Tumor targeted delivery of cytotoxic payloads using affibody

Tumor targeted delivery of cytotoxic payloads using affibody molecules and ABD-derived affinity proteins

Hao Liu

Kungliga Tekniska Högskolan, KTHRoyal Institute of Technology

School of Engineering Sciences in Chemistry, Biotechnology and Health

Stockholm, Sweden 2018

©HaoLiu,Stockholm2018KTHRoyalInstituteofTechnologySchoolofEngineeringScienceinChemistry,BiotechnologyandHealthDepartmentofProteinScienceAlbaNovaUniversityCenterSE-10691Stockholm,SwedenPrintedbyUS-AB,2018ISBN978-91-7729-827-4TRITA-CBH-FOU-2018:26

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AbstractCancertreatmentcostbillionsofdollarseveryyear,butthemortalityrateisstillhigh.An ideal treatment is the so-called “magicbullets” that recognizeandkilltumor cells while leaving normal cells untouched. In recent years, some non-immunoglobulinalternativescaffoldaffinityproteins,suchasaffibodymoleculesand ADAPTs, have emerged and been used to specifically recognize differenttumorantigens.Inthisthesis, Istudiedthepropertiesandanti-tumoractivitiesof affibody andADAPT fusion toxins and affibody drug conjugates. In the firsttwo papers, I studied a panel of recombinant affitoxins (affibody toxin fusionproteins) consisting of an anti-HER2 affibody molecule (ZHER2), an albuminbinding domain (ABD) and a truncated version of Pseudomonas Exotoxin A(PE38X8). The affitoxins demonstrated specific anti-tumor activity on HER2-overexpressing tumor cells in vitro. A biodistribution experiment showed thataddition of an ABD increased the blood retention by 28-fold and a (HE)3 N-terminal purification tag decreased hepatic uptake of the affitoxin comparedwithaHis6tag.InpaperIII, Istudiedimmunotoxinsconsistingofananti-HER2ABD-derived affinity protein (ADAPT), an ABD and a minimized anddeimmunizedversionofPseudomonasexotoxinA (PE25).These immunotoxinsdemonstrated potent and specific cytotoxicity toward HER2 overexpressingtumor cells in vitro similar to affitoxins. In paper IV, I produced a panel ofaffibody drug conjugates consisting of ZHER2, ABD and malemidocaproylmertansine (mc-DM1). The conjugates had selective toxic activity on HER2-overexpressing tumor cells in vitro comparable with the approved drugtrastuzumab emtansine. The conjugate, ZHER2-ZHER2-ABD-mc-DM1was found toprolong the life span of tumor bearing mice and delayed the growth ofxenograftedSKOV-3tumors. Inconclusion,affibodymoleculesandADAPTsarepromisingalternativestoantibodiesfortargetedtumortherapy.

Keywords: Targeted tumor therapy, immunotoxins, ADCs, affibody molecule,ADAPT,pseudomonasexotoxinA,maytansinoid

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PopularScienceSummaryCancerhasbecomeoneoftheleadingcausesofdeathintheworldandthereisnogeneralcureforcanceryet.Canceris intrinsicallyhardtotreatbecauseit isderived fromourowncellsand it ishard for the immunesystemanddrugs todistinguishcancercellsfromhealthyones.Themostcommonlyusedtraditionaltreatmentsforcancerpatientsincludesurgery,chemotherapyandradiotherapy.Surgeryisaneffectivewaytoremovesolidtumors,however,itdamageshealthytissuesurrounding the tumorand it isoftendifficult to removeall tumorcells,whichmayleadtoreoccurringtumors.Chemotherapyandradiotherapykillbothtumorcellsandhealthycells,thusresultinginsideeffects.Anidealtreatmentforcancercouldbeaguidedmissilethatsearchesandthendestroysonlythetumorcellswithoutharmingthehealthyones,socalledtargetedcancertherapy.Researchershavedevelopedavarietyofmoleculesfortargetedtumortherapy.Thelargestclassoftargetingmoleculesisantibodies,oneofthemajormoleculesgeneratedbyourownimmunesystemandnaturallyusedfordefenseagainstinfections.Antibodiesusedfortargetedcancertherapyareoftenengineeredoutsideofhumanbodytoacquirethespecificityforacertaintypeoftumor.Theengineeredantibodiesarealsosometimesfurtherarmedwithtoxicpayloadsforaccuratedeliveryoftoxinstotumorcells.Besidesantibodies,artificialnon-antibodysmalltargetingproteinsmaybeusedtodelivertoxinstotumorsfortargetedcancertherapy.Inthisthesis,thisistheareathatIhaveexplored.Ifoundthatthedesignedsmallproteinscouldspecificallydelivertoxinstotumorcellsandkilltumorcellsgrowinginpetridishesandinmicewithhuman-derivedtumors.

科普总结癌症在全球已经成为⼈类第⼀杀⼿之⼀。迄今为⽌还没有能够根治癌症的⽅法。癌症难以治愈的根本原因是癌细胞来自于⼈类自身的细胞，这使得免疫系统和药物很难将癌细胞同正常细胞区别对待。现在最常用的传统癌症治疗⽅法有：⼿术切除、化疗和放疗。⼿术切除对于固体性肿瘤比较有效，但是会对肿瘤周围组织产⽣损伤，更重要的是⼀般很难保证将所有癌细胞切除。⼀段时间过后，癌症会复发。化疗和放疗都不加区分的杀死癌细胞和正常细胞，因此他们对病⼈身体的损伤很⼤。最理想的癌症药物应该类似于制导导弹，它只搜寻和消灭癌细胞⽽对正常细胞⽆害。研究者们已经开发了⼀系列的药物来实现癌症的靶向治疗。这其中最⼤的⼀类是来自于我们自身免疫系统的抗体。体外⼈⼯改造的抗体可以获得对某种肿瘤的特异性来用于治疗相应的癌症。这类⼈⼯改造的抗体还可以连接上某种毒素分⼦然后精确地投递毒素分⼦到癌细胞中去，从⽽杀死癌细胞。在这篇论⽂里，我探索了试用⼀种比抗体小很多的⼈⼯亲和蛋白来特异地给肿瘤投递两种类型的毒素。我所制备的药物不仅可以杀死在培养⽫中⽣长的肿瘤细胞和在实验小鼠体内⽣长的⼈源肿瘤细胞。

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Listofappendedpapers

Thisthesisisbasedonthefollowingarticlesandmanuscripts.Fullversionsofthesearticlesconcludethisthesis.

I. Liu,H.,Seijsing,J.,Frejd,F.Y.,Tolmachev,V.,&Gräslund,T.(2015).Target-specificcytotoxiceffectsonHER2-expressingcellsbythetripartitefusiontoxinZHER2:2891-ABD-PE38X8,includingatargetingaffibodymoleculeandahalf-lifeextensiondomain.InternationalJournalofOncology,47,601-609.https://doi.org/10.3892/ijo.2015.3027

II. Altai,M.*,Liu,H.*,Orlova,A.,Tolmachev,V.,&Gräslund,T.(2016).InfluenceofmoleculardesignonbiodistributionandtargetingpropertiesofanAffibody-fusedHER2-recognisinganticancertoxin.InternationalJournalofOncology,49,1185-1194.https://doi.org/10.3892/ijo.2016.3614

III. Liu,H.,Lindbo,S.,Ding,H.,Hober,S.,Gräslund,T.FusiontoxinsconsistingofaHER2-interactingABD-derivedaffinityprotein(ADAPT)fusedtotruncatedversionsofPseudomonasexotoxinAwithhighpotencyandspecificity.Manucsript.

IV. Altai,M.*,Liu,H.*,Ding,H.,Mitran,B.,Edqvist,P-H.,Tolmachev,V.,Orlova,A.,&Gräslund,T.Affibody-derivedDrugConjugates:PotentCytotoxicDrugsForTreatmentOfHER2Over-ExpressingTumors.Manucsript.

*ContributedequallyAllarticlesarereprintedbypermissionofthecopyrightholders.

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Respondent’scontributiontoappendedpapers

I. Planned,designedandperformedtheexperimentalworktogetherwith

co-authors.Performeddesign,production,characterizationandinvitrocytotoxicitymeasurementsofrecombinantfusiontoxins.Wrotethemanuscripttogetherwiththeco-authors.

II. Planned,designedandperformedtheexperimentalworktogetherwithco-authors.Performeddesign,production,andcharacterizationofrecombinantfusiontoxins.Wrotethemanuscripttogetherwiththeco-authors.

III. Planned,designedandperformedtheexperimentalworktogetherwithco-authors.Performeddesign,production,characterizationandinvitrocytotoxicitymeasurementsofrecombinantfusiontoxins.Wrotethemanuscripttogetherwiththeco-authors.

IV. Planned,designedandperformedtheexperimentalworktogetherwithco-authors.Performeddesign,production,characterizationandinvitrocytotoxicitymeasurementsofdrugconjugates.Wrotethemanuscripttogetherwiththeco-authors.

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Publicdefenseofdissertation

ThisthesiswillbedefendedJune14th,2018at10:00,OskarKleinsAuditorium,Roslagstullsbacken21,AlbanovaUniversityCenter,Stockholm,forthedegreeof“Teknologiedoctor”(DoctorofPhilosophy,PhD)inBiotechnology.Resopondent:HaoLiu,M.Sci.inGeneticsDepartmentofProteinScience,SchoolofEngineeringSciencesinChemistry,BiotechnologyandHealthKTHRoyalInstituteofTechnology,Stockholm,SwedenFacultyopponent:Prof.NickDevoogdtInvivoCellularandMolecularImagingLaboratory(ICMIC)VrijeUniversiteitBrussel(FreeUniversityofBrussels),Brussels,BelgiumEvaluationcommittee:Prof.ÜloLangel DepartmentofNeurochemistry,StockholmUniversity,Stockholm,SwedenSeniorresearcherKristinaViktorsson DepartmentofOncology-Pathology,KarolinskaInstitute,Solna,SwedenDocentDagSehlinRudbecklaboratory,UppsalaUniversity,Uppsala,SwedenChairman:DocentJohnLöfblomDepartmentofProteinScience,SchoolofEngineeringSciencesinChemistry,BiotechnologyandHealthKTHRoyalInstituteofTechnology,Stockholm,SwedenRespondent’smainsupervisor:Prof.TorbjörnGräslundDepartmentofProteinScience,SchoolofEngineeringSciencesinChemistry,BiotechnologyandHealthKTHRoyalInstituteofTechnology,Stockholm,SwedenRespondent’sco-supervisor:Prof.StefanStåhlDepartmentofProteinScience,SchoolofEngineeringSciencesinChemistry,BiotechnologyandHealthKTHRoyalInstituteofTechnology,Stockholm,Sweden

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Abbreviations

ABD AlbuminbindingdomainADAPT ABD-derivedaffinityproteinADCC Antibodydependentcell-mediatedcytotoxicityALL AcuteLymphocyticleukemiaBiTE Bi-specificTcellengagerCD ClusterofdifferentiationCDC ComplementdependentcytotoxicityCDRs Complementarity-determiningregionsCR CompleteremissionDAR DrugtoantibodyratioDLT DoselimitingtoxicityDVD-Ig DualvariabledomainimmunoglobulinEMA EuropeanMedicinesAgencyEpCAM EpidermalcelladhesionmoleculeER EndoplasmicreticulumErbB EGFRrelatedreceptorsFab Fragment,antigenbindingFc Fragment,crystallizableFcRn NeonatalFcreceptorFv Fragment,variableGSH GlutathioneHER HumanepidermalgrowthfactorreceptorHIC-HPLC HydrophobicinteractionhighperformanceliquidchromatographyHSA Humanserumalbumini.p. Intraperitoneali.v. IntravenousIAA IndoacetamideIC50 HalfmaximalinhibitoryconcentrationMOA MechanismofactionNHL Non-HodgkinlymphomaNHSesters N-hydroxysuccinimideestersNKcell NaturalkillercellPEG PolyethyleneglycolPSMA Prostatespecificmembraneantigens.c. SubcutaneousscFv Single-chainvariablefragmentSPR SurfaceplasmonresonanceTheFDA TheU.S.FoodandDrugAdministrationWHO WorldHealthOrganizationZ Affibodymolecule

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“Thesharpnessofaswordresultsfromrepeatedgrinding.Thefragranceofwintersweetcomesafterthebittercold.”

