The name of the invention: the immunity-inducing agent
Technical field
[0001] The present invention relates to a novel immunity-inducing agent useful for the treatment of cancer and / or prophylactic agent or the like.
[0002] The present invention also provides antigen-presenting cells or cytotoxic T cells for use in cancer immunotherapy, or a method of manufacturing of the cell.
Background technique
[0003] Cancer is a disease among all of the causes of death, those treatments that are currently practiced in combination with radiation therapy and chemotherapy mainly surgical therapy. Despite the discovery in recent years of new surgical methods of development and new anti-cancer drugs, with the exception of the part of the cancer, the treatment results of cancer is the current situation is not much improved. In recent years, with the gene encoding the cancer antigen or a cancer antigen recognized by cytotoxic T cells reactive to cancer advances in molecular biology and cancer immunology has been identified, expectations for antigen-specific immunotherapy It is growing.
[0004] In immunotherapy, to reduce side effects, the peptide or protein is recognized as the antigen, most absent in normal cells, is required to be present specifically in cancer cells. 1991, Belgium Ludwig Institute Boon et al CD8-positive T cells by cDNA expression cloning method using an autologous cancer cell line and cancer-reactive T cells were isolated recognize human melanoma antigen MAGE 1 (Non-Patent Document 1) . Thereafter, the antibody recognizes a tumor antigen which is produced in response to autologous cancer in the body of a cancer patient identified by incorporating the technique of expression cloning of genes, SEREX (serological identification of antigens by recombinant expression cloning) method reported (Patent Document 1, non-Patent Document 2), by this method, some cancer antigens have been isolated. Furthermore, clinical trials for cancer immunotherapy have started to a part of the target.
[0005] On the other hand, as with human breast tumors in dogs and cats are known many tumors such as squamous cell cancer are ranked high in disease statistics dogs and cats. However effective therapeutic agents for cancers in dogs and cats, prophylactic and diagnostic agents currently exists. Tumor of the majority of dogs and cats, in most cases, owners will notice case from the increased progress to tumor, or excised by surgery to visit, or by administration of the human body drugs (such as anti-cancer agents) also, already often died shortly after treatment too late. Such in the current, if available therapeutic and prophylactic agent effective cancer dogs and cats, application to cancers in dogs is expected to be opened.
[0006] Chondroitin Sulfate Proteoglycan 5 (CSPG5) is type 1 transmembrane protein, it is one of the neuregulin family proteins. Also, acting as a binding and growth factors ErbB3, expressed in ovarian cancer with mutations in BRCA1 have been reported to be elevated (Non-patent Documents 3 and 4). Further, CSPG5 is known to be involved in retinal ganglion cells and Purkinje cells, is highly expressed in the nervous system tissues hippocampus etc., extension of axon projections by acting as a growth and differentiation factor for neuronal cells ( non-Patent Document 5, 6 and 7). However, having an immunity-inducing activity CSPG5 protein on cancer cells and thereby no reports that the protein is useful in the treatment or prevention of cancer.
CITATION
Patent Literature
[0007] Patent Document 1: U.S. Patent No. 5,698,396

Non-Patent Document
[0008] Non-Patent Document 1: Bruggen, P. et al, Science, 254:. 1643-1647 (1991)
Non-Patent Document 2: Sahin, U et al, Proc Natl Acad Sci USA, 92:..... 11810-11813 (1995)
.... non-Patent Document. 3: Kinugasa,, Y. et Al, Biochem Biophys Res Commun 321: 1045 (2004)
... non-Patent Document 4: Press, JZ et al, Neoplasia Dec; 12 (12) : 993-1002 (2010).
non-Patent Document 5: Yasuda, Y. et al, Neurosci Res 32:... 313 (1998)
non-Patent Document 6:. Aono, S. et al , J. Neurosci Res.. 83: 110 (2006)
non-Patent Document 7: Nakanishi, K. et al, J. Biol Chem 281:... 24970 (2006)

Summary of the Invention
Problems that the Invention is to Solve
[0009] An object of the present invention is to find a useful novel polypeptides for the treatment of cancer and / or prophylactic agent is to provide the use of the immunity-inducing agent of the polypeptide.
Means for Solving the Problems
[0010] The present invention intensively studied, by SEREX method using a canine testis-derived cDNA library and the cancer-bearing dogs serum, cDNA encoding a protein that binds to antibodies present in serum from cancer-bearing living body acquired, based on the cDNA, canine Chondroitin having the amino acid sequence shown in SEQ ID NO: 2 Sulfate Proteoglycan 5 (hereinafter referred to as "CSPG5") was produced polypeptides. Also, human obtained gene, based on cats and mouse homologous gene, a human having an amino acid sequence shown in SEQ ID NO: 4,6,8,10,12,14 and 16, the cat and mouse CSPG5 polypeptide It was produced. And these CSPG5 polypeptides breast cancer, lung cancer, brain cancer, ovarian cancer, leukemia, malignant lymphoma, adenocarcinoma, mastocytoma, squamous cell carcinoma, that is specifically expressed in tissues or cells of melanoma or neuroblastoma heading was. Furthermore, it was found by administering these CSPG5 the living body, can induce immune cells to CSPG5 in vivo, and that can cause regression of tumors in vivo expressing CSPG5. Furthermore, a polypeptide or expressible recombinant vector a polynucleotide encoding a fragment thereof CSPG5 were found to induce an antitumor effect in vivo against cancers expressing CSPG5.
[0011] Furthermore, CSPG5 polypeptide, are presented by antigen presenting cells, have the ability (immunity-inducing activity) for activation and proliferation of specific cytotoxic T cells to the polypeptide, Therefore, the poly peptides are useful in the treatment and / or prevention of cancer, also and antigen-presenting cells contacted with the polypeptide, the T cells contacted with the antigen presenting cells are useful in the treatment and / or prevention of cancer heading, and have completed the present invention.
[0012] Accordingly, the present invention has the following characteristics.
(1) (i) below (a), poly encoding which is selected from polypeptides, and at least one polypeptide having immunity-inducing activity, or (ii) the polypeptide of (b) and (c) immunity-inducing agent comprising comprise a nucleotide, and the recombinant vector allowing the expression of the polypeptide in vivo, as an active ingredient.
(A) SEQ ID NO 8,4,6,10,12,2 or polypeptide consisting of the amino acid sequence represented by 14, and a polypeptide consisting of not less than 7 consecutive amino acids in the amino acid sequence
(b) sequence number 8,4,6,10,12,2 or polypeptide having an amino acid sequence having 85% or more sequence identity represented by 14, and from less than 7 consecutive amino acids in the amino acid sequence of the polypeptide comprising polypeptide
(c) (a) or (b) a polypeptide comprising a polypeptide as a partial sequence
(2) a polypeptide having the immunity-inducing activity, SEQ ID NO: 8,4,6,10,12,2 or a polypeptide consisting of the amino acid sequence represented by 14, immunity-inducing agent according to (1).
(3) it is for the processing of antigen-presenting cells, immunity-inducing agent according to (1) or (2).
(4) is for the treatment and / or prevention of cancer, the immunity-inducing agent according to (1) or (2).
(5), wherein the cancer is a cancer expressing CSPG5, immunity-inducing agent according to (4).
(6), wherein the cancer is brain tumor, leukemia, malignant lymphoma, a or neuroblastoma, immunity-inducing agent according to (4) or (5).
(7) further comprises an immunopotentiator, the immunity-inducing agent according to any one of (1) to (6).
(8) The immunity enhancing agent, Freund's incomplete adjuvant, Montanide, poly IC and a derivative thereof, CpG oligonucleotide, interleukin 12, interleukin 18, interferon alpha, interferon beta, interferon omega, interferon gamma, and Flt3 ligand is at least one selected from the group consisting of, immunity-inducing agent according to (7).
(9) comprising said an antigen presenting cell-derived polypeptide and the test body defining contacting ex vivo or in vitro to (1), the production of antigen-presenting cells containing a complex of the polypeptide and the MHC molecule Method.
