JP5572937B2 - Immune inducer - Google Patents

Description

  The present invention relates to a novel immunity-inducing agent useful as a therapeutic and / or preventive agent for cancer.

  Cancer is the leading cause of all deaths, and the current treatment is a combination of radiation therapy and chemotherapy, mainly surgery. Despite the recent development of new surgical methods and the discovery of new anti-cancer drugs, the therapeutic results of cancer have not improved much except for some cancers. In recent years, advances in molecular biology and cancer immunology have identified cancer antigens recognized by cytotoxic T cells that respond to cancer and genes encoding cancer antigens, and expectations for antigen-specific immunotherapy have increased. (See Non-Patent Document 1). In immunotherapy, in order to reduce side effects, peptides or proteins that are recognized as antigens are hardly present in normal cells, and are required to be specifically present in cancer cells. In 1991, Boon et al., Ludwig Laboratories, Belgium, isolated human melanoma antigen MAGE1 recognized by CD8 positive T cells by cDNA expression cloning using autologous cancer cell lines and cancer reactive T cells (see Non-Patent Document 2). reference). Subsequently, the SEREX (serological identifications of antigens by recombinant expression cloning) method is used to identify tumor antigens recognized by antibodies produced in response to own cancer in the body of cancer patients by incorporating gene expression cloning techniques. It has been reported (Non-Patent Document 3; Patent Document 1), and various cancer antigens have been isolated (see Non-Patent Documents 4-9). Clinical trials of cancer immunotherapy have been started targeting a part of them.

  On the other hand, as in humans, dogs and cats are known to have a large number of tumors such as breast tumors and squamous cell carcinomas, and are ranked high in the disease statistics of dogs and cats. However, there are currently no effective therapeutic, prophylactic and diagnostic agents for canine and feline cancer. Most dogs and cat tumors are noticed by the owner after the tumor has progressed and the tumor has grown, and even if they come to the hospital and are removed by surgery or administered human drugs (such as anticancer drugs) Already too late to die soon after treatment. Under such circumstances, if cancer therapeutic agents, preventive agents and diagnostic agents effective for dogs and cats become available, it is expected that the use for canine cancer will be opened.

US Pat. No. 5,698,396 Satoshi Akiyoshi, “Cancer and Chemotherapy”, 1997, Vol. 24, p551-519 Bruggen P. et al., Science, 254: 1643-1647 (1991) Proc. Natl. Acad. Sci. USA, 92: 11810-11813 (1995) Int. J. Cancer, 72: 965-971 (1997) Cancer Res., 58: 1034-1041 (1998) Int. J. Cancer, 29: 652-658 (1998) Int.J.Oncol., 14: 703-708 (1999) Cancer Res., 56: 4766-4772 (1996) Hum. Mol. Genet 6: 33-39, 1997 Mol Endocrinol. 9: 243-54 (1995) J Cell Biol. 145: 83-98 (1999)

  An object of the present invention is to provide a novel immunity-inducing agent useful as a therapeutic and / or preventive agent for cancer.

  As a result of diligent research, the inventors of the present application have obtained a cDNA encoding a protein that binds to an antibody present in serum derived from a cancer-bearing organism by the SEREX method using a dog testis-derived cDNA library and the serum of a cancer-bearing dog. Based on the cDNA, a canine thyroid hormone receptor interactor 11 (hereinafter referred to as TRIP11) protein having the amino acid sequence shown in SEQ ID NO: 2 was produced. Moreover, based on the human homologous gene of the acquired gene, human TRIP11 which has an amino acid sequence shown by sequence number 4 was produced. The inventors have found that by administering these TRIP11 to a living body, immune cells can be induced in the living body and that a tumor that has already occurred can be regressed, and the present invention has been completed.

That is, the present invention includes a polypeptide having any of the following (a) and (b) having immunity-inducing activity as an active ingredient or a polynucleotide encoding the polypeptide in vivo. A therapeutic and / or prophylactic agent for cancer comprising a recombinant vector capable of expressing the polypeptide as an active ingredient: (a) a polypeptide having the amino acid sequence shown in SEQ ID NO: 2 or 4 in the sequence listing; (b) (a) polypeptide and 86% or more homology polypeptides have a.

  According to the present invention, a novel immunity-inducing agent useful for cancer treatment and / or prevention has been provided. As specifically shown in the following Examples, when the polypeptide used in the present invention is administered to a cancer-bearing dog, immune cells can be induced in the cancer-bearing dog body, Can be reduced or retracted. Therefore, the polypeptide is useful for treating or preventing cancer.

Examples of the polypeptide contained as an active ingredient in the immunity-inducing agent of the present invention include the following. In the present invention, “polypeptide” refers to a molecule formed by peptide bonding of a plurality of amino acids, and not only a polypeptide molecule having a large number of amino acids but also a low molecular weight molecule having a small number of amino acids. (Oligopeptide) and full-length protein are also included. In the present invention, the full-length protein of TRIP11 having the amino acid sequence shown in SEQ ID NO: 2 or 4 is also included.
(a) possess an amino acid sequence shown in SEQ ID NO: 2 or 4 in the Sequence Listing, the polypeptide having immunity-inducing activity.
(b) has a polypeptide 86% or more homology (a), the polypeptide having an immunity-inducing activity.

  In the present invention, “having an amino acid sequence” means that amino acid residues are arranged in such an order. Thus, for example, “a polypeptide having the amino acid sequence represented by SEQ ID NO: 2” means 1977 amino acids having the amino acid sequence of Met Ser Ser Trp,. By a residue size polypeptide is meant. In addition, for example, “polypeptide having the amino acid sequence represented by SEQ ID NO: 2” may be abbreviated as “polypeptide of SEQ ID NO: 2”. The same applies to the expression “having a base sequence”.

Here, “immunity-inducing activity” means the ability to induce immune cells that secrete cytokines such as interferon in vivo. Whether or not the polypeptide has immunity-inducing activity can be confirmed using, for example, a known ELISPOT assay. Specifically, for example, as described in the Examples below, cells such as peripheral blood mononuclear cells are obtained from a living body administered with a polypeptide whose immunity-inducing activity is to be evaluated, and the cells coexist with the polypeptide. By culturing and measuring the amount of cytokine production from the cells using a specific antibody, the number of immune cells in the cells can be measured, whereby the immunity-inducing activity can be evaluated. In addition, as described in the Examples below, when a recombinant polypeptide prepared based on the amino acid sequence of SEQ ID NO: 2 or 4 is administered to a cancer-bearing organism, the tumor can be regressed due to its immunity-inducing activity. Therefore, the immunity induction activity is evaluated as the ability to suppress the growth of cancer cells expressing the polypeptide of SEQ ID NO: 2 or 4 or to reduce or eliminate the cancer tissue (tumor) (hereinafter referred to as “antitumor activity”). You can also The antitumor activity of a polypeptide is confirmed, for example, by actually administering the polypeptide to a cancer-bearing organism and examining whether the tumor is reduced or the like, as specifically described in the Examples below. be able to. Also, examining whether T cells stimulated with the polypeptide (that is, T cells brought into contact with antigen-presenting cells presenting the polypeptide) exhibit cytotoxic activity against tumor cells in vitro. The antitumor activity of the polypeptide can also be evaluated. As will be described later, the T cell and the antigen-presenting cell can be contacted by co-culturing both in a liquid medium. The cytotoxic activity can be measured by a known method called a ⁵¹ Cr release assay described in, for example, Int. J. Cancer, 58: p317,1994. When the polypeptide is used for cancer treatment and / or prevention, it is not particularly limited, but it is preferable to evaluate immunity induction activity using antitumor activity as an index.

