JP5217502B2 - Cancer treatment and / or prevention agent - Google Patents

Description

  The present invention relates to a novel therapeutic and / or prophylactic 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). A clinical trial of cancer immunotherapy targeting some of them has also started.

  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 cats have a tumor that has progressed to a larger mass, and the owner notices it. If you come to the hospital and have it removed by surgery, or if you are given a human medicine (such as an anticancer drug) 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

  An object of the present invention is to provide a therapeutic and / or prophylactic agent for cancer useful for the treatment or prevention of 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. Obtained and based on the cDNA, the amino acid sequence of the protein was identified. Then, the inventors have found that a recombinant polypeptide produced based on the amino acid sequence can induce antibodies in vivo, and that the already produced tumor can be regressed by administering the recombinant polypeptide to the living body. It was. Further, a polypeptide prepared based on the amino acid sequence derived from dog is presented by human-derived antigen-presenting cells and has the ability to activate and proliferate human-derived cytotoxic T cells specific for the polypeptide. Thus, the present inventors have found that human-derived cytotoxic T cells specific for the polypeptide have the ability to damage human cancer cells that express homologous factors of the polypeptide, thereby completing the present invention.

That is, the present invention relates to a polypeptide represented by any one of SEQ ID NOs: 2, 11, 12 and 13 in the sequence listing, or a polypeptide having 80% or more homology with the polypeptide, containing polypeptide having the activity, or the one of the polypeptides as an active ingredient an expressible recombinant vector in vivo that provides the treatment of cancer and / or prophylactic agent. Further, the present invention is a polypeptide comprising at least 180 or more consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, and comprising a region of amino acids 27 to 206 in the amino acid sequence, or Provided is a therapeutic and / or prophylactic agent for cancer comprising, as an active ingredient, a polypeptide having 80% or more homology with the polypeptide and having anticancer activity. Furthermore , the present invention provides an isolated antigen-presenting cell comprising a complex of any of the following polypeptides (A) to (C) having anticancer activity and an HLA molecule: (A ) A polypeptide comprising 7 or more consecutive amino acids in the amino acid sequence shown in any one of SEQ ID NOs: 11 to 13 in the sequence listing, and (B) having a homology of 80% or more with the polypeptide of (A) A polypeptide comprising 7 or more amino acids, (C) a polypeptide having 8 to 30 amino acid residues, comprising the polypeptide of (A) or (B) as a partial sequence. Furthermore, the present invention provides an isolated T cell that selectively binds a complex of a polypeptide of any one of (A) to (C) above having anticancer activity and an HLA molecule. Furthermore, the present invention provides a medicament comprising the isolated antigen-presenting cell of the present invention or the isolated T cell of the present invention as an active ingredient. Furthermore, the present invention provides a therapeutic and / or prophylactic agent for cancer comprising the isolated antigen-presenting cell of the present invention or the isolated T cell of the present invention as an active ingredient.

  According to the present invention, a novel cancer treatment and / or prevention 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, the tumor that has already occurred can be regressed, so that the treatment and / or prevention of the cancer of the present invention can be achieved. The agent is useful for the treatment and prevention of cancer. In addition, as shown in the Examples below, the above-mentioned polypeptides derived from dogs activate human CD8-positive T cells, which show excellent cytotoxic activity against human cancer cells. Thus, the polypeptide is also useful for the treatment and / or prevention of human cancer.

Examples of polypeptides that can be used as an active ingredient in cancer treatment and / or prevention agents 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, and in the present invention, a protein consisting of the full-length of SEQ ID NO: 2 is also included.
(a) A polypeptide consisting of 7 or more consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing and having anticancer activity.
(b) A polypeptide having anticancer activity, having 80% or more homology with the polypeptide of (a) and consisting of 7 or more amino acids.
(c) A polypeptide comprising the polypeptide of (a) or (b) as a partial sequence and having anticancer 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 206 amino acids having the amino acid sequence of Met Asn Arg Leu,. 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, the “anticancer activity” means the ability to suppress the growth of cancer cells expressing the polypeptide of SEQ ID NO: 2 or a homologous factor thereof, or to reduce or eliminate cancer tissues (tumors). As specifically described in the Examples below, a polypeptide produced based on the amino acid sequence shown in SEQ ID NO: 2 can regress a tumor when administered to a cancer-bearing dog. Whether or not the polypeptide has anticancer activity should be confirmed by examining whether or not the tumor can be reduced by actually administering it to a cancer-bearing organism, as specifically described in the Examples below. Can do. In addition, for example, the anticancer activity of a polypeptide (the ability to suppress the growth of cancer cells) is whether T cells stimulated with the polypeptide can damage cancer cells that express the polypeptide of SEQ ID NO: 2. It can also be evaluated by examining. The cytotoxic activity of T cells can be measured according to a known method called ⁵¹ Cr release assay described in Int. J. Cancer, 58: p317,1994. Can be performed in the manner described in detail below.

  The amino acid sequence shown in SEQ ID NO: 2 in the sequence listing binds to an antibody specifically present in the serum derived from a cancer-bearing dog by the SEREX method using a dog testis-derived cDNA library and the serum of the cancer-bearing dog. It is the amino acid sequence of a novel polypeptide isolated as a polypeptide (see Reference Example 1).

