ATE528396T1 - Look-through-mutagenese zur entwicklung veränderter polypeptide mit verbesserten eigenschaften - Google Patents
Look-through-mutagenese zur entwicklung veränderter polypeptide mit verbesserten eigenschaftenInfo
- Publication number
- ATE528396T1 ATE528396T1 AT05809866T AT05809866T ATE528396T1 AT E528396 T1 ATE528396 T1 AT E528396T1 AT 05809866 T AT05809866 T AT 05809866T AT 05809866 T AT05809866 T AT 05809866T AT E528396 T1 ATE528396 T1 AT E528396T1
- Authority
- AT
- Austria
- Prior art keywords
- polypeptide
- amino acids
- mutagenesis
- libraries
- look
- Prior art date
Links
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
- 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
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/102—Mutagenizing nucleic acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
-
- 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
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
- G01N33/6857—Antibody fragments
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Hematology (AREA)
- Physics & Mathematics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- General Engineering & Computer Science (AREA)
- Urology & Nephrology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Analytical Chemistry (AREA)
- Cell Biology (AREA)
- Plant Pathology (AREA)
- Food Science & Technology (AREA)
- Crystallography & Structural Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Bioinformatics & Computational Biology (AREA)
- Peptides Or Proteins (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US58591804P | 2004-07-06 | 2004-07-06 | |
| PCT/US2005/024140 WO2006023144A2 (en) | 2004-07-06 | 2005-07-06 | Look-through mutagenesis for developing altered polypeptides with enhanced properties |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| ATE528396T1 true ATE528396T1 (de) | 2011-10-15 |
Family
ID=35968026
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AT05809866T ATE528396T1 (de) | 2004-07-06 | 2005-07-06 | Look-through-mutagenese zur entwicklung veränderter polypeptide mit verbesserten eigenschaften |
Country Status (9)
| Country | Link |
|---|---|
| US (1) | US9012369B2 (de) |
| EP (1) | EP1774019B1 (de) |
| JP (2) | JP4939410B2 (de) |
| AT (1) | ATE528396T1 (de) |
| BR (1) | BRPI0513155B1 (de) |
| CA (1) | CA2572917C (de) |
| ES (1) | ES2372503T3 (de) |
| MX (1) | MX2007000105A (de) |
| WO (1) | WO2006023144A2 (de) |
Families Citing this family (75)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9321832B2 (en) | 2002-06-28 | 2016-04-26 | Domantis Limited | Ligand |
| CN104761637B (zh) | 2006-03-31 | 2021-10-15 | 中外制药株式会社 | 调控抗体血液动力学的方法 |
| WO2007127290A2 (en) * | 2006-04-24 | 2007-11-08 | Protelix, Inc. | Method for producing viral vaccine and therapeutic peptide antigens |
| CA2666599A1 (en) | 2006-08-18 | 2008-02-21 | Ablynx N.V. | Amino acid sequences directed against il-6r and polypeptides comprising the same for the treatment of diseases and disorders associated with il-6-mediated signalling |
| US20090118130A1 (en) | 2007-02-12 | 2009-05-07 | Codexis, Inc. | Structure-activity relationships |
| EP2468854B1 (de) * | 2007-07-12 | 2013-09-11 | New England Biolabs, Inc. | Restriktionsendonukleasen mit hoher Genauigkeit |
| CN101939425B (zh) * | 2007-09-26 | 2014-05-14 | 中外制药株式会社 | 抗il-6受体抗体 |
| EP3415529B1 (de) | 2007-09-26 | 2020-11-04 | Chugai Seiyaku Kabushiki Kaisha | Modifizierte antikörperkonstantregion |
| CN101874042B9 (zh) | 2007-09-26 | 2019-01-01 | 中外制药株式会社 | 利用cdr的氨基酸取代来改变抗体等电点的方法 |
