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WO2017170494A1 - Vaccin contre l'obésité - Google Patents

Vaccin contre l'obésité Download PDF

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Publication number
WO2017170494A1
WO2017170494A1 PCT/JP2017/012551 JP2017012551W WO2017170494A1 WO 2017170494 A1 WO2017170494 A1 WO 2017170494A1 JP 2017012551 W JP2017012551 W JP 2017012551W WO 2017170494 A1 WO2017170494 A1 WO 2017170494A1
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WIPO (PCT)
Prior art keywords
ghrelin
seq
mice
pspa
vaccine
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English (en)
Japanese (ja)
Inventor
義和 幸
宏 清野
達彦 畔上
伊藤　裕
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Keio University
University of Tokyo NUC
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Keio University
University of Tokyo NUC
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Priority to JP2018508039A priority Critical patent/JPWO2017170494A1/ja
Publication of WO2017170494A1 publication Critical patent/WO2017170494A1/fr
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology

Definitions

  • the present invention relates to a vaccine preparation for the treatment of obesity.
  • Non-Patent Documents 1 to 3 In addition to the unsatisfactory weight loss effect of these drugs, anti-obesity drugs still remain fat / oil stool, fat-absorbed vitamin-absorbed and liver disorders due to orlistat (Non-Patent Documents 1 to 3), lorcaserin Has been reported to have potential and debatable risks such as carcinogenicity, psychiatric symptoms, valvular disease (Non-patent Document 3). Therefore, it is necessary to develop an effective and safe method for preventing or treating obesity.
  • Ghrelin consists of 28 amino acid residues, and the third serine is octanoylated, and has been identified as a ligand for growth hormone secretagogue receptor (GHSR) (Non-patent Document 5). Ghrelin is mainly produced and secreted by gastric A / X-like cells (Non-patent Document 6), but is also expressed in many tissues such as the intestine, pancreas and pituitary gland (Non-patent Document 7). Octanoylation of the third serine residue is promoted by ghrelin O-acyltransferase (GOAT) and is essential for the activation of its receptor, GHSR (Non-patent Documents 8 and 9).
  • GOAT ghrelin O-acyltransferase
  • Acylated ghrelin (AG, acyl-ghrelin) stimulates hunger and increases food intake through GHSR in afferent neurons of the gastric vagus nerve.
  • unacylated ghrelin (UAG, deacyl-ghrelin) does not affect food intake and GHSR-vagal activity (Non-Patent Document 10).
  • GHSR knockout mice showed a normal phenotype under standard diet conditions, but were resistant to diet-induced obesity (DIO) (Non-patent Document 11).
  • ghrelin is a rational for the treatment of obesity due to the presence of ghrelin in the bloodstream in the light of physiological functions in weight gain and the absence of serious symptoms even if the AG-GHSR signaling system is inhibited. It seems to be a safe target.
  • Treatment of obesity with a vaccine can be an effective treatment method for obesity from the viewpoint of long-term therapeutic effects, low-frequency drug administration, and economic advantages.
  • Vaccination targeting ghrelin has been attempted in rats (Non-Patent Document 12, Patent Document 1), pigs (Non-Patent Document 13) and mice (Non-Patent Documents 14 and 15). These are injectable peptide vaccines and required acylation of the third serine residue (Non-Patent Documents 12 to 15).
  • Systemic immunization of these vaccines suppressed weight gain in rats and pigs (Non-Patent Documents 12 and 13), but no effect on body weight was observed in DIO mice (Non-Patent Documents 14 and 13). 15).
  • Patent Document 2 In ob / ob mice, such as ob / ob mice, due to genetic abnormalities, mice that show progressive and refractory obesity even under standard diet are unclear as to whether weight gain is reduced (Example 5 of patent document 2 etc.). As described above, the development of a vaccine that effectively reduces the increase in body weight against obesity caused by various causes has still been an important solution in the art.
  • an object of the present invention is to provide a vaccine that effectively suppresses weight gain against obesity caused by various causes.
  • the present inventors prepared a vaccine preparation containing a fusion protein of a carrier protein (for example, PspA) and the full length of a plurality of ghrelin polypeptides, and conducted intensive studies on its effects.
  • the vaccine suppresses body weight gain not only in DIO (diet-induced obesity) mice but also in non-obese mice (non-fat mice) and ob / ob mice that develop obesity due to genetic mutations I found out.
  • the ob / ob mouse is suitable for examining the effect of a vaccine against severe obesity in that it exhibits a higher degree of obesity than a DIO mouse due to a genetic abnormality.
  • the present invention includes the following (1) to (15).
  • a vaccine preparation comprising a carrier protein and a fusion protein obtained by fusing the full length of one or a plurality of ghrelin polypeptides.
  • ghrelin polypeptide is any of the following (a) or (b): (A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 (b) one or several in the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 Consisting of an amino acid sequence in which one or several amino acids other than the third serine is substituted, and the third serine is octanoylated.
  • the fusion protein comprises an amino acid sequence represented by SEQ ID NO: 6 Formulation.
  • the anti-obesity vaccine preparation according to any one of (1) to (8) above, wherein the fusion protein forms a complex with cCHP nanogel.
  • ghrelin polypeptide is any of the following (a) or (b): (A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 (b) one or several in the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 Consisting of an amino acid sequence in which one or several amino acids other than the third serine is substituted, and the third serine is octanoylated.
  • the fusion protein according to any one of (10) to (12) above which comprises a polypeptide having a binding activity to GHSR (13) and comprising the amino acid sequence represented by SEQ ID NO: 6.
  • the present invention it is possible to provide a vaccine preparation capable of suppressing weight gain not only in diet-induced obesity but also in genetically-induced obesity and obesity associated with type 2 diabetes.
  • the vaccine according to the present invention significantly reduces the weight gain of ob / ob mice exhibiting obesity due to genetic abnormalities, and thus is effective not only for prevention but also for therapeutic purposes.
  • non-obese mice significantly reduce body weight gain, a therapeutic effect is expected even in an overweight state prior to obesity. Known to be a risk factor). It is also effective in improving glucose tolerance and insulin resistance.
  • the figure which showed the structure of the fusion protein of this invention typically.
  • A Serum IgG antibodies against AG and UAG were induced one week after the final immunization.
  • mice nasally immunized with ghrelin-PspA were fed a high fat diet for 5 weeks, fat accumulation was analyzed by CT scan, perirenal fat was collected, and adipocyte size was measured.
  • CT scan performed the 4th lumbar vertebra level CT, and analyzed the ratio of total fat, subcutaneous fat and visceral fat based on the acquired CT image. Immunized DIO mice had significantly less total fat, subcutaneous fat and visceral fat compared to control DIO mice.
  • B In visceral fat samples stained with HE, 5 consecutive fields were randomly selected and adipocyte size was measured (using a x100 objective). The size of adipocytes in immunized DIO mice was significantly smaller compared to control DIO mice.
  • RNA was prepared from perioscapular fat (brown adipose tissue; BAT), perirenal fat (white adipose tissue; WAT) and quadriceps muscle (skeletal muscle) of DIO mice 5 weeks after the fifth vaccination.
  • BAT perioscapular fat
  • WAT perirenal fat
  • WAT quadriceps muscle
  • DIO mice nasally immunized with ghrelin-PspA were subjected to OGTT (oral glucose tolerance test) and IPITT (intraperitoneal insulin tolerance test). In addition, fasting serum concentrations of LDL, FFA and TG were measured.
  • OGTT oral glucose tolerance test
  • IPITT intraperitoneal insulin tolerance test
  • IPITT mice were fasted for 6 hours, insulin (0.75 IU / 1 kg body weight) was injected intraperitoneally, and blood glucose level was measured.
  • the total amount of IgA in the nasal wash was significantly increased in the immunized mice. Values are shown as mean ⁇ SEM. ** p ⁇ 0.01.
  • the measurement result of AG specific serum IgG antibody before and after immunization of ghrelin-PspA Serum was collected before, during and after immunization of mice nasally immunized 5 times with ghrelin-PspA, and the specific IgG antibody titer against AG was measured by ELISA. The antibody titer increased after 3 times of immunization, reached a peak after 5 times of administration, and then gradually decreased. Values are shown as mean ⁇ SEM.
  • ghrelin-PspA Effect of ghrelin-PspA on weight gain after vaccination in non-obese mice. Results of weekly measurement of body weight of control and immunized mice. Non-obese mice were nasally immunized with ghrelin-PspA every week between 4 and 8 weeks of age, and changes in body weight were observed. Immunization with ghrelin-PspA reduced weight gain in the non-obese state. Values are shown as mean ⁇ SEM. * P ⁇ 0.05; ** p ⁇ 0.01. Effect of nasal immunity of ghrelin-PspA on endogenous ghrelin. RNA was prepared from the stomach of mice 4 weeks after the fifth vaccination.
  • RNA was reverse transcribed, and the resulting cDNA was quantitatively PCR amplified.
  • Relative gene expression data by real-time quantitative PCR was normalized to ⁇ -actin gene. Nasal administration of ghrelin-PspA did not significantly change the expression of ghrelin and GHSR in the stomach. Values are shown as mean ⁇ SEM.
  • Embodiments of the present invention include systemic and mucosal derived vaccine formulations comprising a fusion protein of the full length of one or more ghrelin polypeptides and a carrier protein that serves to enhance the immunogenicity of the ghrelin polypeptide ( Hereinafter, it is described as “the vaccine preparation of the present invention”).
  • Ghrelin consists of 28 amino acid residues, and an active form of ghrelin in which the third serine is octanoylated binds to a growth hormone secretagogue receptor (GHSR), promotes food intake, and reduces energy consumption.
  • GHSR growth hormone secretagogue receptor
  • ghrelin when used as a vaccine, it has been reported that the third serine uses acylated ghrelin such as octanoylation. However, acylation makes ghrelin unstable in vivo.
  • the total length of the ghrelin polypeptide in which the third serine is not acylated (polypeptide consisting of 28 residues) ) was used as an immunogen.
  • a ghrelin polypeptide consisting of 28 residues is sometimes simply referred to as “ghrelin”, but is synonymous with “ghrelin polypeptide” unless otherwise noted.
  • the number of ghrelin polypeptides to be bound to the carrier protein is not particularly limited, but is preferably 1 to 5, for example, and more preferably 2 to 4.
  • the ghrelin polypeptide used in the embodiment of the present invention can be derived from various animal species, and is not limited to, for example, human origin (SEQ ID NO: 1), mouse origin (SEQ ID NO: 2). In addition to dogs (SEQ ID NO: 3), those derived from animals such as rats, pigs, cows and sheep can be used.
  • polypeptide substantially identical to the polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 is represented by, for example, SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.
  • amino acid sequence in which one or several amino acids are deleted, inserted, added, and / or one or several amino acids of amino acid residues other than the third serine are substituted, and A polypeptide having a binding activity to GHSR when the third serine is acylated, such as octanoylated.
  • Carrier protein refers to a protein that functions to enhance the immunogenicity of ghrelin polypeptide.
  • mussel hemocyanin KLH
  • ovalbumin OVA
  • rabbit serum albumin RSA
  • bovine serum Albumin BSA
  • Thyroglobulin TG
  • immunoglobulin tetanus toxoid, diphtheria toxoid, diphtheria toxin mutant CRM197
  • pneumococcal surface antigen A PspA
  • pneumococcal surface antigen C PspC
  • pneumococcal histidine protein A PhpA
  • Hepatitis B virus surface antigens HBs
  • HBc hepatitis B virus core antigen
  • hepatitis E virus capsid protein eg ORF (open reading frame) 2 etc.
  • HIV-1 envelope glycoprotein eg gp41
  • Gp120 and gp160 hepatitis C virus envelope glycoprotein
  • PspA is pneumococcal surface protein A (pneumococcal surface protein A), and is classified into family 1-3 and clade 1-6, but in the embodiment of the present invention, it is classified into any group. Even PspA can be used.
  • the fusion protein used in the embodiment of the present invention is a carrier protein and a plurality of full-length ghrelin polypeptides linked (carrier protein and one or more).
  • a protein in which a linker peptide is optionally linked by a peptide bond eg, a carrier protein and one or more full-length ghrelin polypeptides, and optionally a gene encoding each linker peptide Protein expressed from the fusion gene).
  • Each protein or polypeptide may be linked directly or indirectly via a linker peptide or the like.
  • the carrier protein and ghrelin polypeptide may be arranged in any arrangement.
  • the carrier protein is arranged at the N-terminus or C-terminus, and a plurality of ghrelin polypeptides are continuously linked to form a carrier.
  • a configuration fused to a protein can be illustrated (see, eg, FIG. 1; the amino acid sequence is shown in SEQ ID NO: 4).
  • any linker peptide can be used as long as it is known in the art.
  • linker peptides that can be used in the embodiments of the present invention include, as flexible linkers, peptides that repeat a GGGGS (SEQ ID NO: 5) sequence several times (in the examples of the present invention, peptides that are repeated four times) and GPGP. More specifically, Chen et al., Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev. 2013; 65 (10): 1357-69 as listed in table 3 Things can be used.
  • the fusion protein of the present invention can be easily prepared by a known technique in this technical field.
  • carrier protein eg, diphtheria CRM 197 protein, recombinant hepatitis B vaccine protein (prepared in yeast), PspA, etc.
  • ghrelin gene information can be easily obtained from databases such as GenBank.
  • the reading frame of the nucleic acid sequence is adjusted so that the protein sequence and the nucleic acid sequence encoding the linker peptide can be linked and expressed. Based on the obtained nucleic acid sequence information, DNA encoding the fusion protein is synthesized.
  • the synthesized DNA is inserted into an appropriate expression vector, the protein is expressed and isolated and purified according to a conventional method, and the fusion protein of the present invention can be prepared.
  • the nucleic acid sequence of SEQ ID NO: 5 can be shown as the gene sequence of the fusion protein having the arrangement shown in FIG. 1, and when the DNA having this sequence is expressed, the fusion of the present invention comprising the amino acid sequence represented by SEQ ID NO: 6 Protein can be obtained.
  • Examples of the expression vector for expressing the fusion protein of the present invention include pBR322, pBR325, pUC118, pET and the like (E. coli host), pEGF-C, pEGF-N and the like (animal cell host), pVL1392, pVL1393 and the like ( Insect cell host), pG-1, Yep13, pPICZ, etc. (yeast cell host) can be used.
  • These expression vectors have a replication origin, a selectable marker, and a promoter suitable for each vector, and if necessary, an enhancer, a transcription assembly sequence (terminator), a ribosome binding site, a polyadenylation signal, and the like. You may have.
  • the cells When extracting the expressed polypeptide from cultured cells or cells, the cells are collected by a known method after culturing, suspended in an appropriate buffer, and subjected to ultrasound, lysozyme and / or freeze-thawing. After destroying the cells or cells by, for example, a soluble extract is obtained by centrifugation or filtration.
  • the target polypeptide can be obtained from the obtained extract by appropriately combining known separation and purification methods.
  • Known separation and purification methods include methods that utilize solubility, such as salting out and solvent precipitation, dialysis methods, ultrafiltration methods, gel filtration methods, and methods that mainly use molecular weight differences such as SDS-PAGE, A method using charge difference such as ion exchange chromatography, a method using specific affinity such as affinity chromatography (for example, when a protein is expressed together with a GST tag, a resin in which glutathione is bound to a carrier is used.
  • solubility such as salting out and solvent precipitation
  • dialysis methods such as ultrafiltration methods, gel filtration methods, and methods that mainly use molecular weight differences such as SDS-PAGE
  • a method using charge difference such as ion exchange chromatography
  • a method using specific affinity such as affinity chromatography (for example, when a protein is expressed together with a GST tag, a resin in which glutathione is bound to a carrier is used.
  • Ni-NTA resin or Co-based resin can be used when protein is expressed with His tag, and anti-HA antibody binding column can be used when protein is expressed with HA tag)
  • a method utilizing a difference in hydrophobicity such as phase high performance liquid chromatography, a method utilizing a difference in isoelectric point such as isoelectric focusing, and the like are used.
  • the vaccine formulation of the present invention comprises one or more adjuvants, Freund's incomplete and complete adjuvants, aluminum salts (eg, aluminum hydroxide, aluminum phosphate, aluminum sulfate, aluminum chloride), oil-in-water emulsions (eg, AS03).
