WO2017170365A1 - 免疫誘導剤 - Google Patents
免疫誘導剤 Download PDFInfo
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- WO2017170365A1 WO2017170365A1 PCT/JP2017/012334 JP2017012334W WO2017170365A1 WO 2017170365 A1 WO2017170365 A1 WO 2017170365A1 JP 2017012334 W JP2017012334 W JP 2017012334W WO 2017170365 A1 WO2017170365 A1 WO 2017170365A1
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Definitions
- the present invention relates to a novel immunity-inducing agent useful as an agent for treating and / or preventing cancer.
- MCEMP1 Mast Cell-Expressed Membrane Protein 1
- An object of the present invention is to find a novel polypeptide useful as an agent for treating and / or preventing cancer, and to provide use of the polypeptide as an immunity-inducing agent.
- 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 canine testis-derived cDNA library and the serum of a dog with leukemia.
- a canine Must Cell-Expressed Membrane Protein 1 (hereinafter referred to as MCEMMP1) polypeptide having the amino acid sequence represented by SEQ ID NO: 4 was prepared.
- MCEMMP1 canine Must Cell-Expressed Membrane Protein 1
- MCEMP1 polypeptides are specifically expressed in leukemia, myelodysplastic syndrome, osteosarcoma, thymoma, mastocytoma, and perianal adenocarcinoma. Furthermore, it has been found that by administering these MCEMP1 to a living body, immune cells against MCEMP1 can be induced in the living body, and tumors in the body expressing MCEMP1 can be regressed. Furthermore, it has been found that a recombinant vector capable of expressing a polynucleotide encoding a polypeptide of MCEMP1 or a fragment thereof induces an antitumor effect in vivo against a cancer expressing MCEMP1.
- the present invention includes the following features (1) to (11).
- An immunity-inducing agent comprising a recombinant vector capable of expressing the polypeptide as an active ingredient.
- the immunity-inducing agent according to (1) above which is an active ingredient of an agent for treating and / or preventing cancer.
- the immunopotentiator is incomplete Freund's adjuvant, Montanide, poly IC and its derivatives, CpG oligonucleotide, interleukin 12, interleukin 18, interferon ⁇ , interferon ⁇ , interferon ⁇ , interferon ⁇ , and Flt3 ligand
- the immunity-inducing agent according to (6) above which is at least one selected from the group consisting of: (8) A method for producing an antigen-presenting cell comprising a complex of the polypeptide and an MHC molecule, which comprises contacting the polypeptide defined in (1) with an antigen-presenting cell derived from a subject ex vivo or in vitro. .
- polypeptides selected from the following polypeptides (a) to (d) and having immunity-inducing activity, or a polynucleotide encoding the polypeptide, and the polypeptide in vivo: A method of inducing immunity comprising administering to a subject a recombinant vector capable of expressing.
- SEQ ID NO: 2, 4, 6 in the sequence listing Or a polypeptide consisting of an amino acid sequence in which one to several amino acids are deleted, substituted or added in the amino acid sequence shown in FIG.
- a polypeptide comprising an amino acid sequence having a sequence identity of 90% or more with respect to the amino acid sequence A polypeptide comprising any one of the polypeptides (a) to (c) above as a partial sequence.
- a novel immunity-inducing agent useful for treatment and / or prevention is provided. As specifically shown in the examples described later, when the polypeptide used in the present invention is administered to a subject, immune cells can be induced in vivo, and further, cancer that has already occurred is reduced or regressed. Can be made. Therefore, the polypeptide is useful for treating or preventing cancer.
- polypeptides contained as an active ingredient in the immunity-inducing agent of the present invention include the following polypeptides (a) to (d).
- 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 constituent amino acids but also a low molecular weight having a small number of amino acids. Also included are molecules (oligopeptides) and full-length proteins.
- a polypeptide having an immunity-inducing activity comprising 7 or more consecutive amino acids in the polypeptide having the amino acid sequence shown in SEQ ID NO: 2, 4, 6, or 8 in the sequence listing and not more than the full length.
- B a polypeptide comprising an amino acid sequence in which one to several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2, 4, 6, or 8 in the sequence listing and having immunity-inducing activity .
- “having an amino acid sequence” means that amino acid residues are arranged in such an order.
- a polypeptide having the amino acid sequence represented by SEQ ID NO: 8 refers to Met, His, Ala, Ser, Ala ⁇ (Omitted) ⁇ Gln, Pro, Ser, Thr represented by SEQ ID NO: 8.
- a polypeptide having the amino acid sequence of 183 amino acid residues in size may be abbreviated as “polypeptide of SEQ ID NO: 8”.
- immuno-inducing activity means the ability to induce immune cells that secrete cytokines such as interferon in vivo.
- Whether or not the above polypeptide has immunity-inducing activity can be confirmed using, for example, a known Elispot assay. Specifically, cells such as peripheral blood mononuclear cells are obtained from a living body administered with a polypeptide whose immunity-inducing activity is to be evaluated, the cells are co-cultured with the polypeptide, and the amount of cytokine production from the cells is determined. Since the number of immune cells in the cells can be measured by measuring using a specific antibody, the immunity-inducing activity can be evaluated thereby.
- Elispot assay Specifically, cells such as peripheral blood mononuclear cells are obtained from a living body administered with a polypeptide whose immunity-inducing activity is to be evaluated, the cells are co-cultured with the polypeptide, and the amount of cytokine production from the cells is determined. Since the number of immune cells in the cells can be measured by measuring using a specific antibody, the immunity-inducing activity can be evaluated thereby.