-Theancientaphorisms

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TableofContentsAbstract.....................................................................................................................................................iPopularScienceSummary..............................................................................................................iiListofappendedpapers.................................................................................................................iiiRespondent’scontributiontoappendedpapers..................................................................ivPublicdefenseofdissertation........................................................................................................vAbbreviations......................................................................................................................................vi1 Cancer:causes,diagnosisandtreatments.........................................................11.1 GeneticcauseofCancer..................................................................................................................11.2 Biomarkersofcancer/tumorspecificsurfacereceptors................................................21.3 Cancerdiagnosis................................................................................................................................31.4 Cancertreatment...............................................................................................................................4

2 Antibodiesandotheraffinityproteins...............................................................72.1 Monoclonalantibodies....................................................................................................................72.2 Engineeredantibodyvariants.....................................................................................................92.3 Alternativescaffolds.....................................................................................................................122.3.1 Adnectins(monobody):......................................................................................................132.3.2 Anticalins(Affilins)...............................................................................................................142.3.3 Avimers......................................................................................................................................142.3.4 Knottins(Cystineknotminiproteins)...........................................................................152.3.5 DARPins(Designedankyrinrepeatproteins)...........................................................152.3.6 Affibodymolecules................................................................................................................162.3.7 Albuminbindingdomains(ABDs).................................................................................182.3.8 ABD-DerivedAffinityProteins(ADAPTs)...................................................................18

3 Antibodydrugconjugates(ADCs)andalternativescaffolddrugconjugates..........................................................................................................................203.1 ElementsofanADC.......................................................................................................................213.1.1 TargetsofADCs......................................................................................................................213.1.2 LinkersofADCs......................................................................................................................223.1.3 PayloadsofADCs...................................................................................................................26

3.2 Developmentofalternativescaffolddrugconjugates....................................................294 Immunotoxins...........................................................................................................314.1 Ribosomeinactivatingproteins(RIPs)-basedimmunotoxins....................................324.2 Bacterialmono-ADP-ribosyltransferaseproteinbasedimmunotoxins.................324.3 Alternativescaffoldstoxinsfusions.......................................................................................36

5 Presentinvestigation..............................................................................................385.1 PaperI-Target-specificcytotoxiceffectsonHER2-expressingcellsbythetripartitefusiontoxinZHER2:2891-ABD-PE38X8,includingatargetingaffibodymoleculeandahalf-lifeextensiondomain..........................................................................................................395.2 PaperII-InfluenceofmoleculardesignonbiodistributionandtargetingpropertiesofanAffibody-fusedHER2-recognisinganticancertoxin..................................445.3 PaperIII-FusiontoxinsconsistingofaHER2-interactingABD-derivedaffinityprotein(ADAPT)fusedtotruncatedversionsofPseudomonasexotoxinAwithhighpotencyandspecificity............................................................................................................................495.4 PaperIV-Affibody-derivedDrugConjugates:PotentCytotoxicDrugsForTreatmentOfHER2Over-ExpressingTumors..............................................................................535.5 Conclusionremakesandfutureperspectives....................................................................58

Acknowledgements:........................................................................................................................60Bibliography:......................................................................................................................................61

Cancer:causes,diagnosisandtreatments

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Chapter1

1 Cancer:causes,diagnosisandtreatments1.1 GeneticcauseofCancer

Cancer has become one of the leading causes of death in both developed anddevelopingcounties[1].AccordingtotheWorldHealthOrganization(WHO),8.8millionpeoplediedfromcancermerelyin2015,mostlybylung,liver,colorectal,stomach and breast cancer [2]. To understand the cause and development ofcancerisoffundamental interestandof importanceforitstreatment.Asweallknow, humans are a complex form of life on earth, consisting of about 37.2trillion of the basic building blocks of life: the cells [3].Normally, all cells in ahuman body work together to carry out all tasks needed to orchestrate life.However, some cellsmay start to grow and divide abnormally rapidly. This iswhatwe termcancer.The abnormal growth rate is causedby accumulationofmalignant mutations in the cell’s genome. Since DNA replication is not with100% fidelity, incorporating on average 10.6×10-7mutations per cell division,mutationsgraduallyaccumulatewithsubsequentcelldivisions[4].Althoughtherisk of acquiring mutations leading to malignant transformation is very smallduringeachcelldivision,thenumberofcelldivisionsthroughoutone’slifespanis large. This leads to that approximately 40%of people in thewesternworldwill be diagnosed with cancer. Tumor cells appear when themutations causeabnormalities in tumor suppressor genesor oncogenes, and eventually lead touncontrolledcellgrowth.However,carcinogenesisisnotonlycausedbygeneticchangesbutalsobyepigeneticalternations,whichare functionalmodificationsof the genome that do not alter the actual genomic DNA sequences. TheepigeneticeventspromotingcarcinogenesisincludechangesinDNAmethylation,histonemodifications and expression of small noncodingmicroRNAs (miRNA)andtheyareinheritedduringcelldivision[5].ItislikelythatthecentralroleofepigeneticalterationsincarcinogenesisistodecreaseexpressionofDNArepairgenes[6],[7].


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Thereareanumberofenvironmentalfactorsthatmayaccelerateaccumulationof mutations. According to a statement from the world health organization(WHO), there are ten key factors that increases the risk of acquiring cancer,including tobacco use, obesity, an unhealthy dietwith low intake of fruits andvegetables, a lack of physical activity, alcohol use, HPV-infection, hepatitisinfection, ionizing and ultraviolet radiation, air pollution and indoor smokeexposure. Most of these factors, for example tobacco use, cause continuousdamagetothegenome[8],meaningtheaccumulationofmutationsoccursfasterinpersonswhoareconstantlyexposedtothesefactorsthaninpeopleavoidingthem.

1.2 Biomarkersofcancer/tumorspecificsurfacereceptors

Since tumor cells have mutations resulting in uncontrolled abnormal cellproliferation,theyoftendifferfromnormalcellsinsomeway,whichmayhelpindetection as well as treatment of tumors. Cancer biomarkers are identifiablebiologicalmoleculesthataresignsoftumordevelopment:proteins,smallorganicmolecules,DNA,RNAorevenlipidsetc.,foundinblood,otherbodyfluids,orthetumoritself.Biomarkersmaybeusedforriskprediction,diagnosis,predictionoftreatmentsoutcome,andtreatments[9].HereIwillfocusonproteinbiomarkersexpressed on the cell surface, termed tumor specific or tumor related surfacereceptors,whichareeitheroverexpressed,mutatedorselectivelyexpressedontumorcellscomparedtonormalcells.

Onemajor category of tumor biomarkers is the cluster of differentiation (CD)group,includingCD20,CD30,andCD52[10]–[12]expressedondifferenttypesoflymphoma cells. They are selectively expressed on both lymphomas andlymphocytes.Anothermajorcategoryoftumorbiomarkersisthegrowthfactorreceptors.Theyareoftenoverexpressedontumors,providingabnormalgrowthsignals, which drives tumor growth. The epidermal growth factor receptorfamily, including ERBB1, also known as epidermal growth factor receptor(EGFR); ERBB2, also known as human epidermal growth factor receptor 2(HER2);ERBB3,alsoknownasHER3;andERBB4,alsoknownasHER4,arequiteextensively studied and have proven roles in tumorigenesis [13]. Except forHER2,theepidermalgrowthfactorreceptorshaveligandsthatbindandactivatethereceptors,e.g.EGFforEGFR,heregulinforHER3andHER4[14].HER2doesnot have a ligand and is always in an active state. The active form of theepidermal growth factor receptors is as homo or heterodimers, where theytransmitgrowthpromotingsignals[14].ERBB2,thegeneencodinghumanHER2,is a typical proto-oncogene,which is involved in normal cell development butdrives tumor growth when dysfunctional. A sub-set of patients with breastcancer(approx.20%)[15]andasub-setofpatientswithovariancancer(approx.12%) [16]were found to have tumors overexpressing HER2. HER2overexpressingtumorstendtobemoreaggressivethanHER2-negativetumors,showing the significance of HER2 status for prognosis of outcome [14], [17].AnotherreasonforaparticularinterestinHER2,isthatinmanycasesofbreasttumors,theHER2expressionlevelsaredramaticallyhigherthaninnormalcells[18].ThetumorspecificoverexpressionofHER2makesitagoodbiomarkerfordiagnosis and therapy. Immunohistochemistry staining for HER2 is already apart of the standard breast cancer diagnosis procedure [19]. Antibody drugs,


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such as trastuzumab (commercial name Herceptin®) and pertuzumab(commercialnamePerjeta®),targetingHER2havebeendevelopedandapprovedby the US Food and Drug Administration (FDA) for treatment of HER2overexpressingmetastaticbreastcancerincombinationwithchemotherapeuticdrugs[20],[21].Similarantibodydrugstargetingothertumorspecificortumorrelatedantigenshavealsobeendeveloped.Forexample,theanti-EGFRantibodycetuximab for treatment of metastatic colorectal cancer [22], and the anti-vascularendothelialgrowthfactor(anti-VEGF)antibodybevacizumab,areusedclinicallyfortreatmentofvarioustypesofcancers[23].

1.3 Cancerdiagnosis

Cancerdiagnosis is at least as important as treatment, since the earlier cancergets diagnosed and treated, the higher the survival rate is. The5-year relativesurvival rate for patients with stage 0 breast cancer is almost 100%, whilesurvivaldropstoaround22%forstage4breastcancer[24].Currentlytherearetwostandardwaystodiagnosecancerinclinicalpractice,byimaging procedures or by biopsy. The imaging procedures include computedtomography(CT)scan,ultrasound,magneticresonance imaging(MRI)andPET(positron emission tomography) scan, including various contrasting agents.Theseimagingproceduresarenon-invasive,buttheresultsneedtobecarefullyevaluated to avoid false positive and false negative diagnoses [25]. Althoughcollection of biopsies is invasive, where tissue samples are isolated from thepatientforobservationunderamicroscope,itisalmostalwaysrequiredtogiveadefinitiveresultwhenimagingproceduresshowpossiblepositiveresults.

The traditional imaging diagnosis can be combinedwith assessment of tumorbiomarkers, giving more comprehensive results. For example, if a patient’sbreasttumoroverexpressHER2,thetumorcouldbeimagedbynuclearmedicinescanafter injectionof a radiolabeledHER2specific antibody [26]. In that case,the tumor may be easily distinguished from normal tissue due to a higheraccumulation of radiolabeled HER2 specific antibody in tumor tissue. Thecombinationofsuchmolecularprobesandimagingtechniquesmayalsoprovideinformationofmetastases,whicharedifficulttofindotherwise[27].

Receptorexpressionstatusmayalsobedeterminedaftercollectionofbiopsies.Collected biopsies may for example be stained with a HER2 specific antibodytogether with a color generating secondary antibody, in order to score thetumor’sHER2status,whichisaclueforprognosis[28]–[30].Diagnosticmethodscanalsobeappliedonpeoplewhodonothaveanysymptomofcancer,inordertodetecttumorsataveryearlystage.Thistypeofdiagnosisistermedcancerscreening.Analysisoftraceamountsofprotein,RNAandDNAascancerbiomarkersinbloodorurineispreferredbecausetheyarenon-invasiveandsamplesareeasy toobtain [31].BothcirculatingmicroRNAandmicroRNAdetected in urine, may be used to diagnose breast and ovarian cancer [32].Pilyuginetal.were forexampleable tostage lungcancer fromstage1 to4,bymeasuringthelevelofautoantibodiestoBARD1inserum[33].Ithasevenbeenpossible todetermine theoriginof somecancersbyanalyzing themethylationhaplotypeblocksofcellfreeDNAisolatedfromblood,sincegenomicDNAfromdifferenttissueswerefoundtohavedifferentmethylationpatterns[31].


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Themethodsarenotonlyusedfordiagnosis.Theyarealsousedduringandaftertreatmenttoevaluatetheefficacy.

1.4 Cancertreatment

Doctors generally have five types of treatment possibilities, (i) surgery, (ii)chemotherapy,(iii)radiotherapy,(iv)targetedtherapyand(v)immunotherapy.

(i)Surgeryisaneffectivewaytoremovemanytypesofsolidtumors.However,whencancermetastasizetodistantsites, it isnormally impossibletosurgicallyremoveallmetastases.Forhematopoietic tumors, surgical removal isnotevenpossible.Theaimofthesurgeryistoremoveasmuchaspossibleofthetumor,while minimizing the harm to the patient. Even though the physicians try toremove as much as possible of the tumor, there is always a risk of relapse.Relapses are also sometimes a consequence of surgery, which may lead todisseminationoftumorcells[34].

