JP6910961B2 - 選択細胞への分子送達に適用する遺伝子操作したボツリヌス菌毒素 - Google Patents
選択細胞への分子送達に適用する遺伝子操作したボツリヌス菌毒素 Download PDFInfo
- Publication number
- JP6910961B2 JP6910961B2 JP2017559588A JP2017559588A JP6910961B2 JP 6910961 B2 JP6910961 B2 JP 6910961B2 JP 2017559588 A JP2017559588 A JP 2017559588A JP 2017559588 A JP2017559588 A JP 2017559588A JP 6910961 B2 JP6910961 B2 JP 6910961B2
- Authority
- JP
- Japan
- Prior art keywords
- target cell
- pore
- unit
- forming unit
- polypeptide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/33—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Clostridium (G)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/45—Transferases (2)
-
- 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
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/6415—Toxins or lectins, e.g. clostridial toxins or Pseudomonas exotoxins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
- A61K49/0056—Peptides, proteins, polyamino acids
-
- 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
-
- 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
- A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- 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
-
- 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
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y204/00—Glycosyltransferases (2.4)
- C12Y204/02—Pentosyltransferases (2.4.2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/24—Metalloendopeptidases (3.4.24)
- C12Y304/24069—Bontoxilysin (3.4.24.69), i.e. botulinum neurotoxin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y204/00—Glycosyltransferases (2.4)
- C12Y204/02—Pentosyltransferases (2.4.2)
- C12Y204/02031—NAD+-protein-arginine ADP-ribosyltransferase (2.4.2.31)
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Pharmacology & Pharmacy (AREA)
- Biotechnology (AREA)
- Gastroenterology & Hepatology (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Epidemiology (AREA)
- Microbiology (AREA)
- Toxicology (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Neurosurgery (AREA)
- Neurology (AREA)
- Ophthalmology & Optometry (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicinal Preparation (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Description
本発明は、米国国防総省国防脅威削減局の授与による助成第HDTRA−10−C−0055号の下、政府支援を受けてなされた。米国政府は本発明において一定の権利を有する。
プラスミドpUC57−C2II−C1 HCCは、コドン最適化遺伝子合成品として購入した。これは、BoNT C1のアミノ酸Y1094〜E1291に相当するC1 HCC配列の上流にある、C末端の7個のアミノ酸をトランケートしたC2II遺伝子からなる。プライマーC2IIΔD4FおよびC2IIΔD4−GS(EP)RでC2IIのアミノ酸M1〜T592に相当する遺伝子を増幅し、C1 HCCドメインとのオーバーラッピングPCRに使用する5’BamHI突出部および3’グリシン−セリン(EP)連結領域を加えた。BoNT C1 HCC遺伝子は、3’EcoRI制限部位を含むようにプライマー(EP)GS−C1 HCCFおよびC1 HCCRを使用してPCRで増幅した。二巡目のPCRは、GS(EP)10GSFおよびC1 HCCRを用いて行い、C2IIΔD4−GS(EP)配列の3’を相補するように、C1 HCCの5’増幅産物を伸長させた。得られた2つの断片をオーバーラッピングPCRにより融合し、C2IIΔD4−GS(EP)10GS−C1 HCC(C2II−C1)を得た。C1