The polypeptide (a) is a polypeptide comprising 7 or more consecutive amino acids in the amino acid sequence represented by SEQ ID NO: 2, preferably 9 or more consecutive, more preferably 10 or more consecutive amino acids. Have anti-cancer activity. In addition, preferably, the polypeptide has a sequence located in the region of amino acids 27 to 206 in the amino acid sequence represented by SEQ ID NO: 2. Among the polypeptides, particularly preferred examples include a polypeptide comprising a region of amino acids 27 to 206 in SEQ ID NO: 2 and a polypeptide having the amino acid sequence represented by SEQ ID NO: 2. The anti-cancer activity confirmed in Example 1 below is a recombinant polypeptide comprising a region of amino acids 27 to 206 in the amino acid sequence represented by SEQ ID NO: 2. As is known, antigenicity can be exerted with a polypeptide having about 7 amino acid residues or more. For example, as shown in Example 3 below, the region from amino acid 109 to amino acid 118 (SEQ ID NO: 11) and the region from amino acid 129 to amino acid 137 (SEQ ID NO: 12) in the amino acid sequence shown in SEQ ID NO: 2 ), Or a 9 to 10 amino acid polypeptide consisting of a region of amino acids 177 to 185 (SEQ ID NO: 13) is a human homologous factor of SEQ ID NO: 2 ("homology factor" is derived from mammals other than dogs) A naturally occurring polypeptide having an amino acid sequence identical to or substantially identical to SEQ ID NO: 2 (including an amino acid sequence comprising the same or substantially identical amino acid sequence as SEQ ID NO: 2), A polypeptide that exhibits an action corresponding to the action of a peptide in the mammal.Expression of a homologous factor is described, for example, in Reference Example 1 below. In other words, human cancer cells expressing a primer (SEQ ID NOS: 5 and 6) prepared based on the nucleotide sequence of SEQ ID NO: 1 which is a canine gene sequence can be damaged. it can. Therefore, since a polypeptide having 7 or more amino acid residues can have anticancer activity, it can be used for preparation of a therapeutic and / or prophylactic agent for cancer . However, in the case where the polypeptide exhibits anticancer activity by inducing an antibody against cancer cells, in view of the fact that the antibody produced against the antigenic substance in the living body is a polyclonal antibody, A larger number can induce more types of antibodies that recognize various sites on the antigenic substance, and thus it is considered that anticancer activity can be enhanced. Therefore, in order to enhance anticancer activity, the number of amino acid residues is preferably 30 or more, more preferably 50 or more, still more preferably 100 or more, and even more preferably 150 or more. The amino acid sequence shown in SEQ ID NO: 15 in the sequence listing is an example of the amino acid sequence of the human homologous factor of the polypeptide of SEQ ID NO: 2, and is registered in GenBank with accession number NP_056145.1.

  In addition, as a principle of immune induction by administering a cancer antigen polypeptide, the polypeptide undergoes degradation by peptidase in the body to become a smaller fragment, the fragment is taken up by antigen-presenting cells and presented on the surface, It is known that cytotoxic T cells and the like recognize this and selectively kill cells presenting the antigen. In this case, since the size of the polypeptide taken up by the antigen-presenting cell is relatively small and is about 7 to 30 amino acids, about 7 to 30 consecutive amino acids in the amino acid sequence represented by SEQ ID NO: 2, preferably 9 to A polypeptide comprising about 30 amino acids, more preferably about 10 to 30 amino acids is effective. However, even when the size of the polypeptide is larger than 30 in terms of the number of amino acids, in vivo peptidase undergoes cleavage at random positions to generate various polypeptide fragments, and these polypeptide fragments are taken up by antigen-presenting cells. Therefore, as described above, if a polypeptide having a large size such as one containing the region of amino acids 27 to 206 in the full length of SEQ ID NO: 2 or the amino acid sequence shown in SEQ ID NO: 2 is administered, its life Degradation in the body inevitably results in polypeptide fragments that are effective for inducing immunity through antigen-presenting cells. Therefore, a polypeptide having a large size is also preferable for immunity induction via antigen-presenting cells, and the number of amino acids may be 30 or more, more preferably 50 or more, more preferably 100 or more, more preferably 150 or more, as described above. .

The polypeptide (b) is a polypeptide in which one to several amino acid residues of the polypeptide (a) are substituted, deleted and / or inserted, and having the original sequence 80% or more, preferably 90% or more, more preferably 95% or more, and a polypeptide having anticancer 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 exhibit anticancer activity, it can be used for the preparation of a therapeutic and / or prophylactic agent for cancer . Here, the “homology” of amino acid sequences means that both amino acid sequences are aligned so that as many amino acid sequence residues as possible match (insert a gap if necessary), and the number of matched amino acid residues is This is a percentage expressed by dividing the total number of amino acid residues (the total number of amino acid residues in the longer sequence when the total number of amino acid residues differs between the two sequences). It can be easily calculated using software. 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 replacing the amino acid residues in a polypeptide of the upper Symbol (a), by substituting within the same group, the possibility of maintaining the anticancer activity is increased. In the polypeptide (b), one to several amino acids are substituted and / or deleted in the amino acid sequence represented by SEQ ID NO: 2, and / or one to several amino acids are added to the amino acid sequence. It is also preferred that the polypeptide has a single amino acid inserted therein.

The polypeptide (c) is a polypeptide having the anti-cancer activity, comprising the polypeptide (a) or (b) as a partial sequence. That is, the polypeptide (a) or (b) is obtained by adding another amino acid or polypeptide to one end or both ends of the polypeptide, and has anticancer activity. Such a polypeptide can also be used for the preparation of a therapeutic and / or prophylactic agent for cancer .

Polypeptide as described above, for example, Fmoc method (fluorenyl methyloxy carbonyl method) can be synthesized according to the chemical synthesis methods such as tBoc method (t-butyloxycarbonyl method). Moreover, it can also synthesize | combine by a conventional method using various commercially available peptide synthesizers. Moreover, it can prepare easily using a well-known genetic engineering method. For example, from the RNA extracted from the tissue expressing the gene encoding the polypeptide of SEQ ID NO: 2, cDNA of the gene is prepared by RT-PCR, and the full length or a desired part of the cDNA is incorporated into an expression vector. Thus, the desired polypeptide can be obtained by introducing it into a host cell. Extraction of RNA, RT-PCR, incorporation of cDNA into a vector, and introduction of a vector into a host cell can be performed by well-known methods, for example, as described below. Further, vectors and host cells to be used are well known, and various types are commercially available.