  • adjuvants e.g, Freund's incomplete and complete adjuvants
  • aluminum salts e.g, aluminum hydroxide, aluminum phosphate, aluminum sulfate, aluminum chloride
  • oil-in-water emulsions eg, AS03
  • lipid A e.g , MF59®, Ribi TM, Provax TM ), monophosphoryl lipid A (MPL), potassium alum, saponin (eg QS2, QS21, ISCOM (immunostimulatory complex)) (eg AS01 (QS2, MPL and Including liposomes), AS02 (including squalene, MPL and QS21), AS04 (including MPL and aluminum hydroxide), AS15 (including MPL, QS21, CpG and liposomes), RC-529 (MPL analog), E6020 (including lipid A analog)), toxins (eg mutant CT (non-toxic mutant cholera toxin), mutant LT (non-toxic variant) Type heat-resistant Escherichia coli toxin), pertussis toxin), flagellin, adjuvant composed of nucleic acid components (CpG oligodeoxynucleotide, cyclic di-GMP, cyclic di-AMP, poly (
  • the vaccine preparation of the present invention can be administered by a normal active immunization method, and may be a systemic vaccine administered by injection, or a mucosal-derived vaccine administered by oral or nasal administration or the like without injection.
  • the anti-obesity vaccine preparation of the present invention can be administered one or more times by a method suitable for the dosage form in an effective amount for suppressing weight gain.
  • the anti-obesity vaccine preparation of the present invention can be used as a nasal vaccine administered intradermally, subcutaneously, intramuscularly, intraperitoneally, intravenously, orally, or intranasally. It can also be used as a mixture.
  • the fusion protein of the present invention may be incorporated into a cCHP nanogel and formulated as a fusion protein-cCHP nanogel complex as a vaccine.
  • cCHP nanogel cCHP; cationic type of cholesteryl group-bearing pullulan, self-aggregating nanosize hydrogel
  • cCHP nanogel cCHP; cationic type of cholesteryl group-bearing pullulan, self-aggregating nanosize hydrogel
  • nanogel is efficiently delivered to cells and induces an immune response as an adjuvant-free vaccine (Nochi et al., Nat Mater 9, 572-578 2010: Yuki et al., Biotechnol Genet Eng Rev 29, 61-72 2013).
  • the olfactory bulb can be administered intranasally in mice by administration of cCHP nanogel carrying [ 111 In] -labeled BoHc / A (non-toxic region of heavy chain C-terminal region of botulinum type A toxin) administered intranasally. Did not accumulate in the central nervous system such as the brain and brain (Nochi et al., Nat Mater 9, 572-578 2010).
  • nanogel nasal vaccines have been reported to be safe and induce strong antigen-specific systemic and mucosal antibody immune responses in mice (Kong et al., Infect Immun 81, 1625- 1634 2013).
  • the nanogel is a polymer gel nanoparticle obtained by adding hydrophobic cholesterol as a side chain to a hydrophilic polysaccharide (for example, pullulan).
  • Nanogel can be manufactured based on a well-known method, for example, the method described in international publication WO00 / 12564 etc. Specifically, it is first represented by a hydroxyl group-containing hydrocarbon or sterol having 12 to 50 carbon atoms and OCN-R 1 -NCO (wherein R 1 is a hydrocarbon group having 1 to 50 carbon atoms).
  • a diisocyanate compound is reacted to produce an isocyanate group-containing hydrophobic compound in which one molecule of a hydroxyl group-containing hydrocarbon or sterol having 12 to 50 carbon atoms is reacted.
  • the resulting isocyanate group-containing hydrophobic compound is reacted with a polysaccharide to produce a hydrophobic group-containing polysaccharide containing a hydrocarbon group or steryl group having 12 to 50 carbon atoms.
  • a highly purified hydrophobic group-containing polysaccharide can be produced by purifying the obtained product with a ketone solvent.
  • pullulan amylopectin, amylose, dextran, hydroxyethyldextran, mannan, levan, inulin, chitin, chitosan, xyloglucan, water-soluble cellulose and the like can be used, and pullulan is particularly preferable.
  • Examples of the nanogel used for formulation of the vaccine preparation of the present invention include cholesterol-substituted pullulan (hereinafter referred to as CHP) and CHP derivatives.
  • CHP used in the embodiment of the present invention may appropriately change the amount of cholesterol substitution depending on the size of the antigen and the degree of hydrophobicity. Further, an alkyl group (10 to 30 carbon atoms, preferably about 12 to 20 carbon atoms) may be added to change the degree of hydrophobicity of CHP.
  • the nanogel used in the present invention has a particle size of 10 to 40 nm, preferably 20 to 30 nm. Nanogels are already widely available on the market, and these products may be used in the present invention.
  • the nanogel used here is a nanogel into which a functional group having a positive charge, such as an amino group, is introduced so that the vaccine preparation can enter the negatively charged nasal mucosal surface.
  • a method for introducing an amino group into a nanogel a method using cholesterol pullulan (CHPNH 2 ) to which an amino group has been added can be mentioned. Specifically, CHP (for example, 0.15 g) dried under reduced pressure is dissolved in 15 ml of dimethyl sulfoxide (DMSO), and 1-1′carbonyldiimidazole (for example, 75 mg) is added to this under a nitrogen stream for several hours (for example, For about 1 hour).
  • DMSO dimethyl sulfoxide
  • Ethylenediamine (for example, 300 mg) is gradually added to the reaction solution and stirred for several hours to several tens of hours (for example, about 24 hours).
  • the obtained reaction solution is dialyzed against distilled water for several days.
  • the reaction solution after dialysis is freeze-dried to obtain a milky white solid.
  • the degree of substitution of ethylenediamine can be evaluated using elemental analysis, H-NMR, or the like.