(ii)Chemotherapeuticdrugsaremostly cytotoxic smallorganicmoleculeswithdifferent modes of action (MOA). They may for example damage DNA byalkylation (for example Cisplatin), or inhibit DNA replication (for example 5-fluorouracil), or interfere with enzymes involved in DNA replication (forexample, Doxorubicin), or inhibit mitosis (for example, Mitomycin-C) [35].Although several types of chemotherapeutic drugs are designed to act on fastgrowingtumorcells, theirspecificity isoften lowandnormalcellsmayalsobeaffected.Thereareavarietyofadverseeffects,includingfatigue,hairloss,weightloss and the possibility of getting secondary tumors, when treated with suchagents. For example, the relative risk to develop leukemia or myelodysplasiasyndrome forpatients treatedwithDNAalkylatingdrugswas6.5 timeshighercompared to control patients [36]. Beyond the adverse effects, many patientsalso develop resistance to chemotherapeutics [37], [38]. Chemotherapeuticdrugs can be the only treatment in some cases, but they are often used incombinationwith other types of therapy. For example, they can be given as acomplementarytreatmentincombinationwithsurgerytoreducerelapses.

(iii) Radiation therapy utilizes high-dose radiation to kill cancer cells. Theradiation damages DNA, and the cells die after a while when the damageexceeded the cell’s ability to repair its genome [39]. Radiation therapy can beeitherlocalbyexternalbeamradiationorsystemicbyinfusionofradioisotopesor isotope-labeledcompounds.Both formsofradiationtherapykillnormalandcancercells.Thereisalifetimelimitoftheamountofradiationapersonmaybesubjected towithout severe side effects,which also limits its use. Cancer cellsmayalsodevelopresistancetoradiotherapy[40].


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Figure1.Currentcancertreatmentsoptions.Surgery,chemotherapyandradiotherapyaretraditionalcancertreatments.Immunotherapyandtargetedtherapyaremorerecentcancertreatments.Allthetreatmentsareoftenusedincombinationinclinicalpractice.

(iv)Inthelate1990s,targetedtherapieswereinvestigatedtocircumventthelowspecificityproblemofchemotherapyandradiation therapy.Tamoxifenwas thefirst approved targeted cancer therapeutic, which is used for treatment ofpatientswithestrogenreceptor(ER)positivebreastcancertumors. Itpreventsestrogen from binding to ER in the cytoplasm and therefore inhibits ERstimulationoftumorgrowth[41].Inadditiontosmallmoleculesthatspecificallyact on tumor cells, antibodies play amajor role in targeted therapy since it ispossible to design antibodies with high specificity for differentially expressedtumor antigens. One of the most effective antibody-based cancer drugs istrastuzumab.ItisanFDAapproveddrugfortreatmentofHER2overexpressingmetastatic breast cancer.When it binds to HER2 on the tumor cell surface, itinhibitscellgrowthby inhibitingHER2activation, triggersantibody-dependentcell-mediated cytotoxicity (ADCC) and triggers complement-dependentcytotoxicity(CDC)[42]–[44]Addingtrastuzumabtostandardchemotherapy,ledto 37% of relative improvement in overall survival and all patient subgroupsbenefitedfromit[45].Antibodieshavealsobeenconjugatedwithsmallcytotoxicmolecules,namedantibodydrugconjugates (ADCs),with theaimof increasingtheir potency. T-DM1, trastuzmab in conjugationwithmertansine (DM1), wasapproved by the FDA in 2013 for treatment of metastatic breast tumorsoverexpressingHER2[46].T-DM1retainsallthemodesofactionoftrastuzumab,includingblocking ofHER2,ADCC andCDC, and it directly kills tumor cells byreleasingDM1inthecytoplasm[46].ADCswillbediscussedindetailinchapter3ofthisthesis.

(v) Immunotherapy is a type of cancer treatment utilizing the patient’s ownimmunecellstofightcancer.Thehumanimmunesystemhasbeenfoundtohaveanti-tumoractivity, however it is often inhibitedby the tumor cells and tumormicroenvironment[47],[48].Anothermotivationtoinvestigateimmunotherapytocombatcanceristhattheadaptiveimmunesystemworksspecificallytowardsa particular antigen, which may give, in theory, a highly specific treatmentagainst tumor cells.The ideabehind immunotherapy is tousevariousways to


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activate and direct immune cells to kill tumor cells, which involves bothactivating immunecellsbydifferentroutesand/or todirect themto the tumorcells by ex vivo geneticmodification. Antibodies can be used to block immunecheckpointstoactivateimmunecells.Programedcelldeathprotein1(PD1)andprogrameddeath-ligand1(PD-L1)antibodiesareusedtoenhancethe immuneresponse to tumor cells by releasing the “brake” of the immune system. Theyhaveshownpotentanti-tumoreffects[49].Recently,chimericantigenreceptorTcell (CAR-T) immunotherapy, has drawn a lot of attention, and CTL019(commercialnameKymriah)hasjustbecomethefirstCAR-Ttherapeuticoptionapprovedby theFDA [50].CTL019utilizes thepatient’sownTcellswhicharetransfected with genetic material to express a chimeric antigen receptor,targeting CD19, which is expressed on B cell leukemias. The treatment isapproved for treatment of acute lymphoblastic leukemia (ALL) and chroniclymphocytic leukemia (CLL) [51][52]. The complete remission (CR) rate ofpatientstreatedwithCTL019washigherthan80%[53].Itshouldbenotedthatthesepatientshadaverypoorprognosisotherwise.

Inclinicalpractice,allthetherapiesdescribedaboveareoftenusedincombinationratherthanasmonotherapiestoachievebettertreatmentoutcomesandpreventingrelapses.

Antibodiesandotheraffinityproteins

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Chapter2

2 Antibodiesandotheraffinityproteins2.1 Monoclonalantibodies

The“magicbullet”conceptreferstodrugsdesignedtoonlytakeeffectontheirintendedcell-structuraltargets.PaulEhrlichproposedthisconceptwhensearchingforanti-toxinantibodieswithoutknowingtheactualstructureofantibodies[54].Atfirst,the“magicbullet”drugswerechemotherapeuticsusedtotreatinfectionswithoutcausingmuchharmtothebody,suchasarsphenamine.Monoclonalantibodiesappearedasthe“magicbullet”totreattumorsalmost80yearsaftertheconceptwasproposed.DiscoveryofthechemicalstructureofantibodiesbyGeraldEdelmanandRodneyRobertPorter[55]in1959andtheinventionofhybridomatechnology[56]in1975weretwoimportanteventsthatenabled“magicbullets”derivedfromantibodies.Thehybridomatechnologyallowsimmortalizationofantibody-producingBcellsbyfusingthemtoimmortalizedBcellmyelomas.Thismadeitpossibletoproducelargeamountsofmonoclonalantibodies.However,theearlymonoclonalantibodiesproducedfromhybridomaswereofmurineorigin,andelicitedanimmuneresponse,whichprovokedproductionofneutralizingantibodies,limitingtherapeuticapplication.Theemergenceofantibodyengineeringtechnologiesinthe1990sallowedforconstructionofhumanmurinechimeric,humanizedorevenfullyhumanantibodies,withlowornoimmunogenicity[57].Thesymbolicapprovalofrituximabin1997bytheFDA,indicatedthestartofantibodytargetedtumortherapeutics.

From an immunological point of view, antibodies have the functions ofneutralizingforeignantigensanddirectingotherimmunecellstoclearinfections.WhenBcellsareactivatedbyantigenpresentingcells(APCs),theantibodygenesundergo V(D)J recombination, class switching, somatic hypermutation and


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selection,resultinginacloneproducingaspecificantibodywithhighspecificityand affinity for the antigen [58]. There are several isotypes of antibodiesproducedbyBcells,namelyIgA,IgD,IgE,IgGandIgM,withIgGbeingthemostcommonly used class for therapeutic and biotechnological applications. Tworeasons for the wide-spread use of IgG is that they are the most abundantantibodies in circulation and they are the most diverse antibodies, with foursubclasses [58]. The four IgG subclasses, IgG1, IgG2, IgG3 and IgG4, have so-called effector mechanisms including: facilitation of phagocytosis bymacrophages,elicitingcomplementdependentcytotoxicity(CDC)andantibody-dependent cell-mediated cytotoxicity (ADCC) upon binding to their targets.However,everysubclassdoesnotnecessarilyhavealltheeffectormechanisms.One IgGmolecule consists of four peptides, two identical light chains and twoidenticalheavychains,whichareinterconnectedbydisulfidebridgesformingaYshapedmacromolecule (Figure 2). The IgG structure can be dissected into thefragmentofantigenbinding(Fab)andthefragmentcrystallizable(Fc)regionsbytheir different functions (Figure 2). The ‘head’ of the Fabs containcomplementarity-determiningregions(CDRs),determiningantigenspecificityoftheantibodies.Thediversityofaminoacidsequences intheCDRsisaresultofV(D)J recombination and somatic hypermutations. In theory, antibodiesgeneratedbyB-cellsmaytargetalmostanyantigenpresentedtothehumanbody.TheFcpartofantibodiesinteractwithsomeinnateimmunecells,activatingtheeffector mechanisms. Fc binds to Fcγ receptors (FcγR) expressed on naturalkiller(NK)cells,dendriticcells(DCs),macrophages,neutrophilsandeosinophilsdelivering either inhibitory or activating signals [59]. ADCC is a cell-mediatedimmuneresponsemechanismwherebytypicallyFcγRIIIAreceptorsonNKcellsinteract with Fc in IgGs bound on the cells surface, followed by release ofperforinandgranzymesbytheNK-cellscausinglysisofthetargetcell[58].ADCCisquiteinterestingfromacancertreatmentperspective,sinceithelpsantibodydrugstokill tumorcells.Fccanalsorecruitthecomplementsystemfor lysisoftumor cells, by amechanism called complement-dependent cytotoxicity (CDC).Bindingof complementcomponents toFc initiatesa cascadereaction insertingone ormoremembrane attack complexes (MAC) on pathogens or tumor cells,resultingincelllysisthroughsubsequentinfluxofionsandwater[60].Another important functionof theFcof IgG is tomaintain theconcentrationofIgGincirculation.Theserumhalf-lifeofIgGcanbelongerthanfourweeks,dueto the interaction with the neonatal Fc receptor (FcRn) [61]. FcRn was firstdiscoveredinthenewbornrat’sepithelialcellsinthegut,transportingIgGsfromthe mother’s milk to its bloodstream [61]. Later, it was found that it is alsocrucial for maintaining the concentration of IgG in the bloodstream. FcRn isexpressedonmanytypesofcells,bothnon-hematopoieticandhematopoietic.Itwasfoundthatepithelialandhematopoieticcellsweretheprimarycontributortowild-type IgGhomeostasis invivo[62]. FcRnbinds to Fc in a pHdependentmanner,withlowaffinityatneutralpHandwithhighaffinityinacidicpH.Thisleads to that FcRn binds to antibodies internalized in the endosomalcompartmentswherethepHisacidic(lessthan6.5),FcRnisthenresponsiblefortransporting the bound antibody to the cell surface where a neutral pH isencountered and the antibody is released. This mechanism limits antibodydegradationinthelysosome[63].


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All of above-mentioned properties of antibodies make them well suited fortherapeuticpurposes.Currentlythereare78monoclonaltherapeuticantibodiesapprovedbytheEMAand/ortheFDA[64].

2.2 Engineeredantibodyvariants

Besides the natural formats of antibodies, a variety of engineered antibodies,whichdonotexist innature,havebeendevelopedtoreducethesize, tocreatemulti-valentvariants,orfor“fittopurpose”applications[65].Thefirstwaveofantibody engineering during the 80 to 90s, weremostly aimed at humanizingthemandfocusedoncreatingdifferentvariantsofthevariabledomains.Naturalantibodies have two paratopes, which can bind to twomolecules of the sameantigen simultaneously. One of the paratopes can be engineered to target adifferentantigen,resultinginbi-specificantibodies.Thesebi-specificantibodiesoften have higher anti-tumor activity to the tumor cells expressing the twoantigens by down regulating two growth-promoting receptors simultaneously[66]. In addition, bi-specific antibodies are used to engage T-cells with tumorcellsfortargetedtumortherapies[67].Oneofthefirsttechnologiesdevelopedtoproduce bi-specific antibodies was to use a “knobs into holes” technologycorrectly pairing a half of one antibody with a half of another antibody [68](Figure2).Recently,othermethodshavebeendevelopedtoproducebi-specificantibodies [69], [70]. The specificity of bi-specific antibodies are often wellimprovedcomparedtotheparentalantibodiesandhavebeenshowntopossessamorepotentanti-tumoractivitycomparedwiththeparentalantibodies,sincetwo receptors’ signalingpathways are simultaneously inhibitedby one type ofantibodymolecule[71],[72].