| WO2009068631A1 (de) | 2007-11-27 | 2009-06-04 | Ablynx N.V. | Verfahren und vorrichtung zum herstellen, insbesondere zum runderneuern, von reifen |
| WO2009088924A2 (en) * | 2007-12-31 | 2009-07-16 | Xoma Technology Ltd. | Methods and materials for targeted affinity enhancement |
| EP2275443B1 (de) | 2008-04-11 | 2015-12-02 | Chugai Seiyaku Kabushiki Kaisha | Antigen bindendes molekül, das dazu in der lage ist, wiederholt zwei oder mehr antigenmoleküle zu binden |
| US9296810B2 (en) | 2008-05-02 | 2016-03-29 | Novartis Ag | Fibronectin-based binding molecules and uses thereof |
| TWI440469B (zh) | 2008-09-26 | 2014-06-11 | Chugai Pharmaceutical Co Ltd | Improved antibody molecules |
| WO2010107110A1 (ja) | 2009-03-19 | 2010-09-23 | 中外製薬株式会社 | 抗体定常領域改変体 |
| TWI682995B (zh) | 2009-03-19 | 2020-01-21 | 日商中外製藥股份有限公司 | 抗體恆定區域改變體 |
| US8987433B2 (en) | 2009-03-27 | 2015-03-24 | Sapphire Energy, Inc. | Variant isoprenoid producing enzymes and uses thereof |
| US8748581B2 (en) | 2009-04-10 | 2014-06-10 | Ablynx N.V. | Anti-IL-6R polypeptides and pharmaceutical compositions thereof |
| US10618964B2 (en) | 2009-04-10 | 2020-04-14 | Ablynx N.V. | Nanobody against IL-6R |
| KR101852204B1 (ko) | 2009-06-05 | 2018-04-26 | 아블린쓰 엔.브이. | 기도 감염의 예방 및/또는 치료를 위한 일가, 이가 및 삼가 항 인간 호흡기 세포융합 바이러스 (ｈｒｓｖ) 나노바디 구조물 |
| US8518405B2 (en) * | 2009-10-08 | 2013-08-27 | The University Of North Carolina At Charlotte | Tumor specific antibodies and uses therefor |
| WO2011051327A2 (en) | 2009-10-30 | 2011-05-05 | Novartis Ag | Small antibody-like single chain proteins |
| WO2011051466A1 (en) | 2009-11-02 | 2011-05-05 | Novartis Ag | Anti-idiotypic fibronectin-based binding molecules and uses thereof |
| WO2011064382A1 (en) | 2009-11-30 | 2011-06-03 | Ablynx N.V. | Improved amino acid sequences directed against human respiratory syncytial virus (hrsv) and polypeptides comprising the same for the prevention and/or treatment of respiratory tract infections |
| WO2011092233A1 (en) | 2010-01-29 | 2011-08-04 | Novartis Ag | Yeast mating to produce high-affinity combinations of fibronectin-based binders |
| MX345300B (es) | 2010-07-30 | 2017-01-24 | Novartis Ag * | Moléculas andamio de fibronectina y bibliotecas de las mismas. |
| EP2619226B1 (de) * | 2010-09-22 | 2018-09-12 | Amgen Inc. | Träger-immunglobuline und verwendungen davon |
| US9845362B2 (en) | 2010-10-08 | 2017-12-19 | The University Of North Carolina At Charlotte | Compositions comprising chimeric antigen receptors, T cells comprising the same, and methods of using the same |
| TWI654204B (zh) | 2010-11-30 | 2019-03-21 | Chugai Seiyaku Kabushiki Kaisha | 具有鈣依存性的抗原結合能力之抗體 |
| EP2747782B1 (de) | 2011-09-23 | 2018-01-17 | Ablynx NV | Verlängerte inhibition der interleukin-6-vermittelten signalisierung |
| KR20220116585A (ko) | 2011-10-11 | 2022-08-23 | 비엘라 바이오, 인크. | Cd40l 특이적 tn3 유래 스카폴드 및 이의 사용 방법 |
| CA3233142A1 (en) | 2011-11-30 | 2013-06-06 | Chugai Seiyaku Kabushiki Kaisha | Drug containing carrier into cell for forming immune complex |
| MX2014010495A (es) | 2012-03-02 | 2014-11-14 | Ablynx Nv | Polipeptidos que enlazan a pcrv. |
| WO2014087010A1 (en) | 2012-12-07 | 2014-06-12 | Ablynx N.V. | IMPROVED POLYPEPTIDES DIRECTED AGAINST IgE |
| WO2014116051A1 (ko) * | 2013-01-24 | 2014-07-31 | 스크립스코리아 항체연구원 | 단백질 조합 기반의 Fv 라이브러리 및 이의 제조 방법 |
| EP2989202B1 (de) | 2013-04-25 | 2019-04-10 | Sutro Biopharma, Inc. | Die verwendung des lambda-gam proteins in der ribosomen-display-technologie |