HCCの生成には、プライマーC1 HCCFおよびC1 HCCRを使用してpUC57−C2II−C1 HCCテンプレート上でPCR増幅を行った。C2IIΔD4の生成には、プライマーC2ΔD4FおよびC2ΔD4Rを使用して、ドメイン4をもたないC2II遺伝子を増幅した。プラスミドpUC57−C2Itは、コドン最適化遺伝子合成品として購入した。C2It(C2Iアミノ酸1〜226に相当、PDB 2J3V)は、BamHIおよびEcoRI制限部位を用いてpGex−2Tで直接サブクローニングした。全長のC2I(C2Iアミノ酸1〜431に相当)は、隣接プライマーとしてC2IFとC2IR、オーバーラッピングプライマーとしてC2IOFとC2IORとを用いて、C2Itを合成DNAから増幅したDNAへ融合するオーバーラッピングPCRにより生成した。最終的なPCR産物はすべて、BamHIとEcoRIで消化してpGex−2Tにライゲートした。DH5αを電気穿孔法により形質転換して、N末端GST融合体としてC2II−C1、C1 HCC、C2IIΔD4、C2ItおよびC2Iを増産した。DNA構築物の同一性は配列決定して確認した。プライマー配列を表1に列記する。
リアクリルアミドゲルを用いたSDS−PAGEまたは4〜12%勾配Bis−Trisゲルで分離した。C2II−C1を、抗BoNT C1ポリクローナル抗体(Metabiologics Inc.、Madison、WI)を用いて同定し、陽性対照として精製C1 HCCを、また陰性対照としてC2IIΔD4を用いた。タンパク質をSDS−PAGEで分離し、Towbin緩衝液中でニトロセルロース膜に転写し、5%粉乳PBS−tween緩衝液中でブロッキングし、その後、1μg/ulの抗BoNT C1抗体を0.5%粉乳PBS−tweenで1:5,000に希釈してプローブした。0.5%粉乳PBS−tweenで希釈した抗ウサギHRP二次抗体(1:5,000)をECLブロッティング基質と共に使用してシグナル検出した。
BoNT血清亜型C1神経毒およびADPリボシル化を行うC2毒素ベースの多重組換えタンパク質構築物を、大腸菌を使用して発現させ、精製した。天然のBoNT C1を図2aに示し、天然のC2II結合/トランスロケーションドメインを図2bに示す。対照として使用するために、C末端のドメイン4を欠失するC2毒素(C2IIΔD4)、およびC1神経毒結合ドメインC1 HCCを生産した。C2IIΔD4とBoNT C1のC1 HCC(1094〜1291)をグルタメート−プロリン10回反復ペプチドリンカー、(EP)10で連結させてC2II−C1を生成した(図2c)。これに加え、活性酵素部位を除いた非有毒性C2It(1〜226)を含むペイロード、全長のC2I(1〜431)を含むペイロードの2つのC2Iベースペイロードを構築した(図2d)。
配列番号1−
NNINDSKILSLQNRKNTLVDTSGYNAEVSEEGDVQLNPIFPFDFKLGSSGEDRGKVIVTQNENIVYNSMYESFSISFWIRINKWVSNLPGYTIIDSVKNNSGWSIGIISNFLVFTLKQNEDSEQSINFSYDISNNAPGYNKWFFVTVTNNMMGNMKIYINGKLIDTIKVKELTGINFSKTITFEINKIPDTGLITSDSDNINMWIRDFYIFAKELDGKDINILFNSLQYTNVVKDYWGNDLRYNKEYYMVNIDYLNRYMYANSRQIVFNTRRNNNDFNEGYKIIIKRIRGNTNDTRVRGGDILYFDMTINNKAYNLFMKNETMYADNHSTEDIYAIGLREQTKDINDNIIFQIQPMNNTYYYASQIFKSNFNGENISGICSIGTYRFRLGGDWYRHNYLVPTVKQGNYASLLESTSTHWGFVPVSE
配列番号2−
MLVSKFENSVKNSNKNYFTINGLMGYYFENDFFNLNIISPTLDGNLTFSKEDINSILGNKIIKSARWIGLIKPSITGEYILSTNSPNCRVELNGEIFNLSLNTSNTVNLIQGNVYDIRIEQLMSENQLLKNYEGIKLYWETSDIIKEIIPSEVLLKPNYSNTNEKSKFIPNNTLFSNAKLKANANRDTDRDGIPDEWEINGYTVMNQKAVAWDDKFAANGYKKYVSNPFKPCTANDPYTDFEKVSGQIDPSVSMVARDPMISAYPIVGVQMERLVVSKSETITGDSTKSMSKSTSHSSTNINTVGAEVSGSLQLAGGIFPVFSMSASANYSHTWQNTSTVDDTTGESFSQGLSINTAESAYINPNIRYYNTGTAPVYNVTPTTTIVIDKQSVATIKGQESLIGDYLNPGGTYPIIGEPPMALNTMDQFSSRLIPINYNQLKSIDNGGTVMLSTSQFTGNFAKYNSNGNLVTDGNNWGPYLGTIKSTTASLTLSLPDQTTQVAVVAPNFSDPEDKTPRLTLEQALVKAFRLEKKNGKFYFHGMEISANQKIQVFLDRNTNVDFENQLKNTANKDIMNCIIKRNMNILVKVITFKENISSINIINDTNFGVESMTGLSKRIKGNDGIYRASTKSFSFKSKEIKYPEGFYRMRFVIQSYEPFTCNFKLFNNLIYSNSFDIGYYDEFFYFYCNGSKSFFDISCDIINSINRLSGVFLI
配列番号3−
MPIIKEPIDFINKPESEAKEWGKEEEKRWFTKLNNLEEVAVNQLKNKEYKTKIDNFSTDILFSSLTAIEIMKEDENQNLFDVERIREALLKNTLDRDAIGYVNFTPKELGINFSIRDVELDRDISDETLDKVRQQIINQEYTKFSFISLGLNDNSINESVPVIVKTRVPTTFDYGVLNDKETVSLLLNQGFSIIPESAIITTIKGKDYILIEGSLSQELDFYNKGSEAWGAENYGDYISKLSHEQLGALEGYLHSDYKAINSYLRNNRVPNNDELNKKIELISSALSVKPIPQTLIAYRRVDGIPFDLPSDFSFDKKENGEIIADKQKLNEFIDKWTGKEIENLSFSSTSLKSTPSSFSKSRFIFRLRLSEGAIGAFIYGFSGFQDEQEILLNKNSTFKIFRITPITSIINRVTKMTQVVIDAEGIQNKEI