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Figure 2. Illustrative structure of an antibody and examples engineered antibody variants.The antibody is a “Y” shaped macromolecule consisting of four polypeptide chains; twoidenticalheavychainsandtwoidenticallightchains,interconnectedbydisulfidebonds.Boththe lightandheavychains canbedivided intoavariable region (VLorVH)anda constantregion(CLorCH).AscFv(singlechainvariablefragment)consistsaVLandaVHconnectedbya polypeptide linker. A dsFv (disulfide-stabilized variable fragment) consists a VLanda VHstablizedbyadisulfidebond.Fab(antigenbinding fragment) isonearmof the “Y” shapedantibody.Adiabodyisanon-covalentcrossoverdimeroftwopolypeptides,eachcontainingaVHandaVLfromtwodifferentantibodies.Bi-specificantibodieshaveonespecificityoneacharm,whichisderivedfromtwodifferentantibodies.Atwo-in-oneantibodycross-reactwithtwoantigensoneacharm.DVD-Ig isanartificialantibodyhaving fourvariable fragments.Theupper twohave specificity toone targetand the lower twohave specificity toanothertarget. HcAb (heavy chain only antibody) are isolated from the serum of Camelidae andcontain only heavy chains for interaction with antigens. IgNAR (Immunoglobulin newantigenreceptor)areantibodiesisolatedfromtheserumofsharks,whichalsoonlycontainheavychains.However,theyhavefiveconstantdomains.SdAb(singledomainantibody)canbederivedfromtheVHdomainofHcAbs,IgNARsorcommonIgGs.

An alternativeway tomake an antibody targeting two different antigens is tograft the variable domain of one monoclonal antibody on top of another

scFv dsFv Fab Diabody

Antibody

Bispecific antibody Two-in-one antibody DVD-Ig

Fab

Fc

Light chainHeavy chain CDRs

V L

V H

C L

C H1

CH2

CH3

V H

CH2

CH3

V H

CH1

CH2

CH3

CH4

CH5

V H

HcAb IgNAR sdAb


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monoclonal antibody, which is termed dual-variable-domain immunoglobulin(DVD-Ig) [73], [74](Figure 2). Similar to bi-specific antibodies, a DVD-Ig oftenhas superior anti-tumor activity compared with mono-specific antibodies. Forexample, an anti-EGFR and anti-HER3DVD-Ig showedmore potent anti-tumoractivitybymeansofproliferation inhibition,apoptosis inductionandenhancedinternalization in vitro compared to each parental antibody alone or incombination[74].Unlike the above-mentioned methods, which mainly combine existingmonoclonal antibodies to acquire bi-specificity, a two-in-one antibody wasgeneratedbyphagedisplaytohavecross-activitytotwoantigens[66](Figure2).However, the generation of a two-in-one antibody requires considerableengineeringandisoftentimeconsuming.Apopulartypeofminimizedantibodyisthesinglechainvariablefragment(scFv)consistingofalightchainvariableregion(VL)andaheavychainvariableregion(VH)linkedbyapeptidelinker[75].ThemuchsmallersizeofscFvcomparedtothefull-lengthantibodyresultsinmorerapidtumorpenetration[76].However,scFvsareclearedfromthebloodmuchfasterthanfull-lengthantibodies,becauseofthesmallersizeandlackofFcforFcRn-mediatedprotectionfromintracellulardegradation [76]. ScFvs are in many cases more suitable for diagnosticapplicationsthanfullantibodies,whichwillbediscussedlaterinthischapter.AversionofscFvsarethediabodieswherethelinkerbetweenVHandVLinascFvhasbeenshortenedtoanextentthatitcanonlypairwithanotherscFvmoleculeformingnon-covalentdimers.Thediabodiesmayeitherbebivalentorevenbi-specific [77].Thebi-specificdiabodiesare interestingbecause theyareusuallyeasier to produce than bi-specific antibodies [77]. A version of bi-specificantibodies that connects tumor cells with T cells are termed bi-specific T cellengagers (BiTEs). BiTE diabodies targeting tumor specific antigens (CD19,prostate specific membrane antigen (PSMA) or EGFR) and CD3 on the T cellsurface have been investigated for tumor therapy [78]–[80]. Blinatumomab, aCD19/CD3 BiTE, has been approved by the FDA for treatment of AcutelymphocyticLeukemia(ALL)[81].Otherthantheabove-mentionedantibodyfragmentsconsistingofpairedVLandVH, singlemonomeric variabledomains can also stand alone as binders,whicharenamedsingle-domainantibodies(SdAbs).OnepossiblewaytoproducesuchSdAbs is to isolate VL or VH from conventional human or murine antibodies.However,theyarepronetoaggregationduetotherelativelyhighhydrophobicityof their dimerization surfaces. Another approach to make single-domainantibodiesistoisolateVHfromheavychainonlyantibodies(HcAbs)IgG2orIgG3fromCamelidae[82]andIgNARs(Immunoglobulinnewantigenreceptors)fromsharks [83], which are naturally devoid of a light chain (Fig. 2). The variabledomain of the heavy chain of the heavy chain only camel antibody (VHH) iswidelyknownasananobody,whichisonly~15kDainsize(Fig.2).Sincetheylackalightchain,ananobodyonlycontainsthreecomplementarity-determiningregions (CDRs). However, the CDR1 and CDR3 loops are typically longer thanconventionalVHs, providing adequate antigenbinding surfaceswith an area of600–800Å2[84].


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Nanobodieswith high affinity and desired specificity can be easily selected byphage display techniques [85], [86]. The selected nanobodies can be easilyproduced in bacteria, mammalian cells, or plants [87]. During the last twodecades, nanobodies targeting a variety ofmolecules have been generated forbiotechnologicalandmedicalapplications[88].Eightnanobodiesarecurrentlyinclinicaldevelopment.Theleadingnanobodydrugcaplacizumabfortreatmentofacquiredthromboticthrombocytopenicpurpura(aTTP)hasshownpositivetop-lineresultsinaphaseIIIstudyandisexpectedtobeapprovedbytheauthoritiesin2018[89].

AnotherbranchofantibodyengineeringistomakechangestoFctomodulatethepharmacokinetic properties of antibodies, and the capability to elicit immuneresponses. IgG, themostoftenusedtypeof immunoglobulin,naturallyhas foursubtypes, IgG1, IgG2, IgG3 and IgG4 having different half-life, complementfixationabilityandaffinityforFcγRs[58].Therefore,thechoiceofsubclassofIgGhasgreatinfluenceonthepropertiesofthetherapeuticantibodies.Outofthe78FDAandEMAapprovedtherapeuticmAbs,48areoftheIgG1subtype[64].Thereasons for choosing IgG1 areprobably that theyhave ahigh ability to induceADCCandCDCactivities,andhashighaffinityforFcRn[90].Itisalmostalwaysdesired to have potent ADCC and CDC effects for cancer therapy. In order toenhance theADCC effect, the affinity betweenFcγRs andFcmaybe increased.Engineered fucose deficient glycosylated IgGswas found to have an enhancedADCCeffectoractivity[91],[92].Therearetwoglycol-engineeredantibodiesthathavebeenapprovedforclinicaluse,theanti-CD20mAb,obinutuzumab[93],andthe anti-CCR4 mAb, mogamulizumab [94] A lot of other modifications andmutations to modulate antibody effector functions has been summarized byWangandco-workers[95]

Antibodies are versatile proteins with several different regions binding todifferentmoleculesgivingitdifferentfunctions.Therearenumerousvariantsofantibodiescreated,manyofwhichareaimingforuseindiagnosisortreatmentofcancer.Theproperengineeringofeachpartof anantibodymakes itbetter forthepurpose.

2.3 Alternativescaffolds

Antibodieshaveachievedgreatsuccessintargetedtumortherapy,withover78antibodiesapprovedbytheFDAandalotmoreinclinicaltrials[64],[96],[97].However, antibodies arenotperfect for every application. First, antibodies arequitelargemolecules(150kDa)givingthemaratherpoorpenetrationoftumortissues [98]. Second, the cost-of-goods when producing antibodies is high,because most antibodies have to be produced in mammalian cells to get thecorrectpost-translationalmodificationsuchasglycosylation,pyroglutamylation,deamination of asparagine etc.. The high price of antibody drugsmakes themavailableonlytoasmallportionofallpeopleintheworld.Third,becauseofthecomplex structure of antibodies, it has been proven difficult to producehomogeneousantibodydrugconjugates(ADCs),aclassofdrugs Iwilldescribelaterinthetext.Inthiscase,theconjugationproductsaremixturesofantibodieswithdifferentnumberofdrugsattachedtoit[99].Finally,manyantibodydrugsonthemarketareofmurineorigin,eithermurine-humanchimericantibodiesor


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humanized murine antibodies. It takes a considerable effort to reduce theimmunogenicityoftheoriginalmurineantibodies.With the accumulation of knowledge and the development of technologies tostudy molecular recognition mechanisms in general, we have become able tocreate non-Ig affinity proteins based on scaffolds from various origins [100]Thesealternativenon-Igaffinityproteinscaffoldsareabletospecificallybindtoanantigenwithhighaffinity,similartoantibodies.SincetheydonothaveFc,thealternative scaffolds are beneficial in the cases where ADCC and CDC areundesired.Unlikeantibodies, thealternativescaffoldsareusuallymuchsmaller(<20 kDa), therefore potentially having better tumor penetration [101]. Thesmall size of the alternative scaffolds also results in a fast clearance from thebody, which is not necessarily a disadvantage, and depends on the intendedapplication.Thesmallsizealsomakesitpossibletochemicallysynthesizesomeof the alternative scaffolds such as affibody molecules [102], allowing easyintroduction of unnatural amino acids, if desired. The introduced unnaturalaminoacidscouldbeusedforspecificconjugationofdrugsorimagingprobes.Inaddition, most of the alternative scaffolds exhibit high thermo-stability [100].Another important featureofalternativescaffolds isthattheproductioncost isoftenlowerthanthatofantibodies,becausetheycanoftenbeeasilyproducedinbacterial host cells such as Escherichia coli (E. coli), with a high yield. All thealternativescaffoldsaffinityproteinsaregeneratedbydirectedevolutionusinginvitrodisplaytechnologies,suchasphagedisplay,yeastdisplayandCISdisplayetc,withfewexceptionsthatwildtypesweretakendirectlyasdrug.Generally,atwo-stepprocessisused:step1)generationofalibrarybyrandommutagenesisofaminoacidsinthebindingsurface,step2)isolationofpotentialbindersfromthe library by incubating the library with the target molecule during severalroundsofselection.

Although a number of alternative scaffolds have been developed, they can beclassifiedintotwotypesbasedontheirbindingsurface:i)ligand-bindingaminoacidsinexposedloops,andii)ligand-bindingaminoacidsinsecondarystructureelements. In the thesis I will not cover all the alternative scaffolds in eachcategory,butIwilldiscusssomeimportantexamples.

2.3.1 Adnectins(monobody):AdnectinsarealternativescaffoldproteinsderivedfromthetenthdomainoffibronectintypeIII(10Fn3),whichnaturallybindstointegrins[103].Itfoldsintoaβ-sandwichstructurewithsevenstrandsconnectedbysixloops,similartoanantibody’svariableregionstructure,althoughtheyhaveverylowsequencehomologytoantibodiesandanAdnectinhasnodisulfidebonds[104],[105].Typically,Adnectinshavehighthermo-stabilitywithaTm>80°C[106]inreducingconditionandcanbeproducedwithhighyieldinbacteria.Theloopsresemblethecomplementarity-determiningregions(CDRs)ofantibodies,andarethusobviouscandidatesfordiversificationandselectionofAdnectinsagainstnewtargets.Someofthenon-loopresidueshavealsobeenfoundtobeinvolvedininteractionwiththetargets[107].AdnectinstargetingCD4,tumornecrosisfactor-α(TNF-α)andthevascularendothelialgrowthfactorreceptor2(VEGFR2)withaffinitiesrangingfrompMtonMhasbeengeneratedbymRNAdisplay[108]–[110].TheAdnectintargetingmyostain[111](BMS-986089)for


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treatmentofDuchenne’smusculardystrophy(DMD)iscurrentlybeingevaluatedinaphaseIIclinicaltrial(NCT03039686).

Figure3.Representative3Dstructuresofsomealternativescaffolds(notinscale).Adnectin(PDBID1ttg),anticalin(PDBID3bx7),avimer(PDBID1ajj),knottin(PDBID2it7),DARPin(PDBID4hrn),Affibody(PDBID2kzj)andABD(PDBID1gjs).

2.3.2 Anticalins(Affilins)Anticalins are derived from lipocalins (160-180 amino acids), which exist inmany organisms, including vertebrates, insects, plants and bacteria, serving astransportation or storage proteins of hydrophobic compounds [112]. Thelipocalinsshareaβ-barrelstructureconsistingofeightantiparallelβ-strands;acup-likestructurewithoneendopenandoneendtightlypacked.Thefourloopsat theopenendexhibithighdiversityamong the lipocalins,both in lengthandconformation,despite thehighlyconservedβ-barrelstructure.Normally,16-24residueson the four loops are randomized to generate anAnticalin library forselection of binders [113]. Phage display has been used to select Anticalinsagainstdifferent therapeutic targetsobtainingpicomolaraffinitybinders [114],[115]. LikeAdnectins,Anticalins oftenhavehigh thermo-stability (Tm>70 °C)andgivehighyield inbacterialproduction.Thebi-specificAnticalin (PRS-343)targeting CD137 and HER2 to engage CD137 positive T-cells with HER2overexpressing tumor cells for treatment of HER2 positive advanced ormetastaticsolidtumorshasenteredaphaseIclinicaltrial.

