-4 and IL-10 are Th2 type cytokines inhibiting the activation of CD8⁺ T cells and inducing antibody production by B cells; however, in some recent research, it was found that these cytokines are connected with induction and activity maintenance of cytotoxic CD8⁺, so a new dispute about the function of these cytokines is raised. Although the induction pattern of these cytokines cannot be now clearly illustrated in connection with the induction of anti-tumor immunity by MB-J vaccine according to the present invention, the tumor metastasis inhibitory effect in vivo resulting from the induction of anti-tumor immunity by MB-J vaccine may include, as seen in FIG. 17A, the activation of B cells by Th2 type cytokines such as IL-4 and IL-10 and also the activation of CTL as a complement. In particular, the induction pattern of IL-10, as seen in FIG. 17B, was improved in the case of re-stimulation by live tumor than in the case of spleen cells alone, in all of the normal control group, F-vaccine-immunized control group and MB-J vaccine-immunized group; however, in view of the amount of IL-10 induced, F-vaccine and F/T-vaccine-immunized control groups showed similar results, whereas the MB-J vaccine-immunized group showed more than double the production level of cytokines as compared to these control groups.

Example 18: Proliferation of spleen cells by re-stimulation of vaccine after induction of tumor metastasis in vaccine-immunized mice

From each vaccine-immunized mouse, spleen cells were extracted to perform a re-stimulation experiment. Vaccines used in the experiment were MB-J vaccine, F/T-vaccine and F-vaccine, and immunization was carried out three times for each vaccine. Two weeks after the final immunization, colon 26-M3.1 carcinoma was inoculated into mice, and 2 weeks after the inoculation, the mice were sacrificed to separate the spleen, which was homogenized and aliquoted into 96-well plates at a density of 2.5x10⁵ cells per 100 μl well. As control groups, used were (i) the spleen cells of non-immunized normal mice and (ii) the spleen cells of non-immunized mice and into which tumor cells were inoculated. Experiments were conducted using a group in which spleen cells were treated with only culture medium, a group in which spleen cells were re-stimulated by B-vaccine (10⁴ cells/well), and a group in which spleen cells were re-stimulated by Con-A (final concentration: 5 μg/ml) as a mitigen of T cells. The culture period was 3 days and the proliferation of spleen cells was measured by MTT assay.

Referring to FIG. 18, the result shows the same tendency as after vaccine immunization in Example 16. More specifically, the spleen cells in the normal control group, i.e., culture medium-treated group, expressed the OD value of approximately 0.33, whereas the spleen cells in the other control groups, i.e., tumor-treated control group, F/T-vaccine-immunized control group and F-vaccine-immunized control group, expressed the OD value of approximately 0.37 ~ 0.40, a slightly higher proliferation activity than the normal control group, but not a significant difference. On the other hand, the spleen cells in MB-J vaccine-immunized group expressed the OD value of approximately 0.64, whereby a significant proliferation activity was seen, as compared to the normal control group, regardless of whether re-stimulating agents were present. Since this result was obtained after inoculation of live cancer cells into the vaccine-immunized mice, the spleen cells of MB-J vaccine-immunized group must maintain the already activated condition, even without re-stimulation by vaccine, due to the continuous stimulation by tumor, whereas a significant immune response is not induced against live tumor cells in the case of other vaccines used as control groups.

Meanwhile, according to the result of an experiment testing the re-stimulation of spleen cells with MB-J vaccine in vitro, the spleen cells in the normal control group, tumor control group, F/T-vaccine-immunized group, and F-vaccine-immunized group failed to proliferate, whereas the spleen cells in the MB-J vaccine-immunized group expressed an increased OD value with respect to the re-stimulant, thus it was ascertained that MB-J vaccine has the antigen-specific spleen cell-proliferation activity. In addition, the use of Con-A as a T cell stimulant induced a significant proliferation of immune spleen cells in all groups applied to the present experiment. As a result, it was ascertained that re-stimulation using only MB-J vaccine can induce the tumor-specific proliferation of spleen cells.

Example 19: Production of antibody after induction of tumor metastasis in vaccine-immunized mice

After the cancer metastasis experiment using vaccine-immunized mice was finished, the serum antibody level against tumor antigen was measured using ELISA. More specifically, the aqueous fraction of live colon tumor cells was separated by centrifugation to measure the concentration of protein using Bio-Rad protein assay kit. The supernatant of live colon tumor cells was plated in each well of an ELISA plate, at a protein concentration of 500 μg/ml, using a coating buffer (pH 8.6 bicarbonate buffer) and was fixed with 100% methanol, then serum extracted from mice in each group was added thereto at 300-fold dilution. Then, a conjugate of HRP plus a second antibody against with IgGl or IgG2 was added to examine the colormetric response using TMB solution (Sigma, Ltd.). The reaction was stopped by adding 2M H₂SO₄ to read the absorbance at 450 nm.

