Discovery and Activity of STRO-002, a Novel ADC Targeting Folate Receptor Alpha for Ovarian and Endometrial CancerXiaofan Li, Cristina Abrahams, Sihong Zhou, Stellanie Krimm, Robert Henningsen, Heather Stephenson, Jeffrey Hanson, Mary Rose Masikat, Krishna Bajjuri, Tyler Heibeck, Cuong Tran, Gang Yin, James Zawada, Ganapathy Sarma, Joy Chen, Maureen Bruhns, Willy Solis, Alexander Steiner, Adam Galan, Toni Kline, Ryan Stafford, Alice Yam, Venita I. De Almeida, Mark Lupher, Jr., Trevor Hallam. Sutro Biopharma, South San Francisco, CA, USA

Sutro’s proprietary XpressCF+TM cell-free expression system includes:• incorporationofpAMF,thatenablessite-specificconjugation,• anorthogonaltRNAsynthetasewhichenableshighfidelitypAMFincorporation,• engineeredXpressCF+TMextractsthatutilizeanengineered,attenuatedRF-1whichenables:a.EfficientandmultipleinsertionpointsofpAMFinthesametranslationproductb.PreciseDARrangingfrom1-8inasinglemolecularspeciesADCc.ADCproductioninafewdaysallowingforrapiditerativestructure-activityoptimization

Generation of STRO-002, A Homogenous FolRa-Targeting ADC Through Cell-Free Antibody Synthesis And Site-Specific Conjugation

Selection of Optimum Drug-Linker and DARADC with a Cleavable Drug-Linker at DAR 4 Showed Best Activity

Selection of Top AntibodiesH01 and B10 FolRα Antibodies Exhibited Best Activity in FolRα

Expressing Models

Selection of Optimal LinkerVal-Cit Linker Confered Better Efficacy Than a Val-Ala Linker

Structure of STRO-002Selection of Optimum Conjugation SitesConjugation at Sites Y180/F404 or Y180/K42 Showed Best In Vivo Activity

Selection of Final ADC FormatFolRα antibody H01 Conjugated at Y180 and F404 Induced Significant

Growth Inhibition of Igrov-1 Tumors• FolRαADCvariantswithdifferenttubulin

baseddrug-linkers,anon-cleavablemaytansinoiddrug-linker(SC236)oracleavable2-aminophenylhemiasterlindrug-linker(SC239),conjugatedtoFolRαB10antibodyatDAR=2,4,or6,usingtheY180,F404and/orK42siteswerecompared.

• SC239conjugatesexhibitedbettercytotoxicactivityonIgrov1cellswhichhavelower,butclinicallyrelevantexpressionlevelsofFolRα.

• SC239wasthereforeselectedasthedrug-linkerforthisADC.

• AconjugatewithaDARof4wasselectedsinceaDARof6didnotprovidemuchhighercytotoxicitythantheDAR4version.

• FolRαADCvariantswithdifferentFolRαantibodies(engineeredandisolatedfromFabribosomedisplay)conjugatedtoSC239atpositionsY180andF404werecompared.

• Allvariantshadcomparablein vitro cell killingonhighexpressingFolRαpositiveKBcells.

• Inresponsetoasingle2.5mg/kgdose,ADCvariantswithH01andB10FolRαantibodiesshowedsignificantin vivo activityintheKBxenograftmodel.

• FolRαADCvariantscomposedofB10FolRαantibodyconjugatedtoSC239atthefollowingsitepairsY180/K42,Y180/F404orF404/K42werecompared.

• AllvariantshadcomparableinvitrocellkillingactivityonKBcells.

• Inresponsetoasingledoseat2.5mg/kg,ADCvariantswithSC239conjugatedatY180/K42orY180/F404demonstratedbetter in vivoactivityintheKBxenograftmodelthantheADCconjugatedatF404/K42.

Cell Killing Activity of STRO-002 Is Highly Specific for FolRa Expressing Cells

Summary

In vitro Cell Killing in KB Cells

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In vitro Cell Killing in Igrov1 Cells

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• FolRαADCvariantscomposedofB10orH01FolRαantibodiesconjugatedtoSC239linkerwarheadatY180/K42orY180/F404werecompared.

• H01-Y180/F404showedbestin vitro cytotoxicactivityandin vivo tumor growthinhibitionintheIgrov-1modelfollowingasingledoseat2.5mg/kg.

•STRO-002showedpotentactivityinacellkillingassayonFolRαpositiveIgrov-1andOVSAHOcells,butnoeffectonFolRαnegativeA549cells.

•CytotoxicactivityofSTRO-002wasspecificforcellsexpressingFolRα;andwascompetedwithexcessSP8166(H01).