配列番号4−
MLVSKFENSVKNSNKNYFTINGLMGYYFENDFFNLNIISPTLDGNLTFSKEDINSILGNKIIKSARWIGLIKPSITGEYILSTNSPNCRVELNGEIFNLSLNTSNTVNLIQGNVYDIRIEQLMSENQLLKNYEGIKLYWETSDIIKEIIPSEVLLKPNYSNTNEKSKFIPNNTLFSNAKLKANANRDTDRDGIPDEWEINGYTVMNQKAVAWDDKFAANGYKKYVSNPFKPCTANDPYTDFEKVSGQIDPSVSMVARDPMISAYPIVGVQMERLVVSKSETITGDSTKSMSKSTSHSSTNINTVGAEVSGSLQLAGGIFPVFSMSASANYSHTWQNTSTVDDTTGESFSQGLSINTAESAYINPNIRYYNTGTAPVYNVTPTTTIVIDKQSVATIKGQESLIGDYLNPGGTYPIIGEPPMALNTMDQFSSRLIPINYNQLKSIDNGGTVMLSTSQFTGNFAKYNSNGNLVTDGNNWGPYLGTIKSTTASLTLSLPDQTTQVAVVAPNFSDPEDKTPRLTLEQALVKAFRLEKKNGKFYFHGMEISANQKIQVFLDRNTNVDFENQLKNTANKDIMNCIIKRNMNILVKVITGSEPEPEPEPEPEPEPEPEPEPGSTNVVKDYWGNDLRYNKEYYMVNIDYLNRYMYANSRQIVFNTRRNNNDFNEGYKIIIKRIRGNTNDTRVRGGDILYFDMTINNKAYNLFMKNETMYADNHSTEDIYAIGLREQTKDINDNIIFQIQPMNNTYYYASQIFKSNFNGENISGICSIGTYRFRLGGDWYRHNYLVPTVKQGNYASLLESTSTHWGFVPVSE
配列番号5−
GSEPEPEPEPEPEPEPEPEPEPGS
配列番号6−
MPIIKEPIDFINKPESEAKEWGKEEEKRWFTKLNNLEEVAVNQLKNKEYKTKIDNFSTDILFSSLTAIEIMKEDENQNLFDVERIREALLKNTLDRDAIGYVNFTPKELGINFSIRDVELDRDISDETLDKVRQQIINQEYTKFSFISLGLNDNSINESVPVIVKTRVPTTFDYGVLNDKETVSLLLNQGFSIIPESAIITTIKGKDYILIEGSLSQELDFYNKG
本出願または下記リスト全般にわたり引用可能な引用参考文献のすべて(参考文献、特許、特許出願およびウェブサイトを含む)の内容は、ここに、あらゆる目的のために全体を参照によって本開示に明確に組み入れる。本開示は、別段の指示がない限り、当技術分野で周知の免疫学、分子生物学および細胞生物学の従来技術を採用してもよい。
1. Schiavo, G., Matteoli, M. & Montecucco, C. Neurotoxins affecting neuroexocytosis. Physiol. Rev. 80, 717-766 (2000).
2. Singh, B. R. et al. Clostridial neurotoxins as a drug delivery vehicle targeting nervous system. Biochimie 92, 1252-1259 (2010).
3. Vazquez-Cintron, E. J. et al. Atoxic derivative of botulinum neurotoxin A as
a prototype molecular vehicle for targeted delivery to the neuronal cytoplasm. PLoS ONE 9, doi: e8551710.1371/journal.pone.0085517 (2014).
4. Zhang, P. et al. An efficient drug delivery vehicle for botulism countermeasure. BMC Pharmacol. 9, doi: 10.1186/1471-2210-9-12 (2009).
5. Ho, M. F. et al. Recombinant botulinum neurotoxin A heavy chain-based delivery vehicles for neuronal cell targeting. Protein Eng. Des. Sel. 24, 247-253 (2011).
6. Webb, R. P., Smith, T. J., Wright, P., Brown, J. & Smith, L. A. Production of catalytically inactive BoNT/A1 holoprotein and comparison with BoNT/A1 subunit
vaccines against toxin subtypes A1, A2, and A3. Vaccine 27, 4490-4497 (2009).
7. Mechaly, A., McCluskey, A. J. & Collier, R. J. Changing the receptor specificity of anthrax toxin. mBio 3, e00088-00012, doi: 10.1128/ mBio.00088-12 (2012).8. Fahrer, J. et al. C2-streptavidin mediates the delivery of biotin-conjugated tumor suppressor protein P53 into tumor cells. Bioconjug. Chem. 24, 595-603 (2013).