(10), wherein the antigen presenting cells are dendritic cells or B cells bearing MHC class I molecules The method according to (9).
(11) the (9) or (10) and T cells from antigen-presenting cells and a subject obtained by the method according to be contacted ex vivo or in vitro comprising activating the T cell, wherein the method for manufacturing a cytotoxic T cells specific to the polypeptide as defined in (1).
(12) the (9) or obtained by the method described in (10), and characterized in that it comprises a complex of a polypeptide and MHC molecules as defined above (1), antigen-presenting cells.
(13), wherein the (11) obtained by the method described in, and is specific for a polypeptide as defined above (1), cytotoxic T cells.
[0013] This description includes the disclosure of the priority document of the present application Japanese Patent Application No. 2015-093354.
[0014] The present invention, novel immunity-inducing agent useful for the treatment and / or prevention and the like of the cancer. As specifically shown in Examples below, when a vector encoding the polypeptide or the polypeptide used in the present invention are administered to a subject, it is possible to induce immune cells in vivo in a subject, Furthermore, it is possible to shrink or regress cancers already occurred. Accordingly, the polypeptide or the vector is useful in the treatment or prevention of cancer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] [Figure 1] of the identified CSPG5 gene is a diagram showing the expression patterns of dog tumor tissues or cancer cell lines was shown. Reference numeral 1: the expression pattern in various tissues and cell lines of canine dog CSPG5 gene, reference number 2; expression pattern in various tissues and cell lines of canine dog GAPDH gene.
[Figure 2] of the identified CSPG5 gene, shows the expression pattern in human tumor tissues or cancer cell lines was shown. Expression in each tissue and cell lines of human human GAPDH gene was observed all.
[Figure 3] of the identified CSPG5 gene is a diagram showing the expression patterns in mouse tumor tissue or the cancer cell lines was shown. Reference numeral 3: shows the expression pattern in the mouse GAPDH gene each tissue and cell lines of mouse; mouse CSPG5 mice expression pattern in various tissues and cell lines of the gene, reference number 4.

DESCRIPTION OF THE INVENTION
[0016] A more detailed description of the present invention.
1. Polypeptides
are included as an active ingredient in the immunity-inducing agent of the present invention, the polypeptide having immunity-inducing activity, include those following the (a) ~ (c).
[0017] (A) SEQ ID NO 8,4,6,10,12,2 or polypeptide consisting of the amino acid sequence represented by 14, or a polypeptide consisting of not less than 7 consecutive amino acids in the amino acid sequence
(b) sequence number 8,4,6,10,12,2 or polypeptide having an amino acid sequence at least 85% sequence identity represented by 14, or from less than 7 consecutive amino acids in the amino acid sequence of the polypeptide comprising polypeptide
(c) (a) or a polypeptide comprising the polypeptide as a partial sequence of (b).
[0018] A "polypeptide" as used herein, refers to a molecule multiple amino acids are formed by peptide bonds, not only the number of amino acids is often a polypeptide molecule which constitutes, in the number of amino acids is less low molecular weight molecules (oligo peptides) or full-length protein are encompassed, in the present invention encompasses also the full-length protein CSPG5 having the amino acid sequence represented by SEQ ID NO: 8,4,6,10,12,2 or 14.
[0019] In the present specification, "having an amino acid sequence", unless otherwise specified, means that the amino acid residues are arrayed in the order shown in the amino acid sequence. Thus, for example, a "polypeptide having an amino acid sequence represented by SEQ ID NO: 8", Met Gly Arg Ala shown in SEQ ID NO: 8, Gly · · (Omitted) · · Asn, Asn, Leu, amino acid Thr having the sequence, means a polypeptide the size of 566 amino acid residues. Further, for example, it may be abbreviated to "a polypeptide having the amino acid sequence represented by SEQ ID NO: 8" and "polypeptide of SEQ ID NO: 8". The same applies to the expression "having the nucleotide sequence". In these cases, the term "comprising" in "having the amino acid sequence," or "having the nucleotide sequence", unless otherwise specified, may be replaced with the term "consisting of".
[0020] In the present specification, "immunity-inducing activity" means the ability to induce the immune cells secrete cytokines such as interferon in a living body of a subject.
[0021] As used herein, "subject", refers to a cancer (or tumor) the treatment or prophylaxis an animal in need of immune induction by immunity-inducing agent of the present invention for, the preferably mammalian, such as human, dog including and pet animals such as cats, animals such as pandas are bred in zoos, etc., domestic animals such as cattle, and sports animals such as horses.
[0022] Whether the polypeptide has an immunity-inducing activity can be known ELISPOT assay; can be confirmed using the (ELISpot Assay Enzyme-Linked ImmunoSpot Assay) or the like. More specifically, for example, as described in the Examples below, the polypeptide to be evaluated immunity-inducing activity has been administered animals to obtain cells such as peripheral blood mononuclear cells the cells the polypeptide cocultivated with, cytokine production amount from the cell, by measuring using a specific antibody, it is possible to measure the number of immune cells in the cell, thereby evaluating the immunity-inducing activity .
[0023] Further, as described in the Examples below, the (a) polypeptides (preferably, the recombinant polypeptide) of ~ (c) the administration of the cancer-bearing living body, cause regression of the tumor by its immunity-inducing activity be able to. Therefore, the immunity-inducing activity, the ability to shrink or disappear to inhibit the growth of cancer cells or cancer tissue (tumor) (hereinafter referred to as "antitumor activity") can be evaluated as. Antitumor activity of the polypeptide, for example as specifically described in the Examples below, actually confirm the polypeptide I by examining whether a tumor by administering to a tumor-bearing animal is reduced and the like can do. Also, the polypeptide of the cytotoxic T cells induced by administering to the tumor-bearing animals, to assess the anti-tumor activity of the polypeptide by examining whether shows the cytotoxic activity against tumor it can. Measurement of cytotoxic activity of T cells in vivo, for example, the T cells were administered antibodies to be removed from the body to the tumor-bearing animals, the tumor can be confirmed I'll to examine whether the reduction or the like but it is not limited to this.
[0024] Alternatively, the polypeptides stimulated T cells (i.e., the polypeptide T cells contacted with the antigen presenting cells presenting) is to examine whether showing cytotoxic activity against tumor cells in vitro Accordingly, it is also possible to evaluate the antitumor activity of the polypeptide. Contact between T cells and antigen presenting cells can be as described below, both performed by cocultivation in liquid media. Measurement of cytotoxic activity, for example, D. D. Kharkevitch et al. , Int. J. Cancer, 58: 317-323,1994 listed in 51 can be carried out by known method called Cr release assay. When using the polypeptide for the treatment and / or prophylactic use of cancer is not particularly limited, it is preferable to evaluate the immunity-inducing activity antitumor activity as an index.
[0025] Amino acid sequence represented by SEQ ID NO: 8,4,6,10,12,2 or 14 in the Sequence Listing of the present invention is disclosed, by SEREX method using a canine testis-derived cDNA library and the cancer-bearing dog serum, polypeptides and human binding to specific antibodies present in serum from cancer-bearing dog was isolated cat, and a mouse homologous factor is the amino acid sequence of CSPG5 (see example 1). In human CSPG5 a human homologous factor of the canine CSPG5, sequence identity of nucleotide sequence 87%, the amino acid sequence 87%, cat phase in cats CSPG5 sequence identity is the factor nucleotide sequence 92% at the amino acid sequence 91% There, murine phase murine sequence identity in CSPG5 a same factor nucleotide sequence 84%, the amino acid sequence 85%.