• anti-FolRαantibodiesisolatedusingFab-basedribosomedisplaywereoptimizedfor- Linkerandwarheadattributes- Antibody moiety - Drugantibodyratio- Sitepairsfordrug-linkerattachment

• Folatereceptoralpha(FolRa)isacell-surfaceproteinoverexpressedinovarianandendometrialcancer.• FolRaexpressionishighlyrestrictedonnormaltissues,makingitapromisingtargetforcancertherapy

usingantibodydrugconjugates(ADCs).

• WehaveusedaplatformbasedonSutro’sproprietary XpressCF+TM cell-free expression system and site specificconjugationtodesignanovel,FolRa-targetingADC,STRO-002.

• STRO-002containstheanti-FolRahumanIgG1antibodySP8166(H01)conjugatedtoanovelproprietarycleavabledrug-linker(SC239)atspecificsitesY180andF404ontheantibodyheavychain.

• SP8166(H01)wasdiscoveredandoptimizedusingaFabribosomedisplayselectionandhasfour non-naturalaminoacid,p-azido-phenylalanine(pAMF)residuesincorporatedatpositionsY180andF404oneachheavychain.

• SC239iscomposedofatubulin-targeting3-aminophenylhemiasterlinwarhead,SC209,andacleavablevalinecitrullinep-aminobenzylcarbamatelinkerfunctionalizedwithdibenzocyclooctyne(DBCO).

• TherapidandselectivereactionofDBCOonSC239andpAMFresiduesinSP8166resultsinawell-defined,homogeneousADCwithadrug-antibodyratio(DAR)of~4.

• WehaveleveragedSutro’sXpressCF+TMcell-freetechnologyforrapidinterrogationandoptimizationofparametersforaFolRαtargetingADC,includingchoiceofantibody,conjugationsites,DAR,andlinkerwarhead.

• In vitro and in vivoassessmentshowedthatFolRαADCcomposedofantibodyH01conjugatedtoSC239atpositionsY180andF404,designatedSTRO-002,demonstratedthebestantitumoractivityinFolRαexpressingmodels,andwasselectedastheleadantibodyfortheprogram.

• STRO-002demonstratedgoodpharmacokineticandpharmacologicalpropertiesincludingspecificity,stabilityandsafetyincynomolgusmonkeys(notshownhere),thusmakingitanidealcandidateforclinicaldevelopment.

• IND-enablingstudiessupportingSTRO-002asapotentialtreatmentofFolRαexpressingmalignanciesareon-goingandINDsubmissionisplannedforthesecondhalfof2018.

References•Yin,G.,etal.;(2012)Aglycosylatedantibodiesandantibodyfragmentsproducedinascalableinvitrotranscription-translationsystem.mAbs4:2,217-225.

•Zimmerman,E.S.,etal.;(2014)Productionofsite-specificantibody−drugconjugatesusingoptimizednon-naturalaminoacidsinacell-freeexpressionsystem.BioconjugateChemistry2014,25,351−361.

•Yin,G.,et.al.;(2017)RF1attenuationenablesefficientnon-naturalaminoacidincorporationforproductionofhomogeneousantibodydrugconjugates.ScientificReports7(1):3026.

STRO-002 is Highly Stable In Vitro and In Vivo

• STRO-002ishighlystablein vitro (PBS,cynomolgusorhumanplasmaat50mg/mL)andin vivo(plasmafrommicetreatedwithsingledoseof5mg/kg),withaDARof~4beingretaineduntilday21.

• MinimalreleaseoftheSTRO-002catabolite,SC209,wasobservedaftera4-dayincubationofSTRO-002inPBS,cynoorhumanplasmaat100mg/mL.

• SC209wasaccumulatedintumors,but undetectable in circulation of treatedmicebearingIgrov1tumors.

• STRO-002wasisolatedfromplasmasamplesbyaffinitypull-downandDARmeasuredbyLC-MS.ReleasedSC209wasquantitatedbyLC-MS.

In VitroSC209Release

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Igrov1FolRa Copy # = 1,375,828

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Pharmacokinetic Profile In vitro Cell Killing in Igrov1 Cells

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• FolRαADCvariantscomposedoftheH01-Y180/F404FolRαantibodyconjugatedto3-aminophenylhemiasterlinwitheitheracleavableVal-Citdruglinker,oraVal-Aladruglinker,werecompared.

• BothADCvariantsshowedcomparablepharmacokineticproperties in mice.

• TheSC239conjugateshowedslightlyimprovedinvitrocellkillingactivityandexhibitedsuperiorinvivoefficacythantheSC346conjugateinIgrov-1modelatasingledoseof5and15mg/kg.

• Basedonthecumulativedata,H01conjugatedtoSC239atpositionsY180andF404wasselectedastheleadFolRαtargetingADC,STRO-002..