AntibodyEngineering
MethodsandProtocols
ThirdEdition
Editedby DamienNevoltris
GarvanInstituteofMedicalResearch,ImmunologyDivision,Darlinghurst,NSW,Australia
PatrickChames
InstitutPaoli-Calmettes,CRCM,AixMarseilleUniversity,CNRS,INSERM,Marseille,France
Editors
DamienNevoltris GarvanInstituteofMedicalResearch ImmunologyDivision
Darlinghurst,NSW,Australia
PatrickChames InstitutPaoli-Calmettes
CRCM,AixMarseilleUniversity,CNRS,INSERM
Marseille,France
ISSN1064-3745ISSN1940-6029(electronic)
MethodsinMolecularBiology
ISBN978-1-4939-8647-7ISBN978-1-4939-8648-4(eBook) https://doi.org/10.1007/978-1-4939-8648-4
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Preface
Theuseofantibody-basedtherapeuticshasgrownexponentiallyinthepastfewdecades, nowrepresentingalargecomponentoftherapeuticdrugsthatwasdominatedbysmall organicmoleculesupuntilthelate1990s.Antibodieshaveprovenversatileintreatinga varietyofdiseasesincludingcancer,autoimmunity,infectiousdiseases,orevenneurodegenerativedisorders.Asof2017,70therapeuticantibodieshavebeenapprovedbytheFDA, andmorethan550promisingcandidatesareindifferentphasesofclinicaltrials.They currentlyrepresent20%ofthetop100sellingdrugs,upfromjust1%in2007.However, majorimprovementsandbreakthroughshavebeennecessarytoachievetheseimpressive results.
The1970swerethestartofgreatrevolutionsinthefield:GeraldEdelmanandRodney PorterwereawardedtheNobelPrizefortheirworkonthemolecularstructureof antibodies,thefirstatomicresolutionstructureofanantibodyfragmentwaspublished, followedbythegroundbreakingdevelopmentofhybridomatechnologybyGeorges J.F.Ko ¨ hlerandCe ´ sarMilstein.Thistechnologyallowedantibodiestobeproducedand characterizedasmonoclonals,startingthemoderneraofantibodyengineering.
Despitethisrevolution,thesuccessofantibodiesastherapeuticmoleculeswasnot immediate,andmostclinicalstudiesledtodisappointments.Firstmurineantibodiesused astreatmenthadmanylimitations,suchasashortinvivohalf-life,limitedtumorpenetration,inefficientrecruitmentofhosteffectorfunctions,andmostofall,immuneresponse fromthepatientagainsttheinjectedantibody,alsocalled“HAMA”response,referringto theproductionofneutralizinghumananti-mouseantibodies.
Formanyyears,researchersdevelopedstrategiestoabrogatethisproblem;thejourney towardantibodyhumanizationbegan.Fullymurineantibodiesfirstprogressedtochimeras, wherevariableregionsfrommurineoriginwereassembledontohumanconstantdomains, thentohumanizedantibodiesbyinsertionofonlytherelevantCDRsontohumanantibody scaffolds.Finally,fullyhumanantibodiesweregenerated,directlyingeneticallymodified mice,selectedfromhumansyntheticantibodylibrariesorbysequencingofhumanplasma cells.
However,immunogenicitywasnottheonlyfactorholdingupthedevelopmentof antibodies.Indeed,theclassicalarchitectureofimmunoglobulinmoleculesbearssome inherentlimitations.Manyinnovativeformatshavebeenexploredtoovercomethese majorhurdles,suchasreducingtheantibodytoitsminimalfunctionalsize,modulating thevalency,the(multi)specificity,increasingthehalf-life,andenhancingtherecruitmentof immuneeffectorcells.
Asthedemandformonoclonalantibodiesinresearchandclinicalapplicationscontinues toincrease,thenecessitytodevelopevenmoreefficientmoleculesiscrucial.Antibody engineeringhasbecomeakeydisciplineforgenerationofinnovativeantibodies-based moleculesusedinresearch,diagnostics,andtherapy.
Thisthirdeditionof AntibodyEngineering:MethodsandProtocols remainsinthelineage ofitspredecessorsandgivesthereaderscompleteandeasyaccesstoavarietyofantibody engineeringtechniques.Fromthegenerationofnative,synthetic,orimmuneantibody libraries,theselectionofleadcandidatesthankstodifferentpowerfulandinnovating displaytechnologies,totheirproduction,characterization,andoptimization,thishandbook providesthereaderwithanextensivetoolboxtocreatethepowerfulmoleculesoftomorrow.
