Preclinical Activity and Safety of STRO-002, a Novel ADC

Preclinical Activity and Safety of STRO-002, a Novel ADC Targeting Folate Receptor Alpha for Ovarian CancerCristina Abrahams, Stellanie Krimm, Xiaofan Li, Sihong Zhou, Jeffrey Hanson, Mary Rose Masikat, Krishna Bajjuri, Tyler Heibeck, Dharti Kothari, Abigail Yu, Robert Henningsen, Cuong Tran, Gang Yin, James Zawada, Julie Hang, Maureen Bruhns, Willy Solis, Alexander Steiner, Joy Chen, 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.PreciseDARrangingfrom1-8inawell-definedmolecularspeciesADCc.ADCproductioninafewdaysallowingforrapiditerativestructure-activityoptimization

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

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

Summary

•STRO-002showedpotentactivityinacellkillingassayonFolRαpositiveIgrov-1andOVSAHOcells,butnoeffectonFolRαnegativeA549cells

•CytotoxicactivityofSTRO-002wasspecificforcellsexpressingFolRα;andwascompetedwithexcessSP8166(H01)

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

• Folatereceptoralpha(FolRa)isacell-surfaceproteinoverexpressedinovarianandendometrialcancer• FolRaexpressionishighlyrestrictedonnormaltissues,makingitahighlyrelevanttargetforcancer

therapyusingantibodydrugconjugates(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).

• TherapidandselectivereactionofDBCOonSC239andpAMFresiduesinSP8166resultsinahomogenousADCwiththepredominantspecieshavingadrug-antibodyratio(DAR)of4.

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

• STRO-002demonstratedgoodpharmacologicalpropertiessuchasspecificityandastabledruglinkagewiththecatabolite,SC209,thatisclearedrapidlyfromcirculationandisaweaksubstratefordrug-resistanceeffluxpumps.

• STRO-002exhibitsdose-dependenttumorgrowthinhibitioninovarianandendometrialtumorxenografts,withefficacyappearingtocorelatewithexpressionlevelsofFolRainthexenograftmodel.Inaddition,STRO-002whencombinedwithcarboplatinsignificantlyimprovesefficacycomparedtocarboplatinaloneinIgrov-1tumors.

• FavorablesafetyprofilesforbothSC209andSTRO-002wereobservedinsafetystudiesinratsandincynomolgousmonkeys,respectively.

• STRO-002thereforehasthepotentialforanimprovedsafetyandactivityprofile,andareducedriskoftumordrugresistance,makingitanidealcandidateforclinicaldevelopment.

• IND-enablingstudiessupportingSTRO-002asapotentialtreatmentofFolRaexpressingmalignanciessuchasovarianandendometrialcancerareongoingandINDsubmissionisplannedforsecondhalfof2018.

References•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.ReleasedSC209wasquantitated by LC-MS.

C In VitroSC209Release

A In VitroDARMeasurement

Introduction

D

0

20

40

60

80

100

120

predose day 1 day 3 day 7 predose day 1 day 3 day 7Tumor Levels (ng/g) Plasma Levels (ng/ml)

SC20

9 Le

vel

Tumor Accumulation and SC209 Release In Vivo

Igrov1FolRa Copy # = 1,375,828

0.001 0.01 0.1 1 10 100 10000

20

40

60

80

100

120

nM

Rela

tive

Cell V

iabi

lity

(Cel

lula

r ATP

con

tent

, % o

f con

trol)

OVSAHOFolRa Copy # = 842,703

0.001 0.01 0.1 1 10 100 10000

20

40

60

80

100

120

nM

Rela

tive

Cell V

iabi

lity

(Cel

lula

r ATP

con

tent

, % o

f con

trol)

STRO-002STRO-002 + 0.5uM H01

A549

0.001 0.01 0.1 1 10 100 10000

20

40

60

80

100

120

nM

Rel

ativ

e C

ell V

iabi

lity

(Cel

lula

r ATP

con

tent

, % o

f con

trol) FolRa Negative

B In VivoDARMeasurement

STRO-002 Demonstrates Dose-Dependent Tumor Killing In Igrov-1 Ovarian Tumor Model

A.Igrov-1tumorgrowthcurvesinresponsetoasingledoseofSTRO-002at2.5,5,10and15mg/kg.B.Scatterplotofindividualtumorsizeonday21whencontroltumorsreachedstudyendpoint.C.Tumorgrowthdelayisdefinedastheaveragetime(indays)oftreatmentgrouptoreach300mm3minustimeforvehiclecontrolgrouptoreachthesameendpoint.Statisticalanalysiswasperformedonfinaltumorsizesonday21anddelaytoreach300mm3usingone-wayANOVAwithDunn’sorDunnett’smultiplecomparisonstest,respectively.