  Specifically, the DNA encoding the polypeptide used in the present invention can be prepared, for example, as follows. DNA having the base sequence of SEQ ID NO: 1 is obtained by performing PCR using a pair of primers designed to amplify the base sequence described in SEQ ID NO: 1, using canine chromosomal DNA or cDNA library as a template. Can be prepared. 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 described in SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence listing in the present specification, and a cDNA library such as a dog is prepared using the probe or primer. The DNA can be isolated by screening. The cDNA library is preferably prepared from cells, organs or tissues expressing the DNA. 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 having the base sequence of SEQ ID NO: 1 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 DNA encoding a specific amino acid sequence can be easily specified. Accordingly, since the base sequence of the DNA encoding the polypeptide (b) or the polypeptide (c) described above can be easily specified, such a DNA can be obtained by a conventional method using a commercially available nucleic acid synthesizer. Can be easily synthesized.

  The host cell may be any cell capable of expressing the polypeptide used in the present invention. Examples of prokaryotic cells include Escherichia coli, and examples of eukaryotic cells include monkey kidney cells COS 1 And mammalian cultured cells such as Chinese hamster ovary 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 polypeptide used in the present invention is incorporated into such an expression vector, a prokaryotic host cell is transformed with the vector, and the resulting transformant is cultured, the DNA is encoded. The polypeptide can be expressed in prokaryotic host cells. 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 polypeptide used in the present invention is incorporated into such an expression vector, and after transforming a eukaryotic host cell with the vector, the obtained transformant is cultured. Polypeptides encoded by the DNA 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 ion exchange chromatography, hydrophobic chromatography, affinity chromatography, reverse phase chromatography, and the like.

Polypeptides obtained by the above methods include those in the form of fusion proteins with other arbitrary proteins. For example, glutathione-S-transferase (GST), a fusion protein with a His tag, and the like can be exemplified. Such a polypeptide in the form of a fusion protein is also included in the above-mentioned polypeptide (c) and can be used for preparation of a therapeutic and / or prophylactic agent for cancer . Furthermore, the polypeptide expressed in the transformed cell may be subjected to various modifications in the cell after being translated. Such post-translationally modified polypeptides are also within the scope of the present invention as long as they have anticancer 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 a cancer to be treated as a cancer therapeutic and / or preventive agent of the present invention, a gene encoding the polypeptide of SEQ ID NO: 2 or its homologous factor is expressed (the polypeptide of SEQ ID NO: 2 or its homologous factor is expressed). Cancers (including those expressed as a partial region), including brain tumors, squamous cell carcinoma of the head, neck, lungs, uterus or esophagus, melanoma, lung, breast or uterine adenocarcinoma, renal cancer, etc. However, it is not limited to these. The target animal is a mammal, and dogs, cats, and humans are particularly preferable. Of these, dogs and cats are preferable, and dogs are more preferable. In human cancer, the human homologous factor of the polypeptide of SEQ ID NO: 2 is expressed, taking the polypeptide having the amino acid sequence shown in SEQ ID NO: 15 as an example. A region having high homology with the sequence of No. 2 is included. The therapeutic and / or prophylactic agent for cancer of the present invention is effective for treating and / or preventing human cancer by targeting cancer cells that express the human homologous factor.

  The administration route of the therapeutic and / or prophylactic agent for cancer of the present invention comprising the above polypeptide as an active ingredient may be oral administration or parenteral administration, but intramuscular administration, subcutaneous administration, intravenous administration, intraarterial administration, etc. Parenteral administration is preferred. In order to enhance the anti-cancer effect, as described in the following Examples, it can be administered to regional lymph nodes near the tumor to be treated or subcutaneously near the tumor. The dose may be any amount that is effective for the treatment and / or prevention of cancer, and is appropriately selected according to the size and symptoms of the tumor. Usually, the effective amount of polypeptide per day for the subject animal is 0.0001. μg to 1000 μg, preferably 0.001 μg to 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 following examples, the therapeutic and / or prophylactic agent for cancer of the present invention can regress an already formed tumor. 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 cancer treatment and / or prevention agent of the present invention is useful for both cancer treatment and prevention. In addition, the administration route of the medicine which uses the isolated T cell or isolated antigen-presenting cell of the present invention as an active ingredient, particularly a cancer treatment and / or prevention agent will be described later.

  The therapeutic and / or prophylactic agent for cancer of the present invention may consist of a polypeptide alone, or an additive such as a pharmacologically acceptable carrier, diluent, excipient, etc. suitable for each dosage form. Can be formulated by mixing them as appropriate. 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.

In particular, the treatment and / or prophylactic agent for cancer of the present invention that the polypeptide as an active ingredient, preferably further comprising an adjuvant in addition to the active ingredient. Adjuvants can enhance the immunological response by providing a reservoir of antigen (extracellular or in macrophages), activating macrophages and stimulating a specific set of lymphocytes. The anticancer effect of the therapeutic and / or prophylactic agent can be enhanced. Many types of adjuvants are well known in the art. Specific examples include MPL (SmithKline Beecham), Salmonella minnesota Re 595 lipopolysaccharide of the genus Salmonella and similar products obtained after acid hydrolysis; QS21 (SmithKline Beecham), pure QA- purified from Quillja saponaria extract 21 saponins; DQS21 described in PCT application WO 96/33739 (SmithKline Beecham); QS-7, QS-17, QS-18 and QS-L1 (So, outside 10 persons, “Molecules and Cell ( Molecules and cells), 1997, 7, p. 178-186); Freund's incomplete adjuvant; Freund's complete adjuvant; Vitamin E; Montanide; Alum; CpG oligonucleotide (eg, Kreig, et al. 7 (see “Nature”, Vol. 374, pages 546-549)); Poly IC; Poly ICLC; and various water-in-oil emulsions prepared from biodegradable oils such as squalene and / or tocopherol. As a particularly preferred formulation example, the therapeutic and / or prophylactic agent for cancer of the present invention is administered in a form containing a polypeptide contained as an active ingredient and a combination of DQS21 / MPL. The ratio of DQS21 to MPL is typically about 1:10 to 10: 1, preferably about 1: 5 to 5: 1, more preferably about 1: 1. Typically, for human administration, DQS21 and MPL are present in the formulation in the range of about 1 μg to about 100 μg. Other adjuvants are known in the art and can be used in the present invention (eg, Goding, “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.