[0026] The polypeptide of (a) is SEQ ID NO: 8,4,6,10,12,2 or 14 by 7 or more continuous in polypeptide having an amino acid sequence represented, 8,9 or preferably continuously a polypeptide consisting of 10 or more amino acids, and has an immunity-inducing activity. More preferably, the polypeptide is a polypeptide amino acid sequence with sequence identity to SEQ ID NO: 8 comprises the amino acid sequence 85% or more, particularly preferably, the polypeptide is SEQ ID NO: 8,4 has the amino acid sequence represented by 6,10,12,2 or 14. Incidentally, as known in the art, the antigenicity and immunogenicity if the polypeptide than about 7 amino acid residues can exert. Therefore, if seven consecutive amino acid residues or more polypeptides in the amino acid sequence of SEQ ID NO: 8,4,6,10,12,2 or 14, so may have an immunity-inducing activity, immunity induction of the present invention it can be used to prepare the agent.
[0027] Further, as a principle of immune induction by administration of a cancer antigen polypeptide, the polypeptide is incorporated into antigen-presenting cells, then become smaller fragments undergo degradation by peptidases in said cell, presented on the surface of said cells It is, that it is known that immune cells such as cytotoxic T cells recognize, selectively kill cells presenting the antigen. The size of polypeptides displayed on the surface of antigen-presenting cells are relatively small, about 7 to 30 amino acids number. Therefore, from the viewpoint of presentation on antigen presenting cells, the polypeptide of the (a), successive amino acid sequence represented by SEQ ID NO: 8,4,6,10,12,2 or 14 7 it is about to 30 is one of preferred embodiments, and more preferably it is sufficient as long as it is made of a 8-30 or 9-30 of about amino acid. Polypeptides of relatively small size, without being incorporated into antigen-presenting cells, the program may be presented on the cell surface on direct antigen presenting cells.
[0028] Polypeptides incorporated into the antigen-presenting cell receives the disconnection at random positions by a peptidase in said cell, various polypeptide fragments occurs, these polypeptide fragments are presented on the surface of antigen presenting cells Runode, be administered a large size of the polypeptide as full-length region of SEQ ID NO: 8,4,6,10,12,2 or 14, by degradation with antigen presenting cells, immune induction via antigen-presenting cells valid polypeptide fragments are necessarily occur. Therefore, also for immune induction via the antigen-presenting cell, it can be preferably used a large polypeptide size, the number of amino acid 30 or more, more preferably 100 or more, more preferably 200 or more, more preferably 250 or more, more preferably may be a polypeptide of the full length region of SEQ ID NO: 8,4,6,10,12,2 or 14.
[0029] The polypeptide of (b), the above (a) the description of the sequence listing SEQ ID NO 8,4,6,10,12,2 or fewer of the polypeptide having the amino acid sequence represented by 14 ( preferably, one or several) amino acid residues are substituted, deleted and / or added or inserted and the polypeptide having immunity-inducing activity, or unmodified original sequence and 85%, 90% or more, preferably 95% or more, more preferably 98% or more, more preferably has a 99% or 99.5% or more sequence identity, and a polypeptide having an immunity-inducing activity. In general, the protein antigen, a small number of amino acid residues of the amino acid sequence of the protein is substituted, even if it is deletion or addition or insertion, may have substantially the same antigenicity as the original protein it is well known in the art that. Therefore, since the polypeptide (b) above may exert an immunity-inducing activity, it can be used for preparation of the immunity-inducing agent of the present invention. Also, the polypeptide of (b) above, of the amino acid sequence represented by SEQ ID NO: 8,4,6,10,12,2 or 14, one or several amino acid residues are substituted, deleted, and also preferably a / or addition or insertion polypeptide. The term "several" as used herein, an integer of 2 to 10, preferably an integer of 2 to 6, more preferably an integer of 2-4.
[0030] In the present specification, the amino acid sequence (or a nucleotide sequence) "sequence identity", amino acid residues of the two amino acid sequences to be compared (or base sequence) (or bases) as many matching as both amino acids sequence (or nucleotide sequences) to align the one in which the matched amino acid residues (or the number of matched bases) represents the value obtained by dividing a percentage by total number of amino acid residues (or the total bases). During the alignment, if necessary, to insert the appropriate gaps in one or both of the two sequences to be compared. Alignment of such sequences, e.g. BLAST, it can be performed using FASTA, a known algorithm such as CLUSTALW. If the gap is inserted, the total number of amino acid residues is a number of residues by counting one gap as one amino acid residue. In this way, the total number of amino acid residues counted is, when the different between the two sequences to be compared, sequence identity (%) is the total number of amino acid residues in the longer sequence, matching amino acid residues It is calculated by dividing the number.
[0031] Note that 20 types of amino acids constituting natural proteins, neutral amino acids with side chains having low polarity (Gly, Ile, Val, Leu, Ala, Met, Pro), neutral amino acids (Asn having hydrophilic side chains , those having Gln, Thr, Ser, Tyr, Cys), acidic amino acids (Asp, Glu), basic amino acids (Arg, Lys, His), aromatic amino acids (Phe, Tyr, similar properties as Trp) grouping is possible, that if the nature of the polypeptide is not changed often is known if the substitution between amino acids in the group. Therefore, when replacing the amino acid residues in the polypeptides of (a), by substituting among amino acids within each of these groups, because the possibility of maintaining the immunity-inducing activity increases, which is preferable.
[0032] Polypeptide of (c) comprises a polypeptide of (a) or (b) as a partial sequence, is a polypeptide having an immunity-inducing activity. That is, (a) or (b) the polypeptide one or both ends to the other at least one amino acid residues or other (single or multiple) be one polypeptide is added, immunity-inducing activity a polypeptide having. Such polypeptides can also be used for preparation of the immunity-inducing agent of the present invention.
[0033] The polypeptide, for example, Fmoc method (fluorenyl methyloxy carbonyl method) can be synthesized according to a chemical synthesis method such as tBoc method (t-butyloxycarbonyl method) known peptide synthesis methods such as. It can also be synthesized by a conventional method using various commercially available peptide synthesizers. Further, the using known genetic engineering techniques, to prepare the polynucleotide encoding the polypeptide, is introduced into the host cell incorporating the polynucleotide into an expression vector to produce the polypeptide in said host cell Accordingly, it is possible to obtain the polypeptide of interest.
[0034] The polypeptide encoding polynucleotides, by a conventional method using a known genetic engineering technique or commercially available nucleic acid synthesizer, can be readily prepared. For example, DNA having the nucleotide sequence of SEQ ID NO: 1, PCR is performed using a pair of primers designed to the canine chromosomal DNA or cDNA library was used as a template, can be amplified nucleotide sequence described in SEQ ID NO: 1 it can be prepared by. If DNA having the nucleotide sequence of SEQ ID NO: 3, can be similarly prepared by using a human chromosome DNA or cDNA library as the template. The reaction conditions for PCR can be set appropriately, for example, 30 seconds 94 ° C. (denaturation), 30 seconds to 1 minute at 55 ° C. (annealing), 1 cycle reaction step comprising two minutes at 72 ° C. (elongation) as, after for example 30 cycles, and the like can be mentioned conditions for reacting at 72 ° C. 7 minutes, but is not limited thereto. Further, based on the information of the nucleotide sequence and amino acid sequences as shown in SEQ ID NO: 1 and 3 in the sequence listing herein, to prepare a suitable probe or primer, cDNA live such as dogs and humans therewith by screening rally, it is possible to isolate the desired DNA. cDNA libraries, cells expressing the protein of SEQ ID NO: 2, 4, it is preferably prepared from an organ or tissue. Preparation of the probes or primers, construction of cDNA library, screening cDNA libraries, as well as operations such as cloning the gene of interest are known to those skilled in the art, for example, Molecular Cloning: A Laboratory Manual Second Edition (cold spring Harbor Laboratory Press), it can be carried out according to the method described in current Protocols in molecular Biology (John Uiryi & Sons), and the like. Thus from the obtained DNA as a, can be obtained a DNA encoding a polypeptide of the (a). Moreover, codons encoding each amino acid because it is known, the base sequence of a polynucleotide encoding a specific amino acid sequence can be easily identified. Therefore, since the above (b) or even a nucleotide sequence of the polynucleotide encoding the polypeptide of (c) it can be easily identified, even such polynucleotides, by a conventional method using a commercially available nucleic acid synthesizer it can be readily synthesized.