  The amino acid sequences shown in SEQ ID NOs: 2 and 4, respectively, are antibodies that are specifically present in sera from cancer-bearing dogs by the SEREX method using canine testis-derived cDNA library and sera from cancer-bearing dogs. It is the amino acid sequence of TRIP11 protein isolated as a binding polypeptide and its human homologous factor (see Example 1). TRIP11 (thyroid hormone receptor interactor 11) was originally identified as a factor that interacts with thyroid hormone receptor β, but it was also found that it binds to the Golgi apparatus and microtubules. It is thought to play a role in maintaining the shape of these organelles. However, there is no report that the protein is expressed in cancer and useful for treatment or prevention (Non-patent Document 10 Mol Endocrinol. 9: 243-54 (1995), Non-Patent Document 11 J Cell Biol. 145: 83- 98 (1999)). The homology between the canine TRIP11 gene and the human TRIP11 gene is 88% in terms of base sequence and 86% in terms of amino acid sequence.

The polypeptides of (a) is a polypeptide de having the amino acid sequence shown in SEQ ID NO: 2 or 4, and has an immunity-inducing activity. Na us, as is known in the art, the antigenic can exhibit if the polypeptide than about 7 amino acid residues. Therefore, if the contiguous nine amino acid residues or more polypeptides in the amino acid sequence of SEQ ID NO: 2 or 4, Ru can have an immunity-inducing activity. However, in view of the fact that an antibody produced against an antigenic substance in a living body is a polyclonal antibody, the larger the number of amino acid residues, the more types of antigens that recognize various sites on the antigenic substance. Since antibodies can be induced, immunity-inducing activity can be enhanced .

  In addition, as a principle of immunity induction by administering a cancer antigen polypeptide, the polypeptide is taken up by antigen-presenting cells and then decomposed by peptidase in the cells to become smaller fragments and presented on the surface of the cells It is known that cytotoxic T cells and the like recognize this and selectively kill cells presenting the antigen.

The size of the polypeptide displayed on the surface of the antigen-presenting cell is relatively small, and is about 7 to 30 amino acids. Therefore, from the viewpoint of presentation on antigen presenting cells is sufficient as long as it consists of successive 9 to 30 about amino acids of the amino acid sequence shown in SEQ ID NO 2 or 4. These relatively small polypeptides may be presented directly on the cell surface on antigen-presenting cells without being taken up into antigen-presenting cells.

However, as described above, polypeptides taken into antigen-presenting cells are cleaved at random positions by peptidases in the cells to produce various polypeptide fragments, and these polypeptide fragments are bound to the surface of the antigen-presenting cell. As shown above, when a polypeptide having a large size such as the full length region of SEQ ID NO: 2 or 4 is administered, the polypeptide fragment is effective for inducing immunity via antigen-presenting cells by degradation in antigen-presenting cells Inevitably occurs. Therefore, also for immune induction via antigen-presenting cells, Ru can be preferably used a large polypeptide size.

The polypeptide (b) is a polypeptide in which a small number of amino acid residues in the polypeptide (a) are substituted, deleted and / or inserted, and 86 % or more of the original sequence. The polypeptide has a homology of preferably 90% or more, more preferably 95% or more, and still more preferably 98% or more, and has immunity-inducing activity. In general, a protein antigen may have almost the same antigenicity as the original protein even if a small number of amino acid residues in the amino acid sequence of the protein are substituted, deleted or inserted. It is well known to those skilled in the art. Therefore, since the polypeptide (b) can also exert immunity-inducing activity, it can be used for the preparation of the immunity-inducing agent of the present invention. The polypeptide (b) is a polypeptide in which one to several amino acid residues are substituted, deleted and / or inserted in the amino acid sequence represented by SEQ ID NO: 2 or 4. It is also preferable.

  Here, the “homology” of amino acid sequences means that both amino acid sequences are aligned so that the amino acid residues of the two amino acid sequences to be compared match as much as possible, and the number of matched amino acid residues is the total number of amino acid residues. The percentage divided by is expressed as a percentage. In the above alignment, a gap is appropriately inserted in one or both of the two sequences to be compared as necessary. Such sequence alignment can be performed using a known program such as BLAST, FASTA, CLUSTAL W, and the like. When gaps are inserted, the total number of amino acid residues is the number of residues obtained by counting one gap as one amino acid residue. When the total number of amino acid residues counted in this way is different between the two sequences to be compared, the homology (%) is the total number of amino acid residues in the longer sequence, and the number of amino acid residues matched. Is calculated by dividing.

  The 20 amino acids constituting the natural protein are neutral amino acids having low side chains (Gly, Ile, Val, Leu, Ala, Met, Pro), and neutral amino acids having hydrophilic side chains (Asn , Gln, Thr, Ser, Tyr Cys), acidic amino acids (Asp, Glu), basic amino acids (Arg, Lys, His), and aromatic amino acids (Phe, Tyr, Trp) It is known that the properties of a polypeptide often do not change as long as it can be grouped and the substitution between them. Therefore, when the amino acid residue in the polypeptide (a) of the present invention is substituted, the possibility that the immunity-inducing activity can be maintained is increased by substitution between these groups.

  The above-described polypeptide can be synthesized according to a chemical synthesis method such as Fmoc method (fluorenylmethyloxycarbonyl method) or tBoc method (t-butyloxycarbonyl method). Moreover, it can also synthesize | combine by a conventional method using various commercially available peptide synthesizers. In addition, using a known genetic engineering technique, a polynucleotide encoding the above polypeptide is prepared, the polynucleotide is incorporated into an expression vector and introduced into a host cell, and the polypeptide is produced in the host cell. Thus, the desired polypeptide can be obtained.

The polynucleotide encoding the above-mentioned polypeptide can be easily prepared by a known genetic engineering technique or a conventional method using a commercially available nucleic acid synthesizer. For example, DNA having the base sequence of SEQ ID NO: 1 is subjected to PCR using a canine chromosomal DNA or cDNA library as a template and a pair of primers designed to amplify the base sequence described in SEQ ID NO: 1. Can be prepared. Any DNA having the nucleotide sequence of SEQ ID NO: 3 can be prepared in the same manner by using human chromosomal DNA or cDNA library as the template. PCR reaction conditions can be set as appropriate. For example, one cycle of a reaction step consisting of 94 ° C. for 30 seconds (denaturation), 55 ° C. for 30 seconds to 1 minute (annealing), and 72 ° C. for 2 minutes (extension) Examples of the conditions include, but are not limited to, conditions of reacting at 72 ° C. for 7 minutes after 30 cycles. In addition, appropriate probes and primers are prepared based on the nucleotide sequence and amino acid sequence information shown in SEQ ID NOs: 1 to 4 in the sequence listing in the present specification, and cDNA libraries such as dogs and humans are prepared using the probes and primers. The desired DNA can be isolated by screening for. The cDNA library is preferably prepared from cells, organs or tissues expressing the protein of SEQ ID NO: 2 or 4. Operations such as the preparation of the probe or primer, construction of the cDNA library, screening of the cDNA library, and cloning of the target gene are known to those skilled in the art. For example, Molecular Cloning 2nd Edition, Current Protocols, It can be carried out according to the method described in In-Molecular Biology and the like. From the DNA thus obtained, DNA encoding the polypeptide (a) can be obtained. In addition, since the codons encoding each amino acid are known, the base sequence of a polynucleotide encoding a specific amino acid sequence can be easily specified. Accordingly, since it is possible to easily identify the base sequence of a polynucleotide encoding a polypeptide de of the above (b), also such polynucleotides, easily synthesized by a conventional method using a commercially available nucleic acid synthesizer be able to.

