A tumor vaccine which comprises a microparticle or a lysate prepared from a solidified tumor material selected from the group consisting of a tumor tissue, a tumor cell, and a component thereof, and at least one cytokine and/or cytokine-inducing agent (e.g., a granulocyte-macrophage-colony stimulating factor and/or interleukin-2 and the like), and optionally an adjuvant. The vaccine can be easily prepared and widely applied for prevention of recurrence, inhibition of metastasis and therapeutic treatment regardless of a type of a tumor, and has excellent antitumor effect.

TECHNICAL FIELD

The present invention relates to a tumor vaccine useful for prevention of recurrence, inhibition of metastasis and therapeutic treatment of tumors.

BACKGROUND ART

The tumor vaccine therapy is to activate immune system in vivo, particularly killer lymphocytes that play a key role in cellular immune responses, especially cytolytic T lymphocytes (hereinafter abbreviated as "CTL"), to specifically kill tumor cells without damaging normal cells, and to expect prevention of recurrence of the tumor, inhibition of metastasis, or cure of the established tumor.

Various kinds of tumor vaccines have been developed (Pardoll, D. M., Nature Med., 4(5 Suppl), pp. 525-531, 1998). Roughly tumor vaccines can be categorized depending on tumor-specific materials as follows: (1) vaccines wherein a tumor antigenic peptide with a known property is used; (2) vaccines wherein a tumor tissue extract containing an unidentified tumor antigenic peptide is used; (3) vaccines wherein the above peptide is bound to an antigen-presenting cell, especially a dendritic cell with a strong capability of antigen presentation (Nestle, F. O., et al., Nature Med., 4, pp. 328-332, 1998); (4) vaccines wherein a tumor antigenic protein is taken into a dendritic cell and loaded; (5) vaccines wherein a dendritic cell and a tumor cell are fused; (6) vaccines wherein a tumor antigen is bound to a liposome for uptake together with the liposome (Nakanishi, T., et al., Biochem. Biophys. Res. Comm., 240, pp. 793-797, 1997); (7) vaccines wherein a tumor cell, per se, is treated for inactivation with radiation or a fixing agent before administration; (8) vaccines wherein a cytokine gene, having an antigen-presenting cell stimulating effect or a lymphocyte stimulating effect, is introduced into a tumor cell and the cell is administered as a vaccine for a gene therapy, or wherein a tumor antigenic gene is introduced into a suitable cell and a tumor cell expressing the gene is administered as a vaccine; (9) vaccines wherein a tumor antigenic gene is integrated into a virus or a bacterium for infection of a patient; (10) vaccines wherein a live tumor cell, a tumor antigenic peptide or an extract of a tumor cell is administered, and separately a great amount of a cytokine is administered (Rosenberg, S. A., et al., Nature Med., 4, pp. 321-327, 1998), or wherein a cytokine is formulated into a controlled release preparation and administered (Golumbek, P. T., et al., Cancer Res., 53, pp. 5841-5844, 1993) and the like.

However, any of the above tumor vaccines is advantageous from some aspects while disadvantageous from other points of view. For example, Method (1) can only be applied to tumors which express a specific major histocompatibility complex (hereinafter abbreviated as "MHC", and for Class I referred to as "MHC-I" and for Class II as "MHC-II") that meets to an identified tumor antigenic peptide. The human MHC is highly diverse, and consequently, clinical cases are very limited in which those tumor antigenic peptides can meet the MHC. To overcome the problem, Method (2) using a tumor tissue extract containing an unidentified tumor antigenic peptide has been developed. However, only a trace amount of the tumor antigenic peptide can be extracted from tumor tissues, and it is often impossible to concentrate the extract when the amount of the tumor as a raw material is small. Therefore, the extract cannot be administered in a large amount such as identified and synthesized tumor antigen peptides, and effects are limited.