9. Fahrer, J., Rieger, J., van Zandbergen, G. & Barth, H. The C2-streptavidin delivery system promotes the uptake of biotinylated molecules in macrophages and
T-leukemia cells. Biol. Chem. 391, 1315-1325 (2010).
10. Fahrer, J. et al. Genetically engineered clostridial C2 toxin as a novel delivery system for living mammalian cells. Bioconjug. Chem. 21, 130-139 (2010).
11. Schleberger, C., Hochmann, H., Barth, H., Aktories, K. & Schulz, G. E. Structure and action of the binary C2 toxin from Clostridium botulinum. J. Mol. Biol. 364, 705-715 (2006).
12. Aktories, K. et al. Botulinum-C2 toxin ADP-ribosylates actin. Nature 322, 390-392 (1986).
13. Simpson, L. L. Molecular basis for the pharmacological actions of Clostridium botulinum type C2 toxin. J. Pharmacol. Exp. Ther. 230, 665-669 (1984).
14. Ohishi, I., Iwasaki, M. & Sakaguchi, G. Purification and characterization of 2 components of botulinum C2 toxin. Infect. Immun. 30, 668-673 (1980).
15. Iwasaki, M., Ohishi, I. & Sakaguchi, G. Evidence that botulinum C2-toxin has 2 dissimilar components. Infect. Immun. 29, 390-394 (1980).
16. Ohishi, I. Activation of botulinum C2 toxin by trypsin. Infect. Immun. 55, 1461-1465 (1987).
17. Nagahama, M. et al. Binding and Internalization of Clostridium botulinum C2
Toxin. Infect. Immun. 77, 5139-5148 (2009).
18. Fritz, G., Schroeder, P. & Aktories, K. Isolation and characterization of a
Clostridium botulinum C2 toxin-resistant cell line: evidence for possible involvement of the cellular C2II receptor in growth-regulation. Infect. Immun. 63, 2334-2340 (1995).
19. Pust, S., Barth, H. & Sandvig, K. Clostridium botulinum C2 toxin is internalized by clathrin- and Rho-dependent mechanisms. Cell Microbiol. 12, 1809-1820 (2010).
20. Kaiser, E., Haug, G., Hliscs, M., Aktories, K. & Barth, H. Formation of a biologically active toxin complex of the binary Clostridium botulinum C2 toxin without cell membrane interaction. Biochemistry 45, 13361-13368 (2006).
21. Barth, H. et al. Cellular uptake of Clostridium botulinum C2 toxin requires
oligomerization and acidification. J. Biol. Chem. 275, 18704-18711 (2000).
22. Haug, G. et al. Cellular uptake of Clostridium botulinum C2 toxin: Membrane
translocation of a fusion toxin requires unfolding of its dihydrofolate reductase domain. Biochemistry 42, 15284-15291 (2003).
23. Chaddock, J. A. et al. Inhibition of vesicular secretion in both neuronal and nonneuronal cells by a retargeted endopeptidase derivative of Clostridium botulinum neurotoxin type A. Infect. Immun. 68, 2587-2593 (2000).
24. Blocker, D. et al. The C terminus of component C2II of Clostridium botulinum C2 toxin is essential for receptor binding. Infect. Immun. 68, 4566-4573 (2000).
25. Gill, D. M. Bacterial toxins-a table of lethal amounts. Microbiol. Rev. 46,
86-94 (1982).
26. Montecucco, C. & Schiavo, G. Mechanism of action of tetanus and botulinum neurotoxins. Mol. Microbiol. 13, 1-8 (1994).
27. Strotmeier, J. et al. The biological activity of botulinum neurotoxin type C is dependent upon novel types of ganglioside binding sites. Mol. Microbiol. 81, 143-156 (2011).
28. Simpson, L. L. The origin, structure, and pharmacological activity of botulinum toxin. Pharmacol. Rev. 33, 155-188 (1981).
29. Yowler, B. C. & Schengrund, C. L. Glycosphingolipids-Sweets for botulinum neurotoxin. Glycoconj. J. 21, 287-293 (2004).
30. Karalewitz, A. P. A., Fu, Z. J., Baldwin, M. R., Kim, J. J. P. & Barbieri, J. T. Botulinum neurotoxin serotype C associates with dual ganglioside receptors
to facilitate cell entry. J. Biol. Chem. 287, 40806-40816 (2012).
31. Barth, H., Klingler, M., Aktories, K. & Kinzel, V. Clostridium botulinum C2
toxin delays entry into mitosis and activation of p34(cdc2) kinase and cdc25-C phosphatase in HeLa cells. Infect. Immun. 67, 5083-5090 (1999).
32. Varkouhi, A. K., Scholte, M., Storm, G. & Haisma, H. J. Endosomal escape pathways for delivery of biologicals. J. Control. Release 151, 220-228 (2011).