  The host cell may be any cell that can express the polypeptide. Examples of prokaryotic cells include Escherichia coli, and examples of eukaryotic cells include monkey kidney cell COS 1 and Chinese hamster ovary. Examples include, but are not limited to, cultured mammalian cells such as cell CHO, budding yeast, fission yeast, silkworm cells, and Xenopus egg cells.

  When a prokaryotic cell is used as a host cell, an expression vector having an origin, a promoter, a ribosome binding site, a DNA cloning site, a terminator and the like that can replicate in the prokaryotic cell is used as the expression vector. Examples of the expression vector for E. coli include pUC system, pBluescriptII, pET expression system, pGEX expression system and the like. When the DNA encoding the above polypeptide is incorporated into such an expression vector, a prokaryotic host cell is transformed with the vector, and the resulting transformant is cultured, the polypeptide encoded by the DNA is prokaryotic. It can be expressed in a host cell. In this case, the polypeptide can also be expressed as a fusion protein with another protein.

  When a eukaryotic cell is used as a host cell, an expression vector for a eukaryotic cell having a promoter, a splicing region, a poly (A) addition site and the like is used as an expression vector. Examples of such expression vectors include pKA1, pCDM8, pSVK3, pMSG, pSVL, pBK-CMV, pBK-RSV, EBV vector, pRS, pcDNA3, pMSG, pYES2, and the like. In the same manner as described above, a DNA encoding the above polypeptide is incorporated into such an expression vector, and after transforming a eukaryotic host cell with the vector, the resulting transformant is cultured, and the DNA encodes. Can be expressed in eukaryotic host cells. When pIND / V5-His, pFLAG-CMV-2, pEGFP-N1, pEGFP-C1, etc. are used as an expression vector, fusion with various tags such as His tag, FLAG tag, myc tag, HA tag, GFP The polypeptide can be expressed as a protein.

  For introducing the expression vector into the host cell, a well-known method such as electroporation, calcium phosphate method, liposome method, DEAE dextran method or the like can be used.

  Isolation and purification of the polypeptide of interest from the host cell can be performed by combining known separation operations. For example, treatment with denaturing agents and surfactants such as urea, ultrasonic treatment, enzyme digestion, salting out and solvent fractional precipitation, dialysis, centrifugation, ultrafiltration, gel filtration, SDS-PAGE, isoelectric focusing, Examples include, but are not limited to, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, and reverse phase chromatography.

Polypeptides obtained by the above methods include those in the form of fusion proteins with other arbitrary proteins as described above. For example, glutathione-S-transferase (GST), a fusion protein with a His tag, and the like can be exemplified . Et al is a polypeptide expressed in transformed cells after translation may be subject to various modifications within the cell. Such post-translationally modified polypeptides are also included in the scope of the present invention as long as they have immunity-inducing activity. Examples of such translation modification include elimination of N-terminal methionine, N-terminal acetylation, sugar chain addition, limited degradation by intracellular protease, myristoylation, isoprenylation, phosphorylation, and the like.

As specifically described in the following examples, when a polypeptide having the above-described immunity-inducing activity is administered to a cancer-bearing organism, a tumor that has already occurred can be regressed . The immunity-inducing agent of the present invention is used as a therapeutic and / or prophylactic agent for cancer . Cancers that the Target is a cancer expressing a gene encoding the polypeptide of SEQ ID NO: 2 or 4, brain, head, neck, lung, uterine or esophageal squamous cell carcinoma, melanoma, lung, breast Or adenocarcinoma of the uterus, renal cancer, malignant mixed tumor, hepatocellular carcinoma, basal cell carcinoma, pheochromocytoma-like gingival tumor, oral mass, perianal adenocarcinoma, anal cyst mass, anal sac apocrine adenocarcinoma, Sertoli cell tumor Vaginal vestibular cancer, sebaceous gland cancer, sebaceous gland epithelioma, sebaceous adenoma, sweat gland cancer, intranasal adenocarcinoma, nasal gland cancer, thyroid cancer, colon cancer, bronchial adenocarcinoma, adenocarcinoma, ductal carcinoma, mammary gland cancer, complex type Breast cancer, Breast malignant mixed tumor, Intraductal papillary adenocarcinoma, Fibrosarcoma, Hemangioperiosteum, Osteosarcoma, Chondrosarcoma, Soft tissue sarcoma, Histiocytosarcoma, Myxosarcoma, Undifferentiated sarcoma, Lung cancer, Mast cell tumor, Cutaneous leiomyoma, intraperitoneal leiomyoma, leiomyoma, chronic lymphocytic leukemia, lymphoma, Tubular lymphoma, gastrointestinal lymphoma, small to medium cell lymphoma, adrenal medullary tumor, granulosa cell tumor, pheochromocytoma, bladder cancer (transitional cell carcinoma), purulent inflammation, intraperitoneal liver tumor, liver cancer, Plasmacytoma, malignant adenocarcinoma, angiosarcoma, anal sac adenocarcinoma, oral cancer, metastatic malignant melanoma, melanin-deficient malignant melanoma, cutaneous malignant melanoma, malignant myoepitheloma, malignant testicular epithelioma, Seminoma, colon adenocarcinoma, gastric adenocarcinoma, low-grade sebaceous gland cancer, earwax adenocarcinoma, apocrine adenocarcinoma, poorly differentiated apocrine sweat gland carcinoma, malignant fibrous histiocytoma, multiple myeloma, mesenchymal malignant Tumor, liposarcoma, osteosarcoma, sarcoma of unknown origin, soft tissue sarcoma (spindle cell tumor), poorly differentiated sarcoma, synovial sarcoma, hemangiosarcoma, metastatic malignant epithelial tumor, tubular mammary adenocarcinoma, breast ductal carcinoma, inflammatory Breast cancer, germinoma, leukemia, invasive follicular epithelioma, medium cell Lymphoma, multicentric lymphoma, osteosarcoma (mammary gland), mastocytoma (Patnaik II type), mastocytoma (Grade II), there may be mentioned leiomyosarcoma, and the like. The target animal is a mammal, and humans, dogs and cats are particularly preferable.

  The administration route to the living body of the immunity-inducing agent of the present invention may be oral administration or parenteral administration, but parenteral administration such as intramuscular administration, subcutaneous administration, intravenous administration and intraarterial administration is preferred. When the immunity-inducing agent is used for the purpose of treating cancer, it can be administered to a regional lymph node in the vicinity of the tumor to be treated as described in the following examples in order to enhance the anticancer effect. The dose may be an amount effective for inducing immunity. For example, if it is used for the treatment and / or prevention of cancer, it may be an amount effective for the treatment and / or prevention of cancer. The effective amount for the treatment and / or prevention of cancer is appropriately selected according to the size and symptoms of the tumor, etc., but is usually 0.0001 μg to 1000 μg, preferably 0.001 μg It is 1000 μg and can be administered once or divided into several times. Preferably, it is divided into several times and administered every few days to several months. As specifically shown in the Examples below, the immunity-inducing agent of the present invention can regress tumors that have already formed. Therefore, since anticancer activity can be exerted on a small number of cancer cells at the early stage of development, onset and recurrence of cancer can be prevented if used before onset of cancer or after treatment of cancer. That is, the immunity-inducing agent of the present invention is useful for both treatment and prevention of cancer.