- the tumor when the recombinant polypeptides (a) to (d) above are administered to a cancer-bearing organism, the tumor can be regressed due to its immunity-inducing activity. Therefore, the immunity-inducing activity can also be evaluated as the ability to suppress the growth of cancer cells or to reduce or eliminate cancer tissue (tumor) (hereinafter referred to as “anti-tumor activity”).
- anti-tumor activity The antitumor activity of a polypeptide is confirmed by, for example, examining whether the tumor is actually reduced by administering the polypeptide to a cancer-bearing living body, as specifically described in the examples below. can do.
- cytotoxic T cells induced by administering the polypeptide to a cancer-bearing organism exhibit cytotoxic activity against the tumor. It can. Measurement of the cytotoxic activity of T cells in vivo can be confirmed, for example, by examining whether or not the tumor is increased by administering an antibody that removes T cells from the body, but is not limited thereto. Not.
- immunity induction activity Can be used for the preparation of the immunity-inducing agent of the present invention.
- the polypeptide is taken up by antigen-presenting cells and then decomposed by peptidase in the cells to become smaller fragments and presented on the surface of the cells. It is known that cytotoxic T cells and the like recognize this and selectively kill cells presenting the antigen.
- the size of the polypeptide presented on the surface of the antigen-presenting cell is relatively small, and is about 7 to 30 amino acids. Therefore, from the viewpoint of presentation on antigen-presenting cells, the above-mentioned polypeptide (a) is about 7 to 30 continuous in the amino acid sequence shown in SEQ ID NO: 2, 4, 6, or 8. Is a preferred embodiment, and it is sufficient if it is composed of about 8 to 30 or 9 to 30 amino acids.
- These relatively small polypeptides may be presented directly on the cell surface on antigen-presenting cells without being taken up into antigen-presenting cells.
- Polypeptides taken up by antigen-presenting cells are cleaved at random positions by peptidases in the cells to generate various polypeptide fragments, and these polypeptide fragments are presented on the surface of antigen-presenting cells. Therefore, if a polypeptide having a large size such as the full-length region of SEQ ID NO: 2, 4, 6, or 8 is administered, the polypeptide is effective for inducing immunity via antigen-presenting cells by degradation in the antigen-presenting cells. Fragments are inevitably produced.
- a polypeptide having a large size can be preferably used for immunity induction via antigen-presenting cells, and the number of amino acids is 30 or more, more preferably 100 or more, more preferably 200 or more, and more preferably SEQ ID NOs: 2, 4 , 6 or 8 full length polypeptide.
- the polypeptide (c) is a polypeptide in which a small number (preferably one or several) of amino acid residues in the polypeptide (a) are substituted, deleted and / or inserted. It has 90% or more, preferably 95% or more, more preferably 98% or more, more preferably 99% or more or 99.5% or more sequence identity with the original sequence, and immunity-inducing activity
- a polypeptide having 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.
- the polypeptide (c) can also exert immunity-inducing activity, it can be used for the preparation of the immunity-inducing agent of the present invention.
- the polypeptide (b) one to several amino acid residues in the amino acid sequence represented by SEQ ID NO: 2, 4, 6, or 8 are substituted, deleted and / or inserted. Also preferred are polypeptides. “Several” in the present specification represents an integer of 2 to 10, preferably an integer of 2 to 6, and more preferably an integer of 2 to 4.
- sequence identity of amino acid sequences or base sequences means that both amino acid sequences (or bases) so that the amino acid residues (or bases) of the two amino acid sequences (or base sequences) to be compared match as much as possible. Sequence) and the number of matched amino acid residues (or the number of matched bases) divided by the total number of amino acid residues (or the total number of bases) is expressed as a percentage. In the above alignment, a gap is appropriately inserted in one or both of the two sequences to be compared as necessary. Such alignment of sequences can be performed using a known algorithm such as BLAST, FASTA, or CLUSTALW.
- the total number of amino acid residues is the number of residues obtained by counting one gap as one amino acid residue.
- the sequence identity (%) is the total number of amino acid residues in the longer sequence, and the amino acid residues that match. Calculated by dividing the number.
- the 20 kinds of amino acids constituting the natural protein are neutral amino acids having low polarity side chains (Gly, Ile, Val, Leu, Ala, Met, Pro), 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 if substitution is made between these groups. Therefore, when substituting amino acid residues in the polypeptide of the above (a) of the present invention, substitution between these groups increases the possibility that immunity-inducing activity can be maintained, which is preferable. .
- the polypeptide (d) is a polypeptide having any of the polypeptides (a) to (c) as a partial sequence and having immunity-inducing activity. That is, it is a polypeptide obtained by adding another amino acid or polypeptide to one or both ends of the polypeptide (a) or (b) and having immunity-inducing activity. Such a polypeptide can also be used for the preparation of the immunity-inducing agent of the present invention.
- the above-mentioned polypeptide can be synthesized according to a chemical synthesis method such as Fmoc method (fluorenylmethyloxycarbonyl method) or tBoc method (t-butyloxycarbonyl method). Moreover, it can also synthesize
- a polynucleotide encoding the above polypeptide is prepared, the polynucleotide is incorporated into an expression vector and introduced into a host cell, and the polypeptide is produced in the host cell. Thus, the desired polypeptide can be obtained.
- the polynucleotide encoding the above polypeptide can be easily prepared by a known genetic engineering technique or a conventional method using a commercially available nucleic acid synthesizer.