33. Sandvig, K. & van Deurs, B. Delivery into cells: lessons learned from plant
and bacterial toxins. Gene Ther. 12, 865-872 (2005).
34. Verdurmen, W. P., Luginbuhl, M., Honegger, A. & Pluckthun, A. Efficient cell-specific uptake of binding proteins into the cytoplasm through engineered modular transport systems. J. Control. Release 200, 13-22 (2015).
35. Eckhardt, M., Barth, H., Blocker, D. & Aktories, K. Binding of Clostridium botulinum C2 toxin to asparagine-linked complex and hybrid carbohydrates. J. Biol. Chem. 275, 2328-2334 (2000).
36. Andreu, A., Fairweather, N. & Miller, A. D. Clostridium neurotoxin fragments as potential targeting moieties for liposomal gene delivery to the CNS. ChemBioChem 9, 219-231 (2008).
37. Edupuganti, O. P. et al. Targeted delivery into motor nerve terminals of inhibitors for SNARE-cleaving proteases via liposomes coupled to an atoxic botulinum neurotoxin. FEBS J. 279, 2555-2567 (2012).
38. Tsukamoto, K. et al. Binding of Clostridium botulinum type C and D neurotoxins to ganglioside and phospholipid-Novel insights into the receptor for clostridial neurotoxins. J. Biol. Chem. 280, 35164-35171 (2005).
39. Rummel, A. et al. Botulinum neurotoxins C, E and F bind gangliosides via a conserved binding site prior to stimulation-dependent uptake with botulinum neurotoxin F utilising the three isoforms of SV2 as second receptor. J. Neurochem. 110, 1942-1954 (2009).
40. Muraro, L., Tosatto, S., Motterlini, L., Rossetto, O. & Montecucco, C. The N-terminal half of the receptor domain of botulinum neurotoxin A binds to microdomains of the plasma membrane. Biochem. Biophys. Res. Commun. 380, 76-80 (2009).41. Harper, C. B. et al. Dynamin inhibition blocks botulinum neurotoxin type A endocytosis in neurons and delays botulism. J. Biol. Chem. 286, 35966-35976 (2011).
42. Couesnon, A., Pereira, Y. & Popoff, M. R. Receptor-mediated transcytosis of
botulinum neurotoxin A through intestinal cell monolayers. Cell Microbiol. 10, 375-387 (2008).
43. Simpson, L. L. Identification of the major steps in botulinum toxin action.
Annu. Rev. Pharmacol. Toxicol. 44, 167-193 (2004).
44. Zhao, H. L. et al. Increasing the homogeneity, stability and activity of human serum albumin and interferon-alpha 2b fusion protein by linker engineering. Protein Expr. Purif. 61, 73-77 (2008).
45. Bhandari, D. G., Levine, B. A., Trayer, I. P. & Yeadon, M. E. H-1-NMR study
of mobility and conformational constraints within the proline-rich N-terminal of the LC1 alkali light chain of skeletal myosin. Correlation with similar segments in other protein systems. Eur. J. Biochem. 160, 349-356 (1986).
46. Evans, J. S., Levine, B. A., Trayer, I. P., Dorman, C. J. & Higgins, C. F. Sequenced-imposed structural constraints in the tonB protein of Escherichia coli. FEBS Lett. 208, 211-216 (1986).
47. Roditi, I. et al. Expression of Trypanosoma brucei procyclin as a fusion protein in Escherichia coli. Mol. Biochem. Parasitol. 34, 35-43 (1989).
48. Kroken, A. R. et al. Unique Ganglioside Binding by Botulinum Neurotoxins C and D-SA. FEBS J. 278, 4486-4496 (2011).
49. Heine, K., Pust, S., Enzenmuller, S. & Barth, H. ADP-Ribosylation of Actin by the Clostridium botulinum C2 Toxin in Mammalian Cells Results in Delayed Caspase-Dependent Apoptotic Cell Death. Infect. Immun. 76, 4600-4608 (2008).
50. Barth, H., Roebling, R., Fritz, M. & Aktories, M. The binary Clostridium botulinum C2 toxin as a protein delivery system- Identification of the minimal protein region necessary for interaction of toxin components. J. Biol. Chem. 277, 5074-5081 (2002).
51. Rabideau, A. E., Liao, X., Akcay, G. & Pentelute, B. L. Translocation of Non-Canonical Polypeptides into Cells Using Protective Antigen. Sci. Rep. 5, 11944 (2015).
52. Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685 (1970).
53. Towbin, H., Staehelin, T. & Gordon, J. Electrophoretic transfer of proteins
from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. PNAS 76, 4350-4354 (1979).
54. Burnette, W. N. Western blotting-electrophoretic transfer of proteins from sodium dodecyl sulfate polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein-A. Anal. Biochem. 112, 195-203 (1981).