  The immunity-inducing agent of the present invention may consist only of a polypeptide, or may be appropriately mixed with additives such as pharmacologically acceptable carriers, diluents and excipients suitable for each administration form. It can also be formulated. Formulation methods and usable additives are well known in the field of pharmaceutical formulations, and any method and additive can be used. Specific examples of additives include diluents such as physiological buffers; excipients such as sugar, lactose, corn starch, calcium phosphate, sorbitol, glycine; syrup, gelatin, gum arabic, sorbitol, polyvinyl chloride, tragacanth, etc. Binders such as magnesium stearate, polyethylene glycol, talc, silica and the like, but are not limited thereto. Examples of the dosage form include oral preparations such as tablets, capsules, granules, powders, and syrups, and parenteral preparations such as inhalants, injections, suppositories, and liquids. These preparations can be made by generally known production methods.

  The immunity-inducing agent of the present invention can be used in combination with an immunity enhancing agent that can enhance the immunological response in vivo. The immunopotentiator may be contained in the immunity-inducing agent of the present invention, or may be administered to a patient in combination with the immunity-inducing agent of the present invention as a separate composition.

  Examples of the immune enhancer include an adjuvant. Adjuvants provide a reservoir of antigen (extracellular or within macrophages), enhance immunological responses by activating macrophages and stimulating specific sets of lymphocytes, thus enhancing anticancer effects be able to. Therefore, in particular, when the immunity-inducing agent of the present invention is used for the treatment and / or prevention of cancer, it is preferable that the immunity-inducing agent further contains an adjuvant in addition to the above polypeptide as an active ingredient. Many types of adjuvants are well known in the art, and any adjuvant can be used. Specific examples of adjuvants include MPL (SmithKline Beecham), Salmonella minnesota Re 595 lipopolysaccharide of the genus Salmonella and the like obtained after acid hydrolysis; QS21 (SmithKline Beecham), pure purified from Quillja saponaria extract QA-21 saponin; DQS21 described in PCT application WO96 / 33739 (SmithKline Beecham); QS-7, QS-17, QS-18 and QS-L1 (So, 10 others, “Molecules and Molecules and cells, 1997, 7, p. 178-186); Freund's incomplete adjuvant; Freund's complete adjuvant; Vitamin E; Montanide; Alum; CpG oligonucleotide (eg, Kreig) , And 7 others, see “Nature”, Vol. 374, pages 546-549)); Poly IC Beauty derivatives thereof (poly ICLC, etc.) and various water-in-oil emulsion prepared from squalene and / or biodegradable oil such as tocopherol. Of these, Freund's incomplete adjuvant, montanide, poly IC and derivatives thereof, and CpG oligonucleotides are preferred. The mixing ratio of the adjuvant to the polypeptide is typically about 1:10 to 10: 1, preferably about 1: 5 to 5: 1, more preferably about 1: 1. However, the adjuvant is not limited to the above examples, and other adjuvants known in the art may be used when administering the immunity-inducing agent of the present invention (for example, by Goding, “Monoclonal antibodies: (See Monoclonal Antibodies: Principles and Practice, 2nd edition, 1986). Methods for preparing polypeptide and adjuvant mixtures or emulsions are well known to those skilled in vaccination.

  In addition to the adjuvant, factors that stimulate the immune response of the subject can also be used as the immune enhancer. For example, various cytokines having the property of stimulating lymphocytes and antigen-presenting cells can be used in combination with the immune inducer of the present invention as an immune enhancer. Numerous cytokines capable of enhancing such immunological responses are known to those skilled in the art, such as interleukin-12 (IL-12), which has been shown to enhance the protective effects of vaccines, Examples include, but are not limited to, GM-CSF, IL-18, interferon α, interferon β, interferon ω, interferon γ, and Flt3 ligand. Such factors can also be used as the above-mentioned immunity enhancing agent, and can be administered to patients in the immunity-inducing agent of the present invention or in combination with the immunity-inducing agent of the present invention as a separate composition.

In addition, the polypeptide can be presented to the antigen-presenting cell by contacting the polypeptide and the antigen-presenting cell in vitro. That is, the polypeptides (a) and ( b ) described above can be used as a treatment agent for antigen-presenting cells. Here, dendritic cells or B cells carrying MHC class I molecules can be preferably used as antigen-presenting cells. Various MHC class I molecules have been identified and are well known. MHC molecules in humans are called HLA. Examples of HLA class I molecules include HLA-A, HLA-B, and HLA-C. More specifically, HLA-A1, HLA-A0201, HLA-A0204, HLA-A0205, HLA-A0206, HLA-A0207, HLA-A11, HLA-A24, HLA-A31, HLA-A6801, HLA-B7, HLA-B8, HLA-B2705, HLA-B37, HLA-Cw0401, HLA-Cw0602 and the like.

Dendritic cells or B cells carrying MHC class I molecules can be prepared from peripheral blood by well-known methods. For example, dendritic cells are induced from bone marrow, umbilical cord blood, or patient peripheral blood using granulocyte macrophage colony stimulating factor (GM-CSF) and IL-3 (or IL-4), and tumor-related peptides are introduced into the culture system. Can be added to induce tumor-specific dendritic cells. By administering an effective amount of this dendritic cell, a desired response for cancer treatment can be induced. The cells used can be bone marrow and umbilical cord blood provided by healthy people, the patient's own bone marrow and peripheral blood, etc., but when using the patient's own autologous cells, it is highly safe and has serious side effects. It can also be expected to avoid it. Peripheral blood or bone marrow may be a fresh sample, a cryopreserved sample, or a cryopreserved sample. As the peripheral blood, whole blood may be cultured, or only the leukocyte component may be separated and cultured, but the latter is more efficient and preferable. Furthermore, mononuclear cells may be separated among the leukocyte components. Moreover, when originating in bone marrow or umbilical cord blood, the whole cells constituting the bone marrow may be cultured, or mononuclear cells may be separated and cultured from this. Peripheral blood, its white blood cell components, and bone marrow cells include mononuclear cells, hematopoietic stem cells, immature dendritic cells, CD4 positive cells, and the like that are the origin of dendritic cells. As long as the cytokine used has a property that has been confirmed to be safe and physiologically active, it does not matter whether it is a natural type or a genetically engineered type, and its production method is preferably ensured. Are used in the minimum amount required. The concentration of the cytokine to be added is not particularly limited as long as it is a concentration at which dendritic cells are induced, and is usually preferably about 10 to 1000 ng / mL, more preferably about 20 to 500 ng / mL as the total concentration of cytokines. The culture can be performed using a well-known medium usually used for culturing leukocytes. The culture temperature is not particularly limited as long as leukocyte growth is possible, but is most preferably about 37 ° C. which is the human body temperature. Further, the gas environment during the culture is not particularly limited as long as leukocytes can be grown, but it is preferable to aerate 5% CO ₂ . Further, the culture period is not particularly limited as long as a necessary number of cells are induced, but it is usually performed for 3 days to 2 weeks. As a device used for cell separation and culture, an appropriate device can be used as appropriate, but it is preferable that safety is confirmed for medical use and that the operation is stable and simple. In particular, for cell culture devices, stacked containers, multistage containers, roller bottles, spinner bottles, bag-type incubators, hollow fiber columns, etc. can be used regardless of general containers such as petri dishes, flasks, and bottles. .