The result is disclosed in FIGS. 19A and 19B. The antibody level was the highest in MB-J vaccine-immunized mice group and showed similar pattern in the other control groups. This result was obtained on the basis of the 300-fold dilution of antiserum. As seen in FIGS. 19A and 19B, the antibody levels of IgGl type and IgG2 type, in the normal control group, tumor-treated control group, and F-vaccine-immunized group, were similar, whereas the antibody level in MB-J vaccine-immunized group was significantly higher. In summary, although the present experiment does not show what role the tumor-specific antibody produced plays in vivo, it can be seen at least that MB-J vaccine according to the present invention induces the humoral immunity against tumor.

Example 20: Treatment and survival efficacy of cancer-bearing host by vaccine immunization in spontaneous metastasis model

In a spontaneous metastasis model in which C57BL/6 mice were challenged with B16-BL6 melanoma by footpad inoculation, the inhibitory effect of F/T-vaccine and MB-J vaccine immunization on the proliferation and metastasis of tumors was estimated. For the vaccine immunization, MB-J vaccine prepared using B16-BL6 was administered at 5x10⁵ cells/mouse 3 times at intervals of 2 weeks, and 10 days after the final immunization, live B16-BL6 was subcutaneously injected into the footpad of mice at 5xl0⁵ cells/mouse. 21 days after the inoculation of tumor cells, the tumor-transplanted region ("primary lesion" or "primary tumor") was separated to measure the size of tumor, and after 14 days after the separation of primary lesion, the mice were sacrificed to count the number of tumor colonies having metastasized to the lung. The result is provided in TABLE 8 below.

[TABLE 8]

As seen in TABLE 8 above, F-vaccine and F/T-vaccine partially inhibited the size of primary lesions but not the spontaneous metastasis from the primary lesion. On the other hand, MB-J vaccine significantly inhibited the size of primary lesions and simultaneously the metastasis of tumors by 83.9% as compared to the control groups, whereby it was ascertained that immunization with MB-J vaccine confer a significant inhibition of the proliferation and metastasis of tumors.

Example 21: Prevention of tumor metastasis by the adoptive immunity of spleen cells obtained from vaccine-immunized mice.

In order to confirm that the anti-tumor immunity conferred by MB-J vaccine is induced by tumor-specific effector cells, the adoptive immunization was performed using the spleen cells of vaccine-immunized mice. More specifically, MB-J vaccine and

F/T-vaccine were subcutaneously injected 3 times to Balb/c mice, respectively, and 10 days after the final immunization, live colon 26-M3.1 cells were intravenously injected at 2.5xl0⁴ cells/mouse, and 14 days after the inoculation, the anti-tumor activity granted by the adoptive immunity was estimated using the spleen cells obtained from the mice. For measurement of the anti-tumor activity due to the adoptive immunity, employed was the experimental metastasis model using colon 26-M3.1 carcinoma. More specifically, live colon 26-M3.1 cells were intravenously injected to the normal mice, and 3 days after this inoculation, the spleen cells, which were separated from each vaccine-immunized mice, were intravenously injected at 2xl0⁶ cells/mouse. The result of the experiment is provided in TABLE 9 below.

[TABLE 9]

As seen in TABLE 9, no therapeutic effect by the adoptive immunization, using the spleen cells of normal mice or the spleen cells obtained from the mice 14 days after the inoculation of tumor, was deserved, as compared to the control group.

In the meantime, the adoptive immunization, using the spleen cells obtained from the mice which survived after subcutaneous immunization of MB-J or F/T-vaccine and then intravenous inoculation of tumor cells, was recognized to inhibit tumor metastasis up to 90.9% only in the MB-J vaccine-treated group, whereby it was ascertained that MB-J vaccine induces the tumor-specific cell-mediated immunity. Example 22: Tumor metastasis inhibitory effect in CD4⁺ or CD8⁺ T cell-knockout mice

In the present experiment, in order to elucidate the mechanism of anti-tumor immunity by MB-J vaccine, CD4⁺ or CD8⁺ T cells were removed from mice using antibody against each cell. First, 3 days and 4 days before immunization of MB-J vaccine, respectively, 200 μl of anti-CD4 (rat IgG2b) or anti-CD8 (rat IgG2b) antibody was intravenously injected, respectively. Each antibody was again administered 3 days, 7 days and 10 days after vaccine immunization to entirely remove those cells. Immunization of vaccine was carried out 3 times at intervals of 2 weeks, and isotype-matched rat IgG was administered to the control group.