55. Ma, H. T. & Poon, R. Y. Synchronization of HeLa cells. Methods Mol. Biol. 761, 151-161 (2011).
56. Barth, H., Preiss, J. C., Hofmann, F. & Aktories, K. Characterization of the catalytic site of the ADP-ribosyltransferase Clostridium botulinum C2 toxin by site-directed mutagenesis. J. Biol. Chem. 273, 29506-29511 (1998).
57. US20090269361 A1.
58. WO2013126690 A1.
Claims (17)
- 薬剤を標的細胞に送達するための組成物であって、
標的細胞結合ユニット、
孔形成ユニット、
該標的細胞結合ユニットと該孔形成ユニットとを連結する強固な合成リンカー、および
該薬剤を含むペイロードユニットであって、孔形成ユニットの活性化およびオリゴマー形成時に、孔形成ユニットにより形成される孔に非共有結合するペイロードユニットを含み;
該孔形成ユニットは、非神経毒性の二成分毒素の第1のAB型由来のポリペプチドであり、該標的細胞結合ユニットは、毒素の第2のAB型由来のポリペプチドであり、該第2のAB型は該第1のAB型とは異なっており、
該標的細胞結合ユニットは神経細胞に結合し、および
標的細胞結合ユニットおよび孔形成ユニットは、強固な合成リンカーを介して共有結合で連結して、配列番号4と少なくとも90%の配列同一性を有するポリペプチドを形成する前記組成物。 - 孔形成ユニットは、ボツリヌス菌毒素C2の孔形成ユニット由来のポリペプチドまたはポリペプチドオリゴマーである、請求項1に記載の組成物。
- 薬剤を神経細胞に送達する、請求項1に記載の組成物。
- 標的細胞結合ユニットは、ボツリヌス菌神経毒C1の重鎖の結合ドメイン(C1 Hcc)由来のポリペプチドまたはポリペプチドオリゴマーである、請求項1に記載の組成物。
- 薬剤は、治療薬、診断薬、およびそれらの組合せからなる群から選択される少なくとも1つのメンバーを含む、請求項1に記載の組成物。
- 薬剤は、毒素、細胞周期阻害物質、アポトーシス誘導剤、DNA複製阻害物質、RNA合成阻害物質、タンパク質合成阻害物質、酵素、タンパク質結合薬剤、抗体、中和抗体、標識剤、磁気ビーズ、およびそれらの組合せからなる群から選択される少なくとも1つの
メンバーを含む、請求項1に記載の組成物。 - 薬剤は、ADP−リボシルトランスフェラーゼを含む、請求項1に記載の組成物。
- 薬剤は、ボツリヌス菌毒素C2からのC2Iを含む、請求項1に記載の組成物。
- 薬剤は、蛍光性薬剤を含む、請求項1に記載の組成物。
- 標的細胞結合ユニットは、ボツリヌス菌神経毒C1の重鎖の結合ドメイン(C1 Hcc)を含み、孔形成ユニットは、ボツリヌス菌毒素C2の孔形成ドメインを含み、ペイロードユニットは、ボツリヌス菌毒素C2からのC2Iを含む、請求項1に記載の組成物。
- 薬剤を標的細胞に送達するための組成物であって、
標的細胞結合ユニット、
孔形成ユニット、
該標的細胞結合ユニットと該孔形成ユニットとを連結する強固な合成リンカー、および
該薬剤を含むペイロードユニットであって、孔形成ユニットの活性化およびオリゴマー形成時に、孔形成ユニットにより形成される孔に非共有結合するペイロードユニットを含み;
該孔形成ユニットは、非神経毒性の二成分毒素の第1のAB型由来のポリペプチドであり、該標的細胞結合ユニットは、毒素の第2のAB型由来のポリペプチドであり、該第2のAB型は該第1のAB型とは異なっており、
該標的細胞結合ユニットは神経細胞に結合し、
該標的細胞結合ユニットは、配列番号1のアミノ酸配列230〜426と少なくとも90%の配列同一性を有するポリペプチドを含み、孔形成ユニットは、配列番号2のアミノ酸配列1〜591と少なくとも90%の配列同一性を有するポリペプチドを含み、および
強固な合成リンカーは、配列番号5のアミノ酸配列と少なくとも90%の配列同一性を有するポリペプチドを含む、
前記組成物。 - ペイロードユニットは、配列番号6のアミノ酸配列と少なくとも90%の配列同一性を有するポリペプチドを含む、請求項1に記載の組成物。
- 対象の標的細胞へ薬剤を送達するために使用される組成物であって、該対象は標的細胞を有し、該標的細胞は、脳腫瘍の細胞、神経芽細胞腫の細胞、網膜芽細胞腫の細胞、末梢ニューロン、運動ニューロン、感覚ニューロン、ならびにそれらの組合せからなる群から選択される、請求項1に記載の組成物。
- 標的細胞に薬剤を送達することができるポリペプチドをコードするポリヌクレオチドであって、該ポリペプチドは配列番号4の配列を有するポリペプチドと少なくとも90%の同一性を有するポリペプチドである、前記ポリヌクレオチド。
- 請求項14に記載のポリヌクレオチドを含む宿主細胞。
- 薬剤を標的細胞に送達するための組成物であって、
標的細胞結合ユニット、
孔形成ユニット、
該標的細胞結合ユニットと該孔形成ユニットとを連結する強固な合成リンカー、および
該薬剤を含むペイロードユニットであって、孔形成ユニットの活性化およびオリゴマー形成時に、孔形成ユニットにより形成される孔に非共有結合するペイロードユニットを含
み;
該孔形成ユニットは、非神経毒性の二成分毒素の第1のAB型由来のポリペプチドであり、該標的細胞結合ユニットは、毒素の第2のAB型由来のポリペプチドであり、該第2のAB型は該第1のAB型とは異なっており、
該標的細胞は神経細胞に結合し、および
強固な合成リンカーは、配列番号5のアミノ酸配列と少なくとも90%の配列同一性を有するポリペプチドを含む、
前記組成物。 - 神経細胞は感覚ニューロンである、請求項1に記載の組成物。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201562162582P | 2015-05-15 | 2015-05-15 | |
US62/162,582 | 2015-05-15 | ||
PCT/US2016/032573 WO2016187076A1 (en) | 2015-05-15 | 2016-05-14 | Engineered clostridium botulinum toxin adapted to deliver molecules into selected cells |
Publications (3)
Publication Number | Publication Date |
---|---|
JP2018515542A JP2018515542A (ja) | 2018-06-14 |
JP2018515542A5 JP2018515542A5 (ja) | 2019-07-25 |
JP6910961B2 true JP6910961B2 (ja) | 2021-07-28 |
Family
ID=57320362
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2017559588A Active JP6910961B2 (ja) | 2015-05-15 | 2016-05-14 | 選択細胞への分子送達に適用する遺伝子操作したボツリヌス菌毒素 |
Country Status (8)
Country | Link |
---|---|
US (2) | US10633643B2 (ja) |
EP (1) | EP3294754A4 (ja) |