Where a tumor antigenic peptide is bound to an antigen-presenting cell beforehand, such as in Method (3), a high CTL activating effect is obtained. However, peripheral blood or bone marrow for isolation and preparation of the antigen-presenting cell, especially a dendritic cell having strong antigen presenting capability, should be derived from the patient who bears the tumor and is to be applied with the tumor vaccine therapy to prevent dangerous graft-versus-host-disease (hereinafter abbreviated to "GVHD"), which requires a highly skilled technique and is complicated. Methods (4) and (5) have the same problem as that of Method (3), and in addition, a fusion process is very complicated in Method (5). Although there is no concern about the risk of GVHD in Method (6), an efficiency of the introduction of the tumor antigen into the antigen-presenting cell is sometimes not successfully high, and a relatively great amount of the tumor antigen is required to prepare the tumor vaccine.

Method (7) is also complicated and costly because the tumor cells are obtained by mass culture, and moreover, the method has a problem in that the amount of the tumor antigen contained in the tumor cells per se is very small. This method is known to be successful in tumor cells with high antigenicity when treatment with poly(L-lysine) is applied (Naito, M. and Seno, S., Cell Biol. International Rep., 5, pp. 675-681, 1981). However, the method remains unsuccessful in tumor cells with low antigenicity. Genetic therapies of Methods (8) and (9) are extraordinarily complicated in procedures to obtain approval for the treatment, as well as in therapeutic operations. Method (10) is promising at present; however, especially in the method of Rosenberg et al., a huge amount of interleukin-2 simultaneously administered causes a severe side effect, and clinical results for tumors treatment are sometimes not satisfactory. Even when cytokines are formulated as controlled release preparations by the method of Golumbek et al., a complication still remains in preparation of X-ray-irradiated live tumor cells.

The tumor vaccine is desirably provided in a form that can be handled as easily as possible. From this point of view, methods involving administration of live tumor cells or antigen-presenting cells as a part of a tumor vaccine have problems in that they are technically very complicated as operations under a live state are required. The operations are further complicated for a genetic therapy. When tumor antigenic peptides are known, the peptides can be synthesized in large quantities for administration. However, there are a large variety of tumor antigenic peptides, and additionally, due to restriction from MHC molecules of a patient individual, it often cannot be appropriately determine which of tumor antigenic peptides is applicable to the patient individual, which may limit the application. When a tumor antigenic protein is used instead of the tumor antigenic peptide, the protein is processed in the antigen-presenting cells and then a tumor antigenic peptide that meets the MHC is selected. Accordingly, the method is not restricted by the MHC of a patient individual to be treated. However, this method has a problem in that purification and large-scale preparation of the tumor antigenic protein, per se, is difficult.

As a method for inducing CTL, a method is known in which CTL is induced from peripheral blood mononuclear cells on a fixed tumor tissue obtained by removing paraffin from pathological sections (Liu, S. Q. et al., Nature Med., 2, pp. 1283-1283, 1996). Generally, when an antigenic protein in a soluble state is provided to antigen-presenting cells, the protein has a high stimulating effect on liquid immunity that links to production of antibodies by binding antigenic proteins-derived antigen peptides to MHC-II, whereas the protein has a low stimulating effect on cellular immunity that activates killer cells by binding antigenic proteins-derived antigen peptides to MHC-I. Falo et al. conducted induction of CTL that react to ovalbumin-derived antigenic peptides by binding ovalbumin as a foreign protein with strong antigenicity to iron powder and administering the product to mice without addition of an adjuvant (Falo, Jr., L. D., et al., Nat. Med., 1, pp. 649-653, 1995).