2.3.3 AvimersAvimers are single chains of multimeric A-domains, which exist tandemlyrepeatedinseveralcell-surfacereceptors,suchasinthelowdensitylipoproteinreceptor(LDLR)[116].ThefamilyofnaturallyoccurringA-domainsbindtoover100knowtargets,includingsmallmolecules,proteinsandviruses[117],[118].Atypical A-domain comprises ~35 amino acids (~4 kDa), 12 of which form a


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conservedscaffoldmotif including threedisulfidebondsandacalciumbindingsite[119].Therestoftheaminoacidsareamenableforrandomizationfollowedby selection for Avimers targeting new antigens. Since the A-domains alwaysappear in the formof tandemmulti-domains to increase theavidityeffect, andthesizeofoneA-domainisverysmall,Avimersareoftenselectedinamultimericcontext. After one round of selection, the enriched Avimer monomers aregenetically fused to another pool of Avimer monomers (naïve or previouslyenriched)andthensubjectedtoanotherroundofselection[120].Attheendofasuccessfulselection,aproteincontainingmultipledomains,bindingtodifferentepitopesona single target, isacquired.AnAvimer trimerselectedagainst IL-6with lowpicomolaraffinity [120]enteredphase I clinical trial (NCT00353756)forCrohn’sdiseasein2006,butthereisnofurtherupdatesincethen.MaybeithastodowiththeacquisitionofAvidiaInc.,acompanycommercializingAvimerdrugsandtechnology,byAmgenInc.in2006.

2.3.4 Knottins(Cystineknotminiproteins)Knottins,alsoknownasinhibitorcysteine-knots,aresmallsized,30to50aminoacidslongproteins,whicharenaturallyfoundinavarietyoforganismsservingas protease inhibitors, and antibacterial or antifungal agents [121]. Knottinsadopt a unique structure: one disulfide bond goes through the macro-circleformedbyanothertwodisulfidebondsforminga“knot” likestructure.Owningtothiscysteineknotstructure,knottinsareoftenhighlythermo-andchemicallystable (Tm>80 °C), thusmaking themgoodcandidates fororaladministration[122],[123].Unliketheotheralternativescaffolds,whichareoftenselectedfroma library, the surface exposed loops of knottinsmay be grafted from a knownpeptide to acquire a desired specificity followed by a directed evolutionapproach to optimize the whole structure [124]–[126]. Two naturally derivedknottinsfortreatmentofseverechronicpainandirritablebowelsyndromewithconstipation (IBS-C) and chronic idiopathic constipation (CIC) have beenapprovedbytheFDA[127],[128].

2.3.5 DARPins(Designedankyrinrepeatproteins)The alternative scaffolds discussed above use loops to create the bindingsurfaces. Designed ankyrin repeat proteins (DARPins) can be considered ahybrid between loop-based and secondary structure element-based bindingsurfaces.DARPinsarederivedfromnaturallyoccurringankyrinrepeatproteinsmediating high-affinity protein-protein interactions in almost all organisms[129].DARPinsusually consistof fouror five repeats, includingoneN-cappingrepeat,oneC-cappingrepeatandtwoorthreeinternalrepeats,eachhavingaβ-turn followed by two anti-parallel α-helix-loop structure motifs and a loopconnectingtothenextrepeat[130].Eachrepeatconsistsof33aminoacids,sixofwhich are involved in interaction with the target [130]. The backbone ofDARPinswasdesignedthroughaconsensusstrategy,whereseveralaminoacidsequences of different DARPins of different origins were aligned, followed byidentification of dominantly appearing amino acids in each position [131].DARPinsareoftenhighlythermostablewithupto90°CTmandcanyieldupto200mg/LbyE.coliproductioninshakeflasks[130].DARPinstargetingavarietyof antigens have been selected by phage display and ribosome displaytechnologies [132]. The anti-tumor DARPin drugs that is most advanced in


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clinicaldevelopmentisatri-specificfusionprotein(MP0250)[133]consistingananti-hepatocytegrowthfactor(HGF),ananti-vascularendothelialgrowthfactor-A(VEGF-A),andtwoanti-HSADARPindomains,fortreatmentofrefractoryandrelapsedmultiplemyeloma.ItisinaphaseIIclinicaltrial.TheonlyDARPindrugthat has entered a phase III clinical trial is a PEGylated anti-VEGF DARPin(Abicipar Pegol) for treatment of wet age-related macular degeneration (wetAMD)anddiabeticmacularedema(DME).TheresultsforAbiciparhasshownithas the potential to provide at least equal and potentially higher vision gainswith fewer injections in wet AMD compared to standard of care treatmentLucentis®(ranibizumab)andthat itcanreduceretinaledemainDMEforupto12-16weeks[134].

2.3.6 AffibodymoleculesAffibodymoleculesbelongtoaclassofalternativescaffoldproteins,investigatedin the studies of this thesis. The affibody scaffold, alsodenoteddomainZ,wasoriginallyderivedfromtheBdomainofstaphylococcalproteinA,whichbindstoimmunoglobulins in theFcandFab regions [135]–[137].ProteinA isnaturallyexpressed on the surface of some staphylococci to help them escape fromimmunesurveillancebybinding to immunoglobulins [137].Affibodymoleculesaresmallproteins(~7kDa),consistingof58aminoacidsfoldedintoathreeα-helical bundle structure. The rigidity of its structure makes it possible toaccommodate severalmutationswithoutdestroying its structural integrityanddecreasing the stability toomuch. To create an affibodymolecule binding to adesired target, 13 surface exposed residuals on helix 1 and helix 2, whichnormallybind toFc,are typicallyrandomized togeneratea library [138][136].Thelibrariesaremostlydisplayedonphagebut,insomecasesonstaphylococci,ribosomesoryeast,andthensubjectedtobiopanningorfluorescence-activatedcell sorting (FACS) to enrich binders to various targets [139]. Most of theselectedaffibodymoleculeskeep the threeα-helical structures,however, therearesomeexceptions,wherehelix1becomesaβ-sheet,whichwillbediscussedlater. The scaffold of an affibody molecule has been further optimized usingextensive structural modeling, resulting in molecules with elevated Tm andhydrophilicity[140].Thesmallsizeandfastfoldingnatureofaffibodymoleculesenable chemical synthesis, which may be used to introduce unnatural aminoacids.Thiswillalsobefurtherdiscussedlater.Thereareanumberofaffibodymoleculestargetingaround40diseaserelevanttargets reported in the past 20 years [139]. One affibody molecule has evenreachedaphaseIIIclinicaltrial.Itisananti-HER2affibodymoleculeconjugatedwith radionuclides, denoted ABY-025, for breast cancer imaging [141]. It wasradiolabeledwith 68Ga and 111In forpositronemission tomography (PET) andsingle-photon emission computed tomography (SPECT) imaging, respectively[141],[142].Assoonas1hpostinjection,PETimagingcouldrevealthepositionsof theHER2expressingtumor,andevenmetastaseswith lowHER2expressioncouldbedetected[141],[142].Theanti-HER2affibodymoleculeswereusedastargetingdomains inpaper Iand II in this thesis.Theclinicallymostadvancedtherapeuticaffibodymoleculeisadimericanti-IL17affibodymoleculeinfusionwithABD,denotedABY-035.IthasenteredaphaseIIclinictrialtoevaluatetheeffectiveness to plaque psoriasis [143]. Affibody molecules have severaladvantagesoverantibodies formolecular imagingof tumors.First, theaffibody


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moleculesaremuchsmaller thanantibodies,below thekidney filtrationcutoff,and are thus cleared from circulation very fast. This provides for a lowbackgroundsignalandhigherimagingcontrastonlyashorttimeafterinjectionof the imaging agent. Since the affibody scaffold is cysteine free, it makes itpossible to introduce a unique cysteine at many possible positions, forhomogeneous and site-specific radiolabeling [144]. A recently reported newaffibody imaging concept, is based on affibody-PNA pre-targeting [145]–[147].Instead of direct conjugation of a radionuclide to the affibody molecule, theradionuclides are conjugated on a peptide nucleic acid (PNA) fragment,whichhas a complementary sequence conjugated with the affibody molecule. Thetumor is first saturated with affibody-PNA and then the PNA-radionuclideconjugate is injected for imaging. Because the PNA-radionuclide is small andhighlyhydrophilic, ithaslowunspecificuptakeinmostorgansandisthereforecleared fromthebodyshortlyafter injection.Thebloodandkidneyuptakeare50and2-foldlowercomparedtodirectlylabeledaffibodymolecules[145],[147].Someaffibodymoleculesdonotonlybindtotheirtargetbutalsointerfereswiththe functionof the targets,e.g.by inhibitingsignalingbycellsurfacereceptors.Two affibody molecules binding to two different epitopes on VEGFR2 weregeneratedbyphagedisplay[148].Thebiparatopicheterodimerwithanalbuminbindingdomain(ABD)inbetweenshowed2ordersofmagnitudehigheraffinitythan either of the monomers. In addition, it could inhibit ligand inducedphosphorylationofVEGFR2withapotencyasefficientastheFDAapprovedanti-VEGFR2 antibody, ramucirumab [149]. Besides the anti-VEGFR2 affibodymolecules;anti-HER3,platelet-derivedgrowth factorreceptorβ(PDGFRβ),andinsulin-like growth factor-1 receptor (IGF1R) binding affibodymolecules havealso shown inhibition of tumor cell growth by blocking their ligands binding[150]–[152].

Oneaffibodymoleculewitha“special”structureisthevariantgeneratedtobindto theAlzheimer’sdisease (AD)relatedamyloidβ (Aβ)peptide (denotedZAβ3),whichhasaβsheet-αhelix-αhelixstructureratherthanthenormalthreeα-helixbundle structure [153], [154]. Besides this structural difference, ZAβ3was alsoprobably selected in a disulfide bond bridged dimer format, since cysteineappearedatthesamepositioninalltheselectedclones.ThedimericZAβ3affibodymoleculeshowedmuchhigheraffinityfortheAβpeptidethanmonomericZAβ3.AstructuralanalysisshowedthatthedimericZAβ3boundtoAβinasandwichlikemanner,with Aβ as “meat” in between the ZAβ3 dimer “bread” [153]. The ZAβ3dimerwas lateroptimizedby reformatting into ahead-to-tail dimer, removingunstructuredpartstodecreasethesize,andimprovingtheaffinityto300pMbyaffinitymaturation(denotedZSYM73)[155].ZSYM73fusedwithanalbuminbindingdomain (ABD) has shown significant reduction of the amyloid burden in atransgenic mouse AD model at the start of pathology development with nodetectable toxicity [156]. The anti-Aβ Affibody molecule has not only shownpromising results in preclinical models, but also expanded the structuralpossibilitiesofaffibodymolecules fromanengineeringpointofview.Basedontheanti-Aβaffibodymolecule,affibodymoleculestargetingtau[157],α-synulein[158], and human islet amyloid polypeptide (IAPP) [159], which have similarstructurestotheAβpeptide,havebeengenerated.


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2.3.7 Albuminbindingdomains(ABDs)Albumin binding proteins are expressed on the surface ofmany gram-positivebacteria, functioning as camouflage to evade the host immune system andpotentially also scavenge protein-bound nutrients [160]–[162]. The albuminbinding proteins may contain up to 44 tandemly repeated albumin-bindingdomains(ABDs)[163].TheC-terminalABD3fromStreptococcalproteinG(SPG)(denoted G148-ABD) [164], [165] and protein G-related albumin-binding (GA)modulefromproteinPAB(peptostreptococcalalbumin-binding)oftheanaerobicbacteriumFinegoldiamagna (denotedALB-GA) [166]–[168]are twoABDs thathave been thoroughly investigated both structurally and functionally.Historically,theABDshavebeenusedtoforexamplepurifyordepletealbuminfrom serum to facilitate proteome analysis of biomarker proteins. Later thealbumin-binding region was used as a fusion partner during recombinantproteinproductiontofacilitatetargetproteinpurificationandincreasesolubility,or used for directed immobilization [169]–[171]. Therapeutic relevantapplications of ABDs are to use them for half-life extension of therapeuticproteins. Like immunoglobulins, serum albumin has a long 19 day half-life incirculation,sincethemolecularweightofalbuminisoverthekidneycutoffandalbuminsarealsorescuedbyFcRnfromlysosomaldegradation[172].Therefore,non-covalentbindingtoserumalbuminviaABDmaybeusedtoprolongthehalf-lifeoftherapeutics[173].Althoughithasbeenshownthatincreasingtheaffinityand valency of ABD to serum albumin only resulted in amarginal increase ofhalf-life [174], it is still interesting to increase the affinity, because the affinitymaybereducedinthecontextofafusionprotein.Therefore,G148-ABDwasusedasatemplatetogeneratealibraryfollowedbyselectionforABDswithimprovedaffinity to human serum albumin (HSA) by a combination of combinatorialengineeringandrationaldesign[175].OneselectedvariantdenotedABD035hasanapparentaffinityforHSAintherangeof50-500fM,whichisseveralordersofmagnitude stronger affinity than thewild-typedomain (KD=1nM) [175]. SinceABD is derived from a bacterial protein, which is potentially immunogenic,ABD035 was subjected to a deimmunization procedure and a variant denotedABD094withnoTcellstimulationwasobtained(AffibodyAB,unpublisheddata).