As seen in TABLE 11 below showing the result of the experiment, the cancer metastasis inhibitory effect disappeared almost completely in CD8⁺-knockout mice and by about 59.7% in CD4⁺-knockout mice. This result suggests that the immunity induction by MB-J vaccine is attributable to a mechanism associated with CD4⁺ or CD8⁺ effector T cells.

[TABLE 11]

INDUSTRIAL APPLICABILITY

As described above, the tumor vaccine prepared by the method according to the present invention provides enhanced immunity against tumors, having preventive and therapeutic activity versus diverse tumors. This tumor vaccine enhances mainly the antigen presentation activity of APC to allow the tumor-specific CD4⁺ or CD8⁺ T cell to be activated, thereby inducing the humoral and cell-mediated immune response. Moreover, the tumor vaccine of the present invention has cross-reactivity with respect to tumors so that it can induce the immune response against a variety of tumors, thereby expressing a vaccine effect for preventing the proliferation and metastasis of tumors, and also a therapeutic effect against existing tumors.

Other embodiments and uses of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered exemplary only, with the scope of particular embodiments of the invention indicated by the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method of preparing a tumor vaccine, comprising heating tumor cells derived from a tumor to provide inactive tumor cells and/or tumor antigens obtained from the inactive tumor cells, capable of enhancing immunity against tumors.

2. The method according to claim 1, wherein the tumor is a primary tumor or malignant tumor metastasizing from the primary tumor.

3. The method according to claim 1, wherein the heat treatment is performed by water bath heating, pressure sterilization, or wet sterilization.

4. The method according to claim 1, wherein the heat treatment is performed in the range of more than 45°C and for at least 5 minutes.

5. The method according to claim 4, wherein the heat treatment is performed in the range of 60 ~ 130°C and for 10 ~ 60 minutes.

6. The method according to claim 1, wherein the method further includes one or more steps selected from the group consisting of (a) a step of culturing the tumor cells derived from the tumor under an appropriate condition to proliferate the cells, (b) a step of sonicating the tumor cells during and/or after the heat treatment, and (c) a step of purifying tumor antigens from the inactivated tumor cells.

7. The method according to claim 1, wherein the inactivated tumor cells are in an intact form, maintaining the original cell membrane, or in a lysate form.

8. The method according to claim 1, wherein the inactivated tumor cell includes monovalent or multivalent antigens, which are one or more tumor antigens selected from 45, 57, 62, 74 and 75 kDa proteins obtained after heat treatment, providing immunity against tumors and/or inhibiting tumor proliferation and metastasis.

9. The method according to claim 1, wherein the tumor antigen is an antigen protein having been subjected to denaturation by the heat treatment, or an antigen protein having been newly exposed from the surface of cells by the heat treatment.

10. The method according to claim 1, wherein the tumor antigen serves to present an antigen through MHC of the antigen-presenting cell (APC), thereby inducing mainly the cell-mediated immune response in a subject being immunized.

11. A pharmaceutical composition for prevention and treatment of tumors, comprising (a) a therapeutically effective amount of the inactivated tumor cell and/or tumor antigen obtained therefrom according to claim 1, and (b) a physiologically acceptable carrier, diluent, or excipient, or a combination thereof.

12. A method of enhancing the immunity of a subject being treated against tumors by administering to the subject a therapeutically effective amount of the inactivated tumor cell and/or tumor antigen (tumor vaccine) according to claim 1.

13. The method according to claim 12, wherein the method comprises a step of obtaining tumor cells from a subject and a step of preparing the inactivated tumor cells and/or tumor antigens from the tumor cells according to the preparation method of claim 1 to administer them to a genetically syngeneic subject being treated; or a step of obtaining tumor cells from a subject and a step of preparing the inactivated tumor cells and/or tumor antigens from the tumor cells according to the preparation method of claim 1 to administer them to a genetically allogeneic subject being treated.

14. The method according to claim 12, wherein the subject is a vertebrate animal.

15. The method according to claim 14, wherein the vertebrate animal is a human.

16. The method according to claim 12, wherein the method further includes administering only APC stimulated by the active component, or together with the active component.

17. The method according to claim 12, wherein the method is an immunization method achieved either by one administration of the tumor vaccine or by continuous administration of the tumor vaccine.

18. The method according to claim 12, wherein the tumor vaccine is used for the immunization against different kinds of tumors.

19. A method of administering a therapeutically effective amount of the inactivated tumor cell and/or tumor antigen according to claim 1 to a subject being treated to prevent and/or treat tumors.