JP (1) | JP6910961B2 (ja) |
KR (1) | KR20180021703A (ja) |
CN (1) | CN108137654A (ja) |
CA (1) | CA2986026A1 (ja) |
IL (1) | IL255575A (ja) |
WO (1) | WO2016187076A1 (ja) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11129906B1 (en) | 2016-12-07 | 2021-09-28 | David Gordon Bermudes | Chimeric protein toxins for expression by therapeutic bacteria |
US20240091322A1 (en) * | 2021-04-19 | 2024-03-21 | MVRIX Co., Ltd. | Protein complex including botulinum toxin translocation domain and endolysin and antibacterial composition including same |
Family Cites Families (17)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA2296765A1 (en) * | 1996-08-28 | 1998-03-05 | Ophidian Pharmaceuticals, Inc. | Soluble recombinant botulinum toxin proteins |
US6747137B1 (en) * | 1998-02-13 | 2004-06-08 | Genome Therapeutics Corporation | Nucleic acid sequences relating to Candida albicans for diagnostics and therapeutics |
EP1180999A4 (en) * | 1999-05-12 | 2005-03-30 | Us Army Medical Res And Materi | RECOMBINANT VACCINE AGAINST BOTULINUM NEUROTOXIN |
ATE261494T1 (de) * | 1999-12-02 | 2004-03-15 | Health Prot Agency | Konstrukte zur verabreichung von therapeutischen wirkstoffen an die neuronzellen |
GB0112687D0 (en) * | 2001-05-24 | 2001-07-18 | Microbiological Res Authority | Pharmaceutical use of secreted bacterial effector proteins |
GB0216865D0 (en) * | 2002-07-19 | 2002-08-28 | Microbiological Res Authority | Targetted agents for nerve regeneration |
WO2004076634A2 (en) | 2003-02-24 | 2004-09-10 | Ira Sanders | Cell membrane translocation of regulated snare inhibitors , compositions therefor, and methods for treatment of disease |
EP1452589A1 (en) * | 2003-02-28 | 2004-09-01 | Migragen AG | ADP-ribosyltransferase c3cer |
WO2005090393A2 (en) | 2004-02-09 | 2005-09-29 | The Government Of The United States, As Represented By The Secretary Of Health And Human Services | Multimeric protein toxins to target cells having multiple identifying characteristics |
US7083783B2 (en) * | 2004-02-18 | 2006-08-01 | Wisconsin Alumni Research Foundation | Method for treating glaucoma |
DE102004035606A1 (de) * | 2004-07-22 | 2006-03-30 | Biotecon Therapeutics Gmbh | Carrier für Arzneimittel zur Gewinnung der oralen Bioverfügbarkeit |
DE102004043009A1 (de) * | 2004-09-06 | 2006-03-23 | Toxogen Gmbh | Transportprotein zum Einbringen chemischer Verbindungen in Nervenzellen |
US8795989B2 (en) * | 2007-04-30 | 2014-08-05 | University Of Maryland | Enzymic production of neoagarobiose |
WO2009023549A2 (en) * | 2007-08-09 | 2009-02-19 | University Of Rochester | Vaccine against botulism |
KR20100088683A (ko) * | 2007-10-23 | 2010-08-10 | 알러간, 인코포레이티드 | 변형 클로스트리듐 독소를 사용한 만성 신경성 염증의 치료 방법 |
CA2772400A1 (en) * | 2009-08-27 | 2011-03-17 | Synaptic Research, Llc | A novel protein delivery system to generate induced pluripotent stem (ips) cells or tissue-specific cells |
WO2013126690A1 (en) | 2012-02-23 | 2013-08-29 | President And Fellows Of Harvard College | Modified microbial toxin receptor for delivering agents into cells |
-
2016
- 2016-05-14 US US15/574,151 patent/US10633643B2/en active Active
- 2016-05-14 CN CN201680041056.9A patent/CN108137654A/zh active Pending
- 2016-05-14 WO PCT/US2016/032573 patent/WO2016187076A1/en active Application Filing
- 2016-05-14 CA CA2986026A patent/CA2986026A1/en active Pending