- DNA having the base sequence of SEQ ID NO: 3 is subjected to PCR using a canine chromosomal DNA or cDNA library as a template and a pair of primers designed to amplify the base sequence described in SEQ ID NO: 3.
- PCR reaction conditions can be set as appropriate. For example, one cycle of a reaction step consisting of 94 ° C.
- the conditions include, but are not limited to, conditions of reacting at 72 ° C. for 7 minutes after 30 cycles.
- appropriate probes and primers are prepared based on the nucleotide sequence and amino acid sequence information shown in SEQ ID Nos. 1 and 3 in the sequence listing in the present specification, and cDNA libraries such as humans and dogs are prepared using the probes and primers.
- the desired DNA can be isolated by screening for.
- the cDNA library is preferably prepared from cells, organs or tissues expressing the proteins of SEQ ID NOs: 2, 4.
- the host cell may be any cell that can express the polypeptide.
- prokaryotic cells include Escherichia coli, and examples of eukaryotic cells include monkey kidney cells COS1, Chinese hamster ovary cells. Examples include, but are not limited to, cultured mammalian cells such as CHO, budding yeast, fission yeast, silkworm cells, and Xenopus egg cells.
- 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, pBluescript II, pET expression system, pGEX expression system and the like.
- 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.
- expression vectors include pKA1, pCDM8, pSVK3, pMSG, pSVL, pBK-CMV, pBK-RSV, EBV vector, pRS, pcDNA3.1, pSecTag (A, B, C), pMSG, pYES2, etc. it can.
- a DNA encoding the polypeptide is incorporated into such an expression vector, and after transforming a eukaryotic host cell with the vector, the resulting transformant is cultured, and the DNA encodes. Can be expressed in eukaryotic host cells.
- 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.
- Polypeptides obtained by the above methods include those in the form of fusion proteins with other arbitrary proteins as described above. Examples thereof include glutathione-S-transferase (GST) and a fusion protein with a His tag. Such a polypeptide in the form of a fusion protein is also included in the scope of the present invention as the polypeptide (d). 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 included in the scope of the present invention as long as they have immunity-inducing activity. Examples of such translational modifications include elimination of N-terminal methionine, N-terminal acetylation, sugar chain addition, limited degradation by intracellular protease, myristoylation, isoprenylation, phosphorylation and the like.
- the immunity-inducing agent of the present invention can be used as a therapeutic and / or prophylactic agent for cancer.
- the polypeptide having the above-described immunity-inducing activity can be used in a method for treating and / or preventing cancer by immunity induction.
- the target cancer is not particularly limited as long as it is a cancer that expresses MCEMP1, but is preferably a cancer that expresses MCEMP1 significantly more than normal cells.
- Formation syndrome, osteosarcoma, thymoma, mastocytoma or perianal adenocarcinoma these specific cancers include, for example, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic Granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, leukemic leukemia, leukemia leukemia, basophilic leukemia, blastic leukemia, bovine leukemia, chronic myelocytic Leukemia, skin leukemia, embryonic cell leukemia, eosinophilic leukemia, gross leukemia, leader cell leukemia, shilling leukemia, stem cell leukemia, sub Hematologic leukemia, undifferent
- the subject (animal) to be used is preferably a mammal, more preferably a mammal including a primate, a pet animal, an animal bred in a zoo, a domestic animal, a sport animal, and the like. Particularly preferably a human, a dog or a cat.
- the immunity-inducing agent of the present invention may consist only of a polypeptide, or may be appropriately mixed with additives such as pharmacologically acceptable carriers, diluents and excipients suitable for each administration form. It can also be formulated. Formulation methods and usable additives are well known in the field of pharmaceutical formulations, and any method and additive can be used. Specific examples of additives include diluents such as physiological buffers; excipients such as sugar, lactose, corn starch, calcium phosphate, sorbitol, glycine; syrup, gelatin, gum arabic, sorbitol, polyvinyl chloride, tragacanth, etc.
- 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.
- 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.
- adjuvants include MPL (SmithKline Beecham), Salmonella minnesota Re 595 lipopolysaccharide, and the like obtained after purification and acid hydrolysis; QS21 (SmithKline Beecham), purified from Quillajasapur. QA-21 saponin; DQS21 described in PCT application WO 96/33739 (SmithKline Beecham); QS-7, QS-17, QS-18 and QS-L1 (So, outside, 10 people, “Molecules and Cell (Molecules and cells), 1997, Vol. 7, pp.
- factors that stimulate the immune response of the subject can also be used as the immune enhancer.
- various cytokines having the property of stimulating lymphocytes and antigen-presenting cells can be used in combination with the immune inducer of the present invention as an immune enhancer.
- Numerous cytokines capable of enhancing such immunological responses are known to those skilled in the art, such as interleukin-12 (IL-12), which has been shown to enhance the protective effects of vaccines, Examples include, but are not limited to, GM-CSF, IL-18, interferon ⁇ , interferon ⁇ , interferon ⁇ , interferon ⁇ , and Flt3 ligand.
- IL-12 interleukin-12
- Such factors can also be used as the above-mentioned immunity enhancing agent, and can be administered to patients in the immunity-inducing agent of the present invention or in combination with the immunity-inducing agent of the present invention as a separate composition.
- Antigen-presenting cell or cytotoxic T cell further comprises contacting the polypeptide and an antigen-presenting cell derived from a subject ex vivo or in vitro, and the MHC molecule A method for producing an antigen-presenting cell containing the complex of the present invention is provided.