The inventors of the present invention found that CTL can be induced efficiently from peripheral blood lymphocytes of the same individual by fixing soluble tumor antigenic proteins on fine polystyrene beads and subjected the product as fine solids to phagocytosis by antigen-presenting cells in human peripheral blood mononuclear cells in a cell culture system in vitro (Kim, C., et al., Cancer Immunol. Immunother., 47, pp. 90-96, 1998). It is also known that dead cell-derived antigens can efficiently induce immune responses thousands folds stronger when the antigens are phagocytosed in the state of dead cells by immature dendritic cells than when the antigens are not phagocytosed (Inaba, et al., Lecture SI-3-3, Japanese Immunology Society, Dec. 2, 1998).

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide a tumor vaccine which can be simply handled, generally applied for prevention of tumor recurrence, inhibition of metastasis and therapeutic treatment, regardless of a type of a tumor, and also has a strong antitumor effect.

The inventors of the present invention conducted intensive studies to achieve the foregoing object. As a result, they found that the prevention of tumor recurrence, inhibition of metastasis, and therapeutic treatment can be achieved with high efficiency by using a material solidified from tumor tissues, tumor cells, or components thereof by a fixation operation, and processing the material into microparticles in a size that can be phagocytosed by antigen-presenting cells, or lysing the material by a lysation operation, and then using the resulting product as a tumor vaccine in combination with at least one kind of cytokines.

The present invention thus provides a tumor vaccine which comprises microparticles prepared from a solidified tumor material selected from the group consisting of tumor tissues, tumor cells and components thereof, and at least one kind of cytokines and/or cytokine-inducing agents; and a tumor vaccine which comprises a lysate prepared from a solidified tumor material selected from the group consisting of tumor tissues, tumor cells and components thereof, and at least one kind of cytokines and/or cytokine-inducing agents.

According to preferred embodiments of the present invention, provided are the aforementioned tumor vaccine which further contains an adjuvant unspecifically inducing immune responses; the aforementioned tumor vaccine for administration to an identical site in vivo; the aforementioned tumor vaccine which contains a cytokine-controlled release preparation as the cytokine; and the aforementioned tumor vaccine which contains a granulocyte-macrophage-colony stimulating factor and/or interleukin-2 as the cytokine. From another aspect, there is provided a tumor vaccine for using in combination with at least one kind of cytokines which contains, as an active ingredient, microparticles prepared from a solidified tumor material selected from the group consisting of tumor tissues, tumor cells and components thereof, or a lysate prepared from the tumor material.

From still other aspects, there are provided a method of therapeutic treatment of, prevention of recurrence of, and inhibition of metastasis of a tumor which comprises the step of administering an effective amount of microparticles prepared from a solidified tumor material selected from the group consisting of tumor tissues, tumor cells and components thereof, and at least one kind of cytokines and/or cytokine-inducing agents; a method which comprises the step of administering an effective amounts of a lysate prepared from a solidified tumor material selected from the group consisting of tumor tissues, tumor cells and components thereof, and at least one kind of cytokines and/or cytokine-inducing agents; the aforementioned methods in which the administration is repeatedly made an identical site; and a use of the microparticles or the lysate prepared from the aforementioned solidified tumor material for the manufacture of the aforementioned tumor vaccines.

BEST MODE FOR CARRYING OUT THE INVENTION

The tumor vaccine of the present invention is characterized to comprise microparticles or a lysate prepared from a solidified tumor material selected from the group consisting of tumor tissues, tumor cells and components thereof as a tumor antigen, and further containing at least one kind of cytokines and/or cytokine-inducing agents.

As the tumor cells or tumor tissues, for example, those derived from a mammal, preferably those derived from a human, can be used. Those from any species of organisms may be used so far that the cells or tissues contain a tumor antigen of a tumor to be therapeutically or preventively treated. The types of the tumor tissues are not particularly limited so far that they contain tumor cells. When components of tumor cells or tumor tissues are used, the types of the components are not limited so far that they contain a substance as potential tumor antigen. Fresh specimens such as solid cancer tissues, bone marrow, and white blood cells which contain cancer cells isolated or collected from the living body can be used as tumor materials. As the component of tumor tissues or tumor cells, for example, antigenic peptides or antigenic proteins can be used.