[0035] As the host cells, the long polypeptide and capable of expressing cells may be any one, such as E. coli Examples of prokaryotic cells, monkey kidney cells COS1 Examples of eukaryotic cells, Chinese hamster ovary cells cultured mammalian cells such as CHO, budding yeast, fission yeast, silkworm cells, and the like Xenopus egg cells, but are not limited to.
[0036] When using a prokaryotic cell as a host cell, the expression vector replicable origin in prokaryotic cells, a promoter, Shine - Dalgarno sequence (or ribosome binding site), DNA cloning site, using an expression vector having a terminator. Expression vectors for E. coli, pUC system, pBluescriptII, pET expression system and pGEX expression system. Incorporating DNA encoding the above polypeptide into such an expression vector, after a prokaryotic host cell transformed with the vector, followed by culturing the obtained transformant, prokaryotic polypeptides wherein said DNA encodes it can be expressed in a host cell. In this case, the polypeptide can also be expressed as a fusion protein with another protein.
[0037] When using eukaryotic cells as host cells, the expression vector, a promoter, a splicing region, a eukaryotic expression vector having a poly (A) addition site and the like is used. Such expression vectors, pKA1, pCDM8, pSVK3, pMSG, pSVL, pBK-CMV, pBK-RSV, EBV vector, pRS, pcDNA3, pMSG, pYES2 and the like. Similar to the above, incorporating a DNA encoding the above polypeptide into such an expression vector, after a eukaryotic host cell with the vector and transformed by culturing the obtained transformant, the DNA encodes the by which polypeptides can be expressed in eukaryotic host cells. pIND / V5-His as an expression vector, in the case of using the pFLAG-CMV-2, pEGFP-N1, pEGFP-C1 or the like, His tag, FLAG tag, myc tag HA tag, a fusion protein obtained by adding a variety of tags such as GFP as it can be expressed the polypeptide.
[0038] Introduction into the host cell expression vectors, electroporation, calcium phosphate method, liposome method, it is possible to use known methods such as DEAE-dextran method.
[0039] In order to isolate and purify a polypeptide of interest from the host cells may be carried out by combining known separation operations. For example treatment with denaturing agents such as urea or a surfactant, ultrasonication, enzymatic digestion, salting-out or solvent fractional precipitation, dialysis, centrifugation, ultrafiltration, gel filtration, SDS-PAGE, isoelectric focusing, ion exchange chromatography, hydrophobic chromatography, Africa Niti chromatography, but reverse phase chromatography, and the like, without limitation.
[0040] The polypeptide obtained by the above method, as described above, also include those in the form of a fusion protein with any other protein. For example, such fusion proteins with glutathione -S- transferase (GST) or His tag can be exemplified. Form of the polypeptide such fusion proteins are also within the scope of the present invention as a polypeptide of the (c). Further, polypeptides expressed in transformed cells after translation may be subject to various modifications within the cell. Such post-translationally modified polypeptides are also as long as it has an immunity-inducing activity, it is included within the scope of the present invention. Examples of such translational modification, elimination of N-terminal methionine, N-terminal acetylation, glycosylation, limited degradation by an intracellular protease, myristoylation, isoprenylation, and phosphorylation.
2. Immunity-inducing agent
as specifically described in the Examples below, administration of a polypeptide having an immunity-inducing activity as the tumor-bearing animals, can cause regression of the tumor has already occurred. Therefore, the immunity-inducing agent of the present invention can be used for the treatment and / or prevention of cancer. Further, the polypeptide having immunity-inducing activity described above can be used in the treatment and / or prevention of cancer by inducing immunity.
[0041] As used herein, the term "tumor" and "cancer" refers to malignant neoplasms, are used interchangeably.
[0042] In this case, as the cancer of interest, preferably a cancer expressing CSPG5, more preferably breast, lung, brain, ovarian cancer, leukemia, malignant lymphoma, adenocarcinoma, mastocytoma, squamous cell carcinoma a melanoma or neuroblastoma, particularly preferably breast, lung, brain, leukemia, malignant lymphoma, mastocytoma, melanoma or neuroblastoma.
[0043] Moreover, animals of interest (subject), as described above, preferably a mammal, more preferably a primate, pet animals, animals kept in zoos, etc., livestock, and sports animals a mammal, particularly preferably human, dog or cat.
[0044] Route of administration to the living body of the immunity-inducing agent of the present invention may be either oral administration or parenteral administration, but intramuscular, subcutaneous, intravenous, parenteral administration, such as intraarterial administration is preferred. When using the immunity-inducing agent for therapeutic purposes of cancer, to enhance the anti-cancer effect can also be administered as described in the Examples below, the lymph nodes near the tumor to be treated. The dosage may be an amount effective to induce immunity, for example if used in the treatment and / or prevention of cancer, may be an amount effective for the treatment and / or prevention of cancer. A therapeutically effective amount and / or prophylaxis of cancer is appropriately selected depending on the tumor size and symptoms such as, usually, 0.0001 ~ 1000 [mu] g as an effective amount of one day to the subject animal, preferably 0. is 001 ~ 1000 [mu] g, it can be administered in single or divided doses. Preferably, several times, administered several days to several months apart. As specifically shown in the Examples below, the immunity-inducing agent of the present invention can cause regression of tumors that have already been formed. Accordingly, since the early development of a small number of cancer cells may exert anticancer effects, the use after cancer development before and cancer treatment, it is possible to prevent the onset or recurrence of cancer. That is, the immunity-inducing agent of the present invention is useful for both the prevention and treatment of cancer.
[0045] Immunity-inducing agent of the present invention may be composed of only a polypeptide, suitable for each dosage form, pharmaceutically acceptable carriers, diluents, formulated by mixing appropriate additives such as excipients it is also possible. Here "preparation" may be used "immunity-inducing composition" and a "pharmaceutical for immune induction" interchangeably. Formulation methods and available additives are well known in the art of pharmaceutical formulation, it can be used any method and additives. Specific examples of the additives, diluents such as physiological buffer; sugar, lactose, corn starch, calcium phosphate, sorbitol, excipients such as glycine; syrup, gelatin, gum arabic, sorbitol, polyvinyl chloride, tragacanth and the like binding agents such as; magnesium stearate, polyethylene glycol, talc, and lubricants such as silica include, but are not limited to. The formulation can include tablets, capsules, granules, powders, oral preparations such as syrups, inhalants, injections, suppositories, and the like parenteral preparations such as solutions. These preparations can be produced by production method it is generally known.
[0046] Immunity-inducing agent of the present invention may be used in combination with an immune enhancer that can enhance the immunological response in vivo. Immunopotentiator may be included in the immunity-inducing agent of the present invention may be administered in combination with the immunity-inducing agent of the present invention as separate compositions to the patient.