  Other factors that stimulate the immune response of the subject animal (patient) may also be administered to the patient together with the cancer treatment and / or prevention agent of the present invention. For example, cytokines are useful for enhancing immunological responses as a result of lymphocyte stimulatory properties. Numerous cytokines useful for such purposes are known to those of skill in the art, examples of which include interferon, interleukin-12 (IL-12), which has been shown to enhance the protective effects of vaccines, GM-CSF, IL-18 and Flt3 ligand. Such a factor may be contained in the therapeutic and / or preventive agent for cancer of the present invention, or administered to a patient in combination with the therapeutic and / or prophylactic agent of the present invention as a separate composition. May be.

In addition, the polypeptide used in the present invention can be presented to the antigen-presenting cell by contacting the antigen-presenting cell in vitro. That is, the above-described polypeptides (a) to (c) can be used as a treatment agent for antigen-presenting cells. In this case, the polypeptide is not particularly limited, but preferably has a size of about 30 amino acid residues or less, more preferably 9 to 30 amino acid residues, still more preferably 10 to 30 amino acid residues. As the polypeptide to be presented to the antigen-presenting cell, among the polypeptides (a) to (c) above,
(A) a polypeptide consisting of 7 or more consecutive amino acids in the amino acid sequence shown in any one of SEQ ID NOs: 11 to 13 in the sequence listing and having anticancer activity;
(B) a polypeptide having anticancer activity, having 80% or more homology with the polypeptide of (A) and comprising 7 or more amino acids, and
(C) A polypeptide having an anticancer activity, comprising the polypeptide of (A) or (B) as a partial sequence and having 8 to 30 amino acid residues, particularly any one of SEQ ID NOs: 11 to 13 A polypeptide having the amino acid sequence shown in FIG. As specifically shown in the Examples below, the polypeptide having the amino acid sequence of SEQ ID NOs: 11 to 13 can preferably induce T cells that damage cancer cells. In addition, as described above, a polypeptide having a size of at least 7 amino acid residues can exhibit antigenicity.

  Here, dendritic cells or B cells carrying MHC class I molecules can be preferably used as antigen-presenting cells. In humans, 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, it can be obtained by inducing dendritic cells from bone marrow, umbilical cord blood or patient peripheral blood using granulocyte macrophage colony stimulating factor (GM-CSF) and IL-3 (or IL-4). As a sample for obtaining the above dendritic cells or B cells, bone marrow and umbilical cord blood provided from a healthy living body, patient's own bone marrow and peripheral blood, etc. can be used. Is highly safe and can also be expected to avoid serious side effects. Peripheral blood or bone marrow may be a fresh sample, a cryopreserved sample, or a cryopreserved sample. Peripheral blood, its white blood cell components, and bone marrow cells contain monocytes, hematopoietic stem cells or immature dendritic cells, CD4 positive cells, etc. that are the origin of dendritic cells. The dendritic cells or B cells can be obtained by doing so, but it is more efficient and preferable to separate and culture only the leukocyte component. Furthermore, mononuclear cells may be separated from 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. 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 body temperature of mammals such as humans. 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 itself of contacting the above-described polypeptide used in the present invention with an 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 concentration of the polypeptide 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 polypeptide 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. As described above, the polypeptide to be contacted with the antigen-presenting cell is preferably the above-mentioned polypeptide (A) to (C), and in particular, the polypeptide having the amino acid sequence represented by any of SEQ ID NOs: 11 to 13 is used. More preferred.

  By culturing the antigen-presenting cell in the presence of the above-described polypeptide, the polypeptide 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 polypeptide prepared from the amino acid sequence shown in SEQ ID NO: 2 can induce antibody production in vivo and regress cancer tissue. Therefore, by administering cytotoxic T cells prepared as described above into a living body, cancer cells can be damaged and cancer tissue can be regressed. 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.

  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 polypeptides (a) to (c) as described above, more preferably the polypeptides (A) to (C).

  The administration route of a drug containing an antigen-presenting cell or T cell as an active ingredient, for example, a therapeutic and / or prophylactic agent for cancer, 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.

  In addition, by expressing the polynucleotide encoding the polypeptide of (a) to (c) in the body of the subject animal, antibody production and cytotoxic T cells can be induced in the living body. Accordingly, cancer can be treated and / or prevented. That is, the polynucleotide encoding the polypeptide used in the present invention is also useful as a genetic vaccine. In this case, the polynucleotide encoding the polypeptide is used by being incorporated into a vector that can be expressed in the subject animal cell (preferably in a mammalian cell). Such a vector may be a plasmid vector or a viral vector, and any vector known in the field of gene vaccines may be used. In addition, as described above, a polynucleotide such as DNA encoding the polypeptide can be easily prepared by a conventional method.

  A gene vaccine can be obtained by incorporating a polynucleotide encoding the above polypeptide into a desired vector. Integration into a vector can be performed using methods well known to those skilled in the art.

  The method for administering the gene vaccine may be any method known in the field of gene vaccines, and is not particularly limited. For example, parenteral administration such as intramuscular administration, subcutaneous administration, intravenous administration, and intraarterial administration is preferable. . The dose is appropriately selected according to the type of antigen, symptoms, etc., but is usually about 0.1 μg to 100 mg, preferably about 1 μg to 10 mg, per kg of body weight of the subject animal.

  Examples of the method using a viral vector include retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, poxvirus, poliovirus, Sindbis virus, and other RNA viruses or DNA viruses incorporated into the subject animal. The method of infecting is mentioned. 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 take a pharmaceutical form such as a solution, but is generally an injection containing a DNA encoding the peptide used in the present invention as an active ingredient, etc. If necessary, a conventional carrier 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.