  • the complex of the fusion protein of the present invention which is a vaccine antigen, and the cationic nanogel described above is obtained by allowing the cationic nanogel and the fusion protein of the present invention to coexist and interact with each other and incorporating the fusion protein into the cationic nanogel. Can be produced. Making a composite is called composite.
  • the mixing ratio of PspA and cationic nanogel may be such that, for example, the nanogel: fusion protein has a molar ratio of 1: 1 to 4: 1, preferably 3: 1.
  • the fusion protein and nanogel complex of the present invention is formed by mixing the fusion protein and the nanogel in a buffer, and allowing to stand at 4 to 50 ° C., for example, 46 ° C.
  • the fusion protein of the present invention and the nanogel may be mixed together with a suitable adjuvant.
  • the buffer used for conjugation of the fusion protein and the nanogel is not particularly limited, and can be appropriately prepared depending on the kind of the fusion protein and the nanogel.
  • a Tris-HCl buffer 50 mM, pH 7.6)
  • the prepared fusion protein-nanogel complex can be analyzed for its physicochemical properties by a known method. For example, analysis can be performed by measuring fluorescence response energy transfer (FRET), dynamic light scattering (DLS), and zeta potential.
  • the vaccine preparation of the present invention may contain known pharmaceutically acceptable stabilizers, preservatives, antioxidants and the like.
  • stabilizer include gelatin, dextran, sorbitol and the like.
  • preservatives include thimerosal and ⁇ -propiolactone.
  • antioxidant include ⁇ -tocopherol.
  • the dose of the vaccine preparation can be appropriately determined depending on the age, weight, etc. of the administration target, but is a pharmaceutically effective amount of the vaccine antigen.
  • a pharmaceutically effective amount refers to the amount of antigen necessary to induce an immune response against the vaccine antigen.
  • a dose of vaccine antigen of several ⁇ g to several tens of mg may be administered once to several times a day, and may be administered several times at intervals of 1 to several weeks, for example, 1 to 5 times.
  • Another embodiment of the present invention is a fusion protein of the present invention, a nucleic acid encoding the fusion protein, and a vector containing the nucleic acid.
  • a fusion protein schematically shown in FIG. 1 was prepared, a vaccine preparation containing this fusion protein was prepared, and its effect on weight gain was examined.
  • This example is merely an example of an embodiment of the present invention, and the scope of the present invention is not limited by this example.
  • mice 1-1-1 DIO mice and ob / ob mice Male C57BL / 6JJcl mice were obtained from Clea Japan, and male C57BL / 6JHamSlc-ob / ob mice were obtained from Japan SLC. All mice were bred under constant feeding and watering conditions with a 12-hour to 12-hour light-dark cycle according to the guidelines prescribed by the Animal Experiment Committee of the University of Tokyo Medical Science Institute.
  • Standard chow (CA-1; Japan Marie) and high fat feed (D12451; Research Diets Inc) are 3.47 kcal / g (13.0% kcal (from lipid), 31.2% kcal (from protein) and 55.8% kcal, respectively) (From nitrogen-free extract)) and 4.73 kcal / g (45% kcal (from lipid), 20% kcal (from protein) and 35% kcal (from carbohydrate)).
  • DIO mice were fed a standard diet until 8 weeks of age, and fed a high fat diet after the final immunization. On the other hand, ob / ob mice were fed standard chow throughout the experimental period.
  • Non-obese mice (without high-fat diet) Male C57BL / 6JJcl mice were obtained from Nippon Claire Co., Ltd. All mice were bred under constant feeding and watering conditions with a 12-hour to 12-hour light-dark cycle according to the guidelines prescribed by the Animal Experiment Committee of the University of Tokyo Medical Science Institute. Mice were fed standard chow (CA-1; Japan Marie).
  • Ghrelin-PspA (fusion protein of the present invention)
  • FIG. 1 is a mouse full-length UAG (mouse preproghrelin; amino acids 24-51) (GenBank accession number; AB035701). )
  • SEQ ID NO: 2 three repeats, N-terminal PspA / Rx1 (pUAB055; amino acids 1 to 302) (PspA family 1, clade2) (GenBank accession number; M74122) are arranged.
  • Individual UAG sequences are linked by a flexible (GGGGS) 4 linker. The gene encoding ghrelin-PspA was outsourced to Takara Bio Inc.
  • the synthesized gene was cleaved with Nco I and Xho I (Takara Bio) and then inserted into a pET-20b (+) vector (Novagen) having a His-tag at the C-terminus.
  • Rosetta2 (DE) pLysS competent cells (Novagen) were transformed with a vector into which the ghrelin-PspA gene had been inserted according to the attached instructions.
  • the resulting transformant was inoculated into LB medium containing 100 ⁇ g / ml ampicillin and 34 ⁇ g / ml chloramphenicol and incubated at 37 ° C. until OD 600 reached 0.5-0.8.
  • Ghrelin-PspA protein consists of anti-ghrelin antibody (1: 2000, Abcam) and anti-PspA antibody ((1: 2000, Kong et al., Infect Immun 81, 1625-1634 (2013)), and anti-His- Reacted with a tag antibody (1: 5000, GE Healthcare Japan) and detected with a peroxidase-conjugated anti-rabbit or mouse IgG secondary antibody (1: 5000, JacksonReImmunoResearch Laboratories). It was visualized with (Thermo Fisher Fisher Scientific) and detected with ImageQuant LAS 4000 mini (GE Healthcare Japan).
  • cCHP nanogels were prepared according to previously reported methods (Nochi et al., Nat Mater 9, 572-578 (2010): Ayame et al., Bioconjug Chem 19, 882-890 (2008)), and ghrelin-PspA and Crick di-GMP was mixed at a molecular ratio of 3: 1 (nanogel: ghrelin-PspA) and encapsulated for 1 hour at 46 ° C. for encapsulation in cCHP nanogel, a cationic nanoparticle.
  • Nasal immunization Mice received 5 ghrelin-PspA vaccines consisting of 5 ⁇ g ghrelin-PspA and 10 ⁇ g cyclic di-GMP encapsulated in cCHP (4, 5, 6, 7 and 8 weeks old DIO mice, and 26/27, 28, 29 and 30 week old ob / ob mice), a micropipette was used to drop droplets (7.99 ⁇ l per mouse) on the nostril I immunized my nose.
  • mice C57BL / 6J male mice were injected subcutaneously on the back with a vaccine solution consisting of 10 ⁇ g of ghrelin-PspA and each adjuvant 3 times every 2 weeks (4, 6, 8 weeks old) using a 23 gauge needle did.
  • Non-obese mice To enhance the antigen-specific immune response, 10 ⁇ g cyclic di-GMP (Yamasa Soy Sauce) was added to 5 ⁇ g ghrelin-PspA.
  • cCHP nanogels were prepared according to previously reported methods (Nochi et al., Nat Mater 9, 572-578 (2010): Ayame et al., Bioconjug Chem 19, 882-890 (2008)), and ghrelin-PspA and Crick di-GMP was mixed at a molecular ratio of 3: 1 (nanogel: ghrelin-PspA) and encapsulated for 1 hour at 46 ° C.
  • ghrelin-PspA vaccine composed of 5 ⁇ g ghrelin-PspA and 10 ⁇ g cyclic di-GMP encapsulated in cCHP 5 times every other week (4, 5, 6, 7 and 8 weeks old), micro A drop (droplet administration of 7.99 ⁇ l per mouse) was dropped on the nostril with a pipette, and nasal immunization was performed.
  • Antibody titer 1-4-1 DIO mice and ob / ob mice Antibody titers in serum and nasal washes were measured by ELISA. Each well of a 96 microwell plate (Immulon 1 B; Thermo Fisher Scientific) was coated overnight with BSA-conjugated AG or UAG or 0.1 ⁇ g of PspA at 4 ° C. The plate coated with the antigen was incubated for 1 hour in a blocking buffer containing 1% BSA (PBST; PBS containing Tween20). A blood sample was collected by inserting a capillary tube into the retro-orbital sinus, and blood was centrifuged (3,000 rpm 20 min) to prepare serum.
  • PBST blocking buffer containing 1% BSA
  • the nasal wash was collected by washing the nasal cavity with 200 ⁇ l of PBS. Serum and nasal washes were diluted with blocking buffer (2-fold serial dilution), added to wells coated with AG-BSA, UAS-BSA or PspA and incubated at 25 ° C. for 2 hours. After washing the plate with PBST, diluted HRP-conjugated goat anti-mouse IgG, IgA, IgE and IgM (SouthernBiotech) (1: 2000 [IgE], 1: 4000 [IgA, IgM], 1: 5000 [IgG]) It added to each well and incubated at 25 ° C. for 1.5 hours.
  • reaction product was developed using TMB Microwell peroxidase substrate system (Kirkegaard & Perry Laboratories), and the absorbance at 450 nm wavelength was measured.
  • the endpoint titer was expressed as Reciprocal log 2 at the final dilution showing an OD 450 of 0.1 units higher than the negative control.
  • Non-obese mice Antibody titers in serum and nasal washes were measured by ELISA.
  • serum antibody titer In the measurement of serum antibody titer, each well of a 96 microwell plate (Immulon 1 B; Thermo Fisher Scientific) was coated with 0.1 ⁇ g of acylated ghrelin (AG) conjugated with BSA at 4 ° C. overnight. The plate coated with the antigen was incubated for 1 hour in a blocking buffer containing 1% BSA (PBST; PBS containing Tween20). Blood samples were collected by inserting a capillary tube into the retro-orbital sinus every week at the age of 4 to 11 weeks, and blood was centrifuged (3,000 rpm 20 min) to prepare serum.
  • PBST blocking buffer containing 1% BSA
  • Serum was diluted with blocking buffer (2-fold serial dilution), added to wells coated with AG-BSA, and incubated at 25 ° C. for 2 hours. After washing the plate with PBST, diluted HRP-conjugated goat anti-mouse IgG (SouthernBiotech) (1: 5000) was added to each well and incubated at 25 ° C. for 1.5 hours. After incubation, the reaction product was developed using TMB Microwell peroxidase substrate system (Kirkegaard & Perry Laboratories), and the absorbance at 450 nm wavelength was measured. The endpoint titer was expressed as Reciprocal log 2 at the final dilution showing an OD 450 of 0.1 units higher than the negative control.
  • each well of a 96 microwell plate (F96SoMaxiSorp; Thermo Fisher Scientific) was coated with goat anti-mouse IgA (SouthernBiotech) 0.5 ⁇ g overnight at 4 ° C.
  • the plate coated with IgA was incubated for 1 hour in a blocking buffer (PBST; PBS containing Tween 20) containing 1% BSA.
  • PBST blocking buffer
  • the nasal wash was collected by washing the nasal cavity with 200 ⁇ l of PBS at 13 weeks of age (5 weeks after the final immunization).
  • the nasal wash was diluted with blocking buffer (2-fold serial dilution), added to wells coated with IgA, and incubated at 25 ° C. for 2 hours.
  • the IgA concentration of the nasal cavity washing liquid sample was determined based on a standard curve obtained from an IgA standard product (purified mouseAIgAk isotype standard, PharmMingen).
  • Non-obese mice and ob / ob mice Mouse weights were measured weekly with an accuracy of 0.1 g using a precision scale (GX-6000; A & D Corporation).
  • Respiratory gas analysis Energy consumption was calculated by indirect calorimetry, which measures VO 2 .
  • VO 2 was calculated by the following formula (Ishihara et al., J Nutr 130, 2990-2995 (2000): Nicholson et al., Physiol Meas 17, 43-55 (1996)).
  • VO 2 [(FEN 2 / FIN 2 ) ⁇ FIO 2 -FEO 2 ] ⁇ VT ⁇ 10 (Where FEN 2 and FEO 2 are the N 2 and O 2 concentrations in the exhalation, respectively. FIN 2 and FIO 2 are the N 2 and O 2 concentrations in the inspiration, respectively. VT is the standard condition (STPD; airflow through the chamber, corrected for standard temperature pressure dry).
  • the total amount of mesenteric fat, epididymal fat and perirenal fat was collected and measured at the time of dissection as visceral fat (5 weeks after the final vaccination).
  • RNA was measured 5 weeks after the final vaccination (n 9 / group), BAT (peri-scapular fat), WAT (peri-renal fat) and Isolated from frozen tissue of skeletal muscle (quadriceps) using TRIzol reagent (Life technologies).
  • BAT peri-scapular fat
  • WAT peri-renal fat
  • Prior to quantitative RT-PCR 1 ⁇ g of isolated RNA was reverse transcribed with PrimeScript RT Master Mix (Takara Bio). Quantitative PCR of the synthesized cDNA was performed using StepOnePlus System (Life technologies) using SYBR Green Master Mix (Life technologies). PCR amplification conditions were 95 ° C for 20 seconds, 95 ° C for 3 seconds; 60 ° C for 30 seconds for 40 cycles. After the PCR reaction, melting curve analysis was performed to quantify the PCR product. See Table 1 for primer sequences.
  • RNA was isolated from frozen stomach tissues of mice using TRIzol reagent (Life technologies) 4 weeks after the final vaccination (n 6 / group). Prior to quantitative RT-PCR, 1 ⁇ g of isolated RNA was reverse transcribed with PrimeScript RT Master Mix (Takara Bio). Quantitative PCR of the synthesized cDNA was performed using StepOnePlus System (Life technologies) using SYBR Green Master Mix (Life technologies). PCR amplification conditions were 95 ° C for 20 seconds, 95 ° C for 3 seconds; 60 ° C for 30 seconds for 40 cycles. After the PCR reaction, melting curve analysis was performed to quantify the PCR product. See Table 2 for primer sequences.
  • mice were fasted for 6 hours and administered 2 g glucose per kg body weight. Blood samples were collected from the tail vessel at 0, 15, 30, 60, 90 and 120 minutes after glucose administration.
  • the glucose level was determined by the glucose oxidase method using an automatic glucose meter (Glutest-Ace®; Sanwa Chemical Laboratory).
  • Glutest-Ace® automatic glucose meter
  • mice were fasted for 6 hours in a state where they could drink freely, and intraperitoneally injected with 0.75 IU insulin per kg body weight. Blood glucose levels were measured 0, 15, 30, 45, 60 and 90 minutes after insulin administration.
  • an AG peptide was synthesized (Eurofins Genomics, Tokyo, Japan) and indium-111 ( 111 In) using the chelating agent diethylenetriaminepentaacetic acid (DTPA) (Dojindo Laboratories) (Nippon Mediphysics).
  • DTPA diethylenetriaminepentaacetic acid
  • 1 mg of AG was labeled with 7.4 Mbq of 111 In, and the labeled product was passed through a PD-10 column (GE Healthcare Japan) to remove free 111 In that was not labeled.
  • 111 In-ghrelin was diluted with PBS (containing 50 ⁇ g AG in 100 ⁇ l, radioactivity 5,000,000 cpm).
  • WIZARD Automatic Gamma Counters PerkinElmer
  • radioactivity per 10 ⁇ l of serum collected from each mouse 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours and 4 hours after 111 In-ghrelin administration Measured with For the calculation of t1 / 2 of 111 In-ghrelin, the gamma count number was plotted on a semilogarithmic graph to determine the slope of the straight line.
  • mice GHSR GeneBank accession number AAI37883
  • Thermo Fisher Scientific carrying a neomycin resistance gene with BamHI and XhoI, and then inserted at the cleavage position.
  • the produced expression vector was transduced into Chinese hamster ovary (CHO) cells using Lipofectamine 3000 Transfection Reagent (Thermo Fisher Scientific).
  • the transduced strains were incubated in the presence of 250 ⁇ g / ml Geneticin (Nacalai Tesque). Stable transduced strains were confirmed by PCR (GHSR forward / BGH reverse) using gene-specific primers. See Table 1 for primer sequences.
  • GHSR-CHO cells contain 1.25 mmol / l probenecid and 0.04% pluronic F-127 after culturing in sterile flat-bottom 96-well plates (Thermo Fisher Scientific) at a density of 4.0 ⁇ 10 4 cells / well for 15 hours In the loading medium, Fluo-4-AM fluorescence indicator reagent was added and incubated at 37 ° C. for 1 hour. The intracellular Ca 2+ concentration was measured using a fluorescent microplate reader FLUOstar OPTIMA (BMG LABTECH JAPAN).
  • ghrelin-PspA consisting of a 3 times repeat sequence of full length AG of mouse (SEQ ID NO: 2) and PspA / Rx1 was designed (FIG. 1).
  • the expression vector inserted with ghrelin-PspA was introduced into E. coli competent cells, and the protein purified from the cells was identified as ghrelin-PspA by Western blotting. 2-1-1.
  • C57BL / 6J mice were immunized intranasally with 5 ⁇ g ghrelin-PspA and 10 ⁇ g cyclic di-GMP adjuvant encapsulated in cCHP nanogel.
  • Five vaccinations induced serum IgG against both AG and UAG (FIG. 2A).
  • serum IgG In addition to serum IgG, other immunoglobulin isotypes such as IgM, IgA and IgE were hardly induced (FIG. 2B).
  • the antibody titer of the anti-AG serum IgG antibody rapidly increased and reached a peak one week after the final immunization (9 weeks old).
  • antibody titer gradually decreased over several months, but maintained for over 1 year (FIG. 2C).
  • Non-obese mice C57BL / 6J mice were immunized intranasally 5 times every other week (4, 5, 6, 7 and 8 weeks old) with 5 ⁇ g ghrelin-PspA and 10 ⁇ g cyclic di-GMP adjuvant encapsulated in cCHP nanogel .
  • the antibody titer of the nasal lavage fluid was measured at the age of 13 weeks, the total IgA increased, and ghrelin-specific antibodies were also included (FIG. 11).
  • serum IgG the antibody titer was measured every week at the age of 4 to 11 weeks.
  • the anti-AG antibody titer increased after the third immunization and peaked immediately after the fifth dose. Thereafter, the anti-AG serum IgG antibody titer began to gradually decline (FIG. 12).
  • ghrelin-PspA vaccination also reduced weight gain (FIG. 4B).
  • Non-obese mice C57BL / 6J mice were immunized nasally with ghrelin-PspA five times every other week (4, 5, 6, 7 and 8 weeks of age). Also in non-obese mice, ghrelin-PspA vaccine administration significantly suppressed weight gain (FIG. 13).
  • PPAR ⁇ one of UCP1 upregulators in BAT, was also increased in immunized mice (FIG. 8D).
  • WAT white adipose tissue
  • UCP2 and PPAR ⁇ expression was up-regulated, but UCP3, PPAR ⁇ and PPAR ⁇ in muscle were not different between immunized and control mice (FIGS. 8G to I).
  • FFA serum free fatty acids
  • LDL low density lipoprotein
  • TG triglycerides
  • the vaccine preparation of the present invention can induce systemic immunity and mucosal immunity, and can effectively treat obesity. Therefore, the present invention is expected to be used in the field of obesity prevention and treatment.

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Abstract

La présente invention concerne une formulation vaccinale comprenant une protéine porteuse et une protéine de fusion où sont fusionnés un ou plusieurs polypeptides de ghréline de pleine longueur, la formulation vaccinale induisant une réponse immunitaire générale et une réponse immunitaire muqueuse. Le polypeptide de ghréline peut être, par exemple, un polypeptide comprenant une séquence d'acides aminés représentée par SEQ ID NO : 1, SEQ ID NO : 2, ou SEQ ID NO : 3, ou un polypeptide sensiblement identique à ces polypeptides.
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WO2020027309A1 (fr) * 2018-08-03 2020-02-06 国立大学法人東京大学 Vaccin intranasal induisant une immunité cellulaire
WO2022086300A1 (fr) * 2020-10-23 2022-04-28 에스케이바이오사이언스 주식회사 Composition de vaccin ou kit destiné à réduire la taille ou le volume d'un tissu cible, contenant un matériel génétique qui code pour un antigène étranger
RU2846265C1 (ru) * 2020-10-23 2025-09-02 Ск Биосайенс Ко., Лтд. Композиция вакцины или набор для уменьшения размера или объёма ткани-мишени, содержащие генетический материал, кодирующий чужеродный антиген
US12409135B2 (en) 2021-12-08 2025-09-09 Immunitybio, Inc. Neoepitope vaccine delivery vehicle and methods of making the same

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020027309A1 (fr) * 2018-08-03 2020-02-06 国立大学法人東京大学 Vaccin intranasal induisant une immunité cellulaire
CN112566656A (zh) * 2018-08-03 2021-03-26 国立大学法人东京大学 诱导细胞性免疫的经鼻疫苗
JPWO2020027309A1 (ja) * 2018-08-03 2021-08-12 国立大学法人 東京大学 細胞性免疫を誘導する経鼻ワクチン
US11564993B2 (en) 2018-08-03 2023-01-31 The University Of Tokyo Intranasal vaccine that induces cellular immunity
JP7445897B2 (ja) 2018-08-03 2024-03-08 国立大学法人 東京大学 細胞性免疫を誘導する経鼻ワクチン
WO2022086300A1 (fr) * 2020-10-23 2022-04-28 에스케이바이오사이언스 주식회사 Composition de vaccin ou kit destiné à réduire la taille ou le volume d'un tissu cible, contenant un matériel génétique qui code pour un antigène étranger
RU2846265C1 (ru) * 2020-10-23 2025-09-02 Ск Биосайенс Ко., Лтд. Композиция вакцины или набор для уменьшения размера или объёма ткани-мишени, содержащие генетический материал, кодирующий чужеродный антиген
US12409135B2 (en) 2021-12-08 2025-09-09 Immunitybio, Inc. Neoepitope vaccine delivery vehicle and methods of making the same
US12502428B1 (en) 2025-04-30 2025-12-23 Utopia Therapeutics Pvt Ltd. Immunotherapeutic composition for prevention of obesity, nonalcoholic fatty liver disease and hypertriglyceridemia, and methods of use and preparation thereof

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