Darlinghurst,NSW,AustraliaDamienNevoltris Marseille,FrancePatrickChames
Preface.. ...................................................................v
Contributors .................................................................xi
PART IIN SILICO METHODS
1AntibodyDesignandHumanizationviaInSilicoModeling... ...............3 VinodhB.KurellaandReddyGali
2AntibodyAffinityMaturationbyComputationalDesign .....................15 DaisukeKurodaandKouheiTsumoto
3UseofIMGT® DatabasesandToolsforAntibodyEngineering andHumanization.. ....................................................35 Marie-PauleLefranc,Franc¸oisEhrenmann,SofiaKossida, Ve´roniqueGiudicelli,andPatriceDuroux
PART IIGENERATIONOF DIVERSITY
4ConstructionofHumanNaıveAntibodyGeneLibraries .....................73 MichelaPasello,AlessandraMallano,MichelaFlego,SilviaZamboni, AnnaMariaGiudice,andKatiaScotlandi
5ConstructionofSyntheticAntibodyLibraries ..............................93 De´borahCaucheteur,GautierRobin,VincentParez,and PierreMartineau
6ConstructionofHistidine-EnrichedSharkIgNARVariableDomain AntibodyLibrariesfortheIsolationofpH-SensitivevNARFragments ........109 DoreenKo¨nning,SteffenHinz,JuliusGrzeschik,ChristianSchro¨ter, SimonKrah,StefanZielonka,andHaraldKolmar
7DisplayTechnologiesforGenerationofIgSingleVariableDomains... ........129 VladimirBobkov,BasvanderWoning,andHansdeHaard
8AStreamlinedApproachfortheConstructionofLargeYeast SurfaceDisplayFabAntibodyLibraries ....................................145 SimonKrah,JuliusGrzeschik,SimonRosowski,RamonaGaa, IrisWillenbuecher,DenizDemir,LarsToleikis,HaraldKolmar, StefanBecker,andStefanZielonka
PART IIISELECTIONSOF LEAD CANDIDATE
9PhageDisplayandSelectionsonPurifiedAntigens... .......................165 MagaliColazetandPatrickChames
10SelectionofAntibodiestoTransientlyExpressedMembrane ProteinsUsingPhageDisplay ............................................179 MartinaL.Jones,StephenM.Mahler,andSumukhKumble
11SelectionofAntibodyFragmentsAgainstStructuredDNAby PhageDisplay ..........................................................197
MahdiZeraati,MarcelE.Dinger,andDanielChrist
12SelectionofAntibodyFragmentsbyYeastDisplay... .......................211 NathalieScholler
13RapidSelectionofHigh-AffinityAntibodyscFvFragments UsingRibosomeDisplay .................................................235
BirgitDreierandAndreasPlu ¨ ckthun
14InVitroSelectionofSingle-DomainAntibody(VHH)Using cDNADisplay ..........................................................269
NaotoNemoto,ShigefumiKumachi,andHidenaoArai
15SequencingandAffinityDeterminationofAntigen-SpecificB LymphocytesfromPeripheralBlood. .....................................287
PeterSchofield,RodrigoVazquez-Lombardi,MahmoudAbdelatti, DamienNevoltris,ChristopherC.Goodnow,DanielChrist, andJoanneH.Reed
PART IVPRODUCTIONOF RECOMBINANT ANTIBODIES
16ExpressionofIgGMonoclonalswithEngineeredImmune EffectorFunctions.. ....................................................313 RodrigoVazquez-Lombardi,DamienNevoltris, RomainRouet,andDanielChrist
17AnIRES-MediatedTricistronicVectorforEfficientGenerationof Stable,High-LevelMonoclonalAntibodyProducingCHO DG44CellLines........................................................335 JessnaH.M.Yeo,Mariati,andYuanshengYang
18Production,Purification,andCharacterizationofAntibody-TNF SuperfamilyLigandFusionProteins.. .....................................351 MartinSiegemund,NadineBeha,andDafneMu ¨ ller
PART VFC ENGINEERING
19ChemoenzymaticDefucosylationofTherapeuticAntibodiesfor EnhancedEffectorFunctionsUsingBacterial α-Fucosidases.. ...............367 ChaoLi,TiezhengLi,andLai-XiWang
20FcGlyco-andFcProtein-Engineering:DesignofAntibody VariantswithImprovedADCCandCDCActivity... .......................381 ChristianKellner,StefanieDerer,KatjaKlausz,SophiaRosskopf, TimWirt,ThiesRo¨sner,AnnaOtte,ElisaCappuzzello, andMatthiasPeipp
21FcEngineering:TailoredSyntheticHumanIgG1-FcRepertoire forHigh-AffinityInteractionwithFcRnatpH6.0... .......................399 AbhishekSaxena,BingxinBai,Shin-ChenHou,LianlianJiang, TianleiYing,ShaneMiersch,SachdevS.Sidhu,andDonghuiWu
PART VICHARACTERIZATION,OPTIMIZATION, AND INNOVATIVE FORMATS
22MeasuringAntibody-AntigenBindingKineticsUsingSurface PlasmonResonance. ....................................................421
StephenHearty,PaulLeonard,HuiMa,andRichardO’Kennedy
23ParallelEvolutionofAntibodyAffinityandThermalStability forOptimalBiotherapeuticDevelopment ..................................457
EdwardFranklin,OrlaCunningham,andBrianFennell
24TheUseofSomaticHypermutationfortheAffinityMaturation ofTherapeuticAntibodies.. .............................................479
PeterM.Bowers,WilliamJ.Boyle,andRobertDamoiseaux
25SelectionandUseofIntracellularAntibodies. ..............................491 SandrineMoutel,Cle´mentNizak,andFranckPerez
26Site-SpecificRadioactiveLabelingofNanobodies ...........................505 MaxineCrauwels,SamMassa,CharlotteMartin,CeciliaBetti, StevenBallet,NickDevoogdt,CatarinaXavier,andSergeMuyldermans
Index... ...................................................................541
Contributors
MAHMOUD ABDELATTI GarvanInstituteofMedicalResearch,Sydney,NSW,Australia; FacultyofMedicine,St.Vincent’sClinicalSchool,UniversityofNewSouthWales,Sydney, NSW,Australia
HIDENAO ARAI EpsilonMolecularEngineering,Inc.,Saitama,Japan
BINGXIN BAI LaboratoryofAntibodyEngineering,ShanghaiInstituteforAdvanced ImmunochemicalStudies,ShanghaiTechUniversity,Shanghai,China
STEVEN BALLET ResearchGroupofOrganicChemistry,DepartmentsofChemistryand BioengineeringSciences,VrijeUniversiteitBrussel,Brussels,Belgium
STEFAN BECKER ProteinEngineeringandAntibodyTechnologies,MerckKGaA,Darmstadt, Germany
NADINE BEHA InstituteofCellBiologyandImmunology,UniversityofStuttgart,Stuttgart, Germany
CECILIA BETTI ResearchGroupofOrganicChemistry,DepartmentsofChemistryand BioengineeringSciences,VrijeUniversiteitBrussel,Brussels,Belgium
VLADIMIR BOBKOV argenxBVBA,Zwijnaarde,Belgium
PETER M.BOWERS ClinicalandTranslationalScienceInstitute,UCLADavidGeffen SchoolofMedicine,UniversityofCalifornia,LosAngeles,LosAngeles,CA,USA
WILLIAM J.BOYLE ClinicalandTranslationalScienceInstitute,UCLADavidGeffen SchoolofMedicine,UniversityofCalifornia,LosAngeles,LosAngeles,CA,USA
ELISA CAPPUZZELLO DivisionofStemCellTransplantationandImmunotherapy, DepartmentofMedicineII,UniversityHospitalSchleswig-HolsteinandChristianAlbrechts,UniversityofKiel,Kiel,Germany;DepartmentofSurgery,Oncologyand Gastroenterology,OncologyandImmunologySection,UniversityofPadua,Padua,Italy
DE ´ BORAH CAUCHETEUR InstitutdeRechercheenCance´rologiedeMontpellier(IRCM), Montpellier,France;INSERM,U1194,Montpellier,France;Universite´deMontpellier, Montpellier,France;InstitutRe´gionalduCancerdeMontpellier,Montpellier,France
PATRICK CHAMES InstitutPaoli-Calmettes,CRCM,AixMarseilleUniversity,CNRS, INSERM,Marseille,France
DANIEL CHRIST GarvanInstituteofMedicalResearch,Sydney,NSW,Australia;Facultyof Medicine,St.Vincent’sClinicalSchool,UniversityofNewSouthWales,Sydney,NSW, Australia
MAGALI COLAZET CentredeRechercheenCance´rologiedeMarseille(CRCM),CNRS, INSERM,InstitutPaoli-Calmettes,AixMarseilleUniversity,Marseille,France
MAXINE CRAUWELS CellularandMolecularImmunology,VrijeUniversiteitBrussel,Brussel, Belgium;InVivoCellularandMolecularImagingLaboratory,VrijeUniversiteitBrussel, Brussels,Belgium
ORLA CUNNINGHAM BiomedicineDesign,Pfizer,Dublin,Ireland
ROBERT DAMOISEAUX DepartmentofMolecularandMedicalPharmacology,California NanoSystemsInstitute,UniversityofCalifornia,LosAngeles,LosAngeles,CA,USA
HANSDE HAARD argenxBVBA,Zwijnaarde,Belgium
DENIZ DEMIR ProteinEngineeringandAntibodyTechnologies,MerckKGaA,Darmstadt, Germany
STEFANIE DERER InstituteofNutritionalMedicine,MolecularGastroenterology,University HospitalSchleswig-Holstein,CampusLu¨beck,Lu¨beck,Germany
NICK DEVOOGDT InVivoCellularandMolecularImagingLaboratory,VrijeUniversiteit Brussel,Brussels,Belgium
MARCEL E.DINGER GarvanInstituteofMedicalResearch,Sydney,NSW,Australia; FacultyofMedicine,St.VincentsClinicalSchool,UniversityofNewSouthWales,Sydney, NSW,Australia
BIRGIT DREIER DepartmentofBiochemistry,UniversityofZurich,Zurich,Switzerland
PATRICE DUROUX IMGT®,TheInternationalImMunoGeneTicsInformationSystem®, Laboratoired’ImmunoGe´ne´tiqueMole´culaire(LIGM),InstitutdeGe´ne´tiqueHumaine (IGH),UMR9002CNRS-UM,Universite´deMontpellier,MontpellierCedex,France
FRANC¸OIS EHRENMANN IMGT®,TheInternationalImMunoGeneTicsInformation System®,Laboratoired’ImmunoGe´ne´tiqueMole´culaire(LIGM),InstitutdeGe´ne´tique Humaine(IGH),UMR9002CNRS-UM,Universite´deMontpellier,MontpellierCedex, France;UMR1202BIOGECO,INRA,Universite´Bordeaux,SitederecherchesFore ˆ tBois dePierroton,CestasCedex,France
BRIAN FENNELL BiomedicineDesign,Pfizer,Dublin,Ireland
MICHELA FLEGO NationalCenterforGlobalHealth,TheNationalInstituteofHealth, Rome,Italy
EDWARD FRANKLIN BiomedicineDesign,Pfizer,Dublin,Ireland
RAMONA GAA ProteinEngineeringandAntibodyTechnologies,MerckKGaA,Darmstadt, Germany
REDDY GALI TheHarvardClinicalandTranslationalScienceCenterandCountway LibraryofMedicine,HarvardMedicalSchool,Boston,MA,USA;Departmentof BiomedicalInformatics,HarvardMedicalSchool,Boston,MA,USA
ANNA MARIA GIUDICE CRSDevelopmentofBiomolecularTherapies,Experimental OncologyLab,RizzoliOrthopedicInstitute,Bologna,Italy
VE ´ RONIQUE GIUDICELLI IMGT®,TheInternationalImMunoGeneTicsInformation System®,Laboratoired’ImmunoGe´ne´tiqueMole´culaire(LIGM),InstitutdeGe´ne´tique Humaine(IGH),UMR9002CNRS-UM,Universite´deMontpellier,MontpellierCedex, France
CHRISTOPHER C.GOODNOW GarvanInstituteofMedicalResearch,Sydney,NSW, Australia;FacultyofMedicine,St.Vincent’sClinicalSchool,UniversityofNewSouth Wales,Sydney,NSW,Australia
JULIUS GRZESCHIK InstituteforOrganicChemistryandBiochemistry,Technische Universit € atDarmstadt,Darmstadt,Germany
STEPHEN HEARTY SchoolofBiotechnology,DublinCityUniversity,Dublin,Ireland; NationalCentreforSensorResearch,DublinCityUniversity,Dublin,Ireland
STEFFEN HINZ InstituteforOrganicChemistryandBiochemistry,TechnischeUniversit € at Darmstadt,Darmstadt,Germany
SHIN-CHEN HOU LaboratoryofAntibodyEngineering,ShanghaiInstituteforAdvanced ImmunochemicalStudies,ShanghaiTechUniversity,Shanghai,China
LIANLIAN JIANG LaboratoryofAntibodyEngineering,ShanghaiInstituteforAdvanced ImmunochemicalStudies,ShanghaiTechUniversity,Shanghai,China
MARTINA L.JONES ARCTrainingCentreforBiopharmaceuticalInnovation,Australian InstituteforBioengineeringandNanotechnology(AIBN),TheUniversityofQueensland, St.Lucia,QLD,Australia
DOREEN KONNING InstituteforOrganicChemistryandBiochemistry,Technische Universit € atDarmstadt,Darmstadt,Germany;Antibody-DrugConjugatesandTargeted NBETherapeutics,MerckKGaA,Darmstadt,Germany
CHRISTIAN KELLNER DivisionofStemCellTransplantationandImmunotherapy, DepartmentofMedicineII,UniversityHospitalSchleswig-HolsteinandChristianAlbrechts,UniversityofKiel,Kiel,Germany
KATJA KLAUSZ DivisionofStemCellTransplantationandImmunotherapy,Departmentof MedicineII,UniversityHospitalSchleswig-HolsteinandChristian-Albrechts,Universityof Kiel,Kiel,Germany
HARALD KOLMAR InstituteforOrganicChemistryandBiochemistry,TechnischeUniversitat Darmstadt,Darmstadt,Germany
SOFIA KOSSIDA IMGT®,TheInternationalImMunoGeneTicsInformationSystem®, Laboratoired’ImmunoGe´ne´tiqueMole´culaire(LIGM),InstitutdeGe´ne´tiqueHumaine (IGH),UMR9002CNRS-UM,Universite´deMontpellier,MontpellierCedex,France
SIMON KRAH InstituteforOrganicChemistryandBiochemistry,TechnischeUniversit € at Darmstadt,Darmstadt,Germany;ProteinEngineeringandAntibodyTechnologies,Merck KGaA,Darmstadt,Germany
SHIGEFUMI KUMACHI EpsilonMolecularEngineering,Inc.,Saitama,Japan
SUMUKH KUMBLE ARCTrainingCentreforBiopharmaceuticalInnovation,Australian InstituteforBioengineeringandNanotechnology(AIBN),TheUniversityofQueensland, St.Lucia,QLD,Australia
VINODH B.KURELLA ProteinEngineering,Immuno-OncologyDivision,Intrexon Corporation,Germantown,MD,USA;MerrimackPharmaceuticals,ProteinEngineering, Cambridge,MA,USA
DAISUKE KURODA DepartmentofBioengineering,SchoolofEngineering,TheUniversityof Tokyo,Tokyo,Japan
MARIE-PAULE LEFRANC IMGT®,TheInternationalImMunoGeneTicsInformation System®,Laboratoired’ImmunoGe´ne´tiqueMole´culaire(LIGM),InstitutdeGe´ne´tique Humaine(IGH),UMR9002CNRS-UM,Universite´deMontpellier,MontpellierCedex, France
PAUL LEONARD SchoolofBiotechnology,DublinCityUniversity,Dublin,Ireland;National CentreforSensorResearch,DublinCityUniversity,Dublin,Ireland
CHAO LI DepartmentofChemistryandBiochemistry,UniversityofMaryland,College Park,MD,USA
TIEZHENG LI DepartmentofChemistryandBiochemistry,UniversityofMaryland,College Park,MD,USA
DAFNE MULLER InstituteofCellBiologyandImmunology,UniversityofStuttgart, Stuttgart,Germany
HUI MA SchoolofBiotechnology,DublinCityUniversity,Dublin,Ireland
STEPHEN M.MAHLER ARCTrainingCentreforBiopharmaceuticalInnovation, AustralianInstituteforBioengineeringandNanotechnology(AIBN),TheUniversityof Queensland,St.Lucia,QLD,Australia
ALESSANDRA MALLANO NationalCenterforGlobalHealth,TheNationalInstituteof Health,Rome,Italy
MARIATI BioprocessingTechnologyInstitute,AgencyforScience,TechnologyandResearch (A*STAR),Singapore,Singapore
CHARLOTTE MARTIN ResearchGroupofOrganicChemistry,DepartmentsofChemistryand BioengineeringSciences,VrijeUniversiteitBrussel,Brussels,Belgium
PIERRE MARTINEAU InstitutdeRechercheenCance´rologiedeMontpellier(IRCM), Montpellier,France;INSERM,U1194,Montpellier,France;Universite´deMontpellier, Montpellier,France;InstitutRe´gionalduCancerdeMontpellier,Montpellier,France
SAM MASSA CellularandMolecularImmunology,VrijeUniversiteitBrussel,Brussel, Belgium;InVivoCellularandMolecularImagingLaboratory,VrijeUniversiteitBrussel, Brussels,Belgium
SHANE MIERSCH BantingandBestDepartmentofMedicalResearch,TerrenceDonnelly CentreforCellularandBiomolecularResearch,UniversityofToronto,Toronto,ON, Canada
SANDRINE MOUTEL CNRS,UMR144,Paris,France;InstitutCurie,PSLResearch University,Paris,France
SERGE MUYLDERMANS CellularandMolecularImmunology,VrijeUniversiteitBrussel, Brussel,Belgium
NAOTO NEMOTO GraduateSchoolofScienceandEngineering,SaitamaUniversity, Saitama,Japan;EpsilonMolecularEngineering,Inc.,Saitama,Japan
DAMIEN NEVOLTRIS GarvanInstituteofMedicalResearch,ImmunologyDivision, Darlinghurst,NSW,Australia
CLE ´ MENT NIZAK LaboratoiredeBiochimie,ESPCIParis,PSLResearchUniversity,CNRS UMR8231ChimieBiologieInnovation,Paris,France
RICHARD O’KENNEDY SchoolofBiotechnology,DublinCityUniversity,Dublin,Ireland; NationalCentreforSensorResearch,DublinCityUniversity,Dublin,Ireland;Qatar FoundationandResearchComplex,HamadBinKhalifaUniversity,EducationCity, Doha,Qatar
ANNA OTTE DivisionofStemCellTransplantationandImmunotherapy,Departmentof MedicineII,UniversityHospitalSchleswig-HolsteinandChristian-Albrechts,Universityof Kiel,Kiel,Germany
VINCENT PAREZ InstitutdeRechercheenCance´rologiedeMontpellier(IRCM),Montpellier, France;INSERM,U1194,Montpellier,France;Universite´deMontpellier,Montpellier, France;InstitutRe´gionalduCancerdeMontpellier,Montpellier,France
MICHELA PASELLO CRSDevelopmentofBiomolecularTherapies,ExperimentalOncology Lab,RizzoliOrthopedicInstitute,Bologna,Italy
MATTHIAS PEIPP DivisionofStemCellTransplantationandImmunotherapy,Department ofMedicineII,UniversityHospitalSchleswig-HolsteinandChristian-Albrechts,University ofKiel,Kiel,Germany
FRANCK PEREZ CNRS,UMR144,Paris,France;InstitutCurie,PSLResearchUniversity, Paris,France
ANDREAS PLUCKTHUN DepartmentofBiochemistry,UniversityofZurich,Zurich, Switzerland
THIES ROSNER DivisionofStemCellTransplantationandImmunotherapy,Departmentof MedicineII,UniversityHospitalSchleswig-HolsteinandChristian-Albrechts,Universityof Kiel,Kiel,Germany
JOANNE H.REED GarvanInstituteofMedicalResearch,Sydney,NSW,Australia;Faculty ofMedicine,St.Vincent’sClinicalSchool,UniversityofNewSouthWales,Sydney,NSW, Australia
GAUTIER ROBIN InstitutdeRechercheenCance´rologiedeMontpellier(IRCM),Montpellier, France;INSERM,U1194,Montpellier,France;Universite´deMontpellier,Montpellier, France;InstitutRe´gionalduCancerdeMontpellier,Montpellier,France
SIMON ROSOWSKI InstituteforOrganicChemistryandBiochemistry,TechnischeUniversitat Darmstadt,Darmstadt,Germany
SOPHIA ROSSKOPF DivisionofStemCellTransplantationandImmunotherapy,Department ofMedicineII,UniversityHospitalSchleswig-HolsteinandChristian-Albrechts,University ofKiel,Kiel,Germany
ROMAIN ROUET GarvanInstituteofMedicalResearch,Sydney,NSW,Australia
ABHISHEK SAXENA LaboratoryofAntibodyEngineering,ShanghaiInstituteforAdvanced ImmunochemicalStudies,ShanghaiTechUniversity,Shanghai,China
PETER SCHOFIELD GarvanInstituteofMedicalResearch,Sydney,NSW,Australia;Faculty ofMedicine,StVincent’sClinicalSchool,UniversityofNewSouthWales,Sydney,NSW, Australia
NATHALIE SCHOLLER Kite,aGileadCompany,SantaMonica,CA,USA
CHRISTIAN SCHROTER Antibody-DrugConjugatesandTargetedNBETherapeutics,Merck KGaA,Darmstadt,Germany
KATIA SCOTLANDI CRSDevelopmentofBiomolecularTherapies,ExperimentalOncology Lab,RizzoliOrthopedicInstitute,Bologna,Italy
SACHDEV S.SIDHU LaboratoryofAntibodyEngineering,ShanghaiInstituteforAdvanced ImmunochemicalStudies,ShanghaiTechUniversity,Shanghai,China;BantingandBest DepartmentofMedicalResearch,TerrenceDonnellyCentreforCellularandBiomolecular Research,UniversityofToronto,Toronto,ON,Canada;DepartmentofMolecularGenetics, UniversityofToronto,Toronto,ON,Canada
MARTIN SIEGEMUND InstituteofCellBiologyandImmunology,UniversityofStuttgart, Stuttgart,Germany
LARS TOLEIKIS ProteinEngineeringandAntibodyTechnologies,MerckKGaA,Darmstadt, Germany
KOUHEI TSUMOTO DepartmentofBioengineering,SchoolofEngineering,TheUniversityof Tokyo,Tokyo,Japan;MedicalProteomicsLaboratory,TheInstituteofMedicalScience,The UniversityofTokyo,Tokyo,Japan
BASVANDER WONING argenxBVBA,Zwijnaarde,Belgium
RODRIGO VAZQUEZ-LOMBARDI GarvanInstituteofMedicalResearch,Darlinghurst/ Sydney,NSW,Australia
LAI-XI WANG DepartmentofChemistryandBiochemistry,UniversityofMaryland,College Park,MD,USA
IRIS WILLENBUECHER ProteinEngineeringandAntibodyTechnologies,MerckKGaA, Darmstadt,Germany
TIM WIRT DivisionofStemCellTransplantationandImmunotherapy,Departmentof MedicineII,UniversityHospitalSchleswig-HolsteinandChristian-Albrechts,Universityof Kiel,Kiel,Germany
DONGHUI WU LaboratoryofAntibodyEngineering,ShanghaiInstituteforAdvanced ImmunochemicalStudies,ShanghaiTechUniversity,Shanghai,China
CATARINA XAVIER InVivoCellularandMolecularImagingLaboratory,VrijeUniversiteit Brussel,Brussels,Belgium
YUANSHENG YANG BioprocessingTechnologyInstitute,AgencyforScience,Technologyand Research(A*STAR),Singapore,Singapore
JESSNA H.M.YEO BioprocessingTechnologyInstitute,AgencyforScience,Technologyand Research(A*STAR),Singapore,Singapore
TIANLEI YING KeyLaboratoryofMedicalMolecularVirologyofMOE/MOH,Shanghai MedicalCollege,FudanUniversity,Shanghai,China
SILVIA ZAMBONI DepartmentofNeuroscience,TheNationalInstituteofHealth,Rome, Italy
MAHDI ZERAATI GarvanInstituteofMedicalResearch,Sydney,NSW,Australia;Facultyof Medicine,St.Vincent’sClinicalSchool,UniversityofNewSouthWales,Sydney,NSW, Australia
STEFAN ZIELONKA InstituteforOrganicChemistryandBiochemistry,Technische UniversitatDarmstadt,Darmstadt,Germany;ProteinEngineeringandAntibody Technologies,MerckKGaA,Darmstadt,Germany xviContributors
InSilicoMethods
3Methods
1.Thefirststepistoobtainthemouseantibodyvariableand heavyandlightsequencesandcreateahomologymodelvia anyoftheantibodyhomologymodelingsoftware.Input mouseantibodysequences(VHandVL)intoeitherantibody homologymodelingsoftware(BioLuminate,Discoverystudio, orMOE)oranyofthesepublicwebservers(e.g.,PIGS, Rosetta,SAbPred,orKotai).(Foranexample,refertoSubheading 2, step1,foramouseantibodysequence.)UsePIGS webserverasastartingtoolforthisexercise(Fig. 1a,b).
PIGS—http://circe.med.uniroma1.it/pigs/index.php
Rosetta—http://rosie.rosettacommons.org/antibody SAbPred—http://opig.stats.ox.ac.uk/webapps/sabdabsabpred/WelcomeSAbPred.php
Kotai—https://sysimm.ifrec.osaka-u.ac.jp/kotaiab/
2.Humanframeworkselectioncanbeprimarilyaccomplished fromtwosources:
(a)IMGTdatabasecanbeusedtoobtainmostidentical (percentageidentity)humangermlinerepertoirefor bothVHchainandVLchains.IMGT’sDomainGapAlign toolcanbeusedtoobtainthemostsimilarcorresponding humangermlinesequencesforheavyandlightchainseparately(http://imgt.org/3Dstructure-DB/cgi/Dom ainGapAlign.cgi)(see Fig. 2).Usedefaultsettings;under speciesdrop-downmenu,chooseHomosapiens (Humans),andthenclick“AlignandIMGT-gapmy sequenceforVHandVLseparately.
(b)BlastantibodysequencesVHandVLseparatelytofind themostidenticalhumanframeworkinPDB.Igblasttool canbeutilizedforthisanalysis(https://www.ncbi.nlm. nih.gov/igblast/).
Humanframeworkselectioncriteriacanbemadebased onpercentageidentitytoindividualVHandVLchainsor humanframeworkswithhighestoverallidentityforboth chains(VHandVL).
3.Mutateandreplacemouseresiduesinthehomologymodel createdin step1 tohumanresidues,eitherfoundinthealignmentusingIMGT’sDomainGapAlignalignment(step2a)or humanantibodystructureobtainedfromPDB(step2b). Replacementofmouseresiduestohumanresiduescanbe performedusingPyMOL>wizard> mutagenesistool https:// www.pymol.org/ (see Note3).Inaddition,usercansubject theresultingchimericmodeltoRamachandranplotvalidation usingPDBsumGenerateoption(thisstepisoptional)(https:// www.ebi.ac.uk/thornton-srv/databases/pdbsum/Generate. html).
Fig.1 (a)Antibodyhumanizationstepsaredescribedinthisworkflow.As describedinthisflowchart,therearenumerousstepsinthisprocessthat needusageofdifferentantibodymodelingservers,sequencealignmenttools, andproteinvisualizationsoftware.(b)Visualrepresentationofantibodyhumanizationdesignprocess.Differentstepsinvolvedinantibodyhumanizationare annotatedbasedoneachstageofthedesigndevelopment.Amouseantibody
4.Thishumanizedhomologymodelhashumanframeworksand mouseCDRs(chimeric),whichissubjectedtoenergyminimizationsusingSwiss-PDBViewer(spdbv)software.Upload thishumanizedmodelinthespdbvsoftware,andselectall,and thenundertools,select“computeenergy(ForceField).”The output“energyreport”canbecopiedintoaworddocument forreview.Residueswithhightotalenergiesneedtobeexaminedfurther.ThiscanbecarriedoutusingPyMOLsoftware (see Note4).
5.Energy-minimizedhumanizedmodelisthenexaminedboth visuallyandenergeticallyforstericclashes.Basedonthedegree ofentropiesscore,aswellasextentofstericclashes,someresidues maybereplacedeithertonovelmutationsorbackmutation (parentalmouse)residues.Forexample(Figs. 3 and 4),ifthere isastericclashbetweentworesiduesintheframeworkregions, thentheresiduethatisnotconservedacrossdifferentgermlinesis mutatedtofixthestericclash(see Note5).
6.Onceresiduereplacementiscompleted,humanizedhomology modelisagainsubjectedtoenergyminimization(step4)to examineameliorationofthesestericclashes(Fig. 3b).Ifno furtherstericclashesarefound,thenthishumanizedmodelis designatedasdesignone.
7.Todesignadditionalhumanizedvariants,onecanchoosethe secondhighestidenticalhumanframeworkfrom step2 and carryouttheengineeringuntil step6 toobtaindesigntwo. Repeat steps from 2 to 6 toobtainaminimalsetof20different humanizedvariantsforexperimentaltesting(see Fig. 1a,b).
4Notes
1.Toexploresomeadditionalexamplesofmouseantibodiesfor humanization,referto[14],asitlists17differentmouse antibodiesforhumanizationusingthesamemethodsasmentionedinthischapter.
Fig.1 (continued)sequencewasutilizedtocreatea3Dhomologymodel,CDRs inorange,lightchaininblue,andheavychaininpurple,andsequencesthatare differentinmousearehighlightedinyellow.Oncehumangermlinesequenceis obtainedfromeitherIMGTorAbysisdatabaseandalignedwithmouseantibody sequence,thoseresiduesthatweredifferentarechangedtoreflecthuman sequences(green—step3).ThischimerichumanizedmodelcanalsobevalidatedviaRamachandranplotusingPDBsumGenerateoption(step4—optional). Thehumanizedmodel(humanframeworkandmouseCDRs)wasthensubjected toenergyminimizationusingSwiss-PDBViewer(spdbv)software(step5).Ifno stericclashesarefound,thiswillresultincreationofthefirsthumanized antibodydesign(step7)

Fig.2 MouseantibodyandcorrespondinghumangermlinesequencealignmentusingIMGTDomainGapAlign tool.Mouseantibodysequence(PDBID—3MBX)isalignedwiththemostidenticalhumangermlinesequence. Mouseresiduesinbothheavyandlightchain(yellow)weremutatedtohumangermlineresidues(green), whereasthemouseCDRs(orange)wereunchanged.Mousevariableheavychainis71.4%(identical)tohuman germlineIGHV2-5*09;atotalof24aminoacidsneededtobereplaced.Whereasmousevariablelightchainis 82.3%(identical)tohumangermlineIGKV4-1*01,assuch,only12aminoacidsneededtobereplaced
2.IfusingPIGSforantibodymodelbuildingwithVHandVLas inputsequencefromPDBID—3MBXantibody,therewillbe twowarningsafterthesubmissionfortemplateselection.One being“NoIGsatisfyingsearchcriteriaamongfirst20,and otherisH3canonicalstructurenotdefined.”Tocircumvent firstwarning,underresults,selectthreshold40results,instead of20(default).
3.MutagenesistoolinPyMOLisrecommendedformutatingand replacingmouseantibodyresiduesintheheavychainandlight chaintocorrespondingresiduesinthehumangermlinegene (Fig. 2).InPyMOL,selectwizardandmutagenesis,itwill promptto“pickaresidue”ofthemouseaminoacid,and mutatetocorrespondinghumanaminoacidfromFig. 2. (Ifusing3MBXasanexample,intheheavychain (VH)position1,mouseglutamate(E)needstobemutated toglutamine(Q)forhumanization.)(see Fig. 2).
4.Energyminimizationofthehumanizedchimericmodelusing Swiss-PDBViewer(spdbv).Oncethehybrid-humanizedmodel isopenedusingSwiss-PDBViewer,itmightgiveanerrorof “unrealisticBfactor.”Pleaseignoreandclosethisgenericerror. Computeenergy(forcefield),undertools,isfirstselected. Makesurethepop-upwindowhasalltheboxeschecked, including“showenergyreport.”Theenergyreportisusually savedin“temp”folder,whichcanbefoundinsidetheoriginal folder,whereSwiss-PDBViewerisinstalled (SPDBV_4.1.0_OSX“foldername.”Itisrecommendedto usedesktopfolderasadestinationforsoftwareinstallation). In3MBX,therearemanyresidueswithhightotalenergies.As anexample,onlyacoupleofthemaredescribedhere.Residues intheheavychainposition24(PHE)andinthelightchain position46(LEU)haveveryhighelectrostaticortotalscore.

Fig.3 Energyminimizationofthehumanized3MBXmouseantibodyinSwiss-PDBViewer(invacuo)via GROMOS96forcefield.First30residuesoftheheavychainareonlyshown,asanexample,inthis.(a)Heavy chainresiduephenylalanine(Phe)24hashightotalenergyscore,duetoastericclashwithasparagine(Asn)at position76.(b)Phenylalanineisconserved;assuchasparaginewasmutatedtoserine(mouseresiduenot showninthisfigure)thathadasignificantimpactonloweringtheoveralltotalenergyofthePhe24residue
References
Fig.4 Stericclashesinheavyandlightchainsofthehumanizedmouseantibody 3MBX.(a)Heavychainframework1,residuephenylalanine(Phe)24hasasteric clashwithasparagine(Asn)offramework3atposition76(Pheisnumber24in themodelbutis25inIMGTalignment;Asnnumberis76inthehomologymodel but85intheIMGTalignment).Pheisconservedbetweenmouseandhuman germline;Asnwasreplacedwithserine(Ser)tofixthisstericclash.(b)Inthe lightchainframework2,leucine46(position46inthemodel,whichcorresponds toposition52inIMGTnumberingscheme)hadastericclashwithtryptophan (W)intheCDRH3loop.Tofixthisclash,leucine(Leu)46wasmutatedto isoleucine(Ile)
5.Tofixthesestericclashes,ifusing3MBXasanexample,inthe heavychain(VH)framework1,phenylalanine(Phe)atposition 24hasastericclashwithframework3,asparagine(Asn) 76(homologymodelnumberingisdifferentthanIMGTalignmentnumbering; see Fig. 4 fordetails).AsPheisconserved betweenmouseandhuman,AsnwasbackmutatedtoSer (mouse)residuetorelievethisstericclash.IntheVL,leucine (Leu)46hasastericclashwithtryptophan(W)intheCDRH3 loop.Tofixthisclash,leucine(Leu)46canbemutatedto isoleucine(Ile)(see Fig. 4).
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