- the polypeptide can be presented to the antigen-presenting cell by contacting the polypeptide and the antigen-presenting cell ex vivo or in vitro. That is, the polypeptides (a) to (d) can be used as a treatment agent for antigen-presenting cells.
- dendritic cells or B cells carrying MHC class I molecules can be preferably used as antigen-presenting cells.
- 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.
- HLA-A1 HLA-A0201, HLA-A0204, HLA-A0205, HLA-A0206, HLA-A0207, HLA-A11, HLA-A24, HLA-A31, HLA-A6801, HLA-B7, HLA-B8, HLA-B2705, HLA-B37, HLA-Cw0401, HLA-Cw0602, and the like.
- Dendritic cells or B cells carrying MHC class I molecules can be prepared from peripheral blood by well-known methods. For example, dendritic cells are induced from bone marrow, umbilical cord blood or patient peripheral blood using granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-3 (or IL-4), and tumor-related peptides are introduced into the culture system. Can be added to induce tumor-specific dendritic cells.
- GM-CSF granulocyte-macrophage colony-stimulating factor
- IL-3 or IL-4
- the whole cells constituting the bone marrow may be cultured, or mononuclear cells may be separated and cultured from this.
- Peripheral blood, its white blood cell components, and bone marrow cells include mononuclear cells, hematopoietic stem cells, immature dendritic cells, CD4 positive cells, and the like that are the origin of dendritic cells.
- 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 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.
- 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.
- stacked containers, multistage containers, roller bottles, spinner bottles, bag-type incubators, hollow fiber columns, etc. can be used regardless of general containers such as petri dishes, flasks, and bottles. .
- the method of contacting the polypeptide and the antigen-presenting cell ex vivo or in vitro can be performed by a well-known method. For example, it can be performed by culturing antigen-presenting cells in a culture solution containing the polypeptide.
- the peptide concentration in the medium is not particularly limited, but is usually about 1 to 100 ⁇ g / ml, preferably about 5 to 20 ⁇ g / ml.
- the cell density during the culture is not particularly limited, but is usually about 10 3 to 10 7 cells / ml, preferably about 5 ⁇ 10 4 to 5 ⁇ 10 6 cells / ml. Cultivation is preferably performed according to a conventional method in an atmosphere of 37 ° C. and 5% CO 2 .
- the length of the peptide that can be presented on the surface by antigen-presenting cells is usually about 30 amino acid residues at the maximum. Therefore, although not particularly limited, when the antigen-presenting cell is contacted with the polypeptide ex vivo or in vitro, the polypeptide may be prepared to a length of about 30 amino acid residues or less.
- the present invention further comprises contacting the antigen-presenting cell with a T cell derived from a subject ex vivo or in vitro to activate the T cell, wherein the cytotoxic T specific to the polypeptide described above is activated.
- a method for producing a cell is provided.
- a cytotoxic T specific to the polypeptide is obtained.
- Cells can be induced and 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.
- cytotoxic T cells specific for the polypeptide are induced and proliferated. Therefore, isolated T cells that selectively bind to the complex of the polypeptide and MHC molecule can be prepared using the polypeptide.
- the administration route of the therapeutic and / or prophylactic agent for cancer containing antigen-presenting cells or isolated T cells as an active ingredient is preferably parenteral administration such as intravenous administration or intraarterial administration.
- the dose is appropriately selected according to symptoms, administration purposes, 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.
- one or two or more additives well known in the pharmaceutical field can be added.
- DNA vaccine By producing a polynucleotide encoding the polypeptide of (a) to (d) in the body of a subject animal, antibody production and cytotoxic T cells can be induced in the living body. The same effect as that obtained by administering the peptide can be obtained. That is, the immunity-inducing agent of the present invention comprises a polynucleotide that encodes the polypeptides (a) to (d) above, and contains a recombinant vector capable of expressing the polypeptide in vivo as an active ingredient. May be. As shown in the Examples described later, a recombinant vector capable of expressing such an antigen polypeptide is also called a DNA vaccine.
- the administration route of the DNA vaccine is preferably a parenteral administration route such as intramuscular administration, subcutaneous administration, intravenous administration or intraarterial administration, and the dosage can be appropriately selected according to the type of antigen.
- the weight of the DNA vaccine per kg body weight is about 0.1 ⁇ g to 100 mg, preferably about 1 ⁇ g to 10 mg.
- Examples of methods using viral vectors include retrovirus, adenovirus, adeno-associated (also referred to as “adeno-associated”) virus, herpes virus, vaccinia virus, pox virus, poliovirus, Sindbis virus, and other RNA viruses or DNA viruses. And a method of incorporating a polynucleotide encoding the polypeptide and infecting the subject animal with the polynucleotide.
- retroviruses, adenoviruses, adeno-associated viruses, vaccinia viruses and the like are particularly preferred.
- an in vivo method for directly introducing the gene into the body, and collecting a certain cell from the target animal and transferring the gene to the cell 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 edition, 1994, Vol. 12, No. 15, and references cited therein), in vivo method is more preferable.
- the liposome or membrane-fused liposome containing the DNA can be in the form of a liposome preparation such as a suspension, a freezing agent, or a centrifugal concentrated freezing agent.
- a cDNA phage library was synthesized using the obtained mRNA (5 ⁇ g).