Reference 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 healthy dogs 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 reacted with a sera of a patient dog diluted 250 times at room temperature for 2 to 3 hours.

As the above-mentioned dog serum, serum collected from a dog with squamous cell carcinoma 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 250-fold with TBS-T (0.05% Tween20 / 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 TBST (0.05% Tween20 / TBS), and then diluted 3000 times with TBS containing 0.5% nonfat dry milk as a secondary antibody. Goat anti-dog IgG (Goat anti Dog IgG-h + I HRP conjugated: manufactured by BETHYL Laboratories) was reacted at room temperature for 1 hour, and detected by an enzyme color reaction using an NBT / BCIP reaction solution (manufactured by Roche) Colonies that coincided with the chromogenic reaction positive site were picked 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). Repeat the secondary and tertiary screening in the same way as above until the chromogenic positive colonies are unified, and screen for 35360 phage clones that react with IgG in the serum to 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 Escherichia coli (XL1-Blue MRF ′) prepared to have an absorbance OD ₆₀₀ of 1.0, 100 μl of purified phage solution and 1 μl of ExAssist helper phage (STRATAGENE) are mixed and then 37 After reaction at 15 ° C. for 15 minutes, add 4 ml of LB medium, incubate at 37 ° C. for 2 to 3 hours, immediately incubate in a 70 ° C. water bath for 20 minutes, then centrifuge at 4 ° C. and 5000 rpm for 20 minutes to obtain the supernatant as a phagemid solution As recovered. Next, 100 μl of phagemid host Escherichia coli (SOLR) prepared to have an absorbance OD ₆₀₀ of 1.0 and 100 μl of the purified phage solution were mixed and then inverted at 37 ° C. for 15 minutes, and 100 μl was 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, cultured at 37 ° C. in LB medium containing ampicillin (final concentration 50 μg / ml), and then plasmid DNA having the target insert is prepared using PureLink Quick plasmid Miniprep Kit (manufactured by Invitrogen). Purified.

  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: 3 and the T7 primer shown in SEQ ID NO: 4. 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 a novel gene.

(4) Expression analysis in each tissue Expressions in normal dog tissues and various cancer cell lines were examined by RT-PCR (Reverse Transcription-PCR) for the genes obtained by the above methods. 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. The PCR reaction was performed as follows using the obtained gene-specific primers (described in SEQ ID NOs: 5 and 6). That is, 0.25 μl of the sample prepared by the reverse transcription reaction, 2 μM each of the above primers, 0.2 mM of each dNTP, 0.65 U of 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 a cycler (manufactured by BIO RAD), a cycle of 94 ° C.-1 min, 55 ° C.-1 min, 72 ° C.-30 sec was repeated 30 times. The gene-specific primer was used to amplify a region of bases 40 to 526 in the base sequence of SEQ ID NO: 1. For comparison, β-actin specific primers (described in SEQ ID NOs: 7 and 8) were also used. As a result, as shown in FIG. 1, it was strongly expressed specifically in testis in healthy dog tissues, and strong expression was confirmed in breast cancer cell lines in cancer cell lines. Moreover, when the expression of the homologous factor of the acquired gene was confirmed together, strong expression was detected in human brain tumor, mouse colon cancer, and mouse malignant melanoma.

  In FIG. 1, reference number 1 on the vertical axis indicates the expression pattern of the acquired novel gene, and reference number 2 indicates the expression pattern of the gene for β-actin, which is a comparative control.

Example 1: Production of recombinant protein and confirmation of antitumor effect (1) Production of recombinant protein Based on the gene obtained in Reference Example 1, a recombinant protein was produced by the following method. PCR was prepared from the phagemid solution obtained in Reference Example 1 and subjected to sequence analysis. 1 μl of the vector, BamHI and EcoRI restriction enzyme cleavage sequences and two types of primers containing an enterokinase cleavage site (described in SEQ ID NOs: 9 and 10) ) 1 μM, 0.2 mM dNTP, 1 mM MgSO ₄ , 1 U of KOD polymerase (Toyobo Co., Ltd.), each reagent and attached buffer are added to make a total volume of 50 μl, and Thermal Cycler (BIO RAD Co.) is used. A cycle of 94 ° C. for 15 seconds, 53 ° C. for 30 seconds, and 68 ° C. for 40 seconds was repeated 35 times. The above two types of primers amplify the region encoding the region from the 27th amino acid to the 206th amino acid in the 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 560 bp 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 that matches the target sequence was treated with BamHI and EcoRI restriction enzymes, purified with QIAquick Gel Extraction Kit, and then the target gene sequence was treated with BamHI and EcoRI restriction enzymes pAcGHLT-A (BD Biosciences Pharmingen) Inserted). By using this vector, a GST-fused recombinant protein can be produced. A recombinant virus body containing the target gene was prepared using this plasmid. For the production of a recombinant virus body, an insect cell Sf9 and a BD baculoGold Transfection Kit (manufactured by BD Biosciences Pharmingen) were used to prepare a recombinant virus body containing the target gene sequence according to the protocol attached to the kit. The target protein was expressed in insect cells by infecting insect cells with this virus.

After culturing Sf9 and Sf21 insect cells (about 2 × 10 ⁷ cells) in TFN-FH insect cell medium (BD Biosciences Pharmingen) at 27 ° C. for 3 to 4 days using a 120 cm ² flask, Sf-900 SFM The serum-free medium (manufactured by Invitrogen) was replaced, 50 to 500 μl of the recombinant virus solution was added to the medium, and further cultured at 27 ° C. for 4 days, and then the supernatant was collected.