| US20160069897A1 (en) * | 2013-04-25 | 2016-03-10 | Suthro Biopharma, Inc. | Selection of fab fragments using ribosomal display technology |
| WO2014200018A1 (ja) | 2013-06-11 | 2014-12-18 | 独立行政法人 国立精神・神経医療研究センター | 再発寛解型多発性硬化症（ｒｒｍｓ）患者の治療予後予測方法、及び新規治療適応判断方法 |
| TWI707038B (zh) | 2013-08-05 | 2020-10-11 | 美商扭轉生物科技有限公司 | 重新合成之基因庫 |
| US11554181B2 (en) | 2014-09-05 | 2023-01-17 | The University Of North Carolina At Charlotte | Tumor specific antibody conjugates and uses therefor |
| JP2017534617A (ja) | 2014-10-21 | 2017-11-24 | アブリンクス エン．ヴェー． | Ｉｌ−６ｒ関連疾患の処置 |
| WO2016126882A1 (en) | 2015-02-04 | 2016-08-11 | Twist Bioscience Corporation | Methods and devices for de novo oligonucleic acid assembly |
| TWI873632B (zh) | 2015-02-27 | 2025-02-21 | 日商中外製藥股份有限公司 | Il-6受體抗體用於製備醫藥組成物的用途 |
| CN107428817B (zh) | 2015-03-12 | 2022-07-12 | 免疫医疗有限责任公司 | 纯化白蛋白融合蛋白的方法 |
| US9981239B2 (en) | 2015-04-21 | 2018-05-29 | Twist Bioscience Corporation | Devices and methods for oligonucleic acid library synthesis |
| PL3299810T3 (pl) | 2015-05-19 | 2021-12-13 | National Center Of Neurology And Psychiatry | Sposób określania zastosowania nowej terapii u pacjentów ze stwardnieniem rozsianym (sm) |
| EP3297661B1 (de) | 2015-05-21 | 2021-06-30 | Full Spectrum Genetics, Inc. | Verfahren zur verbesserung der eigenschaften von proteinen |
| KR20180050411A (ko) * | 2015-09-18 | 2018-05-14 | 트위스트 바이오사이언스 코포레이션 | 올리고핵산 변이체 라이브러리 및 그의 합성 |
| CN108698012A (zh) | 2015-09-22 | 2018-10-23 | 特韦斯特生物科学公司 | 用于核酸合成的柔性基底 |
| WO2017095958A1 (en) | 2015-12-01 | 2017-06-08 | Twist Bioscience Corporation | Functionalized surfaces and preparation thereof |
| USD782516S1 (en) | 2016-01-19 | 2017-03-28 | Apple Inc. | Display screen or portion thereof with graphical user interface |
| KR102212257B1 (ko) | 2016-08-22 | 2021-02-04 | 트위스트 바이오사이언스 코포레이션 | 드 노보 합성된 핵산 라이브러리 |
| WO2018057526A2 (en) | 2016-09-21 | 2018-03-29 | Twist Bioscience Corporation | Nucleic acid based data storage |
| EP3554514A4 (de) | 2016-12-16 | 2020-08-05 | Twist Bioscience Corporation | Variante bibliotheken der immunologischen synapse und synthese davon |
| CN110892485B (zh) | 2017-02-22 | 2024-03-22 | 特韦斯特生物科学公司 | 基于核酸的数据存储 |
| AU2018234629A1 (en) | 2017-03-15 | 2019-10-17 | Twist Bioscience Corporation | Variant libraries of the immunological synapse and synthesis thereof |
| US11851486B2 (en) | 2017-05-02 | 2023-12-26 | National Center Of Neurology And Psychiatry | Method for predicting and evaluating therapeutic effect in diseases related to IL-6 and neutrophils |
| WO2018231864A1 (en) | 2017-06-12 | 2018-12-20 | Twist Bioscience Corporation | Methods for seamless nucleic acid assembly |
| JP7169999B2 (ja) | 2017-06-12 | 2022-11-11 | ツイスト バイオサイエンス コーポレーション | シームレス核酸アセンブリのための方法 |
| AU2018328847A1 (en) | 2017-09-11 | 2020-04-23 | Twist Bioscience Corporation | GPCR binding proteins and synthesis thereof |
| US12605469B2 (en) | 2017-10-02 | 2026-04-21 | The University Of North Carolina At Charlotte | Tumor specific antibody conjugates and uses therefor |
| WO2019079769A1 (en) | 2017-10-20 | 2019-04-25 | Twist Bioscience Corporation | HEATED NANOWELLS FOR THE SYNTHESIS OF POLYNUCLEOTIDES |
| US11692037B2 (en) | 2017-10-20 | 2023-07-04 | Hyogo College Of Medicine | Anti-IL-6 receptor antibody-containing medicinal composition for preventing post-surgical adhesion |
| EP4715681A2 (de) | 2018-01-04 | 2026-03-25 | Atlas Data Storage, Inc. | Digitale informationsspeicherung auf dna-basis |
| WO2019151418A1 (ja) | 2018-01-31 | 2019-08-08 | 元一 加藤 | Ｉｌ－６阻害剤を含有する喘息の治療剤 |