- the cDNA phage library was prepared by using cDNA Synthesis kit, Zap-cDNA Synthesis Kit, ZAP-cDNA GigapackIII Gold Cloning Kit (manufactured by STRATAGENE) according to the protocol attached to the kit.
- the size of the prepared cDNA phage library was 1 ⁇ 10 6 pfu / ml.
- coli / phage extract was passed through an NHS column (GE Healthcare Bio-Science) to immobilize the E. coli / phage derived protein.
- Serum dog serum was passed through and reacted with this protein-immobilized column, and antibodies adsorbed to E. coli and phage were removed from the serum.
- the serum fraction passed through the column was diluted 500 times with TBS containing 0.5% nonfat dry milk, and this was used as an immunoscreening material.
- the purified plasmid was analyzed for the full-length insert sequence by the primer walking method using the T3 primer shown in SEQ ID NO: 9 and the T7 primer shown in SEQ ID NO: 10.
- the gene sequence described in SEQ ID NO: 3 was obtained by this sequence analysis.
- a sequence identity search program BLAST search http://www.ncbi.nlm.nih.gov/BLAST/ was used to perform sequence identity searches with known genes. As a result, the obtained gene was found to be the MCEMP1 gene.
- Gene pool cDNA (manufactured by Life technology), QUICK-Clone cDNA (manufactured by Clontech) and Large-Insert cDNA Library (manufactured by Clontech) were used as cDNAs for human normal tissues (brain, testis, colon, placenta).
- the PCR reaction was performed using the obtained gene-specific primers (SEQ ID NOs: 11 and 12 for dog primers, SEQ ID NOs: 13 and 14 for human primers, and SEQ ID NOs: 15 and 16 for mouse primers) as follows.
- the human MCEMP1 plasmid administration group showed a significant antitumor effect compared to the control group.
- the group administered with the human MCEMP1 plasmid showed no significant antitumor effect compared to the control group.
- the present invention is useful for the treatment and / or prevention of cancer because it provides an immunity-inducing agent containing a polypeptide that exhibits antitumor activity against various cancers.
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Abstract
Description
(1)以下の(a)~(d)のいずれかのポリペプチドから選択され、かつ免疫誘導活性を有する少なくとも1つのポリペプチド、又は該ポリペプチドをコードするポリヌクレオチドを含む、かつ生体内で該ポリペプチドを発現可能である組換えベクター、を有効成分として含む免疫誘導剤。
(a)配列表の配列番号2、4、6、又は8に示されるアミノ酸配列中の連続する7個以上から全長以下のアミノ酸からなるポリペプチド
(b)配列表の配列番号2、4、6、又は8に示されるアミノ酸配列において1~数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなるポリペプチド
(c)配列表の配列番号2、4、6、又は8に示されるアミノ酸配列に対して90%以上の配列同一性を有するアミノ酸配列からなるポリペプチド
(d)上記(a)~(c)のポリペプチドを部分配列として含むポリペプチド
(2)抗原提示細胞の処理剤である、上記(1)に記載の免疫誘導剤。
(3)癌の治療及び/又は予防剤の有効成分である、上記(1)に記載の免疫誘導剤。
(4)上記癌がMCEMP1を発現する癌である、上記(3)に記載の免疫誘導剤。
(5)上記癌が白血病、骨髄異形成症候群、骨肉腫、胸腺腫、肥満細胞腫又は肛門周囲腺癌である、上記(3)又は(4)に記載の免疫誘導剤。
(6)免疫増強剤をさらに含む、上記(1)~(5)のいずれかに記載の免疫誘導剤。
(7)上記免疫増強剤が、フロイントの不完全アジュバント、モンタニド、ポリIC及びその誘導体、CpGオリゴヌクレオチド、インターロイキン12、インターロイキン18、インターフェロンα、インターフェロンβ、インターフェロンω、インターフェロンγ、及びFlt3リガンドから成る群より選ばれる少なくとも一つである、上記(6)に記載の免疫誘導剤。
(8)(1)に定義するポリペプチドと被験体由来の抗原提示細胞とをex vivo又はインビトロで接触させることを含む、該ポリペプチドとMHC分子との複合体を含む抗原提示細胞の作製方法。
(9)上記抗原提示細胞が、MHCクラスI分子を保有する樹状細胞又はB細胞である、上記(8)に記載の方法。
(10)上記(8)又は(9)に記載の方法によって得られる抗原提示細胞と被験体由来のT細胞とをex vivo又はインビトロで接触させて該T細胞を活性化することを含む、上記(1)に定義するポリペプチドに特異的な細胞障害性T細胞の作製方法。
(11)以下の(a)~(d)のポリペプチドから選択され、かつ免疫誘導活性を有する少なくとも1つのポリペプチド、又は該ポリペプチドをコードするポリヌクレオチドを含む、かつ生体内で該ポリペプチドを発現可能である組換えベクター、を被験体に投与することを含む、免疫誘導方法。
(a)配列表の配列番号2、4、6、又は8に示されるアミノ酸配列中の連続する7個以上から全長以下のアミノ酸からなるポリペプチド
(b)配列表の配列番号2、4、6、又は8に示されるアミノ酸配列において、1~数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなるポリペプチド
(c)配列表の配列番号2、4、6、又は8に示されるアミノ酸配列に対して90%以上の配列同一性を有するアミノ酸配列からなるポリペプチド
(d)上記(a)~(c)のいずれかのポリペプチドを部分配列として含むポリペプチド
本発明により、癌の治療及び/又は予防等に有用な新規な免疫誘導剤が提供される。後述の実施例において具体的に示されるように、本発明で用いられるポリペプチドを被験体に投与すると、生体内において免疫細胞を誘導することができ、さらに、既に生じている癌を縮小もしくは退縮させることができる。従って、該ポリペプチドは癌の治療や予防に有用である。
1.ポリペプチド
本発明の免疫誘導剤に有効成分として含まれるポリペプチドとしては、以下の(a)~(d)のポリペプチドが挙げられる。なお、本明細書において、「ポリペプチド」とは、複数のアミノ酸がペプチド結合することによって形成される分子をいい、構成するアミノ酸数が多いポリペプチド分子のみならず、アミノ酸数が少ない低分子量の分子(オリゴペプチド)や、全長タンパク質も包含される。
(a)配列表の配列番号2、4、6、又は8に示されるアミノ酸配列を有するポリペプチド中の連続する7個以上から全長以下のアミノ酸からなり、免疫誘導活性を有するポリペプチド。
(b)配列表の配列番号2、4、6、又は8に示されるアミノ酸配列において1~数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ免疫誘導活性を有するポリペプチド。
(c)配列表の配列番号2、4、6、又は8に示されるアミノ酸配列に対して90%以上の配列同一性を有するアミノ酸配列からなり、かつ免疫誘導活性を有するポリペプチド
(d)上記(a)~(c)のいずれかのポリペプチドを部分配列として含み、かつ免疫誘導活性を有するポリペプチド。
後述の実施例に具体的に記載される通り、上記の免疫誘導活性を有するポリペプチドを担癌生体に投与すると、腫瘍を退縮させることができる。従って、本発明の免疫誘導剤は、癌の治療及び/又は予防剤として用いることができる。また、上記の免疫誘導活性を有するポリペプチドは、免疫誘導による癌の治療及び/又は予防方法に用いることができる。
本発明はさらに、上記のポリペプチドと被験体由来の抗原提示細胞とをex vivo又はインビトロ(in vitro)で接触させることを含む、該ポリペプチドとMHC分子との複合体を含む抗原提示細胞の作製方法を提供する。
上記(a)~(d)のポリペプチドをコードするポリヌクレオチドを対象動物の体内で発現させることによっても、該生体内で抗体生産や細胞障害性T細胞を誘導することができ、ポリペプチドを投与するのと同等の効果が得られる。すなわち、本発明の免疫誘導剤は、上記(a)~(d)のポリペプチドをコードするポリヌクレオチドを含み、生体内で該ポリペプチドを発現可能な組換えベクターを有効成分として含むものであってもよい。後述の実施例に示されるように、このような抗原ポリペプチドを発現可能な組換えベクターは、DNAワクチンとも呼ばれる。
[実施例1] SEREX法による新規癌抗原タンパクの取得
(1)cDNAライブラリーの作製
犬の精巣から酸-グアニジウム-フェノール-クロロフォルム法(Acid guanidium-Phenol-Chloroform法)により全RNAを抽出し、Oligotex-dT30 mRNA purification Kit(宝酒造株式会社製)を用いてキット添付のプロトコールに従ってポリA RNAを精製した。
上記作製したcDNAファージライブラリーを用いて、イムノスクリーニングを行った。具体的にはΦ90×15mmのNZYアガロースプレートに約2500クローンとなるように宿主大腸菌(XL1-Blue MRF')に感染させ、42℃、3~4時間培養し、溶菌斑(プラーク)を作らせ、IPTG(イソプロピル-β-D-チオガラクトシド)を浸透させたニトロセルロースメンブレン(Hybond C Extra:GE Healthecare Bio-Sciece社製)でプレートを37℃で4時間覆うことによりタンパク質を誘導・発現させ、メンブレンにタンパク質を転写した。その後メンブレンを回収し0.5%脱脂粉乳を含むTBS(10mM Tris-HCl、150mM NaCl pH7.5)に浸し4℃で一晩振盪することによって非特異反応を抑制した。このフィルターを500倍希釈した患犬血清と室温で2~3時間反応させた。
上記方法により単離した1個の陽性クローンを塩基配列解析に供するため、ファージベクターからプラスミドベクターに転換する操作を行った。具体的には宿主大腸菌(XL1-Blue MRF')を吸光度OD600が1.0となるよう調製した溶液200μlと、精製したファージ溶液100μlさらにExAssist helper phage (STRATAGENE社製)1μlを混合した後37℃で15分間反応後、LB培地を3ml添加し37℃で2.5~3時間培養を行い、直ちに70℃の水浴にて20分間保温した後、4℃、1000×g、15分間遠心を行い、上清をファージミド溶液として回収した。次いでファージミド宿主大腸菌(SOLR)を吸光度OD600が1.0となるよう調製した溶液200μlと、精製したファージ溶液10μlを混合した後37℃で15分間反応させ、50μlをアンピシリン(終濃度50μg/ml)含有LB寒天培地に播き37℃一晩培養した。トランスフォームしたSOLRのシングルコロニーを採取し、アンピシリン(終濃度50μg/ml)含有LB培地37℃にて培養後、QIAGEN plasmid Miniprep Kit(キアゲン社製)を使って目的のインサートを持つプラスミドDNAを精製した。
上記方法により得られた遺伝子に対しイヌ、ヒト及びマウスの各種正常組織、各種腫瘍組織及び各種癌細胞株における発現をRT-PCR(Reverse Transcription-PCR)法により調べた。逆転写反応は以下の通り行った。すなわち、各組織50~100mg及び各細胞株5~10×106個の細胞からTRIZOL試薬(Life technology社製)を用いて添付のプロトコールに従い全RNAを抽出した。この全RNAを用いてSuperscript First-Strand Synthesis System for RT-PCR(Life technology社製)により添付のプロトコールに従いcDNAを合成した。ヒト正常組織(脳、精巣、結腸、胎盤)のcDNAは、ジーンプールcDNA(Life technology社製)、QUICK-Clone cDNA(クロンテック社製)及びLarge-Insert cDNA Library(クロンテク社製)を用いた。PCR反応は、取得した遺伝子特異的なプライマー(イヌプライマーは配列番号11及び12、ヒトプライマーは配列番号13及び14、マウスプライマーは配列番号15及び16に記載)を用いて以下の通り行った。すなわち、逆転写反応により調製したサンプル0.25μl、上記プライマーを各2μM、0.2mMの各dNTP、0.65UのExTaqポリメラーゼ(宝酒造社製)となるように各試薬と添付バッファーを加え全量を25μlとし、Thermal Cycler(BIO RAD社製)を用いて、94℃-30秒、55℃-30秒、72℃-1分のサイクルを30回繰り返して行った。その結果、図1に示すように、イヌMCEMP1遺伝子は、イヌ腫瘍組織では肥満細胞腫、肛門周囲腺癌で強い発現が見られた(図1)。ヒトMCEMP1遺伝子発現においては、健常なヒト組織ではほとんどの組織で発現が見られず、一方ヒト癌細胞では白血病、骨髄異形成症候群、骨肉腫の細胞株で強い発現が見られた(図2)。さらに、マウスMCEMP1遺伝子の発現は、白血病、胸腺腫の細胞株で発現が検出された(図3)。
(1)生体内でマウスMCEMP1を発現する組換えベクターの作製
配列番号7の塩基配列を基に、以下の方法にて、生体内でマウスMCEMP1を発現する組換えベクターを作製した。PCRは、実施例1において発現の見られたマウス白血病細胞株EL4(ATCCより購入)より調製した。cDNAを1μl、EcoRI及びNotI制限酵素切断配列を含む2種類のプライマー(配列番号17及び18に記載)を各0.4μM、0.2mM dNTP、1.25UのPrimeSTAR HSポリメラーゼ(宝酒造株式会社製)となるように各試薬と添付バッファーを加え全量を50μlとし、Thermal Cycler(BIO RAD社製)を用いて、98℃-10秒、55℃-15秒、72℃-1分のサイクルを30回繰り返すことにより行った。なお、上記2種類のプライマーは、配列番号8のアミノ酸配列全長をコードする領域を増幅するものであった。PCR後、増幅されたDNAを1%アガロースゲルにて電気泳動し、QIAquick Gel Extraction Kit(QIAGEN社製)を用いて約550bpのDNA断片を精製した。
5匹のA/Jマウス(7週齢、雄、日本SLC社から購入)に対して、上記で作製したチューブ(マウスMCEMP1/チューブ)を遺伝子銃に固定し、純ヘリウムガスを用いて400psiの圧力で、剃毛したマウスの腹腔にDNAワクチンを7日ごとに計3回、経皮投与した後に(プラスミドDNA接種量は2μg/匹になる)実施例1においてMCEMP1遺伝子の発現の見られたマウス白血病細胞株EL4細胞を移植して抗腫瘍効果を評価した(予防モデル)。なお、コントロールとして、マウスMCEMP1遺伝子が挿入されていないプラスミドDNAを予防モデルで5匹投与した。
5匹のA/Jマウス(7週齢、雄、日本SLC社から購入)に対して、上記で作製したチューブ(マウスMCEMP1/チューブ)を遺伝子銃に固定し、純ヘリウムガスを用いて400psiの圧力で、剃毛したマウスの腹腔にDNAワクチンを7日ごとに計3回、経皮投与した後に(プラスミドDNA接種量は2μg/匹になる)実施例1においてMCEMP1遺伝子の発現の見られたマウス白血病細胞株EG7細胞を移植して抗腫瘍効果を評価した(予防モデル)。なお、コントロールとして、マウスMCEMP1遺伝子が挿入されていないプラスミドDNAを予防モデルで5匹投与した。
配列番号1の塩基配列を基に、以下の方法にて、生体内でヒトMCEMP1を発現する組換えベクターを作製した。PCRは、実施例1において発現の見られたヒト白血病細胞株U937(ATCCより購入)より調製した。cDNAを1μl、EcoRI及びNotI制限酵素切断配列を含む2種類のプライマー(配列番号19及び20に記載)を各0.4μM、0.2mM dNTP、1.25UのPrimeSTAR HSポリメラーゼ(宝酒造株式会社製)となるように各試薬と添付バッファーを加え全量を50μlとし、Thermal Cycler(BIO RAD社製)を用いて、98℃-10秒、55℃-15秒、72℃-1分のサイクルを30回繰り返すことにより行った。なお、上記2種類のプライマーは、配列番号2のアミノ酸配列全長をコードする領域を増幅するものであった。PCR後、増幅されたDNAを1%アガロースゲルにて電気泳動し、QIAquick Gel Extraction Kit(QIAGEN社製)を用いて約550bpのDNA断片を精製した。