  Production of the recombinant protein was confirmed by Western blotting in the following manner. Add an equal volume of 2X running buffer (0.125M Tris-HCl pH6.8, 10% 2ME, 4% SDS, 10% sucrose, 0.01% BPB) to the collected culture supernatant and heat-treat at 95 ° C for 5 minutes went. Thereafter, the sample was separated by SDS-PAGE and transferred to a PVDF membrane. The protein-transferred PVDF membrane was blocked with PBS containing 10% Block Ace (manufactured by Snow Brand) at 4 ° C overnight, and then goat polyclonal antibody against anti-GST (manufactured by GE Healthecare Bio-Science) at room temperature. The reaction was carried out for 1 hour. The plate was washed three times with PBST, and reacted with an HRP-labeled anti-goat IgG antibody (GE Healthecare Bio-Science) for 1 hour at room temperature. After washing with PBST three times, reaction with SuperSignal WestFemto (Pierce) was performed, and the expression of the recombinant protein was confirmed by exposing to X-ray film. The recombinant protein contained a region from the 27th amino acid to the 206th amino acid in the amino acid sequence shown in SEQ ID NO: 2.

  After passing the soluble fraction obtained by the above method through a 5 ml Hi-Trap GST column (GE Healthecare Bio-Science), the non-adsorbed fraction was washed with PBS, and immediately 50 mM Tris containing 20 mM glutathione. After elution with a buffer solution of -HCl pH 8.0, the GST part, which is a fusion protein, was cleaved and purified with a specific enzyme.

Specifically, 200 μl of the purified fraction obtained by the above method was dispensed into 1 ml of a reaction buffer (20 mM Tris-HCl, 50 mM NaCl, 2 mM CaCl ₂ pH 7.4), and then enterokinase (Novagen) After addition of 2 μl, the mixture was allowed to stand overnight at room temperature and reacted to cleave GST, and purified 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.

(2) Evaluation of immunity-inducing ability of recombinant protein in vivo in dogs 1 ml of the purified sample obtained in Reference Example 1 was mixed with an equal amount of incomplete Freund's adjuvant (Wako Pure Chemical Industries, Ltd.) Administered subcutaneously in dogs. In a dog as a comparative control, 1 ml of a physiological phosphate buffer (PBS) and an equal amount of incomplete Freund's adjuvant (manufactured by Wako Pure Chemical Industries) were mixed and administered. After one week, blood was collected from the forelimbs, then the serum was separated, and the antibody titer in the serum against the administered recombinant protein was measured using an immunoplate on which the recombinant protein had been immobilized. Evaluated.

Specifically, the antibody titer was measured by the following method. That is, the recombinant protein purified by the above method (1) was immobilized on a 96-well immunoplate manufactured by Nunc and then blocked with 0.5% BSA-50 mM NaHCO ₃ pH8.3 buffer solution to block excess functional groups. After reacting at room temperature for 1 hour, various sera were reacted by diluting 250 times with a buffer solution of 0.5% BSA-50 mM NaHCO ₃ pH 8.3. After washing with PBS-T (PBS containing 0.05% Tween 20), HRP-conjugated goat anti-dog IgG antibody and substrate O-phenylendiamine were added, and the absorbance was measured using an absorptiometer.

  As a result, as shown in FIG. 2, a significantly higher antibody titer against the recombinant protein was detected in the dog administered with the recombinant protein compared to the control dog administered with PBS as a comparative control. Was shown to have high antigenicity. In FIG. 2, reference numbers 3, 5, and 7 are the results obtained using sera collected on days 0, 4, and 7 after administration of the recombinant protein, respectively. The results are obtained using sera collected 0 days, 4 days, and 7 days after administration of PBS, respectively.

(3) Anti-tumor effect of recombinant protein on cancer-bearing dogs The anti-tumor effect of the above-mentioned recombinant protein was evaluated on 2 cancer-bearing dogs (2 breast tumors) having a tumor on the epidermis. Prior to administration, the antibody titer against the recombinant protein in the serum of each patient dog was first measured by the method described in (2) above. As a result, a higher antibody titer was detected compared to healthy dogs (Fig. 3). This suggests that a protein having the amino acid sequence shown in SEQ ID NO: 2 is expressed as a cancer antigen in the tumor tissue in the body of these cancer-bearing dogs.

2 ml of the recombinant protein purified as described in (1) above and an equal volume of incomplete Freund's adjuvant (Wako Pure Chemical Industries, Ltd.) are mixed to prepare a cancer therapeutic agent, which is added to the regional lymph nodes near the tumor every week. Three doses were given. As a result, cancer therapeutic agent at the time of administration of about 50 mm ³ and 100 mm ³ in size was the tumor, respectively, after each 13 days from the cancer therapeutic agent administered and after 21 days, was completely regressed.

In addition, for each affected dog with adenocarcinoma that developed around the anus and squamous cell carcinoma that developed in the epidermis, MOTANIDE ISA51 (manufactured by SEPPIC) in an amount equivalent to 10 μg (0.5 ml) of the recombinant protein purified as described above. Were mixed to prepare a cancer therapeutic agent, which was administered subcutaneously in the vicinity of the tumor four times every week. As a result, when the cancer therapeutic agent was administered, the tumors whose cancer sizes were about 37 mm ³ and 28 mm ³ were completely regressed 35 days and 42 days after the cancer therapeutic agent administration, respectively.

Example 2: Induction of peptide epitope-reactive CD8-positive T cells (1) In order to predict the HLA-A0201 binding motif in the amino acid sequence shown in SEQ ID NO: 2, a known BIMAS software (http: //bimas.dcrt.nih The amino acid sequence of SEQ ID NO: 2 was analyzed using a computer prediction program using (available at .gov / molbio / hla_bind /), and 20 types of polypeptides predicted to be capable of binding to HLA class I molecules were selected.

(2) Peripheral blood was separated from HLA-A0201-positive healthy individuals, layered on a Lymphocyte separation medium (OrganonpTeknika, Durham, NC), and centrifuged at 1,500 rpm at room temperature for 20 minutes. Fractions containing peripheral blood mononuclear cells (PBMC) were collected and washed three times (or more) in cold phosphate buffer to obtain PBMC. The obtained PBMC was suspended in 20 ml of AIM-V medium (Life Technololgies, Inc., Grand Island, NY, USA), and allowed to adhere in a culture flask (Falcon) for 2 hours under conditions of 37 ° C. and 5% CO ₂ . . Non-adherent cells were used for T cell preparation, and adherent cells were used to prepare dendritic cells.