| IL278771B2 (en) | 2018-05-18 | 2025-09-01 | Twist Bioscience Corp | Polynucleotides, reagents and methods for nucleic acid hybridization |
| WO2020139871A1 (en) | 2018-12-26 | 2020-07-02 | Twist Bioscience Corporation | Highly accurate de novo polynucleotide synthesis |
| WO2020176678A1 (en) | 2019-02-26 | 2020-09-03 | Twist Bioscience Corporation | Variant nucleic acid libraries for glp1 receptor |
| AU2020227802A1 (en) | 2019-02-26 | 2021-10-14 | Twist Bioscience Corporation | Variant nucleic acid libraries for antibody optimization |
| WO2020213665A1 (ja) | 2019-04-17 | 2020-10-22 | 国立大学法人広島大学 | Ｉｌ－６阻害剤及びｃｃｒ２阻害剤を組み合わせて投与することを特徴とする泌尿器がんの治療剤 |
| CA3144644A1 (en) | 2019-06-21 | 2020-12-24 | Twist Bioscience Corporation | Barcode-based nucleic acid sequence assembly |
| US12091777B2 (en) | 2019-09-23 | 2024-09-17 | Twist Bioscience Corporation | Variant nucleic acid libraries for CRTH2 |
| EP4034564A4 (de) | 2019-09-23 | 2023-12-13 | Twist Bioscience Corporation | Variante nukleinsäurebibliotheken für einzeldomänenantikörper |
| BR112022011235A2 (pt) | 2019-12-09 | 2022-12-13 | Twist Bioscience Corp | Bibliotecas de variantes de ácido nucleico para receptores de adenosina |
| TWI872251B (zh) | 2020-05-29 | 2025-02-11 | 日商中外製藥股份有限公司 | 含有抗體製劑 |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0527809B1 (de) * | 1990-04-05 | 1995-08-16 | CREA, Roberto | "walk-through"-mutagenese |
| EP1230269A2 (de) * | 1999-11-03 | 2002-08-14 | Maxygen, Inc. | Erzeugung von antikörperdiversität |
| EP1240319A1 (de) * | 1999-12-15 | 2002-09-18 | Genentech, Inc. | "shotgun scanning", eine methode zur kartierung von funktionellen protein epitopen |
| US7647184B2 (en) | 2001-08-27 | 2010-01-12 | Hanall Pharmaceuticals, Co. Ltd | High throughput directed evolution by rational mutagenesis |
| NZ535927A (en) * | 2002-04-17 | 2007-06-29 | Roberto Crea | Universal libraries for immunoglobulins |
| MXPA04010222A (es) * | 2002-04-17 | 2005-03-07 | Roberto Crea | Dopacion en mutagenesis en ruta. |
| JP4791960B2 (ja) * | 2003-06-27 | 2011-10-12 | バイオレン，インク． | ルックスルー突然変異誘発 |
-
2005
- 2005-07-06 MX MX2007000105A patent/MX2007000105A/es active IP Right Grant
- 2005-07-06 AT AT05809866T patent/ATE528396T1/de not_active IP Right Cessation
- 2005-07-06 CA CA2572917A patent/CA2572917C/en not_active Expired - Lifetime
- 2005-07-06 BR BRPI0513155-3A patent/BRPI0513155B1/pt active IP Right Grant
- 2005-07-06 EP EP05809866A patent/EP1774019B1/de not_active Expired - Lifetime
- 2005-07-06 JP JP2007520506A patent/JP4939410B2/ja not_active Expired - Fee Related
- 2005-07-06 WO PCT/US2005/024140 patent/WO2006023144A2/en not_active Ceased
- 2005-07-06 ES ES05809866T patent/ES2372503T3/es not_active Expired - Lifetime
- 2005-07-06 US US11/571,088 patent/US9012369B2/en active Active
-
2011
- 2011-09-15 JP JP2011201518A patent/JP2012041347A/ja active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| ES2372503T3 (es) | 2012-01-20 |
| WO2006023144A2 (en) | 2006-03-02 |
| JP2008505642A (ja) | 2008-02-28 |
| JP2012041347A (ja) | 2012-03-01 |
| US9012369B2 (en) | 2015-04-21 |
| WO2006023144A3 (en) | 2006-08-17 |
| EP1774019A2 (de) | 2007-04-18 |
| CA2572917A1 (en) | 2006-03-02 |
| BRPI0513155A (pt) | 2008-04-29 |
| US20080214406A1 (en) | 2008-09-04 |
| JP4939410B2 (ja) | 2012-05-23 |
| MX2007000105A (es) | 2007-07-18 |
| CA2572917C (en) | 2012-04-03 |
| BRPI0513155B1 (pt) | 2021-07-20 |
| EP1774019B1 (de) | 2011-10-12 |
| EP1774019A4 (de) | 2009-05-20 |
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