The method of bringing the polypeptide into contact with the antigen-presenting cell in vitro can be performed by a well-known method. For example, it can be performed by culturing antigen-presenting cells in a culture solution containing the polypeptide. The peptide concentration in the medium is not particularly limited, but is usually about 1 μg / ml to 100 μg / ml, preferably about 5 μg / ml to 20 μg / ml. The cell density at the time of culture is not particularly limited, but is usually about 10 ³ cells / ml to 10 ⁷ cells / ml, preferably about 5 × 10 ⁴ cells / ml to 5 × 10 ⁶ cells / ml. Cultivation is preferably carried out in a 37 ° C., 5% CO ₂ atmosphere according to a conventional method. The length of the peptide that can be presented on the surface by antigen-presenting cells is usually about 30 amino acid residues at the maximum. Therefore, although not particularly limited, when the antigen-presenting cell and the polypeptide are contacted in vitro, the polypeptide may be prepared to a length of about 30 amino acid residues or less.

  By culturing the antigen-presenting cells in the presence of the above-described polypeptide, the peptide is taken up by the MHC molecule of the antigen-presenting cell and presented on the surface of the antigen-presenting cell. Therefore, an isolated antigen-presenting cell containing a complex of the polypeptide and MHC molecule can be prepared using the polypeptide. Such antigen-presenting cells can present the polypeptide to T cells in vivo or in vitro, and induce and proliferate cytotoxic T cells specific for the polypeptide.

Antigen-presenting cells containing the above-described polypeptide and MHC molecule complex prepared as described above are contacted with T cells in vitro to induce cytotoxic T cells specific for the polypeptide. And can be grown. This can be done by co-culturing the antigen-presenting cells and T cells in a liquid medium. For example, it can be carried out by suspending antigen-presenting cells in a liquid medium, placing them in a container such as a well of a microplate, adding T cells thereto, and culturing. The mixing ratio of antigen-presenting cells and T cells during co-culture is not particularly limited, but is usually about 1: 1 to 1: 100, preferably about 1: 5 to 1:20 in terms of the number of cells. The density of antigen-presenting cells suspended in the liquid medium is not particularly limited, but is usually about 1 to 10 million cells / ml, preferably about 10,000 to 1 million cells / ml. The coculture is preferably performed in a 37 ° C., 5% CO ₂ atmosphere according to a conventional method. The culture time is not particularly limited, but is usually 2 days to 3 weeks, preferably about 4 days to 2 weeks. The coculture is preferably performed in the presence of one or more interleukins such as IL-2, IL-6, IL-7 and IL-12. In this case, the concentration of IL-2 and IL-7 is usually about 5 U / ml to about 20 U / ml, the concentration of IL-6 is usually about 500 U / ml to about 2000 U / ml, and the concentration of IL-12 is usually about Although it is about 5 ng / ml to 20 ng / ml, it is not limited to these. The above co-culture may be repeated once to several times by adding fresh antigen-presenting cells. For example, the operation of discarding the culture supernatant after co-culture, adding a fresh antigen-presenting cell suspension, and further co-culturing may be repeated once to several times. The conditions for each co-culture may be the same as described above.

  By the co-culture described above, cytotoxic T cells specific for the polypeptide are induced and proliferated. Therefore, an isolated T cell that selectively binds the complex of the polypeptide and the MHC molecule can be prepared using the polypeptide.

  As described in the Examples below, the genes encoding the TRIP11 proteins of SEQ ID NOs: 2 and 4 are specifically expressed in canine and human cancer cells and testis, respectively. Therefore, it is considered that the TRIP11 protein of SEQ ID NO: 2 or 4 is significantly present in cancer cells than in normal cells. When a part of TRIP11 protein present in cancer cells is presented on MHC molecules on the surface of cancer cells and the cytotoxic T cells prepared as described above are administered in vivo, the cytotoxicity Sex T cells can damage cancer cells. In addition, since the antigen-presenting cells presenting the polypeptide can induce and proliferate cytotoxic T cells specific for the polypeptide in vivo, the antigen-presenting cells are administered in vivo. In some cases, cancer cells can be damaged. That is, the cytotoxic T cell and the antigen-presenting cell prepared using the polypeptide are also useful as a cancer treatment and / or prevention agent, like the immunity-inducing agent of the present invention.

When administering the above-mentioned isolated antigen-presenting cells or isolated T cells to a living body, in order to avoid in vivo immune responses that attack these cells as foreign bodies, antigen presentation collected from patients undergoing treatment Cells or T cells are preferably prepared using the polypeptide (a) or ( b ) as described above.

  The administration route of the therapeutic and / or prophylactic agent for cancer containing antigen-presenting cells or isolated T cells as an active ingredient is preferably parenteral administration such as intravenous administration or intraarterial administration. The dose is appropriately selected according to symptoms, purpose of administration, etc., but is usually 1 to 10 trillion, preferably 1 million to 1 billion, and this is once every few days to several months. Administration is preferred. The preparation may be, for example, one in which cells are suspended in physiological buffer saline, and can be used in combination with other anticancer agents, cytokines, and the like. In addition, one or two or more additives well known in the pharmaceutical field can be added.

Further, by expressing a polynucleotide encoding the polypeptide of (a) or ( b ) in the body of the subject animal, antibody production and cytotoxic T cells can be induced in the living body, The same effect as that obtained by administering the peptide can be obtained. That is, the immunity-inducing agent of the present invention comprises a polynucleotide encoding the above-described polypeptide (a) or ( b ), and contains a recombinant vector capable of expressing the polypeptide in vivo as an active ingredient. There may be. Such a recombinant vector capable of expressing an antigen polypeptide is also called a gene vaccine. The vector used for producing the gene vaccine is not particularly limited as long as it can be expressed in the target animal cell (preferably in the mammalian cell), and may be a plasmid vector or a virus vector, and is known in the field of gene vaccines. Any of these vectors may be used. In addition, as described above, a polynucleotide such as DNA or RNA encoding the polypeptide can be easily prepared by a conventional method. In addition, the polynucleotide can be incorporated into a vector by a method well known to those skilled in the art.

  The administration route of the gene vaccine is preferably a parenteral administration route such as intramuscular administration, subcutaneous administration, intravenous administration or intraarterial administration, and the dosage can be appropriately selected according to the type of antigen and the like. Usually, the weight of the genetic vaccine per kg body weight is about 0.1 μg to 100 mg, preferably about 1 μg to 10 mg.

  As a method using a viral vector, for example, a polynucleotide encoding the above polypeptide in RNA virus or DNA virus such as retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, pox virus, poliovirus, Sindbis virus, etc. And a method for infecting a target animal with this. Of these, methods using retroviruses, adenoviruses, adeno-associated viruses, vaccinia viruses and the like are particularly preferred.

  Examples of other methods include a method in which an expression plasmid is directly administered into muscle (DNA vaccine method), a liposome method, a lipofectin method, a microinjection method, a calcium phosphate method, an electroporation method, and the like. The method is preferred.

  In order to cause the gene encoding the above-mentioned polypeptide used in the present invention to actually act as a medicine, an in vivo method in which the gene is directly introduced into the body, and a certain type of cell is collected from the target animal and the gene is extracted outside the body. There is an ex vivo method that introduces the cells into the body and returns the cells to the body (Nikkei Science, April 1994, p20-45, Monthly Pharmaceutical Affairs, 1994, Vol. 36, No. 1, p.23-48, Experimental Medicine Special Issue) 1994, Vol. 12, No. 15, and references cited therein). In vivo methods are more preferred.