[0047] As the immunopotentiating agent can be, for example, an adjuvant. Adjuvants provides reservoir of antigen (extracellularly or within macrophages), activating macrophages and by stimulating specific sets of lymphocytes, so may enhance the immunological response to enhance the anticancer activity be able to. Thus, in particular, when using the immunity-inducing agent of the present invention for the treatment and / or prevention of cancer, the immunity-inducing agent preferably further comprises an adjuvant in addition to serving as an active ingredient the polypeptide. Many types of adjuvants are well known in the art, it can be used in any adjuvant. Examples of adjuvants include, MPL (SmithKline Beecham), Salmonella minnesota Re 595 lipopolysaccharide purification and congeners obtained after acid hydrolysis of Salmonella; QS21 (SmithKline Beecham), pure purified from Quillja saponaria extract QA-21 saponin; PCT application WO96 / 33739 (SmithKline Beecham) has been described in DQS21; QS-7, QS-17, QS-18 and QS-L1 (Seo (So) et al., "Morekyuruzu-and-cell (Molecules and cells) ", 1997, Vol. 7, p.178-186); incomplete Freund's adjuvant; Freund's complete adjuvant; vitamin E; Montanide Alum or aluminum hydroxide (Alum); CpG oligonucleotide (e.g., Craig (Kreig) et al., "Nature (Nature)", 374, pp. See p.546-549); poly IC and a derivative thereof (poly ICLC, etc. ) and squalene and / or various water-in-oil emulsions prepared from biodegradable oils such as tocopherol; and α-galactosylceramide, and the like. Among these, Freund's incomplete adjuvant, Montanide, poly IC and derivatives thereof, and CpG oligonucleotides are preferred. The mixing ratio of the adjuvant and polypeptide is typically about 1:10 to 10: 1, preferably about 1: 5 to 5: 1, more preferably from about 1: 1. However, the adjuvant is not limited to the above examples, adjuvants other than known the art may also be used in the administration of the immunity-inducing agent of the present invention (e.g., Goddingu (Goding) al, "Monoclonal Anti Nanobodies: Principles and practice (Monoclonal Antibodies: Principles and practice) ", second edition, 1986). Process for the preparation of mixtures or emulsions of a polypeptide and an adjuvant are well known to those skilled in the vaccination.
[0048] Further, as the immunopotentiating agent, in addition to the adjuvant, it is also possible to use factors that stimulate the immune response of the subject. For example, it may be used in combination with the immunity-inducing agent of the present invention the various cytokines having a property to stimulate lymphocytes and antigen-presenting cells as an immune enhancer. Are known in a number of cytokines skilled person capable enhance such immunological response, examples, interleukin-12, which can enhance the protective effects of the vaccine are shown (IL-12), GM-CSF, IL-18, interferon alpha, interferon beta, interferon omega, including but interferon γ and Flt3 ligand, but are not limited to. Such factors can also be used as the immunopotentiating agent can be included in the immunity-inducing agent of the present invention, or as separate compositions in combination with the immunity-inducing agent of the present invention is administered to a patient.
[0049] Further, the above-described polypeptide and (from a subject) an antigen-presenting cell by contacting ex vivo or in vitro, the polypeptide can be presented on antigen-presenting cells. Ie, a polypeptide of the above (a), (b) or (c) may be used for treatment of antigen-presenting cells. Examples of the antigen-presenting cells, may be preferably used dendritic cells or B cells bearing MHC class I molecules. Various MHC class I molecules have been identified and are well known. MHC molecules in humans are referred to as HLA. The HLA class I molecules, HLA-A, HLA-B, there may be mentioned 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, such as HLA-Cw0602 and the like.
[0050] As an example of using the polypeptide for the treatment of antigen-presenting cells, the following 3. In as described, to produce the antigen-presenting cell comprising a complex between said polypeptide and the MHC molecule, and, of the polypeptide to such making a cytotoxic T cells specific to the polypeptide mention may be made of the use.
[0051] Dendritic cells or B cells bearing MHC class I molecules can be prepared from peripheral blood by a well-known method. For example, from bone marrow, umbilical cord blood or patient peripheral blood using granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-3 (or IL-4) induces dendritic cells, tumor-associated peptide on the culture system by adding, it can induce tumor-specific dendritic cells.
[0052] The dendritic cells by administering an effective amount, can induce the desired response in the treatment of cancer. Cells used in bone marrow and umbilical cord blood has been provided from healthy people, but it is possible to use a patient's own bone marrow and peripheral blood, etc., if you use the patient's natural autologous cells, high safety, serious side effects can also be expected be avoided. Peripheral blood or bone marrow may be either fresh sample, cold storage sample and cryopreserved sample. Peripheral blood may be cultured whole blood, only white blood cell component may be cultured in isolation, but the latter is preferred and efficient. It may be further separated mononuclear among white blood cell component. Further, in the case of bone marrow or umbilical cord blood origin may be cultured whole cells constituting the bone marrow may be cultured by separating therefrom mononuclear. Peripheral blood and their white blood cell component, the bone marrow cells, which contain dendritic cell origin become mononuclear hematopoietic stem cells or immature dendritic cells and CD4-positive cells and the like. Cytokines used is not particularly limited as long as safety and physiological activity have been confirmed properties, natural or recombinant type, and the like, but not limited for its production method, preferably the quality to be used in medical is ensured the preparation is used in a necessary minimum amount. Concentration of cytokines to be added is not particularly limited as long as the concentration of the dendritic cells is induced, preferably about 10 ~ 1000 ng / mL in a total concentration of normal cytokine, more preferably about 20 ~ 500ng / mL. Culturing can be carried out using well-known media commonly used for culturing leukocytes. While the culture temperature is not particularly limited as long as the proliferation of white blood cells, and most preferably about 37 ° C. which is human body temperature. Also, the gaseous environment in the culture is not particularly limited as long as the proliferation of white blood cells, 5% CO 2 preferably bubbling. Further culture period is not particularly limited as long as a period in which the required number of cells are induced, is usually performed between 3 days to 2 weeks. Equipment to be used for cell separation and culture, can be used as appropriate appropriate, confirmed safety for pharmaceutical use, and it is preferable operation is simple and stable. The particular cell culture device, a petri dish, a flask, regardless generally containers such as bottles, the multilayer container and multistage vessel, roller bottle, spinner type bottle, bag type culturing vessel, may also be used hollow fiber column and the like .
[0053] Furthermore, according to the present invention, the (a), (b) or (c) polypeptides also by expressing in the body of the subject animal a polynucleotide encoding an antibody production and cytotoxic in the living body can induce sex T cells, effects such as the treatment and prevention is obtained for comparable cancer and to administer the polypeptide. That is, the immunity-inducing agent of the present invention, the above (a), as (b) or comprises a polynucleotide encoding the polypeptide of (c), the active ingredient capable of expressing recombinant vectors said polypeptide in vivo it may include. As shown in Examples below, recombinant vectors capable of expressing such antigen polypeptide is also called gene vaccine.
[0054] Vector used to produce the gene vaccine is in the target animal cells (preferably mammalian in animal cells) is not particularly limited as long as it is a vector capable of expressing in may be a viral vector in a plasmid vector, known in the field of genetic vaccines You may use any vector. Incidentally, a polynucleotide such as DNA or RNA encoding the polypeptide, as described above, can be easily prepared by a conventional method. Further, incorporation of the polynucleotide into a vector can be carried out using methods well known to those skilled in the art.
[0055] The route of administration of the gene vaccine is preferably a parenteral route of administration intramuscular administration, subcutaneous administration, intravenous administration, intraarterial administration, etc., the dosage can be appropriately selected depending on the antigen such as the type usually per body weight 1 kg, about 0.1 [mu] g ~ 100 mg by weight of the gene vaccine is preferably about 1 [mu] g ~ 10 mg.
[0056] The method according to viral vectors, for example retroviruses, Sendai virus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, poxvirus, poliovirus, the RNA virus or DNA virus, such as Sindbis virus, encoding the polypeptide incorporate polynucleotides, methods for infecting the like which in the target animal. In this, retrovirus, adenovirus, adeno-associated virus, a method of using vaccinia virus are particularly preferred.
[0057] Other methods include a method of administering an expression plasmid directly into muscle (DNA vaccine method), liposome method, lipofectin method, microinjection method, calcium phosphate method, electroporation method and the like, in particular DNA vaccine method, liposome the law is preferable.
[0058] To act as a medicament in practice a gene encoding the polypeptide used in the present invention, genes in vivo method for introducing into the body directly, and collected certain cells from a subject animal the cells genes in vitro Although there are ex vivo method of returning the cells in the body it is introduced into the (Nikkei Science (Japan), April 1994, p20-45, monthly pharmaceutical (Japan), 1994, Vol. 36, No. 1, p. 23-48, experimental medicine Supplement (Japan), 1994, Vol. 12, No. 15, and such as these references), in vivo method is more preferable.