- 2016-05-14 KR KR1020177035990A patent/KR20180021703A/ko not_active Application Discontinuation
- 2016-05-14 JP JP2017559588A patent/JP6910961B2/ja active Active
- 2016-05-14 EP EP16797057.3A patent/EP3294754A4/en active Pending
-
2017
- 2017-11-09 IL IL255575A patent/IL255575A/en unknown
-
2020
- 2020-04-21 US US16/854,551 patent/US11118170B2/en active Active
Also Published As
Publication number | Publication date |
---|---|
US11118170B2 (en) | 2021-09-14 |
EP3294754A4 (en) | 2019-04-03 |
US20180127734A1 (en) | 2018-05-10 |
JP2018515542A (ja) | 2018-06-14 |
US10633643B2 (en) | 2020-04-28 |
IL255575A (en) | 2018-01-31 |
CN108137654A (zh) | 2018-06-08 |
EP3294754A1 (en) | 2018-03-21 |
CA2986026A1 (en) | 2016-11-24 |
US20200255815A1 (en) | 2020-08-13 |
KR20180021703A (ko) | 2018-03-05 |
WO2016187076A1 (en) | 2016-11-24 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US9827298B2 (en) | Neurotoxins exhibiting shortened biological activity | |
US7514088B2 (en) | Multivalent Clostridial toxin derivatives and methods of their use | |
TW201629092A (zh) | 陽離子神經毒素 | |
TW201130974A (en) | Modified clostridial toxins comprising an integrated protease cleavage site-binding domain | |
WO2018109447A1 (en) | Neurotoxins | |
CN110072880A (zh) | 工程化肉毒杆菌神经毒素 | |
JP4991073B2 (ja) | 神経細胞への治療薬の送達のための構築物 | |
US11118170B2 (en) | Engineered clostridium botulinum toxin adapted to deliver molecules into selected cells | |
EP1523336B1 (en) | Targeted agents for nerve regeneration | |
TW201819403A (zh) | 雜合神經毒素 | |
JP2022549057A (ja) | 分子の細胞内送達のための複合体 | |
Pavlik et al. | Retargeting the Clostridium botulinum C2 toxin to the neuronal cytosol | |
Oloomi et al. | N-terminus leader sequence of Shiga toxin (Stx) 1 is essential for production of active recombinant protein in E. coli | |
JP5889305B2 (ja) | 組換え神経毒ポリペプチドの選択的製造 | |
Park et al. | Development of plug-and-deliverable intracellular protein delivery platforms based on botulinum neurotoxin | |
EP3512956A1 (en) | Method for producing di-chain clostridial neurotoxins | |
Muraro | Studies of Botulinum Neurotoxins Mechanism of Action |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20180309 |
|
A711 | Notification of change in applicant |
Free format text: JAPANESE INTERMEDIATE CODE: A711 Effective date: 20190201 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A821 Effective date: 20190201 |
|
A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20190508 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20190619 |
|
A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20200318 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20200324 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20200619 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20201201 |
|
A601 | Written request for extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A601 Effective date: 20210222 |
|
A601 | Written request for extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A601 Effective date: 20210428 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20210526 |
|
TRDD | Decision of grant or rejection written | ||
A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20210608 |
|
A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20210707 |
|
R150 | Certificate of patent or registration of utility model |
Ref document number: 6910961 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |