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Methods in Molecular Biology 1045 Laurent Ducry Editor Antibody-Drug Conjugates M M B TM ETHODS IN OLECULAR IOLOGY SeriesEditor JohnM.Walker School ofLife Sciences University ofHertfordshire Hatfield, Hertfordshire,AL109AB,UK For further volumes: http://www.springer.com/series/7651 . Antibody-Drug Conjugates Edited by Laurent Ducry Lonza Ltd, Visp, Switzerland Editor LaurentDucry LonzaLtd Visp,Switzerland ISSN1064-3745 ISSN1940-6029(electronic) ISBN978-1-62703-540-8 ISBN978-1-62703-541-5(eBook) DOI10.1007/978-1-62703-541-5 SpringerNewYorkHeidelbergDordrechtLondon LibraryofCongressControlNumber:2013943693 #SpringerScience+BusinessMedia,LLC2013 Thisworkissubjecttocopyright.AllrightsarereservedbythePublisher,whetherthewholeorpartofthematerialis concerned,specificallytherightsoftranslation,reprinting,reuseofillustrations,recitation,broadcasting,reproduction onmicrofilmsorinanyotherphysicalway,andtransmissionorinformationstorageandretrieval,electronicadaptation, computersoftware,orbysimilarordissimilarmethodologynowknownorhereafterdeveloped.Exemptedfromthis legalreservationarebriefexcerptsinconnectionwithreviewsorscholarlyanalysisormaterialsuppliedspecificallyfor thepurposeofbeingenteredandexecutedonacomputersystem,forexclusiveusebythepurchaserofthework. 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Printedonacid-freepaper HumanaPressisabrandofSpringer SpringerispartofSpringerScience+BusinessMedia(www.springer.com) Preface Antibody–drugconjugates(ADCs)representapromisingtherapeuticapproachforcancer patients by combining the antigen-targeting specificity of monoclonal antibodies (mAbs) ® with the cytotoxic potency of chemotherapeutic drugs. The FDA approval of Adcetris ® (brentuximabvedotin)in2011andKadcyla (trastuzumabemtansineorT-DM1)in2013 hasvalidatedtheideaofmaking“armed”antibodies,attractingalotofattentionintothis field. ADC technology has been an active area of research in recent years, resulting in a numberofADCsindevelopmentfor varioustumor types.Thenumberofimmunoconju- gatesorADCsundergoingclinicaltrialwillthusfurtherincrease,possiblyreplacingsome of the existing naked monoclonal antibodies, and becoming the next generation of anticancerbiotherapeutics. AlthoughtheADCconceptisquitesimple,successfullydesigninganddevelopingsuch a“smartbomb”isacomplextask.Despiteatremendousincreaseinourunderstandingin recentyears,alotofworkisnecessaryinordertoidentifyasuitabletarget;properlydesign the mAb, the linker, and the payload; as well as conjugate them in a reproducible and scalablefashion. The success of the current conjugation technologies has been achieved thanks to the developmentofnewmethodologies.Theaimofthisbookistoprovidedetailedprotocols for many of the key ADC techniques necessary for working in the field. Each method is described by an author who has regularly used the technique in his or her laboratory. In addition, several review chapters are included to summarize the current knowledge and results in the ADC area. These should make this book useful to readers with no previous ADC experience as well as those already working in the field. It is my hope that this publicationwill further drive ADC development and thus help towardsimproving cancer treatmentsofthefuture. Visp,Switzerland LaurentDucry v Contents Preface.................................................................... v Contributors............................................................... ix 1 Antibody–DrugConjugate(ADC)ClinicalPipeline:AReview.............. 1 IngridSassoonandVe´roniqueBlanc 2 Antibody–DrugConjugateTargetSelection:CriticalFactors ............... 29 NeilH.Bander 3 SelectinganOptimalAntibodyforAntibody–DrugConjugateTherapy: InternalizationandIntracellularLocalization ............................. 41 JayHarper,ShenlanMao,PatrickStrout,andAdeelaKamal 4 Antibody–DrugConjugatePayloads..................................... 51 JanAnderl,HeinzFaulstich,TorstenHechler,andMichaelKulke 5 LinkerTechnologiesforAntibody–DrugConjugates ...................... 71 BirteNolting 6 InVivoTestingofDrug-LinkerStability ................................. 101 Pierre-YvesAbecassisandCe´lineAmara 7 PharmacokineticsandADMECharacterizations ofAntibody–DrugConjugates.......................................... 117 KedanLin,JayTibbitts,andBen-QuanShen 8 SafeHandlingofCytotoxicCompoundsinaBiopharmaceutical Environment.......................................................... 133 MiriamI.HensgenandBernhardStump 9 Micro-andMid-ScaleMaleimide-BasedConjugationofCytotoxic DrugstoAntibodyHingeRegionThiolsforTumorTargeting ............. 145 JamesE.Stefano,MichelleBusch,LihuiHou,AnnaPark, andDiegoA.Gianolio 10 ProtocolsforLysineConjugation ....................................... 173 Marie-PriscilleBrunandLaurenceGauzy-Lazo 11 EngineeringTHIOMABsforSite-SpecificConjugation ofThiol-ReactiveLinkers............................................... 189 SunilBhakta,HelgaRaab,andJagathR.Junutula 12 EnzymaticAntibodyModificationbyBacterialTransglutaminase............ 205 PatrickDennler,RogerSchibli,andElianeFischer 13 FormulationDevelopmentofAntibody–DrugConjugates ................. 217 WilliamJ.GalushandAdityaA.Wakankar 14 ConjugationProcessDevelopmentandScale-Up ......................... 235 BernhardStumpandJessicaSteinmann 15 MethodsforConjugatingAntibodiestoNanocarriers ..................... 249 AnilWaghandBenedictLaw 16 Drug-to-AntibodyRatio(DAR)byUV/VisSpectroscopy ................. 267 YanChen vii viii Contents 17 Drug-to-AntibodyRatio(DAR)andDrugLoadDistribution byHydrophobicInteractionChromatographyandReversedPhase High-PerformanceLiquidChromatography.............................. 275 JunOuyang 18 Drug-to-AntibodyRatio(DAR)andDrugLoadDistribution byLC-ESI-MS........................................................ 285 LouisetteBasa 19 DeterminationofChargeHeterogeneityandLevelofUnconjugated AntibodybyImagedcIEF.............................................. 295 JoyceLinandAlexandruC.Lazar 20 Risk-BasedScientificApproachforDetermination ofExtractables/LeachablesfromBiomanufacturing ofAntibody–DrugConjugates(ADCs) .................................. 303 WeibingDing Index..................................................................... 313 Contributors PIERRE-YVES ABECASSIS (cid:1) DSAR (Disposition, Safety & Animal Research), Sanofi, Vitry-sur-Seine, France CE´LINE AMARA (cid:1) DSAR (Disposition, Safety & Animal Research), Sanofi, Vitry-sur-Seine, France JAN ANDERL (cid:1) Heidelberg Pharma GmbH, Ladenburg, Germany NEIL H. BANDER (cid:1) Weill-Cornell Medical College, Cornell University, New York, NY, USA LOUISETTE BASA (cid:1) Protein Analytical Chemistry, Genentech Inc., South San Francisco, CA, USA SUNIL BHAKTA (cid:1) Genentech Inc., South San Francisco, CA, USA VE´RONIQUE BLANC (cid:1) Sanofi Oncology, Vitry-sur-Seine, France MARIE-PRISCILLE BRUN (cid:1) Natural Product and Protein Chemistry, Sanofi, Vitry-sur-Seine, France MICHELLEBUSCH (cid:1) TransitionalResearch,Genzyme,aSanofiCompany,Framingham, MA, USA YAN CHEN (cid:1) Protein Analytical Chemistry, Genentech Inc., South San Francisco, CA, USA PATRICK DENNLER (cid:1) Center for Radiopharmaceutical Sciences, Paul Scherrer Institute, Villigen, Switzerland WEIBING DING (cid:1) SLS Global Technical Support, Pall Corporation, Port Washington, NY, USA HEINZ FAULSTICH (cid:1) Heidelberg Pharma GmbH, Ladenburg, Germany ELIANE FISCHER (cid:1) Center for Radiopharmaceutical Sciences, Paul Scherrer Institute, Villigen, Switzerland WILLIAM J. GALUSH (cid:1) Early Stage Pharmaceutical Development, GenentechInc., South San Francisco, CA, USA LAURENCE GAUZY-LAZO (cid:1) Natural Product and Protein Chemistry, Sanofi, Vitry-sur-Seine, France DIEGO A. GIANOLIO (cid:1) Drugs and Biomaterials R&D, Genzyme, a Sanofi Company, Cambridge, MA, USA JAY HARPER (cid:1) Oncology Research, MedImmune Inc., Gaithersburg, MD, USA TORSTEN HECHLER (cid:1) Heidelberg Pharma GmbH, Ladenburg, Germany MIRIAM I. HENSGEN (cid:1) Bioconjugates R&D, Lonza Ltd, Visp, Switzerland LIHUI HOU (cid:1) Transitional Research, Genzyme, a Sanofi Company, Framingham, MA, USA JAGATH R. JUNUTULA (cid:1) Genentech Inc., South San Francisco, CA, USA ADEELA KAMAL (cid:1) Oncology Research, MedImmune Inc., Gaithersburg, MD, USA MICHAEL KULKE (cid:1) Heidelberg Pharma GmbH, Ladenburg, Germany BENEDICT LAW (cid:1) Department of Pharmaceutical Sciences, College of Pharmacy, Nursing and Allied Sciences, North Dakota State University, Fargo, ND, USA ALEXANDRU C. LAZAR (cid:1) Analytical & Pharmaceutical Sciences, ImmunoGen, Inc., Waltham, MA, USA ix