WO2015099167A1 - Il-17aを標的とするワクチン - Google Patents
Il-17aを標的とするワクチン Download PDFInfo
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- WO2015099167A1 WO2015099167A1 PCT/JP2014/084682 JP2014084682W WO2015099167A1 WO 2015099167 A1 WO2015099167 A1 WO 2015099167A1 JP 2014084682 W JP2014084682 W JP 2014084682W WO 2015099167 A1 WO2015099167 A1 WO 2015099167A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
- C07K16/244—Interleukins [IL]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0005—Vertebrate antigens
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/06—Antipsoriatics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/08—Antiallergic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55516—Proteins; Peptides
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
Definitions
- the present invention relates to a vaccine targeting the IL-17A epitope.
- SLE Systemic lupus erythematosus
- SLE is an autoimmune disease that can affect the entire body from the scalp to the footpad, and is a chronic inflammatory disease in which various symptoms and multiple organs are damaged.
- cytokines such as type 1 interferon are involved in the process, but details are unknown.
- SLE patients are women of childbearing age, the relationship between female hormones and etiology is suspected.
- black women are more prevalent in North America than white women, and a genetic predisposition is suspected, but the etiology remains unknown.
- corticosteroids for treatment of SLE, corticosteroids, immunosuppressants, anti-cancer drugs such as cyclophosphamide, etc. are used. Although nonspecific immunosuppressive action can suppress SLE disease, There are problems such as being susceptible to infectious diseases due to a typical immunosuppressive action. Moreover, corticosteroids have side effects such as hypertension, diabetes, dyslipidemia, and depression, and cyclophosphamide has strong side effects such as bone marrow suppression, carcinogenicity, and infertility. The problem of infertility is particularly acute because many SLE patients are women of reproductive age. Under such circumstances, it is desired to elucidate the pathological condition in detail and to develop a molecule-specific treatment method with less side effects based on the pathogenesis.
- Non-patent Document 1 the blood IL-17 concentration in SLE patients was significantly higher than that in the control group.
- Non-patent Document 2 the blood concentration of IL-17 is increased, and when IL-17 neutralizing antibody is administered to the mice, the kidney lesions are Improvement was reported.
- IL-17 is a cytokine secreted from Th17 cells that are immunocompetent cells, macrophages, neutrophils, etc., rheumatoid arthritis, inflammatory bowel disease (Crohn's disease, ulcerative colitis), multiple sclerosis, It has been revealed that in many autoimmune diseases such as psoriasis, it plays an important role in aggravating the pathology. Recently, it has been reported that anti-IL-17 antibody drugs show therapeutic effects on psoriasis patients (Non-patent Document 4). However, antibody drugs are very expensive, and autoantibodies against the antibody drugs themselves are produced in the course of continuous use, and problems such as loss of effectiveness (secondary ineffectiveness) have come to be known.
- Non-patent Document 5 Non-patent Document 5
- immunization of the full length also induces cellular immunity against IL-17, and cells that express IL-17 are attacked by cytotoxic T cells, and there is a risk that harmful side effects are strongly manifested.
- cytotoxic T cells There is also a risk of producing antibodies that cross-react with other cytokines similar to IL-17.
- a vaccine using only an epitope of a part of IL-17 having no homology with other proteins as an immunogen is desirable in terms of safety.
- Non-patent Document 6 It was reported that two types of epitope vaccines for IL-17 were prepared and administered to inflammatory bowel disease model mice (Non-patent Document 6). However, in this report, the pathological condition was not improved in the vaccine administration group, but rather worsened, and inflammation of the large intestine and increased collagen deposition were observed. As described above, although there is a report on an epitope vaccine against IL-17, there is no report that the vaccine has shown a therapeutic effect for some diseases, as far as the applicant knows.
- An object of the present invention is to provide a vaccine against IL-17A, and a therapeutic and / or prophylactic agent for a disease containing the vaccine, such as SLE, in which IL-17A is involved in aggravation of the disease state.
- the present inventors have identified an amino acid site important for binding to the IL-17A receptor from the structure of IL-17A. Then, a part of the amino acid site was used as an antigen, and a polynucleotide encoding it was inserted into a vector encoding hepatitis B virus core antigen polypeptide to prepare a DNA vaccine.
- this DNA vaccine was administered to SLE model mice, NZBWF1 mice, a high increase in antibody titer was observed, and an antibody that correctly recognizes IL-17A was produced by a binding experiment between the antibody and recombinant IL-17A. It was confirmed.
- the present inventors can improve the pathology of colitis model mice, arthritis model mice, colon cancer transplant mice, and lung cancer transplant mice by administering the DNA vaccine or a peptide vaccine encoded by the DNA. It was confirmed. Surprisingly, the present inventors did not see any exacerbation of the pathological condition observed with the epitope vaccine of IL-17 reported in the past in the vaccine administration group found in the present invention. It was confirmed. Based on these findings, the present inventors have completed the present invention.
- a vaccine for preventing or treating a disease involving IL-17A as an exacerbation factor including any of the following (1) to (3): (1) The amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence shown in SEQ ID NO: 8, the amino acid sequence derived from a non-human mammal corresponding to SEQ ID NO: 1, or the non-human mammal corresponding to SEQ ID NO: 8 A polypeptide comprising an amino acid sequence derived from an animal, (2) The amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence shown in SEQ ID NO: 8, the amino acid sequence derived from a non-human mammal corresponding to SEQ ID NO: 1, or the non-human mammal corresponding to SEQ ID NO: 8 In an amino acid sequence derived from an animal, a polypeptide comprising an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted or added, and (3) expressing the polypeptide of (1) or (2) above An expression vector
- [1]-[6] vaccine (1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1, and (2) an expression vector capable of expressing the polypeptide of (1) above, [8]
- the vaccine according to [1]-[6], wherein the disease involving IL-17A as an exacerbation factor is rheumatoid arthritis including any of the following (1) to (3): (1) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1; (2) the amino acid sequence shown in SEQ ID NO: 11 or the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 12; and (3) an expression vector capable of expressing the polypeptide of (1) or (2) above, [9]
- a preventive or therapeutic agent for a disease in which IL-17A is involved as an exacerbation factor comprising an antibody that recognizes the following polypeptide (1) or (2) and inhibits the function of IL-17A: (1) The amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence shown in SEQ ID NO:
- the disease in which IL-17A is involved as an exacerbation factor is rheumatoid arthritis.
- a prophylactic or therapeutic agent of [9]-[11], comprising the polypeptide [14] a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1, [14-1] a polynucleotide encoding [14], I will provide a.
- the vaccine of the present invention can be used for the treatment of diseases such as SLE in which IL-17A is involved as a disease-causing factor.
- the figure shows the binding of recombinant IL-17A protein to antibodies from the serum of Balb / c mice immunized with IL-17A1 DNA vaccine (epitope sequence: RPSDYLNR) or IL-17A2 DNA vaccine.
- Anti-IL-17A antibody was used as the primary antibody.
- C) Anti-BSA antibody was used as the primary antibody. The increase of the antibody titer in the 6th week of the Balb / c mouse immunized with IL-17A1 DNA vaccine is shown. No1 to No4 primary antibodies (mouse antiserum) were diluted 10-fold and further diluted stepwise every 10-fold. No1: IL-17A1 No1 antisera, No2: IL-17A1 No2 antisera, No3: IL-17A1 No3 antisera, No4: IL-17A1 No4 antisera A) IL-17A1 DNA vaccine administration plan to NZBWF1 mice is shown.
- the increase in antibody titer in the IL-17A1 DNA vaccine administration group is shown.
- the figure shows the binding of antisera obtained from mice immunized with IL-17A1 DNA vaccine and BSA-IL-17A1 (RPSDYLNR) conjugate (No5 BSA conjugate) or mouse recombinant IL-17A (No5 rIL-17).
- the primary antibody mouse antiserum was diluted 100 times and further diluted stepwise every 5 times.
- NZBWF1 or MRL / lpr mice administered IL-17A1 DNA vaccine A) Urine protein (NZBWF1 mouse), B) Urinary MCP-1 concentration (pg / ml) (NZBWF1 mouse), C) Blood IL- 17A concentration (pg / ml) (NZBWF1 mouse), D) blood TNF- ⁇ concentration (pg / ml) (MRL / lpr mouse), E) blood IL-1 ⁇ concentration (pg / ml) (NZBWF1 mouse), F) Blood TNF- ⁇ concentration (pg / ml) (NZBWF1 mouse) is shown.
- NZBWF1 mouse or MRL / lpr mouse after IL-17A1 DNA vaccine or Saline administration A) PAS staining photo (weakly enlarged), B) PAS stained photo (strongly enlarged), C) F4 / 80 shows photographs of immunostaining (NZBWF1 mice), D) Pictures of immunostaining of F4 / 80 (MRL / lpr mice).
- mouth is shown.
- the photograph of HE staining of the liver after administering IL-17A1 DNA vaccine or Saline to NZBWF1 mouse or MRL / lpr mouse is shown.
- C57 BL / 6 mice administered with IL-17A1 DNA vaccine or Saline and inoculated with LLC cells B) body weight (g), C) tumor volume (mm 3 ), D) tumor 28 days after LLC cell inoculation Weight (mg), E) Photographic image of lung metastasis, F) Number of cancer lung metastasis (number), G) Number of cancer liver metastasis (number).
- Antisera IL-17 Human obtained from a mouse immunized with a vaccine comprising a peptide comprising the human IL-17A1 epitope (SEQ ID NO: 1) or serum before vaccine administration (Preimmune) and BSA-human IL-17A1 Shows binding to the conjugate.
- the primary antibody (mouse antiserum) was diluted 10 times and further diluted stepwise every 5 times.
- the primary antibody (mouse antiserum) was diluted 10 times and further diluted stepwise every 5 times.
- the primary antibody (mouse antiserum) was diluted 10 times and further diluted stepwise every 5 times. Binding of antiserum obtained from a mouse immunized with a vaccine comprising a peptide comprising the human IL-17A2 epitope (SEQ ID NO: 8) and BSA-human IL-17A2 conjugate (No AF975), or human IL-17A3 The binding between antisera obtained from a mouse immunized with a vaccine containing a peptide consisting of an epitope (SEQ ID NO: 9) and a BSA-human IL-17A3 conjugate (No AF976) is shown. The primary antibody (mouse antiserum) was diluted 10 times and further diluted stepwise every 5 times.
- Vaccine containing peptide consisting of human IL-17A4 epitope (SEQ ID NO: 11), vaccine containing DNA encoding human IL-17A5 epitope (SEQ ID NO: 12), DNA encoding human IL-17A6 epitope (SDY)
- SEQ ID NO: 11 The increase in antibody titer in the sixth week of Balb / c mice immunized with a vaccine containing or a vaccine containing DNA encoding the human IL-17A7 epitope (DYY) is shown.
- Each primary antibody (mouse antiserum) was diluted 10 times and further diluted stepwise every 5 times.
- A) A plan for administration of various IL-17A peptide vaccines and Type II collagen to DBA / 1 mice is shown.
- IL-17 (-) control IL-17 non-added, IL-17 only: recombinant human IL-17A added, IL-17 + antisera: recombinant human IL-17A and anti-human IL-17A1 mouse antibody, AIL-17 + antisera: Recombinant human IL-17A and non-immune mouse antibody
- the present invention provides a vaccine against IL-17A, and a therapeutic and / or prophylactic agent for a disease containing the vaccine, such as SLE, in which IL-17A is involved as an aggravating factor of a disease state.
- the vaccine against IL-17A of the present invention (also referred to as epitope vaccine) is selected from the group consisting of the following (1) to (3).
- the amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence shown in SEQ ID NO: 8, the amino acid sequence derived from a non-human mammal corresponding to SEQ ID NO: 1, or the non-human mammal corresponding to SEQ ID NO: 8 A polypeptide comprising an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted or added in an amino acid sequence derived from an animal, (3) An expression vector capable of expressing the polypeptide of (1) or (2) above.
- the vaccine against IL-17A of the present invention is preferably selected from the group consisting of the following (1 ′) to (3 ′).
- (3 ′) An expression vector capable of expressing the polypeptide of (1 ′) or (2 ′).
- the most preferred vaccine against IL-17A of the present invention is selected from the group consisting of the following (1 ′′) or (2 ′′).
- the disease in which IL-17A is involved as an exacerbation factor is not particularly limited as long as the disease state is exacerbated by IL-17A.
- SLE inflammatory bowel disease (ulcerative colitis, Crohn's disease), psoriasis, rheumatoid arthritis, multiple sclerosis, encephalomyelitis, tumor (lung cancer (non-small cell lung cancer is preferred among them), colon cancer, IL-17A, including blood cell tumors), arteriosclerosis, chronic inflammatory diseases, allergic diseases (delayed type hypersensitivity, contact type hypersensitivity, etc.)
- SLE, inflammatory bowel disease, rheumatoid arthritis, lung cancer, colon cancer, psoriasis and multiple sclerosis are preferable, and SLE, inflammatory bowel disease, rheumatoid arthritis, lung cancer and colon cancer are most preferable.
- the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or the DNA encoding the polypeptide has therapeutic effects on SLE, inflammatory bowel disease, rheumatoid arthritis, lung cancer, and colon cancer. Indicated.
- the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 it encodes the amino acid sequence shown in SEQ ID NO: 11, or the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 12, or the polypeptide DNA also showed a therapeutic effect on rheumatoid arthritis. Exacerbation means that the condition further deteriorates, and the degree of deterioration does not matter.
- the subject of administration of the vaccine of the present invention is any mammal, and is a mammal that has developed a disease in which IL-17A is involved in the progression of the disease state or a mammal that is likely to develop it.
- Mammals include, for example, rodents such as mice, rats, hamsters, guinea pigs, and laboratory animals such as rabbits, pets such as dogs and cats, livestock such as cows, pigs, goats, horses and sheep, humans, monkeys, Examples include primates such as orangutans and chimpanzees, preferably humans.
- the subject of administration may or may not be receiving treatment.
- the substance contained in the vaccine is a substance derived from an administration subject (that is, when administered to a human, the vaccine is a human-derived substance, and when administered to a mouse, The vaccine is preferably a mouse-derived substance).
- Polypeptide of the above (1) (or (1 ′), (1 ′′), the same applies hereinafter) included in the vaccine of the present invention (hereinafter, the above (2) (or (2 ′), the same applies hereinafter)) (Also referred to as a polypeptide of the present invention) is a partial amino acid sequence of IL-17A.
- the amino acid sequence at positions 62 to 69 of human IL-17A is SEQ ID NO: 1
- the corresponding amino acid sequence at positions 65 to 72 of mouse IL-17A is SEQ ID NO: 5.
- Each of these is encoded by a nucleotide sequence represented by SEQ ID NO: 2, SEQ ID NO: 6, for example.
- the amino acid sequence of human IL-17A at positions 102 to 118 is SEQ ID NO: 8, and the corresponding amino acid sequence at positions 105 to 121 of mouse IL-17A is SEQ ID NO: 10.
- Each of these is encoded, for example, by the nucleotide sequence represented by SEQ ID NO: 13 and SEQ ID NO: 14.
- SEQ ID NO: 8 As an amino acid sequence derived from a non-human mammal corresponding to SEQ ID NO: 1 (SEQ ID NO: 8), the sequence information disclosed in SEQ ID NO: 1 (SEQ ID NO: 8) in this specification, a known sequence database Etc. can be used to design appropriate primers and probes, and can be easily obtained using ordinary genetic engineering techniques such as RT-PCR or plaque hybridization.
- the polypeptide of (2) above contained in the vaccine of the present invention has one or several (preferably 1 to several (2 to 5)) amino acids deleted or substituted in the partial sequence of the IL-17A amino acid sequence.
- Amino acid sequence inserted or added Such a polypeptide lacks one or several (preferably 1 to several (2 to 5)) amino acids in the amino acid sequence represented by SEQ ID NO: 1 (SEQ ID NO: 8) in humans.
- Amino acid sequences that are deleted, substituted, inserted or added are also included.
- As the amino acid sequence for example, (1) one or several (preferably 1 to several (2 to 5)) amino acids in the amino acid sequence represented by SEQ ID NO: 1 (SEQ ID NO: 8) are deleted.
- the amino acid sequence contained in the polypeptide of (2) above is one or several (preferably 1 to several) amino acid sequences derived from a non-human mammal corresponding to SEQ ID NO: 1 (SEQ ID NO: 8).
- An amino acid sequence in which several (2 to 5) amino acids have been deleted, substituted, inserted or added is also preferred.
- such a polypeptide lacks one or several amino acids (preferably 1 to several (2 to 5)) in the amino acid sequence represented by SEQ ID NO: 5 (SEQ ID NO: 10). Amino acid sequences that are deleted, substituted, inserted or added are also included.
- amino acid sequence for example, (1) one or several (preferably 1 to several (2 to 5)) amino acids in the amino acid sequence shown in SEQ ID NO: 5 (SEQ ID NO: 10) are deleted. (2) an amino acid sequence in which one or several (preferably 1 to several (2 to 5)) amino acids are added to the amino acid sequence shown in SEQ ID NO: 5 (SEQ ID NO: 10); 3) an amino acid sequence in which one or several (preferably 1 to several (2 to 5)) amino acids are inserted into the amino acid sequence shown in SEQ ID NO: 5 (SEQ ID NO: 10), (4) SEQ ID NO: An amino acid sequence in which one or several (preferably 1 to several (2 to 5)) amino acids in the amino acid sequence shown in 5 (SEQ ID NO: 10) are substituted with other amino acids, or (5) the above ( An amino acid sequence in which mutations 1) to (4) are combined (in this case, Sum, one or several (preferably 1 to several (2 to 5))) are included.
- amino acid residue substitution examples include conservative amino acid substitution.
- Conservative amino acid substitution refers to substitution of a specific amino acid with an amino acid having a side chain having the same properties as the side chain of the amino acid. Specifically, in a conservative amino acid substitution, a particular amino acid is replaced with another amino acid belonging to the same group as the amino acid.
- Groups of amino acids having side chains of similar nature are known in the art. For example, such amino acid groups include amino acids having basic side chains (eg, lysine, arginine, histidine), amino acids having acidic side chains (eg, aspartic acid, glutamic acid), amino acids having neutral side chains.
- An amino acid having a neutral side chain further includes amino acids having a polar side chain (for example, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), and amino acids having a nonpolar side chain (for example, alanine, Valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan).
- amino acids having aromatic side chains for example, phenylalanine, tryptophan, tyrosine
- amino acids having side chains including hydroxyl groups for example, alcoholic hydroxyl groups, phenolic hydroxyl groups) (for example, serine, threonine, Tyrosine) and the like.
- “Deletion of amino acid residue” includes, for example, selecting and deleting an arbitrary amino acid residue from the amino acid sequence represented by SEQ ID NO: 1.
- Examples of such an amino acid sequence include SEQ ID NO: 11, SEQ ID NO: 12, SDY or DYY, and preferably SEQ ID NO: 11 and SEQ ID NO: 12.
- SEQ ID NO: 15 SEQ ID NO: 16, for example.
- an amino acid residue is inserted or added into the amino acid sequence represented by SEQ ID NO: 1, at the N-terminal side or C-terminal side.
- SEQ ID NO: 1 1 to 2 residues of the basic amino acid arginine (Arg) or lysine (Lys) may be added to the N-terminal side or C-terminal side of the amino acid sequence.
- the polypeptide of the present invention may contain an additional amino acid. Such amino acid addition is permissible as long as the polypeptide induces a specific immune response against IL-17A.
- the amino acid sequence to be added is not particularly limited, and examples thereof include a tag for facilitating detection and purification of the polypeptide. Tags include Flag tag, histidine tag, c-Myc tag, HA tag, AU1 tag, GST tag, MBP tag, fluorescent protein tag (eg, GFP, YFP, RFP, CFP, BFP, etc.), immunoglobulin Fc tag, etc. Can be illustrated.
- the position to which the amino acid sequence is added is the N-terminus and / or C-terminus of the polypeptide of the present invention.
- the amino acids used in the polypeptide of the present invention include L-form, D-form and DL-form, but it is usually preferred to be L-form.
- These polypeptides can be synthesized by an ordinary polypeptide synthesis method and used in the present invention, but in the present invention, the production method, synthesis method, procurement method, etc. are not particularly limited.
- a polynucleotide (DNA or RNA, preferably DNA encoding the polypeptide of (1) or (2) above ) Is functionally linked downstream of a promoter capable of exerting promoter activity in mammalian cells to be administered. That is, the expression vector (3) can express the polypeptide (1) or (2) as a transcription product under the control of a promoter.
- the expression vector of (3) is administered to a mammal, and the polypeptide of (1) or (2) is produced in the mammal, and the mammal is specific for the polypeptide of (1) or (2). An immune response is induced.
- the promoter used is not particularly limited as long as it can function in the cells of the mammal to be administered, and polI promoter, polII promoter, polIII promoter, and the like can be used.
- SV40-derived early promoter, viral promoter such as cytomegalovirus (CMV), mammalian constituent protein gene promoter such as ⁇ -actin gene promoter, and the like are used.
- a transcription termination signal that is, a terminator region is contained downstream of the polynucleotide encoding the above-described polypeptide (1) or (2).
- a selection marker gene for selecting transformed cells a gene that imparts resistance to drugs such as tetracycline, ampicillin, and kanamycin, a gene that complements an auxotrophic mutation, and the like may further be included.
- the type of vector used for the expression vector is not particularly limited, but examples of a vector suitable for administration to mammals such as humans include viral vectors and plasmid vectors.
- examples of viral vectors include retroviruses, adenoviruses, adeno-associated viruses, and the like.
- a plasmid vector is preferably used.
- an expression vector containing a polynucleotide encoding a hepatitis B virus core hereinafter referred to as HBc
- HBc hepatitis B virus core
- HBc has the property of self-assembling and becoming spherical, and the IL-17A epitope can be stably presented outside the core particle formed by self-assembly while maintaining its structure.
- the HBc and IL-17A epitopes may be linked directly by covalent bonds or may be linked via a spacer.
- the spacer is only required to be stably presented on the outside of the core particle formed by self-assembly of HBc while maintaining the structure of the IL-17A epitope, and examples thereof include IT, GAT, CGG, It is not limited to these.
- plasmid vectors containing the sequence include, but are not limited to, pCAGGS, pCR-X8, pcDNA3.1, pZeoSV, and pBK-CMV. More preferably, a pcDNA3.1-HBc vector is mentioned. In this vector, a spacer is inserted between the 240th and 241st bases of a polynucleotide encoding HBc corresponding to the 80th and 81st amino acids of HBc.
- nucleotide sequence encoding the polypeptide of (1) or (2) above is represented by nucleotide number: 246 and nucleotide number: An expression vector comprising a nucleotide sequence inserted between 247.
- the vaccine of the present invention can be provided as a pharmaceutical composition containing any carrier, for example, a pharmaceutically acceptable carrier, in addition to the polypeptide of (1) or (2) or the expression vector of (3).
- Examples of pharmaceutically acceptable carriers include excipients such as sucrose and starch, binders such as cellulose and methylcellulose, disintegrants such as starch and carboxymethylcellulose, lubricants such as magnesium stearate and aerosil, citric acid, Fragrances such as menthol, preservatives such as sodium benzoate and sodium bisulfite, stabilizers such as citric acid and sodium citrate, suspensions such as methylcellulose and polyvinylpyrrolide, dispersants such as surfactants, water, Examples include, but are not limited to, diluents such as physiological saline and base wax.
- the vaccine of the present invention may further contain a nucleic acid introduction reagent in order to promote introduction of the expression vector into cells.
- a viral vector is used as an expression vector
- retronectin, fibronectin, polybrene or the like can be used as a gene introduction reagent.
- a plasmid vector is used as an expression vector, lipofectin, lipofectamine, DOGS (transfectum), DOPE, DOTAP, DDAB, DHDEAB, HDEAB, polybrene, poly (ethyleneimine) (PEI), etc. Cationic lipids can be used.
- the vaccine of the present invention may further contain a carrier protein in order to increase the immunogenicity of the polypeptide encoded by the polypeptide of (1) or (2) or the expression vector of (3).
- a carrier protein is a substance that imparts immunogenicity by binding to a molecule that is not immunogenic due to its low molecular weight, and is known in the art. Examples of carrier proteins include bovine serum albumin (BSA), rabbit serum albumin (RSA), ovalbumin (OVA), squash hemocyanin (KLH), thyroglobulin (TG), immunoglobulin and the like.
- BSA bovine serum albumin
- RSA rabbit serum albumin
- OVA ovalbumin
- KLH squash hemocyanin
- TG thyroglobulin
- immunoglobulin immunoglobulin and the like.
- a polynucleotide encoding the carrier protein may be linked to the polynucleotide encoding the polypeptide of (1) or (2).
- the vaccine of the present invention may further contain an adjuvant that is pharmaceutically acceptable and compatible with the active ingredient.
- adjuvants are generally substances that non-specifically enhance the host immune response, and a number of different adjuvants are known in the art. Examples of adjuvants include, but are not limited to: complete Freund's adjuvant, incomplete Freund's adjuvant, aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP) N-acetyl-normuryl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2- (1'-2'-dipalmitoyl -Sn-glycero-3-hydroxyphosphoryloxy) -ethylamine (MTP-PE), Quill A (registered trademark), lysolecithin, sap
- the vaccine of the present invention can be administered to mammals orally or parenterally. Since polypeptides and expression vectors can be degraded in the stomach, they are preferably administered parenterally.
- Preparations suitable for oral administration include liquids, capsules, sachets, tablets, suspensions, emulsions and the like.
- Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions, which include antioxidants. Further, a buffer solution, an antibacterial agent, an isotonic agent and the like may be contained.
- aqueous and non-aqueous sterile suspensions can be mentioned, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives and the like.
- the preparation can be enclosed in a container in unit doses or multiple doses like ampoules and vials.
- the active ingredient and a pharmaceutically acceptable carrier can be lyophilized and stored in a state that may be dissolved or suspended in a suitable sterile vehicle immediately before use.
- the content of the active ingredient in the pharmaceutical composition is usually about 0.1 to 100% by weight, preferably about 1 to 99% by weight, and more preferably about 10 to 90% by weight of the whole pharmaceutical composition.
- the dose of the vaccine of the present invention varies depending on the subject to be administered, the administration method, the dosage form, etc., but when the active ingredient is the polypeptide of (1) or (2) above, usually one polypeptide per adult is used. In the range of 1 ⁇ g to 1000 ⁇ g per dose, preferably in the range of 20 ⁇ g to 100 ⁇ g, usually 2 to 3 times over 4 to 12 weeks. If the antibody titer decreases, add once each time .
- the expression vector per adult is usually in the range of 1 ⁇ g to 1000 ⁇ g, preferably in the range of 20 ⁇ g to 100 ⁇ g, usually for 4 to 12 weeks. Administer 2 to 3 times. If the antibody titer decreases, add 1 dose each time.
- a specific immune response (specific antibody production, specific T cell proliferation, etc.) against IL-17A is induced, and the mammal is neutralizing antibody against IL-17A. And the function of IL-17A is inhibited, so that a preventive or therapeutic effect for a disease associated with IL-17A as an exacerbation factor is exhibited.
- the present invention provides a kit comprising one or more containers including one or more components of the vaccine of the present invention. Even with the kit of the present invention, it is possible to prevent diseases associated with IL-17 as an exacerbation factor, or to treat or reduce the symptoms.
- IL-17A neutralizing antibody for prevention or treatment of diseases associated with IL-17A as an exacerbation factor
- the present invention recognizes the following polypeptide (1) or (2) and inhibits the function of IL-17A And a preventive or therapeutic agent for a disease associated with IL-17A as an exacerbation factor (preventive or therapeutic agent of the present invention).
- the amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence shown in SEQ ID NO: 8, the amino acid sequence derived from a non-human mammal corresponding to SEQ ID NO: 1, or the non-human mammal corresponding to SEQ ID NO: 8 A polypeptide comprising an amino acid sequence derived from an animal; (2) The amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence shown in SEQ ID NO: 8, the amino acid sequence derived from a non-human mammal corresponding to SEQ ID NO: 1, or the non-human mammal corresponding to SEQ ID NO: 8 A polypeptide comprising an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted or added in an amino acid sequence derived from an animal.
- the preventive or therapeutic agent of the invention preferably contains an antibody that recognizes the following polypeptide (1 ′) or (2 ′) and inhibits the function of IL-17A.
- (1 ′) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, or the amino acid sequence shown in SEQ ID NO: 8;
- (2 ′) A polypeptide comprising the amino acid sequence represented by SEQ ID NO: 11 or the amino acid sequence represented by SEQ ID NO: 12.
- the most preferred preventive or therapeutic agent of the invention includes (1 ′′) an antibody that recognizes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 and inhibits the function of IL-17A.
- IL-17A can be an effective preventive and / or therapeutic means for diseases associated with exacerbations. That is, administration of the antibody can be expected to have a therapeutic effect on a patient who has developed a disease associated with IL-17A as an exacerbation factor, and a preventive effect on a subject who may develop the disease.
- IL-17A neutralizing antibody of the present invention can be used for the prevention and / or treatment of colorectal cancer.
- the antibodies of the present invention natural antibodies such as polyclonal antibodies and monoclonal antibodies, chimeric mice that can be produced using transgenic mice and gene recombination techniques, humanized and single chain antibodies, and human antibody production genes are introduced. And human antibodies prepared by mouse, phage display, etc., and fragments thereof.
- the antibody of the present invention is not particularly limited as long as it recognizes the polypeptide of the present invention and inhibits the function of IL-17A, but is preferably a monoclonal antibody from the viewpoint of specificity to IL-17A.
- the antibody of the present invention is preferably a humanized antibody or a human antibody.
- the above antibody fragment means a partial region of the above-described antibody. Specifically, for example, an antibody fragment containing F (ab ′) 2 , Fab ′, Fab, Fc region, Fv (variable fragment of antibody) SFv, dsFv (disulphide stabilized Fv), dAb (single domain antibody) and the like (Exp. Opin. Ther. Patents, Vol. 6, No. 5, p. 441-456, 1996).
- the above-mentioned humanized antibody refers to an antibody produced using a gene recombination technique in which only the antigen recognition site is derived from a non-human gene and the remaining site is derived from a human gene.
- the human antibody is a human antibody produced by a transgenic mouse into which a human antibody-producing gene has been introduced (eg, TransChromo Mouse (trademark)), human B lymphocyte mRNA, VH and VL genes derived from the genome,
- the class of the antibody is not particularly limited, and the antibody of the present invention includes an antibody having any isotype such as IgG, IgM, IgA, IgD, or IgE.
- IgG or IgM is preferable, and IgG is more preferable in consideration of easiness of antibody purification and the like.
- a polyclonal antibody or monoclonal antibody can be produced by a method known per se. That is, the immunogen (polypeptide of the present invention) is optionally combined with Freund's Adjuvant in mammals, for example, polyclonal antibodies, mouse, rat, hamster, guinea pig, rabbit, cat, dog, pig. Immunize mice, rats, hamsters, guinea pigs, goats, horses or rabbits. In the case of a monoclonal antibody, mice, rats, hamsters and the like are immunized by the same method.
- the polypeptide of the present invention can be used as an immunogen as it is, but may be immunized as a complex with a high molecular compound (eg, carrier protein) having a molecular weight of 10,000 or more.
- a high molecular compound eg, carrier protein
- a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 is synthesized according to the method described above, and bovine serum albumin (BSA), rabbit serum albumin (RSA), ovalbumin (OVA), scallop hemocyanin (KLH) ),
- a carrier protein such as thyroglobulin (TG) or immunoglobulin may be formed and used as an immunogen.
- 1 to 2 preferably 1 amino acid can be added to the polypeptide of the present invention.
- the position of the added amino acid may be any position of the polypeptide and is not particularly limited, but the N-terminal or C-terminal of the polypeptide is preferable.
- any method known per se can be applied as long as the antigenicity of the polypeptide of the present invention can be maintained.
- a cysteine residue can be introduced into the polypeptide of the present invention and bound to the amino group of the polymer compound (carrier protein) via the SH group that is the side chain of the cysteine (MBS method).
- amino groups such as ⁇ -amino group and ⁇ -amino group of lysine residues of proteins can be bonded to each other (glutaraldehyde method).
- the polyclonal antibody can be produced as follows. That is, the immunogen is 1 to 3 in the mouse, rat, hamster, guinea pig, goat, horse or rabbit, preferably goat, horse or rabbit, more preferably rabbit subcutaneously, intramuscularly, intravenously, in a food pad or intraperitoneally. Immunization is given by several injections. Usually, immunization is performed 1 to 5 times about every 1 to 14 days from the initial immunization, and serum is obtained from the immunized mammal about 1 to 5 days after the final immunization.
- serum itself can be used as a polyclonal antibody, ultrafiltration, ammonium sulfate fractionation, Euglobulin precipitation method, caproic acid method, caprylic acid method, ion exchange chromatography (DEAE or DE52, etc.), anti-immunoglobulin column
- affinity column chromatography using a protein A / G column, a column to which an immunogen is crosslinked, or the like.
- Examples of the method for producing a monoclonal antibody include the following methods. First, a hybridoma is prepared from the antibody-producing cell obtained from the immunized animal and a myeloma cell (myeloma cell) having no autoantibody-producing ability, and the hybridoma is cloned. That is, using a culture supernatant of a hybridoma as a specimen, a monoclonal antibody that exhibits specific affinity for the peptide of the present invention used for immunization of mammals and does not cross-react with a carrier protein is produced by immunological techniques. Select a clone. Next, an antibody can be produced from the culture supernatant of the hybridoma by a method known per se.
- a monoclonal antibody can be produced as follows. That is, the immunogen can be subcutaneous, intramuscular, intravenous in mice, rats, or hamsters (including transgenic animals created to produce antibodies from other animals such as human antibody-producing transgenic mice). Immunization is carried out by injection or implantation in a food pad or abdominal cavity. Usually, immunization is carried out 1 to 4 times about every 1 to 14 days from the first immunization, and antibody-producing cells are obtained from the spleen of the mammal immunized about 1 to 5 days after the final immunization.
- a hybridoma (fusion cell) secreting a monoclonal antibody can be prepared according to the method of Köhler and Milstein et al. (Nature, Vol. 256, p.495-497, 1975) and a modification method according thereto. That is, antibody-producing cells contained in the spleen, lymph nodes, bone marrow, tonsils, etc., preferably from the spleen obtained from the immunized mammal as described above, and preferably mouse, rat, guinea pig, hamster, rabbit or human
- a hybridoma is obtained by cell fusion with a myeloma cell having no autoantibody-producing ability derived from mammals such as mouse, rat or human.
- myeloma cells used for cell fusion include mouse-derived myeloma P3 / X63-AG8.653 (653; ATCC No. CRL1580), P3 / NSI / 1-Ag4-1 (NS-1), P3 / X63- Ag8.
- human-derived myeloma U-266AR1, GM1500-6TG-A1-2, UC729-6, CEM-AGR, D1R11 or CEM-T15 examples of myeloma cells used for cell fusion.
- the obtained hybridomas are cultured in, for example, a microtiter plate, and the culture supernatants of wells in which proliferation has been observed are used in the above-described immunization.
- the reactivity with respect to a peptide and the reactivity with respect to the carrier protein of the said supernatant can be performed by measuring and comparing by immunoassay methods, such as ELISA, for example.
- the hybridoma cloned by screening is cultured using a medium (for example, DMEM containing 10% fetal calf serum). And the centrifugation supernatant of the culture solution can be used as a monoclonal antibody solution.
- a medium for example, DMEM containing 10% fetal calf serum
- the centrifugation supernatant of the culture solution can be used as a monoclonal antibody solution.
- ascites can be generated in the animal, and the ascites obtained from the animal can be used as a monoclonal antibody solution.
- the monoclonal antibody is preferably isolated and / or purified in the same manner as the polyclonal antibody described above.
- the chimera antibody is disclosed in JP-B-3-73280 and the like
- the humanized antibody is described in, for example, JP-T-4-506458 and JP-A-62-296890
- the human antibody is, for example, Nature Genetics, Vol.15, p.146-156, 1997, Nature Genetics, Vol.7, p.13-21, 1994, JP-T-4-504365, International Publication No.94 / 25585, Nature, Vol.368, p.856-859, 1994, Japanese translation of PCT publication No. 6-500263, etc.
- Antibody production by phage display can be performed, for example, by collecting and concentrating phage having affinity for an antigen by biopanning from a phage library prepared for human antibody screening. Etc. can be easily obtained. In this case, it is preferable to screen an antibody library using the polypeptide of the present invention as an antigen.
- preferred antibody libraries and antibody screening methods see Science, 228: 4075, p.1315-1317, 1985, Nature, 348, p.552-554, 1990, Curr.ProteineptPept.Sci .; 1 (2) , Pp.155-169, 2000, WO 01/062907, and the like.
- the antibody fragment thus obtained may be used, or the antibody may be prepared using DNA possessed by the phage.
- the compounding amount of the antibody contained in the preventive or therapeutic agent of the present invention is not particularly limited as long as the above effects are exhibited, but usually 0.001 to 90% by weight of the entire preventive or therapeutic agent of the present invention. It is preferably 0.005 to 50% by weight, more preferably 0.01 to 10% by weight.
- the preventive or therapeutic agent of the present invention may contain a pharmaceutically acceptable carrier in addition to the antibody which is an active ingredient.
- a carrier usually used in the pharmaceutical field can be used.
- excipients such as sucrose, starch, mannitol, sorbit, lactose, glucose, calcium phosphate, calcium carbonate, sodium benzoate, sulfite Preservatives such as sodium hydrogen, methylparaben, propylparaben, stabilizers such as citric acid, sodium citrate, acetic acid, suspensions such as methylcellulose, polyvinylpyrrolidone, aluminum stearate, dispersants such as surfactants, water, physiological Examples include diluents such as saline, base waxes such as glycerin and polyethylene glycol, but are not limited thereto.
- Examples of the dosage form of the preventive or therapeutic agent of the present invention include, but are not limited to, liquids and injection preparations.
- the preventive or therapeutic agent of the present invention may be a controlled-release preparation such as an immediate-release preparation or a sustained-release preparation. Since an antibody is generally soluble in an aqueous solvent, it can be easily absorbed regardless of the dosage form. Furthermore, the solubility of the antibody can be increased by a method known per se.
- the preventive or therapeutic agent of the present invention that can be used for the prevention, treatment or alleviation of diseases associated with IL-17A as an exacerbation factor uses the antibody as an active ingredient according to means known per se as a pharmaceutical preparation method. Can be manufactured.
- the preventive or therapeutic agent of the present invention suitable for systemic administration can be produced by dissolving an effective amount of the antibody of the present invention in an aqueous or non-aqueous isotonic sterile injection solution (eg, injection preparation). it can.
- the antibody of the present invention may be produced by lyophilizing (eg, lyophilized preparation) and dissolving it in an aqueous or non-aqueous isotonic sterile diluent.
- the preventive or therapeutic agent of the present invention suitable for local administration can be produced by dissolving the antibody of the present invention in a diluent such as water or physiological saline (eg, liquid).
- the liquid can also be used by inhalation therapy to the bronchi, lungs, etc. using a nebulizer.
- These agents may contain antioxidants, buffers, antibacterial agents, isotonic agents and the like.
- These preventive or therapeutic agents of the present invention can be enclosed in a container in unit doses or multiple doses, such as ampoules and vials.
- the dose of the prophylactic or therapeutic agent of the present invention can be appropriately set according to the activity, type or combination amount of the antibody contained as an active ingredient, administration subject, administration route, age and weight of the administration subject, etc.
- the dose (effective amount) per day for an adult (body weight 60 kg) is 0.1 mg to 1000 mg, preferably 0.1 mg to 500 mg, more preferably 0.1 mg to 300 mg as the amount of antibody.
- the prophylactic or therapeutic agent of the present invention may be administered once or divided into several times as needed per day, or may be administered in several days.
- the preventive or therapeutic agent of the present invention can be used in combination with known prophylactic / therapeutic agents effective for diseases associated with IL-17A as an exacerbation factor. These may be used alone or in combination.
- the term “combination” means that the preventive or therapeutic agent of the present invention is used in combination with a known prophylactic / therapeutic agent for a disease associated with IL-17A as an exacerbation factor.
- the form is not particularly limited.
- HBc was amplified by PCR using the above primer set. The HBc obtained above was cloned into pcDNA 3.1 / V5-His TOPO TA Expression Kit (Invitrogen).
- Nucleic acid sequences encoding the following two types of epitope A1 or epitope A2 of mouse IL-17A were respectively inserted into the pcDNA3.1-HBc vector by Mutagenesis.
- a vaccine comprising a pcDNA3.1-HBc vector into which a nucleic acid sequence encoding a mouse IL-17A1 epitope has been inserted is referred to as an IL-17A1 DNA vaccine, a nucleic acid sequence encoding a mouse IL-17A2 epitope.
- a vaccine containing the pcDNA3.1-HBc vector into which is inserted is referred to as IL-17A2 DNA vaccine.
- the structure of the vector is shown in FIG.
- Mouse IL-17A1 epitope RPSDYLNR SEQ ID NO: 5
- Mouse IL-17A2 epitope DHHMNSV SEQ ID NO: 7
- Experiment 2 Administration of DNA vaccine to mice DNA vaccine was injected into the thigh muscle of each of the following 6-week-old male mice by syringe, and electroporation was performed at that site. Three doses were administered every 2 weeks (120 ⁇ g / 60 ⁇ l ⁇ 1 location / time).
- Experiment 1 Antibody titer measurement, various cytokines in blood and urine, survival analysis NZBWF1 mouse (SLE disease model) HBc-IL-17A1 (IL-17A1 DNA vaccine) group: 6 animals HBc (pcDNA3.1-HBc vector) group: 6 animals Saline group: 10 animals Body weight was measured every week, and serum was collected every 4 weeks. The administration plan is shown in FIG. 4A.
- Experiment 2 Antibody titer measurement, analysis of each organ MRL / lpr mouse HBc-IL-17A1 (IL-17A1 DNA vaccine) group: 6 Saline group: 6 mice Body weight was measured every week, and serum was collected every 4 weeks It was.
- Example 1 Preparation of anti-IL-17A antibody titer measurement plate by antigenic ELISA of mouse IL-17A peptide : 96 wells of mouse IL-17A1 epitope + BSA conjugate, mouse IL-17A2 epitope + BSA conjugate at a concentration of 10 ⁇ g / ml It dispensed to the plate and left still at 4 degreeC overnight. Recombinant mouse IL-17A was dispensed into a 96-well plate at a concentration of 0.25 ⁇ g / ml and allowed to stand at 4 ° C. overnight. The plate was washed once with 200 ⁇ l of PBS and then blocked with 5% skim milk in PBS for 2 hours.
- the primary antibody (mouse antiserum) was serially diluted with 5% skim milk in PBS, 50 ⁇ l was applied to a 96-well plate, and incubated overnight at 4 ° C.
- the plate was washed 7 times with 200 ⁇ l of PBS-T (0.05% Tween), diluted with secondary antibody (anti-mouse IgG Ab-HRP label) 1/1000 (5% skim milk), added 50 ⁇ l at room temperature, 3 hours at room temperature Incubated.
- the plate was washed three times with 200 ⁇ l of PBS-T (0.05% Tween), 50 ⁇ l of TMB solution was added, and the mixture was incubated at room temperature for 30 minutes while protected from light.
- Lane 1 Recombinant mouse IL-17A
- Lane 2 Mouse IL-17A1 epitope + BSA conjugate
- Lane 3 Mouse IL-17A2 epitope + BSA conjugate
- Primary antibody A Antiserum of Balb / c mice administered with IL-17A1 DNA vaccine
- B Commercially available anti-mouse IL-17A antibody
- C Commercially available anti-BSA antibody
- mice administered with IL-17A1 DNA vaccine bound recombinant mouse IL-17A in the same manner as commercially available anti-mouse IL-17A antibody.
- the antiserum of mice administered IL-17A1 DNA vaccine specifically recognized the mouse IL-17A1 epitope + BSA conjugate and did not react with the mouse IL-17A2 epitope + BSA conjugate.
- Recombinant mouse IL-17A contains a large amount of BSA for stabilization, and commercially available anti-BSA antibodies include mouse IL-17A1 epitope + BSA conjugate and mouse IL-17A2 epitope + BSA conjugate.
- Example 2 Effect of IL-17A vaccine in SLE model mice Measurement of blood IL-1 ⁇ , TNF- ⁇ and IL-17A concentrations and urinary MCP-1 concentrations Using a Quantikine ELISA kit, IL was determined according to the manufacturer's protocol. -1 ⁇ , TNF- ⁇ , IL-17A and MCP-1 concentrations were measured. Serum was used at 25 ⁇ l each. 50 ⁇ l of urine was used. A standard curve was prepared using the standard sample attached to the kit, and the concentration of each cytokine was quantified. A significant decrease in blood IL-1 ⁇ was observed in the IL-17A1 DNA vaccine administration group to NZBWF1 mice (FIG. 6E).
- Urine qualitative examination Urine was collected at any time from anesthetized mice. Urine protein, urine creatinine, urinary albumin, urine occult blood, urine specific gravity and the like were measured using a urine multi-stick test paper. A decrease in urine protein was observed in the IL-17A1 DNA vaccine administration group to NZBWF1 mice (FIG. 6A).
- the IL-17A1 DNA vaccine was administered to a NZBWF1 mouse, a model mouse with a survival rate of SLE, and observed daily, and the day when the mouse died was recorded. The number of dead mice was compiled every week to create a survival graph. As a result of long-term observation of the IL-17A1 DNA vaccine administration group, a significant extension of the survival period of the vaccine administration group was observed (FIG. 7A). Further, in the group administered with the DNA vaccine to MRL / lpr mice described in Experiment 1, a tendency to extend the lifespan was also observed (FIG. 8B).
- the statistical method survival rate was statistically processed by Kaplan-Meier method.
- each excised organ was fixed in 4% paraformaldehyde for 24 hours, embedded in paraffin, and cut into 4 ⁇ m sections. The sections were reacted with a primary antibody (anti-F4 / 80 antibody) and a secondary antibody (HRP-labeled anti-rat IgG antibody). Slides were counterstained with hematoxylin and used for microscopic observation.
- kidneys, submandibular glands and liver were dissected and fixed overnight in 4% paraformaldehyde and embedded in paraffin. A 4 ⁇ m section of the kidney was stained with PAS staining. 4 ⁇ m sections of submandibular gland and liver were stained with HE staining.
- the degree of renal lesion in NZBWF1 mice or MRL / lpr mice was confirmed by renal PAS staining. The results are shown in FIGS. 11A and 11B. In the vaccine administration group, glomerular and interstitial destruction was suppressed. The degree of macrophage infiltration was confirmed by immunostaining with F4 / 80. The results are shown in FIGS. 11C and 11D. In the vaccine administration group, suppression of macrophage infiltration around the glomeruli and stroma was observed.
- the degree of submandibular adenitis in NZBWF1 mice was confirmed by submandibular gland HE staining. The results are shown in FIG. Suppression of submandibular adenitis was observed in the vaccine administration group.
- Example 3 Effect of IL-17A vaccine in colitis model mice
- a 6-week-old male mouse was enemamed every week with a TNBS solution (2 mg / 100 ⁇ l / dose), and the femoral muscle was electroporated with IL-17A1 DNA vaccine.
- Mice were sacrificed on week 8.
- the administration plan is shown in FIG.
- the length of the large intestine of the mouse 8 weeks after administration of TNBS was examined in the Balb / c mouse described in the experiment 4 of the large intestine.
- the length of the large intestine became shorter with the induction of colitis by TNBS (Saline group), but this effect was suppressed in the vaccine group (FIG. 14 (C)).
- the large intestine of the sacrificed large intestine was dissected, fixed overnight in 4% paraformaldehyde, and embedded in paraffin. A 4 ⁇ m section of the large intestine was stained with HE and examined histologically. Pathological findings such as infiltration of inflammatory cells were observed in the Saline group (FIG. 14D). In addition, the pathological findings of HE-stained sections were scored. A decrease in H & E score was observed in the vaccine group (FIG. 14 (E)).
- IL-17A vaccine showed an inhibitory effect on colitis in TNBS-induced colitis model mice.
- Example 4 Effect of IL-17A Vaccine in Arthritis Model Mice
- Mouse IL-17A1 DNA vaccine was administered 3 times every 2 weeks using electroporation to the femoral muscle of each of the following 6-week-old male mice (120 ⁇ g / 60 ⁇ l). ⁇ 1 location / time).
- Arthritis was induced by administering Type II collagen and CFA (complete Freund's adjuvant) 28 days after the first vaccine administration, and Type II collagen and IFA (incomplete Freund's adjuvant) 42 days after the first vaccine administration, and 3 times weekly thereafter
- the degree of arthritis was observed and scored.
- the administration plan is shown in FIG.
- IL-17A vaccine showed an arthritis-suppressing effect in arthritis model mice with Type II collagen.
- Example 5 Effect of IL-17A Vaccine in Colorectal Cancer Model Mice
- a vaccine (KLH conjugated) containing a peptide comprising mouse IL-17A1 epitope was administered 3 times every 2 weeks to the femoral muscle of each of the following 6-week-old male mice. (25 ⁇ g / 25 ⁇ l + adjuvant 25 ⁇ l / dose (adjuvant is CFA at first dose, IFA at third dose)).
- mouse colon cancer cell line CT26 cells (5 ⁇ 10 5 cells / Body) were inoculated, and thereafter tumor volumes (0.5 ⁇ major axis ⁇ minor axis ⁇ minor axis) were measured every week.
- IL-17A vaccine showed a tumor growth inhibitory effect and a survival time extension effect in a colon cancer model mouse inoculated with CT26 cells.
- mice IL-17A1 DNA vaccine was administered 3 times every 2 weeks to the femoral muscle of each of the following 6-week-old male mice (120 ⁇ g / 60 ⁇ l). ⁇ 1 location / time).
- mouse lung cancer cell line LLC cells (5 ⁇ 10 5 cells / Body) were inoculated, and thereafter tumor volumes (0.5 ⁇ major axis ⁇ minor axis ⁇ minor axis) were measured every week.
- tumor volumes 0.5 ⁇ major axis ⁇ minor axis ⁇ minor axis
- mice were sacrificed in the ninth week, the tumor weight, lung metastasis, and liver metastasis were confirmed.
- the administration plan is shown in FIG.
- Experiment 7 C57 BL / 6 mice Vaccine group (HBc-IL-17A1 group): 5 Saline group (saline group): 5
- Tumor volume was examined weekly after inoculating LLC cells into C57 BL / 6 mice described in Tumor Volume Experiment 7. It was recognized that the vaccine group had an effect of suppressing tumor growth (FIG. 17C).
- IL-17A vaccine is a lung cancer model mouse inoculated with LLC cells, It showed tumor growth inhibitory effect and metastasis inhibitory effect.
- Example 7 Anti-IL-17A antibody titer measurement by antigenic ELISA of human IL-17A peptide
- a vaccine comprising a peptide comprising the following human IL-17A1 epitope corresponding to mouse IL-17A1 epitope (RPSDYLNR (SEQ ID NO: 5)) (KLH conjugated) was prepared and administered intradermally to Balb / c mice three times at 2-week intervals, and the serum antibody titer was measured according to the method described in Example 1 6 weeks after the first administration.
- Human IL-17A1 epitope RSSDYYNR SEQ ID NO: 1
- an increase in the antibody titer of Balb / c mice administered with the vaccine was observed (FIG. 18).
- the serum also showed cross-reactivity with the mouse IL-17A1 epitope (FIG. 19 (A)). Furthermore, sera obtained by administering a vaccine containing a peptide consisting of the mouse IL-17A1 epitope also showed cross-reactivity with the human IL-17A1 epitope (FIG. 19 (B)).
- a vaccine (KLH conjugated) containing a peptide consisting of the following two types of epitopes of human IL-17A (human IL-17A2 epitope and human IL-17A3 epitope) was prepared, and the vaccine was applied to Balb / c mice at intervals of 2 weeks. Serum antibody titers were measured according to the method described in Example 1 6 weeks after the first administration and 6 weeks after the first administration.
- a vaccine (KLH conjugated) containing a peptide consisting of human IL-17A4 which is a partial sequence of the following human IL-17A1 epitope was prepared.
- Balb / c mice were intradermally administered 3 times at 2-week intervals, and serum antibody titers were measured according to the method described in Example 1 6 weeks after the initial administration.
- Example 8 Effect of Human IL-17A Vaccine in Arthritis Model Mice
- a vaccine comprising a peptide comprising human IL-17A1 epitope, human IL-17A2 epitope or human IL-17A4 epitope on the thigh muscle of each of the following 6-week-old male mice ( KLH conjugated) was administered 3 times every 2 weeks (120 ⁇ g / 60 ⁇ l ⁇ 1 location / time).
- Arthritis was induced by administering Type II collagen and CFA (complete Freund's adjuvant) 28 days after the first vaccine administration, and Type II collagen and IFA (incomplete Freund's adjuvant) 42 days after the first vaccine administration, and 3 times weekly thereafter The degree of arthritis was observed and scored.
- the administration plan is shown in FIG.
- the clinical score of arthritis of DBA / 1 mice described in clinical score experiment 8 was examined. It was confirmed that the vaccine group consisting of human IL-17A1 epitope, human IL-17A2 epitope or human IL-17A4 epitope has an effect of suppressing the onset and progression of arthritis (FIG. 22 (B)).
- human IL-17A vaccine showed an arthritis inhibitory effect in arthritis model mice with Type II collagen.
- Example 9 In vitro neutralization activity against IL-17A The amount of IL-6 secreted when recombinant human IL-17A was added to normal human skin fibroblasts (NHDF) in a medium was measured by ELISA. Addition of 17A promoted IL-16 secretion (IL-17 only). However, when IgG purified from the antiserum of a mouse immunized with a vaccine containing a peptide consisting of the human IL-17A1 epitope was added simultaneously with IL-17A, IL-6 secretion was suppressed (IL-17 + antisera) . Moreover, when IgG purified from the antiserum of non-immunized mice was added simultaneously with IL-17A, IL-6 secretion was hardly suppressed (IL-17 + control sera). These results are shown in FIG.
- the antibody obtained by immunization with a vaccine containing a peptide consisting of the human IL-17A1 epitope suppressed IL-6 secretion from cultured cells in vitro Therefore, it was suggested that the antibody has a neutralizing action on IL-17A activity, and it was suggested that IL-17A is effective for the therapeutic effect of diseases related to aversion.
- the vaccine of the present invention increases the antibody titer against IL-17A, not only SLE, but also rheumatoid arthritis, inflammatory bowel disease, cancer, psoriasis, multiple sclerosis, arteriosclerosis, etc.
- IL-17A can also be used for other diseases that are involved in the progression of disease states, and can greatly contribute to the treatment of these diseases.
- This application is based on Japanese Patent Application No. 2013-273133 (filing date: December 27, 2013) filed in Japan, the contents of which are incorporated in full herein.
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Abstract
Description
また、SLE患者の多くは妊娠可能な年齢の女性であることから、女性ホルモンと病因との関連が疑われている。一方、北米では黒人女性の方が白人女性より有病率が高いとされており、遺伝的素因の存在が疑われているが、病因は依然として不明である。
このような現状において、病態のより詳細な解明と、その病因に基づいた副作用の少ない分子特異的治療法の開発が望まれている。
最近、乾癬患者に対して抗IL-17抗体医薬品が治療効果を示すことが報告された(非特許文献4)。しかし抗体医薬品は非常に高価であり、また継続使用の過程で抗体医薬品自身に対する自己抗体が産生され、有効性が失われる(二次無効)などの問題が知られるようになった。これらのことからIL-17を標的としたワクチンはこれらの疾患に有効であり、かつ安価で長期に渡り有効な治療剤となるものと考えられる。
IL-17に対するワクチンとしては、IL-17全長を免疫原としたペプチドワクチンの研究が報告されている(非特許文献5)。しかし、全長を免疫すると、IL-17に対する細胞性免疫も惹起され、細胞障害性T細胞によりIL-17を発現する細胞が攻撃され、有害な副作用が強く表れる危険性がある。また、IL-17に類似する他のサイトカインに交差反応を示す抗体も産生される危険性がある。そのため、他のタンパク質との相同性がないIL-17の一部分のエピトープだけを免疫原とするワクチンの方が安全面で望ましいと考えられる。そして、IL-17のエピトープワクチンを2種類作製して炎症性腸疾患モデルマウスに投与したとの報告がなされた(非特許文献6)。ところが、該報告では、ワクチン投与群で病態が改善されるのではなく、むしろ増悪し、大腸の炎症やコラーゲン沈着の増加が認められた。
このように、IL-17に対するエピトープワクチンについての報告は存在するものの、該ワクチンにより、何らかの疾患の治療効果が示されたという報告は、出願人の知る限り、皆無である。
また、本発明者らは、大腸炎モデルマウス、関節炎モデルマウス、大腸癌移植マウス、肺癌移植マウスにおいても、該DNAワクチンや該DNAによってコードされるペプチドワクチンを投与することによって、病態を改善できることを確認した。
そして、驚くべきことに、本発明者らは、本発明で見出されたワクチン投与群では、従来報告されていたIL-17のエピトープワクチンで観察されるような、病態の増悪が見られないことを確認した。
本発明者らは、これらの知見に基づき本発明を完成させるに至った。
[1]以下の(1)~(3)のいずれかを含む、IL-17Aが増悪因子として関与する疾患の予防または治療用ワクチン:
(1)配列番号:1に示されるアミノ酸配列、配列番号:8に示されるアミノ酸配列、配列番号:1に対応する非ヒト哺乳動物由来のアミノ酸配列、又は配列番号:8に対応する非ヒト哺乳動物由来のアミノ酸配列を含むポリペプチド、
(2)配列番号:1に示されるアミノ酸配列、配列番号:8に示されるアミノ酸配列、配列番号:1に対応する非ヒト哺乳動物由来のアミノ酸配列、又は配列番号:8に対応する非ヒト哺乳動物由来のアミノ酸配列において、1又は数個のアミノ酸残基が置換、欠失、挿入又は付加されたアミノ酸配列を含むポリペプチド、及び
(3)上記(1)又は(2)のポリペプチドを発現し得る発現ベクター、
[2]以下の(1)~(3)のいずれかを含む、[1]のワクチン:
(1)配列番号:1に示されるアミノ酸配列、又は配列番号:8に示されるアミノ酸配列からなるポリペプチド;
(2)配列番号:11に示されるアミノ酸配列、又は配列番号:12に示されるアミノ酸配列からなるポリペプチド;及び
(3)上記(1)又は(2)のポリペプチドを発現し得る発現ベクター、
[3]発現ベクターが、B型肝炎ウイルスコア(HBc)をコードするヌクレオチド配列を含む、[1]又は[2]のワクチン、
[4]発現ベクターが、該(1)又は(2)のポリペプチドをコードするヌクレオチド配列が配列番号:17で示されるヌクレオチド配列の塩基番号:246と塩基番号:247の間に挿入されたヌクレオチド配列を含む、[1]又は[2]のワクチン、
[5]キャリアタンパク質および/又はアジュバントを含む、[1]-[4]のワクチン、
[5-1][1]-[5]のいずれかのワクチンを含む組成物、
[6]IL-17Aが増悪因子として関与する疾患が、SLE、炎症性腸疾患、関節リウマチ、腫瘍、乾癬、及び多発性硬化症からなる群から選択される、[1]-[5]のワクチン、
[7]以下の(1)又は(2)を含む、IL-17Aが増悪因子として関与する疾患が、SLE、炎症性腸疾患、関節リウマチ、大腸癌、及び肺癌からなる群から選択される、[1]-[6]のワクチン:
(1)配列番号:1に示されるアミノ酸配列からなるポリペプチド、及び
(2)上記(1)のポリペプチドを発現し得る発現ベクター、
[8]以下の(1)~(3)のいずれかを含む、IL-17Aが増悪因子として関与する疾患が、関節リウマチである、[1]-[6]のワクチン:
(1)配列番号:1に示されるアミノ酸配列からなるポリペプチド;
(2)配列番号:11に示されるアミノ酸配列又は配列番号:12に示されるアミノ酸配列からなるポリペプチド;及び
(3)上記(1)又は(2)のポリペプチドを発現し得る発現ベクター、
[9]以下の(1)又は(2)のポリペプチドを認識し、IL-17Aの機能を阻害する抗体を含む、IL-17Aが増悪因子として関与する疾患の予防または治療剤:
(1)配列番号:1に示されるアミノ酸配列、配列番号:8に示されるアミノ酸配列、配列番号:1に対応する非ヒト哺乳動物由来のアミノ酸配列、又は配列番号:8に対応する非ヒト哺乳動物由来のアミノ酸配列を含むポリペプチド、
(2)配列番号:1に示されるアミノ酸配列、配列番号:8に示されるアミノ酸配列、配列番号:1に対応する非ヒト哺乳動物由来のアミノ酸配列、又は配列番号:8に対応する非ヒト哺乳動物由来のアミノ酸配列において、1又は数個のアミノ酸残基が置換、欠失、挿入又は付加されたアミノ酸配列を含むポリペプチド、
[10]以下の(1)又は(2)のポリペプチドを認識し、IL-17Aの機能を阻害する抗体を含む、[9]の予防または治療剤:
(1)配列番号:1に示されるアミノ酸配列、又は配列番号:8に示されるアミノ酸配列からなるポリペプチド;
(2)配列番号:11に示されるアミノ酸配列、又は配列番号:12に示されるアミノ酸配列からなるポリペプチド、
[10-1]IL-17Aが増悪因子として関与する疾患が、SLE、炎症性腸疾患、乾癬、関節リウマチ、多発性硬化症、脳脊髄炎、腫瘍(非小細胞性肺癌、大腸癌、血球系腫瘍などを含むIL-17Aが増悪に関与する種々の腫瘍性疾患)、動脈硬化症、慢性炎症性疾患、及び、アレルギー疾患(遅延型過敏症、接触型過敏症)からなる群から選択される、[9]又は[10]の予防または治療剤、
[11]IL-17Aが増悪因子として関与する疾患が、SLE、炎症性腸疾患、関節リウマチ、腫瘍、乾癬、及び多発性硬化症からなる群から選択される、[9]又は[10]の予防または治療剤、
[11-1]IL-17Aが憎悪因子として関与する疾患がSLEである、[9]又は[10]の予防または治療剤、
[12]IL-17Aが増悪因子として関与する疾患が、SLE、炎症性腸疾患、関節リウマチ、大腸癌、及び肺癌からなる群から選択される、配列番号:1に示されるアミノ酸配列からなるポリペプチドを認識し、IL-17Aの機能を阻害する抗体を含む、[9]-[11]の予防または治療剤、
[13] IL-17Aが増悪因子として関与する疾患が、関節リウマチであり、配列番号:1に示されるアミノ酸配列、配列番号:11に示されるアミノ酸配列又は配列番号:12に示されるアミノ酸配列からなるポリペプチドを含む、[9]-[11]の予防または治療剤、
[14]配列番号:1に示されるアミノ酸配列からなるポリペプチド、
[14-1][14]をコードするポリヌクレオチド、
を提供する。
本発明のIL-17Aに対するワクチン(エピトープワクチンともいう)は、以下の(1)~(3)からなる群より選択される。
(1)配列番号:1に示されるアミノ酸配列、配列番号:8に示されるアミノ酸配列、配列番号:1に対応する非ヒト哺乳動物由来のアミノ酸配列、又は配列番号:8に対応する非ヒト哺乳動物由来のアミノ酸配列を含むポリペプチド、
(2)配列番号:1に示されるアミノ酸配列、配列番号:8に示されるアミノ酸配列、配列番号:1に対応する非ヒト哺乳動物由来のアミノ酸配列、又は配列番号:8に対応する非ヒト哺乳動物由来のアミノ酸配列において、1又は数個のアミノ酸残基が置換、欠失、挿入又は付加されたアミノ酸配列を含むポリペプチド、
(3)上記(1)又は(2)のポリペプチドを発現し得る発現ベクター。
その中でも、本発明のIL-17Aに対するワクチンは、好ましくは、以下の(1’)~(3’)からなる群より選択される。
(1’)配列番号:1に示されるアミノ酸配列、又は配列番号:8に示されるアミノ酸配列からなるポリペプチド、
(2’)配列番号:11に示されるアミノ酸配列、又は配列番号:12に示されるアミノ酸配列からなるポリペプチド、
(3’)上記(1’)又は(2’)のポリペプチドを発現し得る発現ベクター。
最も好ましい本発明のIL-17Aに対するワクチンは、以下の(1’’)または(2’’)からなる群より選択される。
(1’’)配列番号:1に示されるアミノ酸配列、配列番号:11に示されるアミノ酸配列、又は配列番号:12に示されるアミノ酸配列からなるポリペプチド、
(2’’)上記(1’’)のポリペプチドを発現し得る発現ベクター。
本発明のワクチンが投与される場合、該ワクチンに含まれる物質は、投与対象に由来する物質(すなわち、ヒトに投与する場合、該ワクチンはヒト由来の物質であり、マウスに投与する場合、該ワクチンはマウス由来の物質である)であることが好ましい。
本発明では、ヒトIL-17Aの62~69番目のアミノ酸配列を配列番号:1とし、それに対応するマウスIL-17Aの65~72番目のアミノ酸配列を配列番号:5とする。これらはそれぞれ、例えば、配列番号:2、配列番号:6で表されるヌクレオチド配列によりコードされる。また、ヒトIL-17Aの102~118番目のアミノ酸配列を配列番号:8とし、それに対応するマウスIL-17Aの105~121番目のアミノ酸配列を配列番号:10とする。これらはそれぞれ、例えば、配列番号:13、配列番号:14で表されるヌクレオチド配列によりコードされる。
配列番号:1(配列番号:8)に対応する非ヒト哺乳動物由来のアミノ酸配列としては、本明細書中の配列番号:1(配列番号:8)に開示された配列情報、公知の配列データベース等を利用して、適切なプライマーやプローブを設計し、RT-PCR又はプラークハイブリダイゼーション等、通常の遺伝子工学的手法を用いて容易に取得することができる。
このようなポリペプチドとしては、マウスの場合、配列番号:5(配列番号:10)で表されるアミノ酸配列において1又は数個(好ましくは1~数(2~5)個)のアミノ酸が欠失、置換、挿入又は付加されたアミノ酸配列も含まれる。該アミノ酸配列としては、例えば、(1)配列番号:5(配列番号:10)に示されるアミノ酸配列中の1又は数個(好ましくは1~数(2~5)個)のアミノ酸が欠失したアミノ酸配列、(2)配列番号:5(配列番号:10)に示されるアミノ酸配列に1又は数個(好ましくは1~数(2~5)個)のアミノ酸が付加されたアミノ酸配列、(3)配列番号:5(配列番号:10)に示されるアミノ酸配列に1又は数個(好ましくは1~数(2~5)個)のアミノ酸が挿入されたアミノ酸配列、(4)配列番号:5(配列番号:10)に示されるアミノ酸配列中の1又は数個(好ましくは1~数(2~5)個)のアミノ酸が他のアミノ酸で置換されたアミノ酸配列、又は(5)上記(1)~(4)の変異が組み合わせられたアミノ酸配列(この場合、変異したアミノ酸の総和が、1又は数個(好ましくは1~数(2~5)個))が含まれる。
本発明のワクチンの投与量は、投与する対象、投与方法、投与形態等によって異なるが、有効成分が上記(1)又は(2)のポリペプチドの場合は、通常成人1人当たりポリペプチドを、一回当たり1μg~1000μgの範囲、好ましくは20μg~100μgの範囲で、通常4週間から12週間に亘って、2回から3回投与し、抗体価が低下した場合にはその都度1回追加投与する。有効成分が上記(3)の発現ベクターの場合は、通常成人1人当たり発現ベクターを、一回当たり1μg~1000μgの範囲、好ましくは20μg~100μgの範囲で、通常4週間から12週間に亘って、2回から3回投与し、抗体価が低下した場合にはその都度1回追加投与する。
本発明は、以下の(1)又は(2)のポリペプチドを認識し、IL-17Aの機能を阻害する抗体を含む、IL-17Aが増悪因子として関連する疾患の予防又は治療剤(本発明の予防又は治療剤)を提供する。
(1)配列番号:1に示されるアミノ酸配列、配列番号:8に示されるアミノ酸配列、配列番号:1に対応する非ヒト哺乳動物由来のアミノ酸配列、又は配列番号:8に対応する非ヒト哺乳動物由来のアミノ酸配列を含むポリペプチド;
(2)配列番号:1に示されるアミノ酸配列、配列番号:8に示されるアミノ酸配列、配列番号:1に対応する非ヒト哺乳動物由来のアミノ酸配列、又は配列番号:8に対応する非ヒト哺乳動物由来のアミノ酸配列において、1又は数個のアミノ酸残基が置換、欠失、挿入又は付加されたアミノ酸配列を含むポリペプチド。
また、発明の予防又は治療剤は、好ましくは、以下の(1’)又は(2’)のポリペプチドを認識し、IL-17Aの機能を阻害する抗体を含む。
(1’)配列番号:1に示されるアミノ酸配列、または配列番号:8に示されるアミノ酸配列からなるポリペプチド;
(2’)配列番号:11に示されるアミノ酸配列、または配列番号:12に示されるアミノ酸配列からなるポリペプチド。
最も好ましい発明の予防又は治療剤は、(1’’)配列番号:1に示されるアミノ酸配列からなるポリペプチドを認識し、IL-17Aの機能を阻害する抗体を含む。
上述のIL-17Aが憎悪因子として関連する疾患の中でも、SLE、炎症性腸疾患、関節リウマチ、肺癌、大腸癌、乾癬、多発性硬化症、特に、SLE、炎症性腸疾患、関節リウマチ、肺癌、大腸癌の予防及び/又は治療に本発明のIL-17A中和抗体を用いることができる。
ポリクローナル抗体又はモノクローナル抗体は、自体公知の方法によって製造することができる。すなわち、免疫原(本発明のポリペプチド)を、必要に応じてフロイントアジュバント(Freund’s Adjuvant)と共に、哺乳動物、例えば、ポリクローナル抗体の場合、マウス、ラット、ハムスター、モルモット、ウサギ、ネコ、イヌ、ブタ、ヤギ、ウマ又はウシ等、好ましくはマウス、ラット、ハムスター、モルモット、ヤギ、ウマ又はウサギに免疫する。モノクローナル抗体の場合は、同様の方法で、マウス、ラット、ハムスター等に免疫する。
DNAワクチンの作製
DNAワクチンベクター
B型肝炎ウイルスコア抗原(HBc)を単離するため、BCCM/LMBP Plasmid Collectionよりplasmid pPLc3(Accession number LMBP 2470)を購入した。以下のプライマーを設計・合成した(国際公開第2012/141280号)。
HBcF 5’-gcc atg gat atc gat cct tat aaa gaa ttc gga gc-3’(配列番号3)
HBcR 5’-ggc ctc tca cta aca ttg aga ttc ccg aga ttg aga-3’(配列番号4)
上記のプライマーセットを使用して、PCRによりHBcを増幅した。
pcDNA 3.1/V5-His TOPO TA Expression Kit(Invitrogen)に前記で得られたHBcをクローニングした。MutagenesisによりpcDNA3.1-HBcベクターに、以下のマウスIL-17Aの2種類のエピトープA1又はエピトープA2をコードする核酸配列をそれぞれ挿入した。以下の実施例では、特に言及しない限り、マウスIL-17A1エピトープをコードする核酸配列を挿入したpcDNA3.1-HBcベクターを含むワクチンをIL-17A1 DNAワクチン、マウスIL-17A2エピトープをコードする核酸配列を挿入したpcDNA3.1-HBcベクターを含むワクチンをIL-17A2 DNAワクチンと表記する。ベクターの構造を図1に示す。
マウスIL-17A1エピトープ RPSDYLNR(配列番号:5)
マウスIL-17A2エピトープ DHHMNSV(配列番号:7)
DNAワクチンのマウスへの投与
6週齢の下記各雄マウスの大腿筋肉にDNAワクチンをシリンジで注射し、その部位にエレクトロポレーションを施行することで投与した。2週間毎に3回投与した(120μg/60μl×1カ所/回)。
実験1:抗体価測定、血中・尿中各種サイトカイン測定、生存率解析
NZBWF1マウス(SLE疾患モデル)
HBc-IL-17A1 (IL-17A1 DNAワクチン)群:6匹
HBc (pcDNA3.1-HBcベクター)群:6匹
Saline群:10匹
毎週体重を測定し、4週間ごとに血清採取を行った。
投与プランを図4Aに示す。
MRL/lprマウス(SLE疾患モデル)
HBc-IL-17A1(IL-17A1 DNAワクチン)群:9匹
Saline群:9匹
毎週体重を測定し、4週間ごとに血清採取を行った。
実験2:抗体価測定、各臓器の解析
MRL/lprマウス
HBc-IL-17A1(IL-17A1 DNAワクチン)群:6匹
Saline群:6匹
毎週体重を測定し、4週間ごとに血清採取を行った。
ELISAによる抗IL-17A抗体価測定
プレートの作製:マウスIL-17A1エピトープ+BSAコンジュゲート、マウスIL-17A2エピトープ+BSAコンジュゲートを10μg/mlの濃度で96ウェルプレートに分注し、4℃で1晩静置した。組み換えマウスIL-17Aを0.25μg/mlの濃度で96ウェルプレートに分注し、4℃で1晩静置した。
前記プレートをPBS 200μlで1回洗浄後、5%スキムミルクin PBSで2時間ブロッキングを行った。1次抗体(マウス抗血清)を5%スキムミルクin PBSで段階希釈し、50μlずつ96ウェルプレートにアプライ、4℃で1晩インキュベートした。
前記プレートをPBS-T(0.05% Tween) 200μlで7回洗浄し、2次抗体(抗マウスIgG Ab-HRP標識)を1/1000希釈(5%スキムミルク)し、50μlずつ添加、常温で3時間インキュベートした。該プレートをPBS-T(0.05% Tween) 200μlで3回洗浄し、TMB溶液を50μl添加、遮光して常温で30分間インキュベートした。0.5N H2SO450μlを添加し反応停止させ、450nmの吸光度を測定した。
Balb/cマウスをIL-17A1 DNAワクチンまたはIL-17A2 DNAワクチンで免疫した予備実験では、IL-17A1 DNAワクチンを投与したBalb/cマウスの抗体価の上昇が認められた(図3)。そして、図4Aに示す投与プランでNZBWF1マウスにIL-17A1 DNAワクチンを投与した結果、第6週で抗体価の上昇が認められ(図4B)、組み換えマウスIL-17Aを正しく認識する抗体が産生されたことを確認した(図5)。しかも、抗IL-17A1抗体価は長時間持続することを確認した(図7B)。
常法に従い、各レーンに蛋白質をアプライして電気泳動を行った。泳動後、メンブレンにトランスファーし、ブロッキング後、各1次抗体を添加して4℃で1晩インキュベートした。続いて2次抗体を反応させ、発色反応により抗体の結合を検出した。結果を図2に示す。
レーン1:組み換えマウスIL-17A
レーン2:マウスIL-17A1エピトープ+BSAコンジュゲート
レーン3:マウスIL-17A2エピトープ+BSAコンジュゲート
1次抗体
A:IL-17A1 DNAワクチン投与したBalb/cマウスの抗血清
B:市販の抗マウスIL-17A抗体
C:市販の抗BSA抗体
IL-17A1 DNAワクチン投与したマウスの抗血清は、マウスIL-17A1エピトープ+BSAコンジュゲートを特異的に認識し、マウスIL-17A2エピトープ+BSAコンジュゲートとは反応しなかった。
なお、組み換えマウスIL-17Aには、安定化のために大量のBSAが添加されており、市販の抗BSA抗体は、マウスIL-17A1エピトープ+BSAコンジュゲート、マウスIL-17A2エピトープ+BSAコンジュゲートとともにこれを認識していた。
血中IL-1β、TNF-α及びIL-17A濃度、尿中MCP-1濃度の測定
Quantikine ELISAキットを用いて、メーカーの指定するプロトコールに従いIL-1β、TNF-α、IL-17A及びMCP-1の濃度測定を行った。血清は25μlずつ使用した。尿は50μlずつ使用した。キットに添付の標準試料を用いて検量線を作製し、各サイトカイン濃度を定量した。NZBWF1マウスへのIL-17A1 DNAワクチン投与群で血中IL-1βの有意な減少が認められた(図6E)。血中IL-17A濃度、尿中MCP-1濃度、血中TNF-α濃度の減少傾向も認められた(図6C、B、F)。また、実験1に記載したMRL/lprマウスへのIL-17A1 DNAワクチン投与群でも、TNF-αの有意な減少が認められた(図6D、8A)。
麻酔したマウスから随時尿を採取した。尿マルチスティック試験紙を用いて、尿蛋白、尿クレアチニン、尿アルブミン、尿潜血、尿比重等を測定した。NZBWF1マウスへのIL-17A1 DNAワクチン投与群で尿タンパク質の減少傾向が認められた(図6A)。
SLEのモデルマウスであるNZBWF1マウスにIL-17A1 DNAワクチンを投与後、毎日観察し、マウスが死亡した日を記録した。死亡したマウスの数を1週間毎にまとめて、生存率のグラフを作成した。IL-17A1 DNAワクチン投与群の長期観察の結果、該ワクチン投与群の生存期間の有意な延長を認めた(図7A)。また、実験1に記載したMRL/lprマウスへのDNAワクチン投与群でも、寿命の延長傾向が認められた(図8B)。
実験2に記載したMRL/lprマウスにIL-17A1 DNAワクチンを投与後、臓器の重量変化を調べた。HBc-IL-17A1群及びSaline群の2群間で、体重に有意差は無かった(図9A)。対照として、全体重当たりの心臓重量を測定したが、有意差は無かった(図9B)。一方、IL-17A1 DNAワクチンを投与後の脾臓重量の有意な減少が認められた(図9C)。そして、全体重当たりの脾臓重量の割合も有意な減少が認められた(図9D)。SLE患者では脾腫がみられ、これらのモデルマウスにおいて全体重当たりの脾臓重量の増加はSLEの増悪を反映する。そのため本結果より、IL-17A1 DNAワクチンがSLE治療効果を有していることが明らかとなった。
生存率はKaplan-Meier法により統計処理を行った。
実験3
NZBWF1マウス
HBc-IL-17A1(IL-17A1 DNAワクチン)群:9匹
Saline群:9匹
毎週体重を測定し、4週間ごとに血清採取を行った。
MRL/lprマウス
HBc-IL-17A1(IL-17A1 DNAワクチン)群:9匹
Saline群:9匹
毎週体重を測定し、4週間ごとに血清採取を行った。
免疫組織染色解析では、摘出した各臓器を4%パラホルムアルデヒド中で24時間固定し、パラフィン中に埋包し、4μmの切片に切り出した。切片を1次抗体(抗F4/80抗体)および2次抗体(HRP標識抗ラットIgG抗体)で反応させた。スライドをヘマトキシリンで対比染色し、顕微鏡観察に用いた。組織検査アッセイでは、腎臓、顎下腺及び肝臓を解剖し、4%パラホルムアルデヒド中で一晩固定し、パラフィン中に埋包した。腎臓の4μm切片をPAS染色で染色した。顎下腺および肝臓の4μm切片をHE染色で染色した。
PAS染色でNZBWF1マウス、又はMRL/lprマウスでの腎病変の程度を確認した。結果を図11A、Bに示す。ワクチン投与群で、糸球体、間質の破壊が抑制されていた。F4/80の免疫染色でマクロファージの浸潤の程度を確認した。結果を図11C、Dに示す。ワクチン投与群で糸球体周囲や間質へのマクロファージ浸潤の抑制が認められた。
HE染色でNZBWF1マウスでの顎下腺炎の程度を確認した。結果を図12に示す。ワクチン投与群で顎下腺炎の抑制が認められた。
ワクチンの安全性の確認のために肝臓の組織切片をHE染色し、観察した。結果を図13に示す。NZBWF1マウス(図13A)及びMRL/lprマウス(図13B)いずれのマウスにおいても、ワクチン投与群及びSaline投与群ともに、病的所見が無いことを確認した。
6週齢の下記各雄マウスにTNBS溶液(2mg/100μl/回)を毎週注腸し、大腿筋肉にIL-17A1 DNAワクチンをエレクトロポレーションを用いて2週間毎に3回投与した(120μg/60μl×1カ所/回)。第8週にマウスを犠死させた。投与プランを図14(A)に示す。
実験4
Balb/cマウス
Normal群(TNBS(-)):1匹
Vaccine群(HBc-IL-17A1群;TNBS(+)):3匹
Saline群(TNBS(+)):3匹
実験4に記載したBalb/cマウスにTNBS投与8週後のマウスの体重の変化を調べた。マウスの体重増加量は、TNBSによる大腸炎の誘導に伴い減少した(Saline群)が、この減少効果はワクチン群では見られなかった(図14(B))。
実験4に記載したBalb/cマウスにTNBS投与8週後のマウスの大腸の長さを調べた。大腸の長さは、TNBSによる大腸炎の誘導に伴い短くなった(Saline群)が、この効果はワクチン群で抑制された(図14(C))。
犠死させたマウスの大腸を解剖し、4%パラホルムアルデヒド中で一晩固定し、パラフィン中に埋包した。大腸の4μm切片をHE染色で染色し、組織学的検討を行った。Saline群で炎症細胞の浸潤などの病理所見が認められた(図14(D))。また、HE染色切片の病理学的所見をスコア化した。ワクチン群でH&Eスコアの低下を認めた(図14(E))。
6週齢の下記各雄マウスの大腿筋肉にマウスIL-17A1 DNAワクチンをエレクトロポレーションを用いて2週間毎に3回投与した(120μg/60μl×1カ所/回)。ワクチン初回投与の28日後にType IIコラーゲンとCFA(完全フロイントアジュバント)、ワクチン初回投与の42日後にType IIコラーゲンとIFA(不完全フロイントアジュバント)を投与することによって関節炎を誘導し、以後毎週3回関節炎の程度を観察し、スコア化した。投与プランを図15(A)に示す。
実験5
DBA/1マウス
Vaccine群(HBc-IL-17A1群):6匹
Saline群(生理食塩水群):6匹
実験5に記載したDBA/1マウスの関節炎の臨床スコアおよび臨床所見を調べた。ワクチン群は関節炎の発症と進行の抑制効果を有することが認められた(図15(B)、(C))。
6週齢の下記各雄マウスの大腿筋肉にマウスIL-17A1エピトープからなるペプチドを含むワクチン(KLH conjugated)を2週間毎に3回投与した(25μg/25μl +アジュバント25μl /回(アジュバントは、初回投与時はCFA、2、3回目はIFA))。ワクチン初回投与の5週後にマウス大腸癌細胞株CT26細胞(5×105細胞/Body)を接種し、以後毎週腫瘍体積(0.5×長径×短径×短径)を計測した。また、全マウスが死亡するまで観察を続け、生存期間を確認した。投与プランを図16(A)に示す。
実験6
Balb/cマウス
Vaccine群(IL-17A1-KLH群):6匹
Saline群(生理食塩水群):6匹
実験6に記載したBalb/cマウスにCT26細胞を接種8日および15日後のマウスの腫瘍体積を調べた。ワクチン群は腫瘍体積の増大抑制効果を有することが認められた(図16(B))。
ワクチンを投与したBalb/cマウスにCT26細胞を接種後、毎日観察し、マウスが死亡した日を記録した。ワクチン投与群の長期観察の結果、生存期間の有意な延長を認めた(図16(C))。
6週齢の下記各雄マウスの大腿筋肉にマウスIL-17A1 DNAワクチンをエレクトロポレーションを用いて2週間毎に3回投与した(120μg/60μl×1カ所/回)。ワクチン初回投与の5週後にマウス肺癌細胞株LLC細胞(5×105細胞/Body)を接種し、以後毎週腫瘍体積(0.5×長径×短径×短径)を計測した。第9週にマウスを犠死させた後、腫瘍の重量、肺転移、肝転移の有無を確認した。投与プランを図17(A)に示す。
実験7
C57 BL/6マウス
Vaccine群(HBc-IL-17A1群):5匹
Saline群(生理食塩水群):5匹
実験7に記載した犠死させた後のマウスの体重を調べた。マウスの体重は、両群間で有意差は認められなかった(図17(B))。
実験7に記載したC57 BL/6マウスにLLC細胞を接種後、毎週腫瘍体積を調べた。ワクチン群は腫瘍の増大を抑制する効果を有することが認められた(図17(C))。
実験7に記載した犠死させた後のマウスの腫瘍重量を調べた。ワクチン群は腫瘍重量の増大を抑制する効果を有することが認められた(図17(D))。
ワクチン群において、肺転移、肝転移を認めなかった。ワクチン非投与群においては肺転移が認められた(図17(E)、(F)、(G))。
腫瘍増大抑制効果および転移抑制効果を示した。
ELISAによる抗IL-17A抗体価測定
マウスIL-17A1エピトープ(RPSDYLNR(配列番号:5))に対応する以下のヒトIL-17A1エピトープからなるペプチドを含むワクチン(KLH conjugated)を作製し、Balb/cマウスに2週間隔で3回皮内投与し、初回投与の6週後に実施例1に記載の方法に従って血清抗体価を測定した。
ヒトIL-17A1エピトープ RSSDYYNR(配列番号:1)
その結果、ワクチンを投与したBalb/cマウスの抗体価の上昇が認められた(図18)。また、前記血清は、マウスIL-17A1エピトープに対しても交差反応性を示した(図19(A))。さらに、マウスIL-17A1エピトープからなるペプチドを含むワクチンを投与することによって得られた血清も、ヒトIL-17A1エピトープに対して交差反応性を示した(図19(B))。
ヒトIL-17A2エピトープ ADGNVDYHMNSVPIQQE(配列番号:8)
ヒトIL-17A3エピトープ LRREPPHCPNSFRL(配列番号:9)
その結果、ヒトIL-17A2エピトープからなるペプチドを含むワクチンを投与したBalb/cマウスの抗体価の上昇が認められた(図20)。
ヒトIL-17A4エピトープ SDYYN(配列番号:11)
ヒトIL-17A5エピトープ SDYY(配列番号:12)
ヒトIL-17A6エピトープ SDY
ヒトIL-17A7エピトープ DYY
その結果、ヒトIL-17A4エピトープからなるペプチドを含むワクチン、ヒトIL-17A5エピトープ、ヒトIL-17A6エピトープまたはヒトIL-17A7エピトープをコードするDNA含むワクチンを投与したBalb/cマウスの抗体価の上昇が認められた(図21)。
6週齢の下記各雄マウスの大腿筋肉にヒトIL-17A1エピトープ、ヒトIL-17A2エピトープまたはヒトIL-17A4エピトープからなるペプチドを含むワクチン(KLH conjugated)を2週間毎に3回投与した(120μg/60μl×1カ所/回)。ワクチン初回投与の28日後にType IIコラーゲンとCFA(完全フロイントアジュバント)、ワクチン初回投与の42日後にType IIコラーゲンとIFA(不完全フロイントアジュバント)を投与することによって関節炎を誘導し、以後毎週3回関節炎の程度を観察し、スコア化した。投与プランを図22(A)に示す。
実験8
DBA/1マウス
H17(ヒトIL-17A1エピトープ)群:3匹
AF4(ヒトIL-17A4エピトープ)群:3匹
AF5(ヒトIL-17A2エピトープ)群:3匹
Control(KLH)群:4匹
Collagen(-)群:3匹
実験8に記載したDBA/1マウスの関節炎の臨床スコアを調べた。ヒトIL-17A1エピトープ、ヒトIL-17A2エピトープまたはヒトIL-17A4エピトープからなるワクチン群は関節炎の発症と進行の抑制効果を有することが認められた(図22(B))。
培地中の正常ヒト皮膚線維芽細胞(NHDF)に組み換えヒトIL-17Aを添加した時のIL-6の分泌量をELISAで測定した結果、IL-17Aの添加によってIL-16の分泌が促進された(IL-17 only)。しかし、ヒトIL-17A1エピトープからなるペプチドを含むワクチンで免疫したマウスの抗血清から精製したIgGをIL-17Aと同時に添加した場合、IL-6の分泌が抑制された(IL-17 + antisera)。また、非免疫マウスの抗血清から精製したIgGをIL-17Aと同時に添加した場合、IL-6の分泌はほとんど抑制されなかった(IL-17 + control sera)。これらの結果を、図23に示す。
本出願は、日本で出願された特願2013-273133(出願日:2013年12月27日)を基礎としており、その内容はすべて本明細書に包含されるものとする。
Claims (13)
- 以下の(1)~(3)のいずれかを含む、IL-17Aが増悪因子として関与する疾患の予防または治療用ワクチン:
(1)配列番号:1に示されるアミノ酸配列、配列番号:8に示されるアミノ酸配列、配列番号:1に対応する非ヒト哺乳動物由来のアミノ酸配列、又は配列番号:8に対応する非ヒト哺乳動物由来のアミノ酸配列を含むポリペプチド;
(2)配列番号:1に示されるアミノ酸配列、配列番号:8に示されるアミノ酸配列、配列番号:1に対応する非ヒト哺乳動物由来のアミノ酸配列、又は配列番号:8に対応する非ヒト哺乳動物由来のアミノ酸配列において、1又は数個のアミノ酸残基が置換、欠失、挿入又は付加されたアミノ酸配列を含むポリペプチド;及び
(3)上記(1)又は(2)のポリペプチドを発現し得る発現ベクター。 - 以下の(1)~(3)のいずれかを含む、請求項1に記載のワクチン:
(1)配列番号:1に示されるアミノ酸配列、又は配列番号:8に示されるアミノ酸配列からなるポリペプチド;
(2)配列番号:11に示されるアミノ酸配列、又は配列番号:12に示されるアミノ酸配列からなるポリペプチド;及び
(3)上記(1)又は(2)のポリペプチドを発現し得る発現ベクター。 - 発現ベクターが、B型肝炎ウイルスコア(HBc)をコードするヌクレオチド配列を含む、請求項1または2に記載のワクチン。
- 発現ベクターが、該(1)又は(2)のポリペプチドをコードするヌクレオチド配列が配列番号:17で示されるヌクレオチド配列の塩基番号:246と塩基番号:247の間に挿入されたヌクレオチド配列を含む、請求項1または2に記載のワクチン。
- キャリアタンパク質および/又はアジュバントを含む、請求項1-4のいずれか1項に記載のワクチン。
- IL-17Aが増悪因子として関与する疾患が、SLE、炎症性腸疾患、関節リウマチ、腫瘍、乾癬、及び多発性硬化症からなる群から選択される、請求項1-5のいずれか1項に記載のワクチン。
- 以下の(1)または(2)を含む、IL-17Aが増悪因子として関与する疾患が、SLE、炎症性腸疾患、関節リウマチ、大腸癌、及び肺癌からなる群から選択される、請求項1-6のいずれか1項に記載のワクチン:
(1)配列番号:1に示されるアミノ酸配列からなるポリペプチド;及び
(2)上記(1)のポリペプチドを発現し得る発現ベクター。 - 以下の(1)~(3)のいずれかを含む、IL-17Aが増悪因子として関与する疾患が、関節リウマチである、請求項1-6のいずれか1項に記載のワクチン:
(1)配列番号:1に示されるアミノ酸配列からなるポリペプチド;
(2)配列番号:11に示されるアミノ酸配列又は配列番号:12に示されるアミノ酸配列からなるポリペプチド;及び
(3)上記(1)又は(2)のポリペプチドを発現し得る発現ベクター。 - 以下の(1)又は(2)のポリペプチドを認識し、IL-17Aの機能を阻害する抗体を含む、IL-17Aが増悪因子として関与する疾患の予防または治療剤:
(1)配列番号:1に示されるアミノ酸配列、配列番号:8に示されるアミノ酸配列、配列番号:1に対応する非ヒト哺乳動物由来のアミノ酸配列、又は配列番号:8に対応する非ヒト哺乳動物由来のアミノ酸配列を含むポリペプチド;
(2)配列番号:1に示されるアミノ酸配列、配列番号:8に示されるアミノ酸配列、配列番号:1に対応する非ヒト哺乳動物由来のアミノ酸配列、又は配列番号:8に対応する非ヒト哺乳動物由来のアミノ酸配列において、1又は数個のアミノ酸残基が置換、欠失、挿入又は付加されたアミノ酸配列を含むポリペプチド。 - 以下の(1)又は(2)のポリペプチドを認識し、IL-17Aの機能を阻害する抗体を含む、請求項9に記載の予防または治療剤:
(1)配列番号:1に示されるアミノ酸配列、又は配列番号:8に示されるアミノ酸配列からなるポリペプチド;
(2)配列番号:11に示されるアミノ酸配列、又は配列番号:12に示されるアミノ酸配列からなるポリペプチド。 - IL-17Aが増悪因子として関与する疾患が、SLE、炎症性腸疾患、関節リウマチ、腫瘍、乾癬、及び多発性硬化症からなる群から選択される、請求項9または10に記載の予防または治療剤。
- IL-17Aが増悪因子として関与する疾患が、SLE、炎症性腸疾患、関節リウマチ、大腸癌、及び肺癌からなる群から選択される、配列番号:1に示されるアミノ酸配列からなるポリペプチドを認識し、IL-17Aの機能を阻害する抗体を含む、請求項9-11のいずれか1項に記載の予防または治療剤。
- IL-17Aが増悪因子として関与する疾患が、関節リウマチであり、配列番号:1に示されるアミノ酸配列、配列番号:11に示されるアミノ酸配列又は配列番号:12に示されるアミノ酸配列からなるポリペプチドを認識し、IL-17Aの機能を阻害する抗体を含む、請求項9-11のいずれか1項に記載の予防または治療剤。
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AU2014370875A AU2014370875B2 (en) | 2013-12-27 | 2014-12-26 | Vaccine targeting IL-17A |
CA2935046A CA2935046C (en) | 2013-12-27 | 2014-12-26 | Vaccine targeting il-17a |
US15/108,415 US11421025B2 (en) | 2013-12-27 | 2014-12-26 | Treatment of IL-17A diseases |
JP2015555069A JP6164593B2 (ja) | 2013-12-27 | 2014-12-26 | Il−17aを標的とするワクチン |
CN201480075747.1A CN106132433B (zh) | 2013-12-27 | 2014-12-26 | 以il-17a作为靶标的疫苗 |
EP14873679.6A EP3088000A4 (en) | 2013-12-27 | 2014-12-26 | Vaccine targeting il-17a |
KR1020167020472A KR101836756B1 (ko) | 2013-12-27 | 2014-12-26 | Il-17a를 표적화하는 백신 |
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Citations (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS62296890A (ja) | 1986-03-27 | 1987-12-24 | メディカル リサーチ カウンスル | 組換えdna生産物及び製造法 |
JPH0373280B2 (ja) | 1984-08-15 | 1991-11-21 | Shingijutsu Kaihatsu Jigyodan | |
JPH04504365A (ja) | 1990-01-12 | 1992-08-06 | アブジェニックス インコーポレイテッド | 異種抗体の生成 |
JPH04506458A (ja) | 1989-12-21 | 1992-11-12 | セルテック リミテッド | Cd3特異的組換え抗体 |
JPH06500233A (ja) | 1990-08-29 | 1994-01-13 | ジェンファーム インターナショナル,インコーポレイティド | 異種免疫グロブリンを作る方法及びトランスジェニックマウス |
WO1994025585A1 (en) | 1993-04-26 | 1994-11-10 | Genpharm International, Inc. | Transgenic non-human animals capable of producing heterologous antibodies |
WO2001062907A1 (en) | 2000-02-22 | 2001-08-30 | Medical & Biological Laboratories Co., Ltd. | Antibody library |
WO2012045848A1 (en) * | 2010-10-08 | 2012-04-12 | Novartis Ag | Methods of treating psoriasis using il-17 antagonists |
WO2012141280A1 (ja) | 2011-04-15 | 2012-10-18 | 国立大学法人 大阪大学 | Dnaワクチン |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20070160576A1 (en) * | 2001-06-05 | 2007-07-12 | Genentech, Inc. | IL-17A/F heterologous polypeptides and therapeutic uses thereof |
EP3366702B1 (en) | 2005-12-13 | 2023-08-09 | Eli Lilly And Company | Anti-il-17 antibodies |
BRPI0708902A2 (pt) | 2006-03-16 | 2011-06-14 | Genentech Inc | mÉtodos de tratar lupus usando anticorpos cd4 |
KR102468907B1 (ko) | 2016-03-25 | 2022-11-18 | 고꾸리쯔 다이가꾸 호우징 오사까 다이가꾸 | 질환의 요인이 되는 생체내 단백질을 표적으로 하는 컨쥬게이트 백신 |
-
2014
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- 2014-12-26 KR KR1020167020472A patent/KR101836756B1/ko active IP Right Grant
- 2014-12-26 JP JP2015555069A patent/JP6164593B2/ja active Active
- 2014-12-26 EP EP14873679.6A patent/EP3088000A4/en active Pending
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- 2014-12-26 AU AU2014370875A patent/AU2014370875B2/en active Active
- 2014-12-26 US US15/108,415 patent/US11421025B2/en active Active
Patent Citations (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH0373280B2 (ja) | 1984-08-15 | 1991-11-21 | Shingijutsu Kaihatsu Jigyodan | |
JPS62296890A (ja) | 1986-03-27 | 1987-12-24 | メディカル リサーチ カウンスル | 組換えdna生産物及び製造法 |
JPH04506458A (ja) | 1989-12-21 | 1992-11-12 | セルテック リミテッド | Cd3特異的組換え抗体 |
JPH04504365A (ja) | 1990-01-12 | 1992-08-06 | アブジェニックス インコーポレイテッド | 異種抗体の生成 |
JPH06500233A (ja) | 1990-08-29 | 1994-01-13 | ジェンファーム インターナショナル,インコーポレイティド | 異種免疫グロブリンを作る方法及びトランスジェニックマウス |
WO1994025585A1 (en) | 1993-04-26 | 1994-11-10 | Genpharm International, Inc. | Transgenic non-human animals capable of producing heterologous antibodies |
WO2001062907A1 (en) | 2000-02-22 | 2001-08-30 | Medical & Biological Laboratories Co., Ltd. | Antibody library |
WO2012045848A1 (en) * | 2010-10-08 | 2012-04-12 | Novartis Ag | Methods of treating psoriasis using il-17 antagonists |
WO2012141280A1 (ja) | 2011-04-15 | 2012-10-18 | 国立大学法人 大阪大学 | Dnaワクチン |
Non-Patent Citations (18)
Title |
---|
CURR. PROTEIN PEPT. SCI., vol. 1, no. 2, 2000, pages 155 - 169 |
EUR J IMMUNOLOGY, vol. 36, no. 11, 2006, pages 2857 - 2867 |
EUR J IMMUNOLOGY, vol. 42, no. 9, 2012, pages 2274 - 2284 |
EXP. OPIN. THER. PATENTS, vol. 6, no. 5, 1996, pages 441 - 456 |
GUAN Q. ET AL.: "An IL -17 peptide-based and virus-like particle vaccine enhances the bioactivity of IL -17 in vitro and in vivo", IMMUNOTHERAPY, vol. 4, no. 12, 2012, pages 1799 - 1807, XP008183808 * |
IMMUNOTHERAPY, vol. 4, no. 12, 2012, pages 1799 - 1807 |
J DERMATOL, vol. 41, 2014, pages 1039 - 1046 |
J IMMUNOLOGY, vol. 188, no. 8, 2012, pages 3567 - 3571 |
KOHLER; MILSTEIN ET AL., NATURE, vol. 256, 1975, pages 495 - 497 |
MA Y. ET AL.: "Targeting TGF-betal by employing a vaccine ameliorates fibrosis in a mouse model of chronic colitis", INFLAMM.BOWEL DIS., vol. 16, no. 6, 1 June 2010 (2010-06-01), pages 1040 - 1050, XP055354000, DOI: 10.1002/IBD.21167 * |
NATURE GENETICS, vol. 15, 1997, pages 146 - 156 |
NATURE GENETICS, vol. 7, 1994, pages 13 - 21 |
NATURE IMMUNOLOGY, vol. 10, no. 7, 2009, pages 778 - 785 |
NATURE, vol. 348, 1990, pages 552 - 554 |
NATURE, vol. 368, 1994, pages 856 - 859 |
ROHN T.A. ET AL.: "VACCINATION AGAINST IL-17 SUPPRESSES AUTOIMMUNE ARTHRITIS AND ENCEPHALOMYELITIS", EUR. J.IMMUNOL., vol. 36, no. 11, 1 November 2006 (2006-11-01), pages 2857 - 2867, XP055162840 * |
SCIENCE, vol. 228, no. 4075, 1985, pages 1315 - 1317 |
See also references of EP3088000A4 |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2017164409A1 (ja) | 2016-03-25 | 2017-09-28 | 国立大学法人大阪大学 | 疾患の要因となる生体内タンパク質を標的とするコンジュゲートワクチン |
KR20180123064A (ko) | 2016-03-25 | 2018-11-14 | 고꾸리쯔 다이가꾸 호우징 오사까 다이가꾸 | 질환의 요인이 되는 생체내 단백질을 표적으로 하는 컨쥬게이트 백신 |
CN108883166A (zh) * | 2016-03-25 | 2018-11-23 | 国立大学法人大阪大学 | 以成为疾病主要原因的生物体内蛋白质为靶标的结合疫苗 |
US10980876B2 (en) | 2016-03-25 | 2021-04-20 | Osaka University | Conjugate vaccine targeting a disease-causing biological protein |
CN108883166B (zh) * | 2016-03-25 | 2023-06-02 | 国立大学法人大阪大学 | 以成为疾病主要原因的生物体内蛋白质为靶标的结合疫苗 |
EP4194007A1 (en) | 2016-03-25 | 2023-06-14 | Osaka University | Conjugate vaccine targeting disorder-causing in vivo protein |
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