  When administered by an in vivo method, it can be administered by an appropriate route according to the disease, symptom or the like for the purpose of treatment. For example, it can be administered intravenously, artery, subcutaneously, intramuscularly. When administered by an in vivo method, for example, it can be in the form of a pharmaceutical preparation such as a solution, but is generally an injection containing a DNA encoding the above-mentioned peptide of the present invention, which is an active ingredient. Depending on the case, customary carriers may be added. In addition, the liposome or membrane-fused liposome (Sendai virus (HVJ) -liposome etc.) containing the DNA can be in the form of a liposome preparation such as a suspension, a freezing agent, a centrifugal concentrated freezing agent and the like.

  In the present invention, the term “base sequence shown in SEQ ID NO: 1” includes the base sequence actually shown in SEQ ID NO: 1 as well as a complementary sequence thereto. Therefore, when saying “polynucleotide having the base sequence shown in SEQ ID NO: 1”, the single-stranded polynucleotide having the base sequence actually shown in SEQ ID NO: 1, its complementary base sequence Single-stranded polynucleotides having and double-stranded polynucleotides comprising these are included. When preparing a polynucleotide encoding the polypeptide used in the present invention, any base sequence is appropriately selected, but those skilled in the art can easily select it.

  Hereinafter, the present invention will be described more specifically based on examples.

Example 1: Acquisition of novel cancer antigen protein by SEREX method (1) Preparation of cDNA library Total RNA was extracted from the testis tissue of a healthy dog by the acid-guanidium-phenol-chloroform method (Acid guanidium-Phenol-Chloroform method). PolyA RNA was purified using Oligotex-dT30 mRNA purification Kit (Takara Shuzo) according to the protocol attached to the kit.

A dog testis cDNA phage library was synthesized using the obtained mRNA (5 μg). The cDNA phage library was prepared using cDNA Synthesis Kit, ZAP-cDNA Synthesis Kit, and ZAP-cDNA Gigapack III Gold Cloning Kit (manufactured by STRATAGENE) according to the protocol attached to the kit. The size of the prepared cDNA phage library was 1.3 × 10 ⁶ pfu / ml.

(2) Screening of cDNA library with serum Immunoscreening was performed using the canine testis-derived cDNA phage library prepared above. Specifically, Φ90 × 15 mm NZY agarose plate was infected with host E. coli (XL1-Blue MRF ′) so as to become 2340 clones, cultured at 42 ° C. for 3 to 4 hours, and lysed plaques were made. Proteins are induced and expressed by covering the plate with nitrocellulose membrane (Hybond C Extra: GE Healthecare Bio-Science) impregnated with IPTG (isopropyl-β-D-thiogalactoside) at 37 ° C. for 4 hours. Transcribed protein. Thereafter, the membrane was recovered, immersed in TBS (10 mM Tris-HCl, 150 mM NaCl pH 7.5) containing 0.5% nonfat dry milk, and shaken at 4 ° C. overnight to suppress nonspecific reaction. This filter was allowed to react with the serum of a patient dog diluted 500 times at room temperature for 2 to 3 hours.

As the above-mentioned patient serum, serum collected from a dog with breast cancer was used. These sera were stored at −80 ° C. and pretreated immediately before use. The serum pretreatment method is as follows. That is, λ ZAP Express phage into which no foreign gene was inserted was infected with host E. coli (XL1-BLue MRF ′), and then cultured overnight at 37 ° C. on NZY plate medium. Next, 0.2 M NaHCO ₃ pH 8.3 buffer containing 0.5 M NaCl was added to the plate, and the mixture was allowed to stand at 4 ° C. for 15 hours, and then the supernatant was recovered as an E. coli / phage extract. Next, the recovered E. coli / phage extract was passed through an NHS-column (GE Healthecare Bio-Science) to immobilize the E. coli / phage derived protein. Serum dog serum was passed through this protein-immobilized column and reacted, and antibodies adsorbed to E. coli and phage were removed from the serum. The serum fraction passed through the column was diluted 500 times with TBS containing 0.5% nonfat dry milk, and this was used as an immunoscreening material.

The membrane on which the treated serum and the fusion protein were blotted was washed 4 times with TBS-T (0.05% Tween20 / TBS), and then diluted 5000 times with TBS containing 0.5% nonfat dry milk as a secondary antibody. Goat anti dog IgG-h + I HRP conjugated: manufactured by BETHYL Laboratories was reacted for 1 hour at room temperature and detected by enzyme color reaction using NBT / BCIP reaction solution (Roche) Then, colonies that coincide with the positive color reaction site were collected from a Φ90 × 15 mm NZY agarose plate and dissolved in 500 μl of SM buffer (100 mM NaCl, 10 mM MgClSO ₄ , 50 mM Tris-HCl, 0.01% gelatin pH 7.5). It was. Repeat the secondary and tertiary screening in the same way as above until the chromogenic positive colonies are unified, screen 30940 phage clones that react with IgG in serum, and isolate one positive clone did.

(3) Homology search of isolated antigen gene In order to use one positive clone isolated by the above method for base sequence analysis, an operation of converting a phage vector into a plasmid vector was performed. Specifically, 200 μl of a host E. coli (XL1-Blue MRF ′) prepared to have an absorbance OD ₆₀₀ of 1.0 is mixed with 100 μl of a purified phage solution and 1 μl of ExAssist helper phage (manufactured by STRATAGENE). After reaction at 15 ° C. for 15 minutes, 3 ml of LB medium was added, cultured at 37 ° C. for 2.5 to 3 hours, immediately incubated in a 70 ° C. water bath for 20 minutes, and then centrifuged at 4 ° C., 1000 × g for 15 minutes. The supernatant was collected as a phagemid solution. Next, 200 μl of a phagemid host Escherichia coli (SOLR) solution having an absorbance OD ₆₀₀ of 1.0 was mixed with 10 μl of the purified phage solution and reacted at 37 ° C. for 15 minutes, and 50 μl was treated with ampicillin (final concentration 50 μg / ml). ) Seeded on LB agar medium and cultured overnight at 37 ° C. A single colony of transformed SOLR is collected and cultured at 37 ° C in LB medium containing ampicillin (final concentration 50 µg / ml), and then the plasmid DNA having the target insert is purified using the QIAGEN plasmid Miniprep Kit (Qiagen). did.

  The purified plasmid was analyzed for the full-length insert sequence by the primer walking method using the T3 primer shown in SEQ ID NO: 5 and the T7 primer shown in SEQ ID NO: 6. By this sequence analysis, the gene sequence described in SEQ ID NO: 1 was obtained. Using the base sequence and amino acid sequence of this gene, a homology search program BLAST search (http://www.ncbi.nlm.nih.gov/BLAST/) was performed, and homology search with a known gene was performed. The obtained gene was found to be the TRIP11 gene. The human homologous factor of canine TRIP11 was human TRIP11 (homology: base sequence 88%, amino acid sequence 86%). The nucleotide sequence of human TRIP11 is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO: 4.