  Meanwhile, adherent cells were cultured in AIM-V medium in the presence of IL-4 (1000 U / ml) and GM-CSF (1000 U / ml). 6 days later, IL-4 (1000 U / ml), GM-CSF (1000 U / ml), IL-6 (1000 U / ml, Genzyme, Cambridge, MA), IL-1β (10 ng / ml, Genzyme, Cambridge, MA) The non-adherent cell population obtained after the AIM-V medium supplemented with TNF-α (10 ng / ml, Genzyme, Cambridge, MA) and further cultured for 2 days was used as dendritic cells.

(3) The prepared dendritic cells are suspended in AIM-V medium at a cell density of 1 × 10 ⁶ cells / ml, the selected polypeptide is added at a concentration of 10 μg / ml, and 37 cells are added using a 96-well plate. ° C., and cultured for 4 hours under the conditions of 5% CO _2. After incubation, X-ray irradiation (3000 rad), washed with AIM-V medium, 10% human AB serum (Nabi, Miami, FL), IL-6 (1000 U / ml) and IL-12 (10 ng / ml, Genzyme) , Cambridge, MA) and suspended in AIM-V medium and added at 1 × 10 ⁵ cells per well of a 24-well plate. Further, the prepared T cell population was added at 1 × 10 ⁶ cells per well, and cultured under conditions of 37 ° C. and 5% CO ₂ . Seven days later, each culture supernatant was discarded, and the dendritic cells treated with each polypeptide obtained in the same manner as described above and then irradiated with X-rays were treated with 10% human AB serum (Nabi, Miami, FL), IL-7 (10 U). / Ml, Genzyme, Cambridge, MA) and AIM-V medium containing IL-2 (10 U / ml, Genzyme, Cambridge, MA) (cell density: 1 × 10 ⁵ cells / ml), 24-well plate 1 × 10 ⁵ cells were added per well and cultured further. The same operation was repeated 4 to 6 times every 7 days, and then stimulated T cells were collected, and induction of CD8 positive T cells was confirmed by flow cytometry.

Example 3: Determination of cytotoxic T cell antigen epitope in SEQ ID NO: 2 that stimulates HLA-A0201-positive CD8 positive T cells (1) Among the T cells in each well induced above, SEQ ID NO: 11, 12 or The proliferation of T cells stimulated with the polypeptide having the amino acid sequence shown in FIG. 13 was confirmed by counting the number of cells under a microscope. In order to examine the specificity for each polypeptide used for pulsing for each T cell in which proliferation was observed, T2 cells expressing the HLA-A0201 molecule pulsed with the polypeptide (Salter RD et al., Immunogenetics, 21: 235-246 (1985), purchased from ATCC) (each polypeptide was added at a concentration of 10 μg / ml in AIM-V medium and cultured for 4 hours under conditions of 37 ° C. and 5% CO ₂ ) 5 × 10 ^{For 4} cells, 5 × 10 ³ T cells were added, and cultured in a 96-well plate in AIM-V medium containing 10% human AB serum for 24 hours. The supernatant after the culture was taken, and the amount of IFN-γ produced was measured by ELISA. As a result, in the culture supernatant of the hole using the T2 cell pulsed with the polypeptide of SEQ ID NO: 11, 12 or 13, compared to the culture supernatant of the hole using the T2 cell not pulsed with the polypeptide, IFN -Γ production was confirmed (FIG. 4). Therefore, the polypeptides of SEQ ID NOs: 11, 12, and 13 were found to be T cell epitope peptides having the ability to specifically stimulate HLA-A0201-positive CD8-positive T cells to proliferate and induce IFN-γ production.

  In FIG. 4, reference numbers 14, 15, and 16 on the vertical axis indicate the results for the polypeptides having the amino acid sequences represented by SEQ ID NOs: 11, 12, and 13, respectively. Reference number 17 is one of the polypeptides derived from SEQ ID NO: 2, but shows the results for the polypeptide SSLQEDLAH (SEQ ID NO: 14) outside the scope of the present invention (Comparative Example 1). Reference number 18 shows the results for the case where the above treatment was performed without adding the polypeptide (Comparative Example 2).

(2) Next, the polypeptide of SEQ ID NO: 11, 12 and 13 which is one of the polypeptides used in the present invention is HLA-A0201 positive and on the tumor cell expressing the protein of SEQ ID NO: 2 or a homologous factor thereof. CD8-positive T cells that are presented on the HLA-A0201 molecule or stimulated with this polypeptide damage HLA-A0201-positive tumor cells expressing the protein of SEQ ID NO: 2 or a homologous factor thereof We examined whether it was possible. A malignant brain tumor cell line, T98G (Stein GH et al., J. Cell Physiol., 99: 43-54 (), in which expression of the homologous factor of the protein of SEQ ID NO: 2 was confirmed by the same method as in Reference Example 1 (4) 1979), ATCC the purchased from) collected 10 ⁵ 50ml ml centrifuge tube and incubated for 2 hours at 37 ° C. was added chromium 51 100 .mu.Ci. Then washed 3 times with AIM-V medium containing 10% human AB serum, was added 10 ^three increments per 96 well V-bottom plates 1 well, suspended further in AIM-V medium containing post 10% human AB serum to Nigosa was ^{10 5,} 5x10 4, was added 2.5 × 10 ⁴ and 1.25 × 10 ⁴ pieces of SEQ ID NO: 11, 12 and 13 each were stimulated with a polypeptide of the HLA-A0201-positive CD8-positive T cells, respectively, The cells were cultured at 37 ° C. and 5% CO ₂ for 4 hours. Cell damage of CD8 positive T cell cells stimulated with each peptide of SEQ ID NO: 11, 12 and 13 by measuring the amount of chromium 51 in the culture supernatant released from the damaged tumor cells after culture Activity was calculated. As a result, it was found that HLA-A0201-positive CD8-positive T cells stimulated with the peptides of SEQ ID NOs: 11, 12, and 13 have cytotoxic activity against T98G (FIG. 5). Therefore, each polypeptide of SEQ ID NO: 11, 12 and 13 which is a polypeptide used in the present invention is HLA-A0201 positive and is expressed on the HLA-A0201 molecule on the tumor cell expressing the homologous factor of the protein of SEQ ID NO: 2. In addition, these polypeptides have been shown to be capable of inducing CD8 positive cytotoxic T cells capable of damaging such tumor cells. In FIG. 5, reference numbers 19, 20, and 21 on the horizontal axis indicate the results for the polypeptides of SEQ ID NOs: 11, 12, and 13, respectively. Further, the reference number 22 on the horizontal axis is one of the protein-derived polypeptides of SEQ ID NO: 2, but the result for the polypeptide of SEQ ID NO: 14 which is a polypeptide outside the scope of the present invention (Comparative Example 3), the horizontal axis Reference numeral 23 in FIG. 6 shows the results when the above treatment was performed without adding the polypeptide (Comparative Example 4).