[0059] When administered by in vivo methods it can be administered disease therapeutic purposes, via a suitable administration route depending on the condition and the like. For example, it may be administered via intravenous, intraarterial, subcutaneous, etc. intramuscularly. When administered by in vivo method, for example, but may take dosage forms such as solution, in general, it is the form of injection containing DNA encoding the peptide of the present invention as an active ingredient, needs in response, a pharmaceutically acceptable carrier (e.g., physiological saline, buffer, etc.) may be added. Also, the liposome or membrane-fused liposomes containing the DNA (Sendai virus (HVJ) - liposome, etc.) in the suspension, frozen drug, may be in the form of liposomal formulation such as centrifugation-concentrated frozen drug.
[0060] In the present invention, when the said "base sequence shown in SEQ ID NO: 1" is other actually the indicated nucleotide sequence in SEQ ID NO: 1, sequences complementary thereto encompass. Therefore, when the said "polynucleotide having a nucleotide sequence shown in SEQ ID NO: 1" is a single-stranded polynucleotide having the nucleotide sequence actually shown in SEQ ID NO: 1, its complementary nucleotide sequence single-stranded polynucleotide having, and double-stranded polynucleotide composed of these are included. When preparing a polynucleotide encoding the polypeptide used in the present invention is the selecting any one of the nucleotide sequences can be appropriately and easily the selected by those skilled in the art.
3. Antigen presenting cells or cytotoxic T cells
present invention further comprises contacting the antigen-presenting cells from above polypeptides and subject ex vivo or in vitro (in vitro), and the polypeptide and the MHC molecule It provides methods of making antigen-presenting cells containing the complex.
[0061] The present invention is also obtained by this method, and characterized in that it comprises a complex between the polypeptide and the MHC molecule, to provide antigen-presenting cells.
[0062] The method itself of contacting the said polypeptide with antigen-presenting cells ex vivo or in vitro can be carried out by well known methods. For example, can be carried out by antigen presenting cells, are cultured in a culture medium containing the polypeptide. The medium may be a commercially available antigen-presenting cell culture medium. Peptide concentration in the medium is not particularly limited, usually about 1 ~ 100 [mu] g / ml, preferably about 5 ~ 20μg / ml. Although cell density is not particularly limited in the culture, usually 10 3 ~ 10 7 cells / ml, preferably about × 10 5 4 ~ 5 × 10 6 , about cells / ml. Culture is carried out according to a conventional method, 37 ° C., 5% CO 2 preferably performed in an atmospheric. The length of the peptide antigen-presenting cells can present on the surface is usually up to about 30 amino acid residues. Therefore, although not particularly limited, when contacting the antigen presenting cells with the polypeptide ex vivo or in vitro, it may be prepared in the following length approximately 30 amino acid residues the polypeptide.
[0063] By culturing antigen-presenting cells in the presence of the polypeptide, the polypeptide is incorporated into MHC molecules of antigen presenting cells and presented on the surface of antigen-presenting cells. Thus, using the above-described polypeptide, comprising the complex between the polypeptide and the MHC molecule, the isolated antigen-presenting cells can be prepared. Such antigen-presenting cells, in vivo or in ex vivo or in vitro, the polypeptide is presented to T cells to induce cytotoxic T cells specific to the polypeptide, it is grown it can.
[0064] The present invention further provides a T cell-derived antigen-presenting cells and subject of the is contacted ex vivo or in vitro comprising activating the T cells, specific cytotoxic to the polypeptide T to provide a method for manufacturing a cell.
[0065] The present invention also can be obtained by this method, and is characterized in that is specific to the polypeptides described above, provides a cytotoxic T cell.
[0066] Are prepared as described above, the polypeptide and the antigen-presenting cell comprising a complex of MHC molecules, by contacting with T cells and ex vivo or in vitro, specific cytotoxic T to the polypeptide induced cells can be grown. This, and the antigen-presenting cells and T cells can be conducted by co-culturing in a liquid medium. For example, antigen-presenting cells suspended in a liquid medium, placed in a container such as wells of a microplate, can be performed by culturing with the addition of T cells to this. The mixing ratio of antigen-presenting cells and T cells upon cocultivation is not restricted, and is usually about at a ratio of cell number 1: 1 to about 1: 100, preferably from about 1: 5 to about 1:20 . The density of the antigen-presenting cells suspended in the liquid medium is not particularly limited, usually, about 100 to 10,000,000 cells / ml, preferably from about 10,000 to 1,000,000 cells / ml. Coculture is carried out according to a conventional method, 37 ° C., 5% CO 2 preferably performed in an atmospheric. The medium may be a commercially available antigen-presenting cells / T cell culture medium. Culture time is not particularly limited, usually, from about 2 days to 3 weeks, preferably about 4 days to 2 weeks. Moreover, co-culture is, IL-2, IL-6 , it is preferably carried out in one or more of the presence of interleukin such as IL-7 and IL-12. In this case, the concentration of IL-2 and IL-7 is usually about 5 ~ 20 U / ml, the concentration of IL-6 is usually about 500 ~ 2000 U / ml, the concentration of IL-12 is usually about 5 ~ 20 ng / ml some, but not limited thereto. The above cocultivation may be repeated once or several times by adding fresh antigen-presenting cells. For example, discarded culture supernatant after coculture, an operation of performing further co-culture was added to a suspension of fresh antigen-presenting cells, it may be repeated once or several times. Conditions for each cocultivation may be the same as described above.
[0067] The cocultivation above, is induced cytotoxic T cells specific to the polypeptide, it is grown. Thus, using the polypeptide, that selectively binds the complex between the polypeptide and the MHC molecule, the isolated T cells can be prepared.
[0068] As described in the Examples below, CSPG5 gene, breast cancer cells, breast tissue, lung cancer cells, lung cancer tissues, liver cancer cells, liver cancer tissue, brain tumor cells, brain tumor tissue, ovarian carcinoma cells, ovarian carcinoma tissue, leukemia, malignant lymphoma, adenocarcinoma cells, adenocarcinoma tissue, mastocytoma, squamous cell carcinoma cells, is specifically expressed in melanoma cells or neuroblastoma cells. Thus, in these carcinomas, it believed CSPG5 exists significantly more than normal cells. Therefore, a part of CSPG5 polypeptides present in the cancer cells to be presented to the MHC molecules on the surface of cancer cells, were prepared as described above cytotoxic T cells are administered in vivo, This cytotoxic T cells can be impaired cancer cells as a marker. Furthermore, antigen-presenting cells that present some CSPG5 polypeptides also induce cytotoxic T cells specific to the polypeptide in vivo, it is possible to grow, the antigen presenting cells live by administering into the body, it can be impaired cancer cells. That is, the cytotoxic T cells and the antigen-presenting cells prepared using the polypeptide are also similar to the immunity-inducing agent of the present invention are useful for the treatment and / or prevention of cancer.
[0069] When administering the isolated antigen-presenting cells or isolated T cells as described above to a living body, in order to avoid the immune response in vivo to attack these cells as foreign, antigen presentation taken from the patient to be treated cells or T cells, the (a) as described above, it is preferable that was prepared using the polypeptide of (b) or (c).
[0070] The route of administration of the therapeutic and / or prophylactic agent for cancer comprising the antigen-presenting cells or isolated T cells as an active ingredient, parenteral administration, such as intravenous or intraarterial administration is preferred. The dose is appropriately selected depending on the symptoms and the purpose of administration, etc., 1 to 10 trillion usually preferably from 100 million to 1 billion, every several days to every several months preferably administered. Preparations, for example, the cells may be such as those suspended in physiological buffered saline, it may be used in combination with other anticancer agents and cytokines like. It is also possible to add a known one or more additives in the pharmaceutical field.
Example [0071] Hereinafter, although now be described by way of the present invention examples, the scope of the present invention shall not be the examples limit.
[Example
1] (1) Preparation of cDNA libraries
acid testicular dog guanidinium - phenol - extracted total RNA by chloroform method (Acid guanidium-Phenol-Chloroform method) , Oligotex-dT30 mRNA purification kit (Takara Shuzo Co. Ltd., Kyoto, Japan) poly a RNA was purified according to the protocol attached to the kit used.
[0072] CDNA was synthesized phage library using the obtained mRNA (5 [mu] g). The preparation of cDNA phage library using cDNA Synthesis kit, Zap-cDNA Synthesis Kit, a ZAP-cDNA GigapackIII Gold Cloning Kit ( STRATAGENE Co.), to prepare a library in accordance with the protocol attached to the kit. The size of the cDNA phage library prepared in × 10 1 6 was pfu / ml.
[0073] (2) Screening of cDNA library with sera
using a cDNA phage library prepared above, immunoscreening was carried out. Specifically, infected host E. coli cells (XL1-Blue MRF ') such that 2,340 clones should appear on an NZY agarose plate Φ90 × 15mm, 42 ℃, 3 and incubated to 4 hours to allow the phage to form plaques. IPTG (isopropyl-beta-D-thiogalactoside) penetration is allowed nitrocellulose membrane (Hybond C Extra: GE Healthecare Bio -Sciece Co.) plate to induce and express proteins covering 4 hours at 37 ° C. with, the membrane proteins were transferred. To suppress non-specific reactions by subsequently the membrane is recovered TBS containing 0.5% non-fat dry milk (10mM Tris-HCl, 150mM NaCl pH7.5) and shaken overnight at 4 ° C. immersed in. This filter was allowed to react for 2-3 hours at room temperature and the 500-fold diluted canine patient serum.
[0074] As the above-described canine patient serum, using a serum collected from canine patients suffering from breast cancer. The serum was stored at -80 ℃, and pretreated immediately before use. Pretreatment method of serum, according to the following method. In other words, they were infected with lambda ZAP Express phage to which no foreign gene was inserted into a host E. coli (XL1-BLue MRF '), 37 ℃ on NZY plate medium and incubated overnight. 0.2 M NaHCO containing 0.5M NaCl in the following 3 buffers of pH8.3 was added to the plate, 15 hours after standing at 4 ° C., the supernatant was collected as an E. coli / phage extract. Then, the recovered E. coli / phage extract NHS- was passed through a column (GE Healthecare Bio-Science Co., Ltd.) to immobilize proteins derived from the E. coli phage. This protein-immobilized column to canine patient serum was allowed to flow through and react remove antibodies adsorbed on E. coli and phage from the serum. The serum fraction that passed through the column was diluted 500-fold with TBS containing 0.5% non-fat dry milk, which was used as immunoscreening material.
[0075] After the membrane was blotted the thus treated serum and the above-described fusion proteins were washed 4 times with TBS-T (0.05% Tween20 / TBS), 0.5% non-fat dry milk as a secondary antibody at free TBS 5000 fold dilution was performed with goat anti-dog IgG: the (goat anti dog IgG-h + I HRP conjugated BETHYL Laboratories , Inc.), reacted for 1 hour at room temperature, followed by detection by the enzyme coloring reaction using the NBT / BCIP reaction solution (Roche, Inc.), colonies positions where a positive coloring reaction was observed were collected from Φ90 × 15mm of NZY agarose plate, SM buffer (100 mM NaCl, 10 mM MgClSO 4 dissolved in, 50mM Tris-HCl, 0.01% gelatin pH 7.5) 500 [mu] l It was. In the same manner as described above until the color reaction positive colonies unifies, secondary, repeated tertiary screening to screen 9110 phage clones that react with IgG in the serum, isolating one positive clone did.
[0076] (3) sequence identity search for isolated antigen gene
for providing a single positive clone isolated by the above method to sequence analysis, performed an operation to convert the phage vector to a plasmid vector. Specifically, host E. coli (XL1-Blue MRF ') absorbance OD of 600 is 1.0 so as a solution 200μl prepared, phage solution 100μl further ExAssist helper phage (STRATAGENE Co.) and purified 37 ° C. After mixing 1μl in 15 minutes after the reaction, of LB medium was 2.5 to 3 hours incubation at 3ml added 37 ° C., was incubated for 20 minutes at immediately 70 ° C. water bath, 4 ° C., then centrifuged 1000 × g, 15 min , and the supernatant was collected as a phagemid solution. Phagemid host E. coli (SOLR) absorbance OD then 600 and solution 200μl which was prepared to become 1.0, allowed to react for 15 minutes at 37 ° C. After mixing phage solution 10μl purified, 50 [mu] l ampicillin (final concentration 50 [mu] g / ml ) and incubated 37 ° C. overnight plated on LB agar medium containing. Single colonies of SOLR was transformed harvested after cultured in ampicillin (final concentration 50 [mu] g / ml) containing LB medium 37 ° C., followed by purification of plasmid DNA having an insert of interest using QIAGEN plasmid Miniprep Kit (Qiagen) .
[0077] Purified plasmid using the T7 primer described in T3 primer SEQ ID NO: 18 as set forth in SEQ ID NO: 17, were analyzed insert the full-length sequence by a primer walking method. And obtaining a gene sequence described in SEQ ID NO: 1 by the sequence analysis. Using a base sequence and amino acid sequence of this gene, the sequence was identical search against known genes perform sequence identity search program BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) the results, obtained gene was found to be CSPG5 gene. In human CSPG5 a human homologous factor of the canine CSPG5, sequence identity of nucleotide sequence 87%, the amino acid sequence 87%, the cat CSPG5, sequence identity of nucleotide sequence 92%, the amino acid sequence 91%, mouse homologous in mice CSPG5 a factor, sequence identity of nucleotide sequence 84% were amino acid sequence 85%. Sequence The nucleotide sequence of human CSPG5 numbers 3, 5, 7, 9 or 11, in the amino acid sequence SEQ ID NO: 4, 6, 8, 10 or 12, the nucleotide sequence SEQ ID NO: 13 cats CSPG5, the amino acid sequence SEQ ID NO: 14 to, respectively the nucleotide sequence of mouse CSPG5 SEQ ID NO: 15, the amino acid sequence in SEQ ID NO: 16.
[0078] (4) expression analysis in each tissue
by the dog to the gene obtained by the method, human and murine various human normal tissues and various cancer tissues and expression in cancer cell lines RT-PCR (Reverse Transcription-PCR ) method Examined. Reverse transcription was performed as follows. That is, each tissue 50 ~ 100 mg and each cell line 5 ~ 10 × 10 6 total RNA was extracted according to the attached protocol using cells from TRIZOL reagent (invitrogen Corp.). CDNA was synthesized according to the attached protocol by the total RNA Superscript First-Strand Synthesis System for RT-PCR using the (invitrogen, Inc.). CDNA of human normal tissues (brain, hippocampus, testis, colon and placenta), Gene Pool cDNA (invitrogen, Inc.) was used QUICK-Clone cDNA (Clontech) and Large-Insert cDNA Library (Clontech). PCR reactions obtained gene-specific primers (dog primers SEQ ID NO: 19 and 20, the human primers SEQ ID NO: 21 and 22, mice primers SEQ ID NO: 23 and 24) were carried out as follows using. That is, the sample 0.25μl prepared by reverse transcription reaction, the above primers each 2 [mu] M, of each dNTP 0.2 mM, the total volume added to the buffer attached to each reagent so that ExTaq polymerase 0.65u (Takara Shuzo) 25 [mu] l and then, using a Thermal Cycler (BIO RAD Co.), 94 ° C. -30 seconds, 55 ° C. -30 seconds, it was carried out with 30 cycles of 72 ° C. -1 minutes. For comparison, (described in canine and human GAPDH primers SEQ ID NO: 25 and 26, mouse GAPDH primers SEQ ID NO: 27 and 28) GAPDH specific primers were used simultaneously. As a result, as shown in FIG. 1, dogs CSPG5 gene, healthy showed no expression in most tissues in dogs tissue, whereas strong expression in canine tumor tissue was observed. Expression of human and mouse CSPG5 gene, as is the case with the canine CSPG5 gene, not confirmed rarely expressed in human and mouse normal tissues, breast cancer in cancer cells, lung cancer, brain cancer, ovarian cancer, leukemia, it is expressed in cell lines of malignant lymphoma was detected (FIG. 2, FIG. 3).