STRO-002 Co-administration with Carboplatin Confers Added Benefit in Igrov-1 Ovarian Tumor Xenografts Compared to Carboplatin Alone

A.Igrov-1tumorgrowthcurvesinresponseto2.5mg/kgSTRO-002withandwithout60mg/kgcarboplatin.B.Scatterplotofindividualtumorsizeonday21whencontroltumorsreachedstudyendpoint.Statisticalanalysiswasperformedonfinaltumorsizesonday21usingone-wayANOVAwithTukey’smultiplecomparisonstest.

STRO-002 Showed Significant Anti-Tumor Activity in Established and Large OVCAR-3 Ovarian Tumor Xenografts

OVCAR-3tumorgrowthcurvesinresponsetoindicateddoseofSTRO-002administeredoncewhenwhentumorswere150mm3(A)or400mm3(B).C.Scatterplotofindividualtumorsizeonday55whencontroltumorsreachedstudyendpoint.Statisticalanalysiswasperformedontumorsizeonday55usingone-wayANOVAwithDunnett’smultiplecomparisonstest.

SC209 is a Poor Substrate for Permeability Glycoprotein 1 (PgP) Efflux Pumps

• MES-SA/MX2amitoxantroneresistantderivativeoftheMES-SAcellline,overexpressP-gp.

• ThecytotoxiceffectsofthethreedrugsonMES-SA/MX2andMES-SAcellsweremeasured in the presence or absence of 5mMofP-gpinhibitorGF120918.

• MES-SA/MX2cellsshoweddifferentlevelsofresistancetothefreewarheadsSC209,DM4andMMAE.ComparedtoDM4andMMAE,MES-SA/MX2cellsshowedmuchlowerresistancetoSC209.

• AdditionofthePgPInhibitorGF120918reversedthedrugresistanceinMES-SA/MX2cellsindicatingthatdrugresistancewasmediatedbyP-gp.

STRO-002 Demonstrates Optimal Stability and a Favorable Safety Profile in Cynomolgus Monkeys

STRO-002wastoleratedat0.9,2.9,and8.7mg/kgfollowingtwoIVdosesonDays1and22.Arepresentativeexposureprofileat8.7mg/kgisshown.TotalAbandADClevelsweresimilaronDay1andDay22andSC209levelsweredetectedatlowlevelsincirculation(<10ng/mL),indicatingoptimalinvivostability.ThemostprominentSTRO-002-relatedtoxicityconsistedofdose-relatedhematologicaltoxicity(neutropenia),thatwasreversible.Nooculartoxicitywasobservedfollowingtwodosesatalltolerateddoses,asassessedbyslitlampmicroscopyandhistopathology.Thesefindingsareconsideredantigen-independent(attributedtothecytotoxiccomponentofSTRO-002),reversible,andclinicallymanageable.0 10 20 30 40

0.0000010.00001

0.00010.001

0.010.1

110

1001000

Time (days)

8.7 mg/kg STRO-002Total AbADCSC209

SC209 Catabolite has Similar Safety and TK Profiles to Other Tubulin-Targeting Drugs in Rats Following a Single IV Dose

Toxicokineticanalysisshowsafastclearanceandlargevolumeofdistribution(notshown).SC209exposuresat1mg/kgcorrespondto~102-foldhigherCmaxand~10.3-foldhigherAUCthanthoseofSTRO-002-derivedSC209atthetolerateddoseof8.7mg/kginmonkeys.Themostprominentdose-relatedtoxicityfindingattolerateddoseswasslighttoseverebonemarrowhypocellularitywhichcorrelatedwithhematologicaltoxicity.

SC209 Dose (mg/kg) Tolerated? Cmax (ng/mL) AUC0-24

(ng.hr/mL) Half-life (hr) CL (mL/hr/kg)

0.33 Yes 25.3 64.4 2.52 6930 1.10 Yes 73.6 244 4.30 6000 2.84 No 267 756 NC NC 7.95 No 2210 NC NC NC

NC–notcalculatedduetomissingTKsamplesduetomortalitiesand/orinabilitytoestimaterateconstants

DM4

0.0001 0.01 1 100 100000

20

40

60

80

100

120

nM

Rel

ativ

e C

ell V

iabi

lity

(Cel

lula

r ATP

con

tent

, % o

f con

trol)

SC209

0.0001 0.01 1 100 100000

20

40

60

80

100

120

nM

Rel

ativ

e C

ell V

iabi

lity

(Cel

lula

r ATP

con

tent

, % o

f con

trol)

MES-SA/MX2 (PgP +++) + PgP inhibitor (GF120918)

MES-SA/MX2 (PgP +++) + No PgP inhibitor

MES-SA (PgP -) + No PgP inhibitor

MMAE

0.0001 0.01 1 100 100000

20

40

60

80

100

120

nM

Rel

ativ

e C

ell V

iabi

lity

(Cel

lula

r ATP

con

tent

, % o

f con

trol)

111x17x8x

A B C

AcknowledgementsCrownBioscience:MinxiaWang EnsignaBiosystems:YuChienChouandMichelFaure.ExperimentalPathologyLaboratories,Inc.:W.MichaelPeden.QuintaraDiscovery:JulieRen