The fixation method to prepare the solidified tumor material is not particularly limited, and any means available to those skilled in the art may be applied. For example, when a tissue fixing agent is used, neutral formalin, glutaraldehyde, an alcohol such as methanol and ethanol and the like can be used. Besides the aforementioned methods, any method may be used so far that fresh tissues or cells or components thereof can be solidified. Tumor materials may be solidified by a method such as paraffin embedding, freezing and the like. When tissues originally in a solid state such as bone tissues are used as the solidified tumor material, it is preferably to apply an appropriate fixation method.

The preparation method of microparticles is not particularly limited, and applicable methods include, for example, a method of grinding the solidified tumor tissues to prepare microparticles of fine fragments, as well as a method of lysing ground fragments of tumor tissues or tumor cells to fix the lysate to solid microparticles, a method of fixing soluble tumor antigens such as antigenic peptides and antigenic proteins to solid microparticles and the like. As the solid microparticles, for example, iron powder, carbon powder, polystyrene beads and the like from about 0.05 to 1,000 .mu.m in diameter can be used. Usable microparticles include ground tissue fragments, tumor cells or soluble tumor antigens bound to lipid particles such as liposomes so as to be recognized as microparticles by the antigen-presenting cells to allow phagocytosis, or a microparticles obtained by binding soluble tumor antigens, per se, to each other by using a binder or a crosslinking agent.

Sizes of microparticles are not especially limited, however, a size that allows phagocytosis by cells with phagocytic ability in vivo is desirable. It is not necessary to grind fixed tumor cells that are originally in a state of small single cells. However, it is desirable to apply grinding or dispersing treatment when the cells aggregate during the fixation operation. For the grinding or dispersing treatment, treatment with a homogenizer, ultrasonic treatment, partial digestion with a digestive enzyme and the like can be used. The microparticles can also be prepared by passing through a screen having a pore size of not more than 1,000 .mu.m, preferably not more than 380 .mu.m. The preparation of these microparticles is well known to those skilled in the art, and the skilled artisan can prepare the microparticles by a single appropriate method or a combination of plural methods.

As a method to prepare the lysate from solidified tumor materials, for example, a method using a proteolytic enzyme can be applied. An example of the proteolytic enzyme includes proteinase K. A method employing an appropriate combination of an enzyme other than the proteolytic enzyme, an acid, an alkali and the like may also be utilized. Any method that can achieve lysis of the solidified tumor material may be employed, and those skilled in the art can choose an appropriate method. The lysate may be fixed to the solid microparticles mentioned above.

The term "lysate" used in the specification means a state of dispersion of the solidified tumor material in an aqueous medium such as water, physiological saline, and a buffer solution to an extent that any solid mass cannot be observed with naked eyes, and to an extent that the dispersoids can be phagocytosed by the antigen-presenting cells. However, the term should not be construed in any limiting way. The details of the preparations of the fixed tumor materials, the preparations of the microparticles, and the preparations of lysates are specifically described in the examples of the present specification. Accordingly, those skilled in the art can prepare the desired microparticles or the lysates by referring to the above general explanations and specific explanations in the examples, and appropriately modifying or altering those methods, if necessary.

The type of cytokines contained in the tumor vaccine of the present invention is not especially limited, and one or more kinds of cytokines can be used. For example, granulocyte-macrophage-colony-stimulating factor (hereinafter abbreviated as "GM-CSF"), or interleukin-2 (hereinafter abbreviated as "IL-2") may preferably be used, and a combination of GM-CSF and IL-2 may also preferably be used. In addition, other cytokines or cytokine-inducing agents can be used which stimulate the local immune cells in vivo, and consequently achieve the same conditions as those achieved by GM-CSF and/or IL-2 administration. As cytokines or cytokine-inducing agents besides these two kinds of cytokines, examples include interleukin-12, interleukin-18, interferon category and the like. However, cytokines or cytokine-inducing agents are not limited to these examples.

These cytokines or inducing agents may preferably be prepared as controlled-release preparations so that concentrations at sites received administration can be kept at a high level as long as possible. Such means for preparing controlled-release preparations is, for example, reported by Golumbek et al (Golumbek, P. T., et al., Cancer Res., 53, pp. 5841-5844, 1993). Various methods for preparation of controlled release preparations are known in the field of the art, and any method can be applied.

The tumor vaccine of the present invention may contain an adjuvant that induces non-specific immune responses. The adjuvant can be used alone or in combination of two or more kinds. As the adjuvant, examples include Freund complete adjuvant, Freund incomplete adjuvant, bacterial preparations such as BCG, bacterial component preparations such as tuberculin, natural macromolecular substances such as keyhole limpet hemocyanine and yeast mannan, Alum, synthetic adjuvant preparations such as Titer Max Gold and the like. However, the adjuvants are not limited to these specific examples, and any substances may be used so far that they are effective as adjuvants. Whether an adjuvant should be used or not can be judged by intensity of inflammatory reaction at a site of administration or intensity of antitumor effect induced as a result of the administration as a standard. For example, alternate administrations of the tumor vaccine containing an adjuvant and the vaccine without adjuvant can be applied to the same site.

Forms of preparation of the tumor vaccine of the present invention are not particularly limited, and desirably, the forms of preparation may be suitable for local administration. The methods for manufacturing pharmaceutical preparations are not particularly limited, and a preparation in a desired form can be prepared by applying a single method available in the field of the art or methods in an appropriate combination. For the manufacture of pharmaceutical preparations, aqueous media such as distilled water for injection and physiological saline, as well as one or more kinds of pharmaceutical additives available in the field of the art can be used. For example, buffering agents, pH adjusting agents, solubilizing aids, stabilizing agents, soothing agents, antiseptics and the like can be used, and specific ingredients thereof are well known to those skilled in the art. The tumor vaccine can also be prepared as a solid preparation such as a lyophilized preparation, and then prepared as an injection by adding a solubilizing agent such as distilled water for injection before use.

When vaccine therapy is carried out using the tumor vaccine of the present invention, the tumor vaccine may be administered only once. However, it is desirable to repeat the administration to the same site of a body to achieve coexistence of a tumor antigen and a cytokine or a cytokine-inducing agent as long as possible. For example, both components may preferably coexist for 3 hours or more so that inflammatory reaction at the site of administration can be induced and conditions can be achieved wherein immune cells are concentrated and cells are kept at the site. When a tumor vaccine without adjuvant is administered, an adjuvant may be administered to the same site. Generally, the tumor vaccine can be administered to a patient from which the tumor material is derived; however, the vaccine can also be administered to a patient bearing a tumor that contains, from a viewpoint of pathological diagnosis, the same or relative species of a tumor antigen as that contained in the tumor material.

The site to be administered is not particularly limited. Preferred sites include those where cytokines are hardly be diffused and disappeared, for example, intradermal, subcutaneous or intramuscular sites, in lymphnodes, and in a main organ such as spleen. However, by choosing a dosage form which prevents ready diffusion of the active ingredients of the tumor vaccine, local administrations may sometimes be performable to any site of a body, or by applying a drug delivery system, the systemic administration may sometimes be possible. The dose and administration period of the tumor vaccine of the present invention are not particularly limited. It is desirable to determine an appropriate dose and administration period by observing effects of the vaccine therapy. The administration can be made, for example, by injections and the like.
 

Claim 1 of 18 Claims

1. A tumor vaccine comprising: (A) a microparticle comprising a fragment of solidified, chemically fixed tumor tissues or cells, said fragment being of a size so as to allow phagocytosis of the fragment; and (B) at least one isolated cytokine, at least one isolated compound that is a cytokine-inducing agent, or a combination thereof.
 

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