Example
2 (1) Preparation of recombinant vector expressing CSPG5 in vivo
based on the nucleotide sequence of SEQ ID NO: 15, by the following method, raw to prepare a recombinant vector expressing CSPG5 in the body. PCR is a mouse neuroblastoma cell line 1 seen Expressed in Example 1 (N2a: purchased from ATCC) was prepared from. 1μl of cDNA, the two primers each 0.4μM (described in SEQ ID NO: 29 and 30), 0.2mM dNTP, 1.25U PrimeSTAR HS polymerase (Takara Shuzo Co., Ltd.) containing a HindIII and XbaI restriction enzyme cleavage sequence the total volume of 50μl was added to the buffer attached to each reagent as, using Thermal Cycler (BIO RAD Co.), 98 ° C. -10 seconds, 55 ° C. -15 seconds, repeating 30 cycles of 72 ° C. -4 minutes by went. The above-described two kinds of primers were those which amplify the region encoding the entire amino acid sequence of SEQ ID NO: 15. After PCR, the amplified DNA was electrophoresed on 1% agarose gel to purify a DNA fragment of about 1000bp using QIAquick Gel Extraction Kit (QIAGEN Inc.).
[0079] Were ligated purified DNA fragment into a cloning vector pCR-Blunt (invitrogen Corp.). This plasmid was recovered after transformation into E. coli, amplified gene fragments were confirmed by sequencing that it matches the sequence of interest. The matching plasmid sequence of interest was treated with HindIII and XbaI restriction enzyme, purified using QIAquick Gel Extraction Kit, a target gene sequence, HindIII, and inserted into the mammalian expression vector PCDNA3.1 treated with XbaI restriction enzyme (Invitrogen) . CSPG5 proteins in mammalian cells by the use of this vector is produced.
[0080] Gold particles 50μg plasmid DNA100μg produced above (Bio Rad Co.), spermidine 100 [mu] l (SIGMA Co.), 1M CaCl 2 was added 100 [mu] l (SIGMA Co.), and allowed to stand in a stirred for 10 minutes at room temperature by vortexing (hereinafter " described as a gold -DNA particles "). After centrifugation for 1 minute at 3000 rpm, washed three times with 100% ethanol supernatant discarded (WAKO Co., Ltd.). After thoroughly stirred by vortexing adding 100% ethanol 6ml gold -DNA particles, pouring gold -DNA particles Tefzel Tubing (Bio Rad Co.) was precipitated on the wall. Ethanol Tefzel Tubing gold -DNA particles adhered was air dried and cut to a length suitable for gene gun.
[0081] (2) Anti-tumor effect of CSPG5 by DNA vaccination
ten A / J mice against (7 weeks old, male, Nippon purchased from SLC Inc.), the tube prepared above was fixed on the gene gun, pure helium at a pressure of 400psi using a gas, a total of 3 times every 7 days DNA vaccine into the peritoneal cavity of mice were shaved, after administered transdermally (plasmid DNA inoculum becomes 2 [mu] g / mouse) mouse neuroblastoma cell lines transplanted N2a cells were evaluated antitumor effects (prevention model). As a control, plasmid DNA CSPG5 gene is not inserted was administered 10 rats in each model.
[0082] Antitumor effect, tumor size (longer diameter × shorter diameter 2 /2) and was evaluated at a ratio of surviving mice. The results of this study, the prevention model, after 21 days, the control group and CSPG5 plasmid administered group, respectively, 1866Mm 3 , 459Mm 3 becomes a significant regression of tumors was observed in CSPG5 plasmid treated group. Observation of the course of survival in the prevention model, whereas the all cases died 54 days after the administration in the control group, the CSPG5 plasmid treated group, 60% of the mice were alive. These results, CSPG5 plasmid treated group significant antitumor effect was observed as compared to the control group.
Industrial Applicability
[0083] The present invention is to provide an immunity-inducing agent comprising a polypeptide exhibiting an anti-tumor activity against various cancers, it is useful for the treatment and / or prevention of cancer.
[0084] SEQ ID NO 17: T3 primer
SEQ ID NO 18: T7 primer
SEQ ID NO 19: Dog RT primer sense
SEQ ID NO: 20: Dog RT primer antisense
SEQ ID NO: 21: human RT primer sense
SEQ ID NO 22: human RT primer antisense
SEQ ID NO: 23: mouse RT primer sense
SEQ ID NO: 24: mouse RT primer antisense
SEQ ID NO: 25 and 26: GAPDH primers
SEQ ID NO: 27 and 28: GAPDH primers
SEQ ID NO 29: mus-fullCSPG5 primer sense
SEQ ID NO 30: mus-fullCSPG5 primer antisense
hereby all publications cited in writing, patents and patent applications are intended to directly incorporated herein by reference.
The scope of the claims
[Claim 1] (I) below (a), comprises a polynucleotide encoding is selected from polypeptides, and at least one polypeptide having immunity-inducing activity, or (ii) the polypeptide of (b) and (c) and immunity-inducing agent comprising a recombinant vector allowing the expression of the polypeptide in vivo, as an active ingredient.
(A) SEQ ID NO 8,4,6,10,12,2 or polypeptide consisting of the amino acid sequence represented by 14, and a polypeptide consisting of not less than 7 consecutive amino acids in the amino acid sequence
(b) sequence number 8,4,6,10,12,2 or polypeptide having an amino acid sequence having 85% or more sequence identity represented by 14, and from less than 7 consecutive amino acids in the amino acid sequence of the polypeptide comprising polypeptide
(c) (a) above or a polypeptide comprising the polypeptide as a partial sequence of (b)
[Claim 2] It said polypeptide having an immunity-inducing activity is a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 8,4,6,10,12,2 or 14, immunity-inducing agent according to claim 1.
[Claim 3] Is for processing of antigen presenting cells, immunity-inducing agent according to claim 1 or 2.
[Claim 4] Is for treatment and / or prevention of cancer, the immunity-inducing agent according to claim 1 or 2.
[Claim 5] Wherein the cancer is a cancer expressing CSPG5, immunity-inducing agent according to claim 4.
[Claim 6] Wherein the cancer is a brain tumor, leukemia, malignant lymphoma, or neuroblastoma, immunity-inducing agent according to claim 4 or 5.
[Claim 7] Further comprising an immune enhancer, immunity-inducing agent according to any one of claims 1 to 6.
[8.] The immunopotentiating agent, Freund's incomplete adjuvant, Montanide, poly IC and a derivative thereof, CpG oligonucleotide, interleukin 12, interleukin 18, interferon alpha, the group consisting of interferon-beta, interferon omega, interferon gamma, and Flt3 ligand is at least one more selected, immunity-inducing agent according to claim 7.
[Claim 9] An antigen presenting cell-derived polypeptide and a subject as defined in claim 1 comprising contacting ex vivo or in vitro, method for producing antigen-presenting cell comprising a complex between said polypeptide and the MHC molecule.
[Claim 10] Wherein the antigen presenting cells are dendritic cells or B cells bearing MHC class I molecules The method of claim 9.
[Claim 11] Claim 9 or 10 and T cells from antigen-presenting cells and a subject obtained by the method according to be contacted ex vivo or in vitro comprising activating the T cells, as defined in claim 1 Poly the method for manufacturing a cytotoxic T cells specific to the peptide.
[Claim 12] Obtainable by a method according to claim 9 or 10, and characterized in that it comprises a complex between the polypeptide and the MHC molecule as defined in claim 1, the antigen-presenting cells.
[Claim 13] Obtained by the method according to claim 11, and characterized in that the polypeptide as defined in claim 1 which is specific, cytotoxic T cells.