(4) Expression analysis in each tissue Expressions in normal tissues and various cell lines of dogs and humans were examined by RT-PCR (Reverse Transcription-PCR) for the genes obtained by the above method. The reverse transcription reaction was performed as follows. That is, total RNA was extracted from 50-100 mg of each tissue and 5-10 × 10 ⁶ cells of each cell line using TRIZOL reagent (manufactured by Invitrogen) according to the attached protocol. Using this total RNA, cDNA was synthesized by Superscript First-Strand Synthesis System for RT-PCR (manufactured by Invitrogen) according to the attached protocol. Gene pool cDNA (manufactured by Invitrogen), QUICK-Clone cDNA (manufactured by Clontech), and Large-Insert cDNA Library (manufactured by Clontech) were used as cDNAs for normal human tissues (brain, hippocampus, testis, colon, placenta). . The PCR reaction was performed as follows using the obtained gene-specific primers (described in SEQ ID NOs: 7 and 8). That is, 0.25 μl of a sample prepared by reverse transcription reaction, 2 μM each of the above primers, 0.2 mM of each dNTP, 0.65 U ExTaq polymerase (manufactured by Takara Shuzo Co., Ltd.), and each reagent and attached buffer were added to make a total volume of 25 μl. Using Cycler (manufactured by BIO RAD), a cycle of 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 1.5 minutes was repeated 30 times. The gene-specific primer comprises a region of bases 1519 to 2957 in the base sequence of SEQ ID NO: 1 (canine TRIP11 gene) and 1872 to 3310 bases in the base sequence of SEQ ID NO: 3 (human TRIP11 gene). Amplification was performed, and the expression of both the canine TRIP11 gene and the human TRIP11 gene could be examined using the primers. For comparison, GAPDH specific primers (described in SEQ ID NOs: 9 and 10) were also used at the same time. As a result, as shown in FIG. 1, the canine TRIP11 gene was strongly expressed in the testis in healthy dog tissues, whereas it was strongly expressed in the canine breast cancer cell line. Similar to the canine TRIP11 gene, the expression of the human gene was confirmed only in the testis, but in human cancer cells, various types of human cancer cells such as brain tumors, leukemia, breast cancer, lung cancer, and esophageal cancer cell lines were found. Expression was detected in cancer cell lines, and it was confirmed that the human TRIP11 gene was also expressed specifically in testis and cancer cells.

  In FIG. 1, the reference number 1 on the vertical axis indicates the expression pattern of the TRIP11 gene, and the reference number 2 indicates the expression pattern of the GAPDH gene as a comparative control.

Example 2: Production of canine and human TRIP11 protein (1) Production of recombinant protein Based on the gene of SEQ ID NO: 1 obtained in Example 1, a recombinant protein was produced by the following method. For PCR, 1 μl of a vector prepared from the phagemid solution obtained in Example 1 and subjected to sequence analysis was used, and two types of primers containing SalI and XhoI restriction enzyme cleavage sequences (described in SEQ ID NOs: 11 and 12) were each 0.4 μM. , 0.2 mM dNTP, 1.25 U PrimeSTAR HS polymerase (Takara Shuzo), add each reagent and attached buffer to make a total volume of 50 μl, and use Thermal Cycler (BIO RAD), 98 ° C. for 10 seconds, It was performed by repeating a cycle of 55 ° C. for 5 seconds and 72 ° C. for 6 minutes 30 times. The two types of primers were those that amplify the region encoding the entire amino acid sequence of SEQ ID NO: 2. After PCR, the amplified DNA was electrophoresed on a 1% agarose gel, and a DNA fragment of about 6.0 kbp was purified using QIAquick Gel Extraction Kit (manufactured by QIAGEN).

  The purified DNA fragment was ligated to the cloning vector pCR-Blunt (manufactured by Invitrogen). After transforming this into E. coli, the plasmid was recovered, and it was confirmed by sequencing that the amplified gene fragment matched the target sequence. A plasmid corresponding to the target sequence was treated with SalI and XhoI restriction enzymes, purified with the QIAquick Gel Extraction Kit, and then inserted into the expression vector pET30b (Novagen) treated with SalI and XhoI restriction enzymes. . By using this vector, a His-tagged recombinant protein can be produced. This plasmid was transformed into E. coli BL21 (DE3) for expression, and the target protein was expressed in E. coli by inducing expression with 1 mM IPTG.

  Further, based on the gene of SEQ ID NO: 3, a recombinant protein of human homologous gene was prepared by the following method. For PCR, 1 μl of cDNA confirmed to be expressed by RT-PCR method from various tissue / cell cDNA prepared in Example 1 and two kinds of primers containing NdeI and KpnI restriction enzyme cleavage sequences (described in SEQ ID NOs: 13 and 14). ) Each 0.4 μM, 0.2 mM dNTP, 1.25 U PrimeSTAR HS polymerase (Takara Shuzo Co., Ltd.), each reagent and attached buffer are added to make a total volume of 50 μl, and Thermal Cycler (BIO RAD) is used, 98 A cycle of 10 ° C.-10 seconds, 55 ° C.-5 seconds, 72 ° C.-6 minutes was repeated 30 times. The two types of primers were those that amplify the region encoding the entire amino acid sequence of SEQ ID NO: 4. After PCR, the amplified DNA was electrophoresed on a 1% agarose gel, and a DNA fragment of about 6.0 kbp was purified using QIAquick Gel Extraction Kit (manufactured by QIAGEN).

  The purified DNA fragment was ligated to the cloning vector pCR-Blunt (manufactured by Invitrogen). After transforming this into E. coli, the plasmid was recovered, and it was confirmed by sequencing that the amplified gene fragment matched the target sequence. A plasmid corresponding to the target sequence was treated with NdeI and KpnI restriction enzymes, purified with the QIAquick Gel Extraction Kit, and then inserted into the expression vector pET30b (Novagen) treated with NdeI and KpnI restriction enzymes. . By using this vector, a His-tagged recombinant protein can be produced. This plasmid was transformed into expression Escherichia coli BL21 (DE3), and the target protein was expressed in Escherichia coli by inducing expression with 1 mM IPTG.

(2) Purification of recombinant protein Recombinant Escherichia coli expressing SEQ ID NO: 1 and SEQ ID NO: 3 obtained as described above was absorbed in LB medium containing 30 μg / mL kanamycin until the absorbance at 600 nm was around 0.7. After culturing at 37 ° C., isopropyl-β-D-1-thiogalactopyranoside was added to a final concentration of 1 mM, followed by culturing at 30 ° C. for 20 hours. Thereafter, the cells were collected by centrifugation at 4800 rpm for 10 minutes. The cell mass was suspended in phosphate buffered physiological saline, and further centrifuged at 4800 rpm for 10 minutes to wash the cell.

  The obtained cell mass was suspended in phosphate buffered physiological saline, and ultrasonically crushed on ice. The E. coli sonication solution was centrifuged at 7000 rpm for 15 minutes, and the resulting supernatant was used as a soluble fraction and the precipitate was used as an insoluble fraction.

  The insoluble fraction was suspended in 4% Triton X-100 solution and centrifuged at 7000 rpm for 10 minutes. This procedure was repeated twice to perform a protease removal operation. Thereafter, the residue was suspended in phosphate buffered saline and subjected to desurfactant operation.

  This residue was suspended in 6 mM guanidine hydrochloride-containing 20 mM phosphate buffer (pH 8.0) and allowed to stand at 4 ° C. for 20 hours to denature the protein. After this denaturation operation, the soluble fraction obtained by centrifugation at 7000 rpm for 20 minutes was prepared by a nickel chelate column (carrier Chelateing Sepharose (trademark) Fast Flow (GE Health Care)), column volume 5 mL, equilibration buffer. 6M guanidine hydrochloride-containing 20 mM phosphate buffer (pH 8.0)). After washing the unadsorbed fraction with 20 mM phosphate buffer (pH 8.0) containing 6M guanidine hydrochloride 10 times the column volume and 20 mM phosphate buffer (pH 8.0) containing 10 mM imidazole, Immediately, elution was performed with a 50 mM-500 mM imidazole stepwise concentration gradient divided into 4 steps to obtain a purified fraction, and this purified fraction was used as a material for administration tests. The target protein in the eluted fraction was confirmed by Coomassie staining performed according to a conventional method. Of these, canine TRIP11 protein is shown in FIG.

Reaction buffer 1ml of 200μl of purified preparation obtained by the above method (20mM Tris-HCl, 50mM NaCl , 2mM CaCl 2 pH7.4) after dispensing the enterokinase (Novagen Co.) 2 [mu] l After the addition, the mixture was allowed to stand and react overnight at room temperature, the His tag was cleaved, and purification was performed using the Enterokinase Cleavage Capture Kit (Novagen) according to the attached protocol. Next, 1.2 ml of the purified sample obtained by the above method was replaced with a physiological phosphate buffer (manufactured by Nissui Pharmaceutical) using ultrafiltration NANOSEP 10K OMEGA (manufactured by PALL), and then HT tough Sterile filtration was performed with 0.22 μm Linacrodisc (manufactured by PALL), and this was used in the following experiments.

Example 3: Administration test of recombinant protein to cancer-bearing dogs (1) Anti-tumor evaluation Two kinds of groups purified as described above for two dogs with cancer on the epidermis (2 breast tumors) The antitumor effect of the replacement protein was evaluated.

Recombinant canine TRP11 and human TRIP11 proteins purified as described above were mixed with 100 μg (0.5 ml) each of 0.5 ml of incomplete Freund's adjuvant (manufactured by Wako Pure Chemical Industries, Ltd.) to prepare a cancer therapeutic agent. A total of 3 doses were given to the regional lymph nodes near the tumor after 7 days. As a result, cancer therapeutic agent at the time of administration, the size of about 75 mm ³ and 102 mm ³ and was the mass respectively, the each of the 10 days after the first administration of the therapeutic agent 63 mm ³ and 85 mm ^3, respectively, after 20 days 35mm ³ and 42mm ^3, after 30 days was reduced respectively to 15 mm ³ and 19 mm ^3.

In addition, to dogs with mastocytoma, 100 μg (0.5 ml) of canine TRP11 protein mixed with 0.5 ml of incomplete Freund's adjuvant was administered three times in the same manner as described above. At the same time, 100 μg of canine interleukin 12 was subcutaneously administered. As a result, the tumor, which had a size of about 165 mm ³ at the time of administration of the cancer therapeutic agent, was completely regressed 23 days after the initial administration of the cancer therapeutic agent.

(2) Evaluation of immunity induction ability Blood of dogs with mammary gland tumors for which antitumor effects were obtained in the administration test in (1) above was collected, and peripheral blood mononuclear cells were isolated according to a conventional method. Evaluation of immunity induction with respect to the administered protein was performed by the IFNγ ELISPOT assay method.

Add 100 μl / well of 70% ethanol to a 96-well plate (MultiScreen-IP, MAIPS4510) manufactured by Millipore, leave it for 5 minutes, remove by suction, wash with sterile water, and wash with 200 mM Sodium Bicarbonate (pH 8.2). After adding 300 μl / well and allowing to stand for 5 minutes, the plate was washed by suction removal. Next, 0.5 μg / well of anti-canine interferon γ monoclonal antibody (R & D, clone 142529, MAB781) added to 200 mM Sodium Bicarbonate was added and incubated overnight at 37 ° C. to immobilize the primary antibody. After the primary antibody was removed by suction, 300 μl / well of blocking solution (1% BSA-5% sucrose-200 mM Sodium Bicarbonate (pH 8.2)) was added and incubated overnight at 4 ° C. to block the plate. After removing the blocking solution by suction, 300 μl / well of RPMI medium (Invitrogen) containing 10% fetal calf serum was added and allowed to stand for 5 minutes to remove the medium by suction. After that, each canine peripheral blood mononuclear cell suspended in RPMI medium containing 10% fetal bovine serum was added to the plate at 5x10 ⁵ cells / well, and the canine TRIP11 or human TRIP11 protein used for each administration was added to this plate. By adding 10 μl / well and culturing under conditions of 37 ° C. and 5% CO ₂ for 24 hours, interferon γ was produced from immune cells that could be present in peripheral blood mononuclear cells. After culturing, the medium was removed, and the wells were washed 6 times with a washing solution (0.1% Tween 20-200 mM Sodium Bicarbonate (pH 8.2)). Rabbit anti-dog polyclonal antibody diluted 1000-fold with the blocking solution was added to each well in an amount of 100 μl and incubated overnight at 4 ° C. After washing the wells with the washing solution three times, 100 μl of HRP-labeled anti-rabbit antibody diluted 1000 times with the blocking solution was added to each well and reacted at 37 ° C. for 2 hours. After washing the well three times with the above washing solution, color was developed with Konica Immunostain (manufactured by Konica), and the well was washed with water to stop the reaction. After stopping the reaction, the membrane was dried, the wells were imaged, and spot-forming cells (SFC) were counted with a KS ELISPOT compact system (Carl Zeiss, Germany).

  As a result, no spots were detected in peripheral blood mononuclear cells before administration in dogs treated with either canine TRIP11 protein or human TRIP11 protein. On the other hand, in the dogs to which canine TRIP11 was administered, 26 spots and 65 spots were detected in peripheral blood mononuclear cells 10 days and 30 days after administration, respectively. In the dogs to which human TRIP11 was administered, 31 spots and 72 spots were detected in peripheral blood mononuclear cells 10 days and 30 days after administration, respectively.

  From the above results, it can be confirmed that in any administration dog, immune cells producing interferon γ reacting specifically with the administered recombinant protein are induced. It is considered that the antitumor effect shown in (1) above was exerted by the immune reaction.

It is a figure which shows the expression pattern in the normal tissue and tumor cell line of the gene which codes TRIP11 protein. Reference number 1; expression pattern of gene encoding TRIP11 protein; reference number 2; expression pattern of GAPDH gene. It is the figure which detected one canine TRIP11 protein of the polypeptide used by this invention manufactured and refine | purified in Escherichia coli by Coomassie staining. Reference number 3; shows the band of canine TRIP11 protein.

Claims (6)

The polypeptide of any one of the following (a) and (b) can be expressed in vivo using a polypeptide having immunity-inducing activity as an active ingredient or a polynucleotide encoding the polypeptide. A therapeutic and / or prophylactic agent for cancer comprising a recombinant vector as an active ingredient.
(a) A polypeptide having the amino acid sequence shown in SEQ ID NO: 2 or 4 in the sequence listing.
(b) (a) polypeptide and 86% or more homology polypeptides have a.   The agent for treating and / or preventing cancer according to claim 1, wherein the polypeptide (b) is a polypeptide having 95% or more homology with the polypeptide (a). Treatment and / or prophylactic agent for cancer according to claim 1, wherein the polypeptide polypeptide having the amino acid sequence shown in SEQ ID NO: 2 or 4 in the Sequence Listing having the immunity-inducing activity. The agent for treating and / or preventing cancer according to any one of claims 1 to 3 , which is for humans, dogs or cats. The therapeutic and / or prophylactic agent for cancer according to any one of claims 1 to 4 , further comprising an immune enhancing agent. The immunopotentiator comprises a group consisting of Freund's incomplete adjuvant, Montanide, poly IC and derivatives thereof, CpG oligonucleotides, interleukin 12, interleukin 18, interferon α, interferon β, interferon ω, interferon γ and Flt3 ligand. The therapeutic and / or prophylactic agent for cancer according to claim 5 , which is at least one selected.