Incidentally, cytotoxic activity, as described above, and 10 ^three malignant brain tumor cell line T98G that has each polypeptide stimulated induced incorporation of CD8-positive T cells 10 ⁵ and chromium 51 used in the present invention The results are shown in FIG. 5, wherein the amount of chromium 51 released into the culture medium after measurement after culturing was measured and the cytotoxic activity against T98G of CD8 positive T cells calculated by the following calculation formula ^* was shown. ^* Formula: cytotoxic activity (%) = chromium 51 release from T98G and U87 MG when CD8 positive T cells are added ÷ chromium 51 release from target cells added with 1N hydrochloric acid × 100

It is a figure which shows the expression pattern in the canine normal tissue and tumor cell line of the novel gene identified by the reference example. Reference number 1; expression pattern of novel gene, reference number 2; expression pattern of β-actin gene. It is a figure which shows the antibody titer with respect to recombinant protein in the serum extract | collected from the healthy dog which administered recombinant protein. Reference numbers 3, 5 and 7 are the results for sera collected at 0 days, 4 days and 7 days after administration of the recombinant protein, respectively. Reference numbers 4, 6 and 8 are 0 days and 4 days after administration of PBS, respectively. The results for the sera collected after 7 days are shown. It is a figure which shows the reactivity of the antibody in the serum derived from a cancer bearing dog with respect to the recombinant protein prepared in the Example. Reference numbers 9 to 11 show the results for healthy dogs, and reference numbers 12 and 13 show the results for cancer-bearing dogs (breast tumors). The figure which shows that the CD8 positive T cell specific to each polypeptide which has an amino acid sequence shown to sequence number 11 thru | or 13 recognizes the composite_body | complex of this polypeptide and HLA-A0201, and produces IFN-gamma. is there. It is a figure which shows the damage activity with respect to a cancer cell of the CD8 positive T cell specific to each peptide which has an amino acid sequence shown to sequence number 11 thru | or 13.

Claims (10)

A polypeptide represented by the amino acid sequence of SEQ ID NO: 2, 11, 12, or 13 in the sequence listing, or a polypeptide having 80% or more homology with the polypeptide and having anticancer activity , Alternatively , a therapeutic and / or prophylactic agent for cancer comprising a recombinant vector capable of expressing any of the aforementioned polypeptides in vivo as an active ingredient. As an active ingredient a polypeptide represented by the amino acid sequence shown in SEQ ID NO: 2 of the sequence table, the treatment and / or prophylactic agent for cancer according to claim 1. A polypeptide comprising at least 180 or more contiguous amino acids in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, and comprising a region of amino acids 27 to 206 in the amino acid sequence , or 80% with the polypeptide A therapeutic and / or prophylactic agent for cancer comprising a polypeptide having the above homology and having anticancer activity as an active ingredient. As an active ingredient a polypeptide represented by the amino acid sequence depicted in SEQ ID NO: 11, the treatment and / or prophylactic agent for cancer according to claim 1. As an active ingredient a polypeptide represented by the amino acid sequence depicted in SEQ ID NO: 12, the treatment and / or prophylactic agent for cancer according to claim 1. As an active ingredient a polypeptide represented by the amino acid sequence depicted in SEQ ID NO: 13, the treatment and / or prophylactic agent for cancer according to claim 1. An isolated antigen-presenting cell comprising a complex of any of the following polypeptides (A) to (C) having anticancer activity and an HLA molecule.
(A) A polypeptide comprising 7 or more consecutive amino acids in the amino acid sequence shown in any one of SEQ ID NOs: 11 to 13 in the Sequence Listing.
(B) A polypeptide having 80% or more homology with the polypeptide of (A) and comprising 7 or more amino acids.
(C) A polypeptide having 8 to 30 amino acid residues, comprising the polypeptide of (A) or (B) as a partial sequence. An isolated T cell that selectively binds a complex of a polypeptide having any of the following (A) to (C) and having anticancer activity and an HLA molecule.
(A) A polypeptide comprising 7 or more consecutive amino acids in the amino acid sequence shown in any one of SEQ ID NOs: 11 to 13 in the Sequence Listing.
(B) A polypeptide having 80% or more homology with the polypeptide of (A) and comprising 7 or more amino acids.
(C) A polypeptide having 8 to 30 amino acid residues, comprising the polypeptide of (A) or (B) as a partial sequence. Isolated T cells of a medicament comprising as an active ingredient isolated antigen presenting cell or claim 8, wherein according to claim 7 wherein. Treatment and / or prophylactic agent for cancer containing isolated T cells as an active ingredient of isolated antigen presenting cell or claim 8, wherein according to claim 7 wherein.