上記で作製したヒトMCEMP1/pcDNA3.1をマウス神経芽細胞腫細胞株N2a細胞(ATCC社)へリポフェクション法により導入し、500μg/mlのG418(ナカライ社)による選抜により、全長ヒトMCEMP1を定常的に発現するN2a細胞株を樹立した(N2a-ヒトMCEMP1)。また、ヒトMCEMP1をコードするcDNAが挿入されていない発現ベクター(以下、emp/pcDNA3.1)を上記と同様に導入して選抜した細胞をコントロール細胞とした(以下、N2a-emp)。
5匹のA/Jマウス(7週齢、雄、日本SLC社から購入)に対して、上記で作製したチューブ(ヒトMCEMP1/チューブ)を遺伝子銃に固定し、純ヘリウムガスを用いて400psiの圧力で、剃毛したマウスの腹腔にDNAワクチンを7日ごとに計3回、経皮投与した後に(プラスミドDNA接種量は2μg/匹になる)上記で作製したN2a-ヒトMCEMP1を、またコントロール細胞としてN2a-empをそれぞれ移植して抗腫瘍効果を評価した(予防モデル)。なお、コントロールとして、ヒトMCEMP1遺伝子が挿入されていないプラスミドDNAを予防モデルで5匹投与した。
Claims (11)
- 以下の(a)~(d)のいずれかのポリペプチドから選択され、かつ免疫誘導活性を有する少なくとも1つのポリペプチド、又は該ポリペプチドをコードするポリヌクレオチドを含む、かつ生体内で該ポリペプチドを発現可能である組換えベクター、を有効成分として含有する免疫誘導剤。
(a)配列表の配列番号2、4、6、又は8に示されるアミノ酸配列中の連続する7個以上から全長以下のアミノ酸から成るポリペプチド
(b)配列表の配列番号2、4、6、又は8に示されるアミノ酸配列において1~数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなるポリペプチド
(c)配列表の配列番号2、4、6、又は8に示されるアミノ酸配列に対して90%以上の配列同一性を有するアミノ酸配列からなるポリペプチド
(d)前記(a)~(c)のいずれかのポリペプチドを部分配列として含むポリペプチド - 抗原提示細胞の処理剤である、請求項1に記載の免疫誘導剤。
- 癌の治療及び/又は予防剤の有効成分である、請求項1に記載の免疫誘導剤。
- 前記癌がMCEMP1を発現する癌である、請求項3に記載の免疫誘導剤。
- 前記癌が白血病、骨髄異形成症候群、骨肉腫、胸腺腫、肥満細胞腫又は肛門周囲腺癌である、請求項3又は4に記載の免疫誘導剤。
- 免疫増強剤をさらに含む、請求項1~5のいずれかに記載の免疫誘導剤。
- 前記免疫増強剤が、フロイントの不完全アジュバント、モンタニド、ポリIC及びその誘導体、CpGオリゴヌクレオチド、インターロイキン12、インターロイキン18、インターフェロンα、インターフェロンβ、インターフェロンω、インターフェロンγ並びにFlt3リガンドから成る群より選ばれる少なくとも一つである、請求項6記載の免疫誘導剤。
- 請求項1に定義するポリペプチドと被験体由来の抗原提示細胞とをex vivo又はインビトロで接触させることを含む、該ポリペプチドとMHC分子との複合体を含む抗原提示細胞の作製方法。
- 前記抗原提示細胞が、MHCクラスI分子を保有する樹状細胞又はB細胞である、請求項8に記載の方法。
- 請求項8又は9に記載の方法によって得られる抗原提示細胞と被験体由来のT細胞とをex vivo又はインビトロで接触させて該T細胞を活性化することを含む、請求項1に定義するポリペプチドに特異的な細胞障害性T細胞の作製方法。
- 以下の(a)~(d)のポリペプチドから選択され、かつ免疫誘導活性を有する少なくとも1つのポリペプチド、又は該ポリペプチドをコードするポリヌクレオチドを含む、かつ生体内で該ポリペプチドを発現可能である組換えベクター、を被験体に投与することを含む、免疫誘導方法。
(a)配列表の配列番号2、4、6、又は8に示されるアミノ酸配列中の連続する7個以上から全長以下のアミノ酸からなるポリペプチド
(b)配列表の配列番号2、4、6、又は8に示されるアミノ酸配列において、1~数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなるポリペプチド
(c)配列表の配列番号2、4、6、又は8に示されるアミノ酸配列に対して90%以上の配列同一性を有するアミノ酸配列からなるポリペプチド
(d)前記(a)~(c)のいずれかのポリペプチドを部分配列として含むポリペプチド
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Also Published As
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AU2017241477B2 (en) | 2022-02-17 |
RU2018137802A3 (ja) | 2020-12-23 |
KR20180123126A (ko) | 2018-11-14 |
AU2017241477A1 (en) | 2018-10-18 |
US11045534B2 (en) | 2021-06-29 |
JPWO2017170365A1 (ja) | 2019-02-07 |
JP7035534B2 (ja) | 2022-03-15 |
CN108697780A (zh) | 2018-10-23 |
EP3437653A1 (en) | 2019-02-06 |
RU2018137802A (ru) | 2020-04-29 |
CA3017711A1 (en) | 2017-10-05 |
CN108697780B (zh) | 2022-07-22 |
BR112018069468A2 (pt) | 2019-07-30 |
EP3437653A4 (en) | 2019-11-27 |
MX2018011113A (es) | 2018-11-09 |
KR102520880B1 (ko) | 2023-04-12 |
US20190091315A1 (en) | 2019-03-28 |
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