2.3.8 ABD-DerivedAffinityProteins(ADAPTs)Besides binding to serum albumin, ABD has also been shown to be a goodalternativescaffoldaffinityprotein,aclasstermedABD-DerivedAffinityProteins(ADAPTs).TheG148-ABDscaffold,whichadoptsathreeα-helixbundlestructuresimilartoaffibodymolecules,wasusedasatemplateforengineering.However,itisonly46aminoacids(~5kDa),evensmallerthanaffibodymolecules(~7kDa)[164]. The binding surface for albumin inG148-ABD resides on helix 2 and theloop connecting helix 2 and helix 3 [161], [176]. In order to engineer mono-specificADAPTs,11surfaceexposedresiduesdistributedonhelix2,helix3andthe interconnecting loop, covering the original albumin binding surface, wererandomized and the library was screened for binders to interferion-γ usingribosome display [177]. The acquired binders had low nanomolar bindingaffinitytointerferon-γandlosttheaffinitytoalbumin.Amorechallengingworkwas to introduce a new binding site while retaining the affinity for albumin.Sinceonlyhelix2andtheloopfollowingitareessentialforbindingtoalbumin,atotal of 11 residues on helix 1 and 3 were subjected to randomization and


19

screenedforbinderstoseveraltargets.ThisresultedinADAPTswithnanomolaraffinity for tumor necrosis factor α (TNF-α), HER2 and HER3 and retainedaffinity forHSA [177]–[179]. Although binding to albuminmay be a beneficialfeaturefortherapyapplications,itraisesthebackgroundsignalifemployedfortumor imaging. An ADAPT targeting HER2 with abolished affinity to albumin,denoted ADAPT6, was conjugated to 68Ga for HER2-expressing tumor imaging[180]. ADAPT6 has an even faster clearance from circulation than affibodymolecules,enablinghighcontrastimaging1hpostinjection.ADAPT6isusedasatargetingdomaininoneofthestudiesinthisthesis(PaperIII).

Antibodydrugconjugates(ADCs)andalternativescaffolddrugconjugates

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Chapter3

3 Antibody drug conjugates (ADCs) andalternativescaffolddrugconjugates

Asdiscussedinthelastchapter,monoclonalantibodieshavebecomeoneofthemainstreamtypesofdrugsfortargetedcancertherapy.However,onlymAbsfortreatmentofhematologicalcancershaveshownstrongeffectsasasingleagent.For example, rituximab treatment for B-cell lymphomas [181].When they areusedasthesingleagentfortreatmentofsolidtumors,trastuzumabforexampletreatmentforbreastcancer,theyonlyshowedmodestanti-tumoractivity[182].ThesemAbsareusuallyusedincombinationwithtraditionalchemotherapeuticdrugsintheclinictoachievecompellingresults.Astudyofpharmacokineticsofchemotherapeutic drugs, such as doxorubicin, illustrated that the accumulateddose in the tumor was just corresponding to 5-10% of the accumulation inhealthytissue[183].OnepossiblewaytoincreasethetherapeuticeffectofmAbswhile minimizing systemic toxicity is to arm the naked mAbs with cytotoxicdrugs.Suchmoleculesarecalledantibodydrugconjugates(ADCs).TheideaistousemAbs to deliver the cytotoxic drug to the tumor and then the drug takeseffectinthetumorcells.ItcombinesspecificityandfavorablepharmacokineticsofmAbswiththehighcytotoxicityofthesmall-moleculecytotoxicdrug.Inorderto fulfill this idea, several factors should be taken into consideration. 1) Asuitable target should be selected. The targets are most often cell surfacereceptorswithdifferential expressionon tumor cells, directing thedrug to thetumor.Sinceallcytotoxicdrugsneedtotakeactionintracellularly,itisbeneficialto target receptors with a fast internalization rate. 2) The linker and thechemistry used to covalently attach the payload. A simple, fast, efficient andhomogeneous conjugation procedure is desired. They also need to be stableenoughunderphysiologicalconditionstoavoidprematurereleaseofthepayloadin circulation. 3) The payloads should be highly toxic (pico to nanomolar IC50


21

values)andamenabletoconjugation,retainingthecytotoxicityevenwithsomeextentofmodification(e.g.linkerattachment).Actually,theideaofADCsisnotnew,thefirstADCwasproducedinlate1950sandthefirstcovalentlylinkedADCtestedinananimalwasinjectedinthe1970s[184].However, early attempts of generatingADCswerenot successful due tomanyreasons,includingimmunogenicityofthechimericantibodies,insufficientpotencyofthepayloads,instabilityofthelinkersandbadchoiceoftargets[185].Based on the lessons learned from the first generation ADCs, the secondgeneration ADCs, had much better therapeutic profile and four of them havebeen approved by the FDA: ado-trastuzumab emtansine (T-DM1, commercialnameKadcyla®)[46],brentuximabvedotin(commercialnameAdcetris®)[186],inotuzumabozogamicin(commercialnameBesponsa®)[187]andgemtuzumabozogamicin(commercialnameMylotarg®)[188].SincetheapprovalofthesefourADCs,thisfieldhasattractedalotattentionfrombothacademicsandindustries,with more than 30 ADCs entered clinical development and more than 60 inclinicaltrials[189]. It isalsoanattractiveideatoconjugatealternativescaffoldaffinityproteinswithdrugsasalternativesofADCs.

3.1 ElementsofanADC

AnADCconsistsofthreecomponents,anantibody,a likerandacytotoxicdrug(Figure4).Theantibodyhasthefunctionofdeliveringthecytotoxicdrugtothetumorcellsandmayalsohaveananti-tumoreffectbyitself.Thecytotoxicdrugsare the effectors, which kill the tumor cells. There are cytotoxic drugs withseveraldifferentmechanismsofaction(MOA).Linkersactaschemicalhandlestojoinantibodieswithdrugs.Althoughitmightseemeasytomakeacombinationof three components into an ADC, in practice, it is not easy to generate aneffectiveADC.ThedetailedinfluencingfactorsofADCswillbediscussedhere.

Figure4. Illustrative structureofADCs.Theantibodybinds to tumor specific orassociatedantigens. The linker joins the cytotoxic drug and the antibody. The payload is a cytotoxicdrugtakingactioninthecytoplasmoftumorcells,causingcelldeath.

3.1.1 TargetsofADCsTheclassiccriteriaofagoodtumorantigenforADCswere:(i)higherexpressionin tumor versus normal tissues, (ii) cell surface expressed and (iii)internalization into cells. The tumor antigens should ideally exist only, or in


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higheramountson tumorcells thanonnormalcells,orat leasthaverestrictedexpressioninaparticulartypeoftissue,sothattheoff-targeteffectislow[190].The tumor antigens expressedon the cell surface aremore accessible toADCsthan targets in cytoplasm. Nearly all the cytotoxic drugs take effects in thecytoplasm; therefore, the tumor antigensmust be internalized, preferably fast.However,ADCscanworkregardlessofsomeofthesecriteria.Asurveyoftumor-andnormal tissueexpressionprofilesofpotential targetsshowedaverybroadrange of expression,with a 64-fold difference betweenmedianCD70 in tumorversus normal and less than 2-fold difference betweenmedian HER2 levels intumor versus normal [190]. The linkers and the cytotoxic drugs furthercomplicate theefficacyandsafetyprofileofanADC,since theyhave impactonthepharmacokineticsandpharmacodynamicsofADCs.Although the median HER2 levels of tumor versus normal is very low,trastuzumab-DM1 (Kadcyla®) targeting HER2was the first ADC that the FDAapprovedfortreatmentofasolidtumor.OneofthereasonsforchoosingHER2as target was probably that HER2 overexpressing tumor cells have adramaticallyhighHER2expressioninaround20-30%ofallbreastcancercases[191], which enables Kadcyla® to deliver a sufficient amount of the cytotoxicpayloadDM1toHER2high-expressiontumorcellswhilesparingthenormalcellswith lowerHER2expression.Thepropertiesof thecytotoxicpayloadDM1andthelinkerSMCCalsohavegreatinfluenceonthedosethatcanbeadministratedbeforeseveresideeffectsappear,ordoselimitingtoxicity(DLT).

MostADCsneedtobeinternalizedtoreleasethecytotoxicdrugs.However,thereare exceptions that ADCs targeting non-internalizing antigens displayedsignificant toxicity by strong “bystander effect” [192]. This will be discussedfurtherinthelinkerdesignsection.

3.1.2 LinkersofADCsSincetherearelimitedtypesofreactivefunctionalgroupsinnaturalaminoacids,a linker is needed to connect the antibodies with various drugs. The mostcommonlyusedsidechainsfordrugattachmentaretheprimaryaminegrouponlysines and sulfhydryl group on cysteines. It is also possible to geneticallyintroduce unnatural amino acid into antibodies and have an unique chemistryreaction connecting the linker to the antibody [193], [194]. The choice of thelinker conjugation chemistrydetermines thedrug to antibody ratio (DAR) andthehomogeneityoftheADCs.Actually,oneofthemajoreffortsoflinkerdesignistoachievehomogeneousconjugation.Linkersalsodeterminethestabilityofthedrug-antibodybond,whichshouldbestableincirculationandefficientlycleavedupon delivery to the target cells. The linker design could also contribute toovercoming multidrug resistance. There are a variety of linkers available foroptimal performance of an ADC for a certain tumor, however, a lot ofexperimentsneedtobedonetofindout.

3.1.2.1 Non-cleavablelinkersAnon-cleavable linkermeans that the linker is still attached to thedrugwhenthe antibody is degraded in the lysosomes of the tumor cells. N-hydroxysuccinimide esters (NHS esters) are themost often used to form non-cleavable amide bonds with lysines on antibodies (figure 5). And a thioetherbondisthemostcommonlyusednon-cleavablechemicalbondbetweenlinkers,


23

drugsand cysteinesonantibodies.Themaleimideorhaloacetamidegroupsonthe linker reactwitha sulfhydrylgroupeitheron thedrugoron theantibody.Twoexamplesofnon-cleavablelinkersaresuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC)used inT-DM1 [195] andmaleimidocaproicacidused inanti-CD30-mcMMAF [196].TheSMCC linker formsanamidebondwith primary amine groups on trastuzumab and forms a thioether bond withDM1. There are an average of 3.5 DM1 molecules on each T-DM1 [195]. Onaverage4moleculesofmcMMAFareconjugatedwithcysteinesofoneanti-CD30antibody,viaathioetherbond[196].WhenT-DM1andanti-CD30-mcMMAFgetinternalized anddegraded in the lysosome, their degradation products, lysine-SMCC-DM1andcysteine-mcMMAF,isreleasedanddiffuseintothecytoplasmtoinhibit cell division [196], [197]. The lysosomal degradation products of theseADCs cannot diffuse to neighboring cells, because charged lysines or cysteinesare inconnectionwiththedrug-linkercomplexandunabletopassthroughthecell membrane [198], leading to that non-cleavable linkers normally do notendowADCswitha“by-stander”effect.

The non-cleavable linker was designed to minimize premature release of thedrugwhenincirculation,however,themaleimide-basedlinkerwasstillfoundtogradually lose payloads via a retro-Michael type reaction [199]. The loss ofpayload in circulation could lead to decreased efficacy and tolerance [200]. Itwas found that the retro-Michael type reaction cannot happen when themaleimide-thioetherbondslowlyhydrolyzesbyaring-openingreaction[199].Amild method, for example incubation of ADC under 37 °C, pH=9.2, wasdemonstratedtospeedupthehydrolysisreactiontoimprovethestabilityofthemaleimide-thioether conjugation [199]. There are also modified maleimide-basedlinkers[201],[202],withincreasedrateofhydrolysis,andnon-maleimide-basedlinkers[200][203]toaddressthestabilityissue.


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Figure5.LinkersemployedinADCsunderclinicaldevelopments.

3.1.2.2 Cleavablelinkers

3.1.2.2.1 Disulfidecleavablelinkers:

Thedrugscanbeattachedtoantibodiesviadisulfidebondssimilartodisulfidebridgesinproteins.Thedisulfidebondisstabletosomeextentincirculationbutis reduced upon internalization to release the drug, since the intracellularreducing agent glutathione (GSH) is about 1,000-fold higher in concentrationthan the most abundant reducing agent cysteine, in blood [204], [205]. Thesusceptibility of the disulfide linker to reducing agents can be tuned byincorporationofmethylgroupsoncarbonatomsadjacenttothedisulfidebondto increase the steric hindrance for reducing agents. Themaximumnumberofmethylgroupsthatcanbeintroducedisfour,twoontheantibodysideandtwoonthecytotoxicdrugside.Therelativestabilityofadisulfidebondsurroundedby fourmethyl groups to the reducing agent DTT, is more than 22,000 times


25

higherthanadisulfidebondwithnomethylgroup,demonstratingalargerangeofadjustabilityofthedisulfidebondlinkerstability[206].Oneadvantageofthecleavable disulfide linker is that the released cytotoxic drug may diffuse toneighboring tumor cells, which is the so-called ‘by-stander’ effect. The “by-stander” effect is especially desirable when the tumors express the tumorantigen heterogeneously. It was observed that the anti-CanAg-DM1 conjugate(targeting a novel glycoform of MUC1, which is strongly expressed in mostpancreatic, biliary and colorectal cancers) with a disulfide linker, had the ‘by-stander’effectwhiletheonewithanon-cleavablelinkerhadnot[207].

3.1.2.2.2 Cleavablepeptidelinker:

TheonlycleavablepeptidelinkerusedinADCssofarconsistsofvaline-citrulline(vc), a protease-sensitive dipeptide. The FDA approved ADC, brentuximabvedotin(tradenameAdcetris®),containsthisvclinker.ThesynonymcAC10-mc-vc-PABC-MMAEmoreexplicitlyshowsthecomponentsofbrentuximabvedotin.The cAC10 is the name of an antibody targeting CD30.Maleimidocaproyl (mc) reacts with interchain cysteines of the antibody to attach drug linker complexes to the antibody and also act as a spacer to create more room for the enzyme cathepsin B in the lysosome to recognize the vc linker. After cleavage by cathepsinB, thePABC-MMAE is released and PABC undergoes self-immolation and then the intactMMAE is released [208]. Brentuximab vedotin is approved for treatment ofHodgkinlymphoma(HL),systemicanaplasticlargecelllymphoma(ALCL)[208]andfortreatmentofpatientswithcutaneousT-celllymphoma(CTCL)whohavereceived prior systemic therapy [209]. Thismc-vc-PABC-MMAE linker payloadcombinationwasalsoconjugatedwithF16,anantibodytargetingtheA1domainofthenon-internalizingextracellularmatrixproteintenascinC[192].InthiscasetheADCreleasesdrugsintheextracellularmatrixsurroundingthetumor,duetocleavageof thevc linkerbyunidentifiedserineproteasesandcarboxylesterase1Cinmouseplasma,followedbydiffusionofthedrugintothetumorcells[210],[211]. This non-internalizing ADC showed potent anti-tumor activity in threexenografted tumor models in mice [192]. However, it would probably havelimitedeffectinhumans,sincehumanplasmaonlyshowedmarginalactivityforcleavage of the vc linker [210], [211]. The peptide cleavable linker has thecombined properties of both cleavable and non-cleavable linkers, maintaininghighstabilityincirculationandboostingdrugreleaseinthelysosome.

3.1.2.2.3 Acid-cleavablehydrazonelinkers:

Therationalebehindthedevelopmentofacid-cleavablelinkersisthatthetumorextracellular microenvironment is acidic [212] and that the low pH in thelysosomecanbeutilizedto facilitatereleaseofdrugs fromADCs.Ahydrozone-based bond is the most commonly used acid-cleavable linker used fordevelopmentofADCs.ThehydrozonebondisnotabsolutestableatphysiologicalpH. The hydrozone bond has a plasma half-life of 183 h at pH 7.2 whilesignificantly shorter half-life (4.4 h) at pH 5, which is the pH of the lysosome[213]. The first generation ADCs using a hydrazone linker was an anti-CD33antibody-calicheamicin conjugate (Gemtuzumab Ozogamicin, Mylotarg®). Thecleavageofthehydrazonelinkerisnecessaryforittoexertitscytotoxicity[214].ThefateofMylotarg®hasbeentortuous.ItwasapprovedbytheFDAin2000forpatients over 60 with relapsed acute myeloid leukemia (AML), or those who


26

were not considered for standard chemotherapy [215]. However, it wasvoluntarily withdrawn from themarket in 2010 due to the lack of significantclinicalbenefit and increasedearlydeaths [216]. In2017, it regainedapprovalfrom the FDA for treatment of adult patients with newly-diagnosed CD33-positive AML [188]. Like gemtuzumab ozogamicin, inotuzumab ozogamicin(Besponsa®), which targets CD22, has the same linker and payload. It wasapprovedbytheEMAandFDAin2017fortreatmentofrelapsedorrefractoryB-cell precursor acute lymphoblastic leukemia [187]. Another example of ahydrozone linker is BR96-doxorubincin targeting Lewis-Y, which showed veryencouragingresultsonxenografted tumors inmiceandrats [217].However, itshowed low tolerability due to gastrointestinal toxicity in a Phase II humanclinical trial in metastatic breast cancer [218]. The stability of the hydrazonelinkermightbean issue causingundesired toxicity, becausehydrazone linkersslowly hydrolyze and release free drugs in circulation. Improvement to thestabilityofthelinkermightresultinabettersafetyprofile.

3.1.2.3 FutureperspectivesoflinkersInordertofurtherimprovetheefficacyandsafetyofADCs,onemajoreffortistodevelopsite-specificconjugationtotheantibody,producinghomogeneousADCshaving adefineddrug to antibody ratio (DAR) [99].One studyhas shown thatspecies with different DARs have varied efficacy, pharmacokinetics andtolerability[219].Manysite-specificconjugationtechnologieshaverecentlybeendeveloped by biotechnological and pharmaceutical companies, includingTHIOMAB[220],cysteinerebridging[221],non-naturalaminoacidconjugation[193], [194], enzyme-assisted ligation [221] and glycoconjugation [222] etc.,indicating the importance and fierce competition within this field. Severalstudies have shown the advantages of hydrophilic linkers, including reducedmultidrug resistance, improved solubility, prolonged pharmacokinetics andincreased therapeutic index [223], [224]. The hydrophilicity of linkersmay beincreased by incorporating PEG molecules into existing linkers [224] orincorporatinghydrophilicpyrophosphate[225].

3.1.3 PayloadsofADCsClinically used chemotherapy drugs, such as doxorubicin, vinblastine ormethotrexate, were the payloads of early ADCs. It was believed that tumorspecific mAbs could deliver more drugs to tumor cells than to healthy cells,resulting inhigherspecificity.However,onlymarginalornoactivitywasfoundwith these ADCs [226]. One of the major reasons for the failure is the lowpotencyofthesechemotherapeuticdrugs.Sinceonly1-2%oftheadministrateddoseofanADCreachedthetumor,andtherearesometimesalimitednumberoftumorantigensonthecellsurface,payloadsofADCsneedtobeextremelypotent,with nanomolar or picomolar IC50 values. The payloads of ADCs under clinicaldevelopment have high potencywith IC50 values in the range of 10-10-10-12M[227]. Besides high potency, the payloads need to be stable throughout ADCpreparation, storage, in circulation, in the lysosome and in the cytoplasm.Payloadsalsoneedtohaveareasonablesolubilityinwaterorbesolublewiththeaddition of a small amount of water-miscible organic solvent to perform theconjugation reaction, because an antibody needs to maintain its nativeconformation in aqueous solution.Althoughanexcessof organic solvent could


27

dissolvemorehydrophobicdrugs, itmaydamage theantibodyand the formedADCismorepronetoaggregateandhaveunfavorablepharmacokinetics [219].Theuseofhydrophilic linkerscouldcounteract thehydrophobicityofpayloads[224].Hydrophobicityofpayloadsalsohas todowithmembranepermeability,which may be translated into ‘by-stander’ effect. Most payloads of ADCs inclinical development fall into two categories, anti-mitotic and DNA damagingagents.

3.1.3.1 Anti-mitoticpayloadsMicrotubulesarepartofthecytoskeleton,whichphysicallysupportthedefinedshape of cells. More importantly, they are the main component of mitoticspindles separating the two sets of chromosomes during mitosis. Themicrotubule binding molecules, suppressing microtubule formation orstabilizing microtubules, may cause a G2/M phase arrest during cell division[228].ThecellsthatfallintoG2/Mphasearrestwilleitherdie,stopdividing,orhave unequal division [229]. Anti-mitotic payloads are good candidates forcancer therapy, since they are selectivelymore toxic to fast-diving tumor cellsthan tonormal cells.Nearly all anti-mitoticpayloads currently in clinical trialsareauristatinsormaytansinoids[227].

3.1.3.1.1 Auristatins:

Dolastatine 10 is a very potent anti-mitotic peptide isolated from the marineshell-less mollusk Dolabella auricularia [230]. Its anti-mitotic activity comesfrom the inhibition of tubulin polymerization, vincristine binding and GTPhydrolysis[230].MonomethylauristatinE(MMAE)andmonomethylauristatinF(MMAF)arethemostpopulardolastatine10derivativesusedasADCpayloads.ThedifferencebetweenthemisthatMMAEcandiffusethroughcellmembranestokillneighboringcellswhileMMAFcannot,duetoanadditionalcarboxylgroup.The combination of MMAE with the mc-vc-PABC linker is used in the FDAapprovedADCbrentuximabvedotinandmanyotherADCsinclinicaltrials[227].On the other hand, theMMAF payload ADC using the non-cleavablemc linkerwas shown to be better tolerated than the corresponding ADC using thecleavablemc-vc-PABClinker[196].

3.1.3.1.2 Maytansinoids:

Maytansineisanaturalbenzoansamacrolideproductfirstisolatedfromthebarkof the African shrub Maytenus ovatus [231]. Maytansine binds to the twovinblastine binding sites on tubulin, with one low affinity site and one highaffinity site, which inhibits microtubule polymerization [231]. Maytansine byitself failed to demonstrate clinical benefit in phase 2 clinical trials againstseveraldifferenttypesoftumors[232]–[234].Therearemainlytwomaytansinederivatives,DM1andDM4,withasulfhydrylgroupasconjugationhandle,usedforgeneratingADCs. It isshownthat thecellularmetabolitesofDM1andDM4(S-methyl-DM1andS-methyl-DM4)interactswithmicrotubulesaseffectivelyas,or evenmore effectively, thanmaytansine [235]. DM1 has been conjugated totrastuzumab via both cleavable disulfide linkers and non-cleavable thioetherSMCClinkersfortreatmentofHER2-overexpressingbreastcancer.Althoughtheinvitrocytotoxicityassayresultsoftrastuzumab-DM1conjugateswithdifferentlinkers were similar, the SMCC trastuzumab-DM1 conjugate (Kadcyla®)


28

demonstrated significantly better anti-tumor activity in an in vivo xenografttumor study [195]. The lysosomal degradation product of Kadcyla® is lysine-MCC-DM1,whichdiffuses into thecytoplasmand isable to inhibitmicrotubulepolymerization[197].Ontheotherhand,all theDM4ADCsunderclinicaltrialsare still using disulfide linkers [227], which is probably due to the higherstability of the disulfide bond connectingDM4 to antibodies compared to thatconnectingDM1.ThetwoadditionalmethylgroupsnexttothedisulfidebondonDM4stericallyhindersandthusslowsthereductionreaction[206].

Figure6.CytotoxicpayloadsemployedinADCsunderclinicaldevelopment.

3.1.3.2 DNAdamagingpayloadsAnother major group of anti-tumor drugs used for creation of ADCs, is DNAdamaging drugs, either by alkylation, single or double strand cleavage, or bychangingthethreedimensionalstructureofDNAbyintercalatingintotheminorgroove.DNAdamagingdrugsaretoxictoalltypesofcells,however,moretoxictorapidlydividingtumorcellthantoslowlydividingcells,possiblyduetohigheraccessibilitytoDNAduringcelldivision[236]andthelessefficientrepairofDNAdamages in tumorcells [237]. In this thesis, I onlygivea short introductionofcalicheamicin, which is the payload of the approved ADCs (inotuzumabozogamicinandgemtuzumabozogamicin).


29

Calicheamicinγ1 isahighlypotentantitumorDNAcleavageantibiotic, isolatedfrombrothextractofthebacteriumMicromonosporaechinospora.Calicheamicininteractswith theminor groove of TCCT/AGGA sequences in DNA and causesdouble-stranded cleavage [238]. Usually, N-Acetyl-γ-calicheamicin is used forcreation of ADCs instead of calicheamicin, because it demonstrated betterefficacy than calicheamicin [239]. N-Acetyl-γ-calicheamicin is used ingemtuzumabozogamicinand inotuzumabozogamicin conjugates.GemtuzumabozogamicinistargetinghumanCD33andwasapprovedbytheFDAin2000fortreatmentofthefirstrecurrenceofacutemyeloidleukemia(AML)butwaslatervoluntarilywithdrawnfromthemarket[214],[216].ThefailureofgemtuzumabozogamicindidnotpreventthesuccessofN-Acetyl-γ-calicheamicinasapayloadin other ADCs. Recently, inotuzumab ozogamicin, targeting human CD22, wasapprovedbytheEMAfortreatmentofadultswithrelapsedorrefractoryCD22-positiveB-cellprecursoracutelymphoblasticleukaemia(ALL)[187].

3.1.3.3 NovelADCpayloadsThereareanumberofpromisingnewADCpayloadsunderpreclinicalorclinicaldevelopment.SomeofthemhavesimilarMOAsasthepayloadsdiscussedabove,e.g. they inhibit tubulin polymerization or damages DNA. Rhizoxin is amicrotubulepolymerizationinhibitorthatsharesthesamepharmacophorewithmaytansinoids [240]. Cryptophycins are a family of tubulin polymerizationinhibitorswhichareanorderofmagnitudemorepotent thanMMAEandDM1[241]. Tubulysins are a family of antimitotic tetrapeptides that have a MOAresemblingdolastatin10[242].SomeofthepayloadshavenovelMOAs,suchasα-amanitin that inhibits RNA polymerase II activity [243], and spliceostatins[244]andthailanstatins[245]thatareRNAspliceosomeinhibitors.

3.2 Developmentofalternativescaffolddrugconjugates

AsseveralADCswereapprovedfortreatmentofcancersandalotmoreADCsareunder clinical development, many researchers started to develop alternativescaffolddrugconjugatesforcancertherapy.AlternativescaffolddrugconjugatesmayhaveadvantagescomparedwithmAbs.All thealternativescaffoldshaveamuch smaller size compared with mAbs, which might be translated to forexamplebetterpenetrationofsolidtumors.TheyareusuallyeasilyexpressedinE. coli and may thus be produced at a lower cost than mAbs. Some of thealternative scaffolds do not contain any cysteine, which make it possible tointroduce a unique cysteine andmake site-specific conjugation. The generallyhigher thermo- and chemical stability and typically fast and easy refolding ofalternative scaffold proteins, possibly allow for a more expanded toolbox ofchemical conjugationreactions.However,alternativescaffoldproteinsoftendonot have any anti-tumor effectormechanism, such as eliciting ADCC and CDC,whichmaybedesirable fromanefficacypointof view,butmaynot alwaysbedesired from a safety point of view. Antibodies also have a much longercirculation half-life, contributing to therapeutic efficacy, but also leaves moretime for uptake in non-targeted tissue aswell as premature release if a labilelinkerisemployed.

Sincealternative scaffolds are relativelynewandmanyof themare still underdevelopment, there are not many alternative scaffolds drug conjugate studies


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publishedyet.DARPintargetingtheepidermalcelladhesionmolecule(EpCAM)wasconjugatedtoMMAFviaathioetherbondbetweenaC-terminalcysteineanda maleimide-group in linker. In the other end of the DARPin, the non-naturalamino acid azidohomoalanine had been introduced onto whichdibenzocyclooctyne-modifiedmouse serumalbumin (MSA)was attached for invivo half-life extension [246]. The resulting conjugate, designated MSA-Ec1-MMAF, selectively killed EpCAM overexpressing cell lines and had a 22-foldincreaseof circulationretentioncompared to the sameconjugatewithoutMSA[246]. An anti-HER2 (ZHER2) affibodymolecule has previously been conjugatedwith three different payloads for selective HER2-positive cancer treatment[247]–[249].PaperIIIinthisthesisisastudyontheanti-tumoractivityofZHER2-DM1conjugates.TheZHER2affibodymoleculehasalsobeenconjugatedwiththepayloadMMAE.AuniquecysteinewasintroducedattheC-terminalendaswellasattwopositionsonhelix3forconjugationwithvcMMAE.ThevariantwithaC-terminal cysteine had the highest conjugation efficiency and the conjugatedaffibody could be easily separated from the unconjugated affibody byhydrophobic interaction high performance liquid chromatography (HIC-HPLC)[249]. The affibody-vcMMAE conjugate showed specific activity on HER2overexpressingtumorcelllinesoverHER2low-expressingcelllines[249].Inanother study by the same group, a drug conjugating sequence containing threecysteineswasgeneticallyfusedtotheC-terminalofadimericanti-HER2affibodyto make a conjugate with vcMMAE [248]. The conjugation products were amixtureofdimericZHER2with0,1,2,or3moleculesofvcMMAE,andtheywereunable to separate the different species to homogeneous compounds by HIC-HPLC. The in vitro cytotoxicity of this dimeric affibody vcMMAE conjugatemixturewas foundtobesimilar to themonomericaffibodyvcMMAEconjugate[248].

Immunotoxins

31

Chapter4

4 ImmunotoxinsA different approach to achieve targeted cancer therapy is to employ fusionproteins,consistingofatargetingdomaincoupledtoatoxicpeptideorprotein.Immunotoxins (ITs) are such a class of designed proteins, consisting ofantibodies or antibody fragments as targeting moiety, coupled to cytotoxicproteins, usually of plant or bacterial origin, as payloads for cancer treatment.LikeADCs, theyare internalizedand transported to thecytosol todeliver theircytotoxic activity. Usually, these protein toxins are enzymes that catalyzereactionscausingcelldeath.Theproteintoxinsareoftenevenmorepotentthandrugs used in ADCs. Another difference between ADCs and ITs is that toxicproteinsneedtogothroughseveraltransportationstepstoenterthecytosol,notsimplydiffusethroughtheendosomalor lysosomalmembranes.Onedrawbackof the toxicproteins is that theyareoften largeand immunogenic, andwill berecognized by the immune system and is therefore often neutralized byantibodies. Typically, the generated neutralizing antibodies prevent furthertreatmentafter1to4cycles[250].There are mainly two types of cytotoxic proteins used in immunotoxins,ribosome inactivating proteins (RIPs), mostly plant derived, and mono-ADP-ribosyltransferaseproteins,derivedfrombacteria.Bothofthemcausecelldeathby stopping protein synthesis in the cell. In the early ITs, whole cytotoxicproteinswere chemically conjugated to full antibodies.However, the cytotoxicproteins are of low specificity because of the toxic proteins’ own cell bindingdomains.Later, thenativebindingdomainsofthetoxicproteinswereremovedtoincreaseselectivity.Withthedevelopmentofrecombinantproteintechnology,immunotoxins are nowmainly produced as genetic fusions between antibodyfragments and toxic proteins, where the latter has the cell-binding domain

Immunotoxins

32

removed.Besidesthesetwocategoriesofprotein-synthesis-inhibitiontoxins,theporeforminganthraxtoxinhasalsobeenusedfortargetedkillingoftumorcells.

4.1 Ribosomeinactivatingproteins(RIPs)-basedimmunotoxins

RIPs are a family of toxic proteins irreversibly inhibiting eukaryotic proteinsynthesisbymodifying ribosomalRNA [251].Theyaremostly found inplants,andarethoughttohaveadefensivefunction,fortheirexpressionisupregulatedafter infections caused by viruses andmicroorganisms [252]. Currently, therearealmost250RIPsdescribed in literatureand theycanbeclassified into twocategories:type1RIPs(RIP1),suchassportinandgelonin,andtype2RIPs(RIP2),suchasricin[253].AlltheRIPshaveN-glycosidaseactivity,removingasingleadenineresiduefromeukaryoticribosomesatthespecificposition,A4324in28SrRNA[254].DepurinationofrRNAinhibitsitsbindingtoelongationfactorsandtherebyproteinsynthesisisirreversiblyinhibited,causingcelldeath[255].Type1RIPsonlycontainasinglepolypeptidechainandtype2RIPsareheterodimericproteins, with chain A having enzymatic activity and chain B binding topolysaccharidesonglycoproteins,similartolectins[253].

Initially, type 2 RIPswere conjugatedwith antibodies tomake immunotoxins,buttype1RIPsbecamemorepopularfortheirsimplersinglechainstructureandlowerunspecificactivity.Saporin,atype1RIP,hasbeenconjugatedwithmanyantibodiesorantibodyfragmentstargetinghematologicalandsolidtumors.Theyhavedemonstratedgreatefficacyforhematologicaltumorsbutarelesseffectivefor treatment of solid tumors in pre-clinical studies [256]. However, thegenerationofneutralizingantibodiesagainsttheimmunotoxinsandsideeffectsincluding promotion of vascular leakage syndrome (VLS) has decreased theinterestinthembybothacademiaandindustry[257].Bouganin is a type 1RIP,with picomolar cytotoxicity, that is expressed in theplantBougainvilleaspectabilisWilld.Asmentionedabove,theimmunogenicityofsaporinisthemainreasonunderminingitsclinicaldevelopment.Bouganinwasdeimmunized by removing T-cell epitopes and the resulting toxin is calleddeBouganin [258]. deBouganin has been fused with antibodies, Fabs anddiabodies targeting different receptors for treatment of EpCAM and HER2overexpressingtumors[259],[260][261].DeBouganinITshavedemonstratedaminimal anti-deBouganin antibody response in a Cynomolgus monkey study[261]. Moreover, trastuzumab-deBouganin was also shown to be cytotoxic totrastuzumab-DM1resistanttumorcelllines[259],[260],sinceproteintoxinsaregenerallynotaffectedbymulti-drugresistantproteins(MDRs),whichprotectedthecelllinesfromtrastuzumab-DM1actioninthiscase.

4.2 Bacterialmono-ADP-ribosyltransferaseproteinbasedimmunotoxins

Some bacteria have evolved different ways to inhibit protein synthesis of theintoxicatedcellsbymodificationofeukaryoticelongationfactors.Thethreemostinvestigatedprotein toxinssecretedbybacteriaarediphtheria toxin(DT) fromCorynebacterium diphtheriae, pseudomonas exotoxin A (ExoA or PE) fromPseudomonasaeruginosa andcholix toxin (CT) fromVibriocholera[262].All ofthesetoxinshaveanenzymaticdomain,whichhastheactivityoftransferringan

Immunotoxins

33

ADPgroupfromNAD+toadiphthamideresidueoneukaryoticelongationfactor2 (eEF2) [263]–[265]. Therefore, they were named mono-ADP-ribosyltransferases. The ADP-ribosylated eEF2s are not able to bind toribosomes and protein synthesis is stopped, eventually causing cell death.Although thesemono-ADP-ribosyltransferases have the same catalytic activity,they differ in structure.Here I focusmydiscussion on the PE toxin, since it isusedinthisthesis.

Figure 7. Representative structures of cytotoxic proteins employed in immunotoxins.PseudomonasexotoxinAdomainIII(PDBID:1aer),diphtheriatoxin(PDBID:1sgk),saporin(PDBID:1qi7),andbouganin(PDBID:3ctk).

PE belongs to the two-component AB toxin family, which has an enzymatic Acomponent and a cell binding B component [266]. It is a 613 amino acid longprotein composed of three domains: a receptor binding domain (domain I), atranslocation domain (domain II) and the ADP-ribosyltransferase domain(domain III) [267]. The intoxication process starts with removal of the C-terminallysineinPEoutsidethecell,presumablybyplasmacarboxypeptidasesof the host, changing the C-terminal amino acids REDLK to REDL [268]. Thebinding domain of PE binds to CD91, which is also known as the alpha2-macroglobulin receptor/low-density lipoprotein receptor-related protein(α2MR/LRP) [269]. After binding to CD91, PE is transported into the cytosolthrough twodifferentpathways, theKDEL-receptormediatedpathwayand thelipiddependentsortingpathway.ThroughtheKDEL-receptormediatedpathway,PE is internalized via clathrin-coated pits into early endosomes. In theendosomes, PE undergoes a conformational change and is cleaved by furin indomainIIfollowedbyreductionofdisulfidebondsindomainII[270],[271].TheC-terminal37kDafragment,includingpartofdomainII,domainIb,anddomainIII, is then transported to the trans Golgi network (TGN) through the Rab9-

Pseudomonas exotoxin A doamin III Diphtheria toxin

Saporin 6 Bouganin

Immunotoxins

34

regulated pathway [272]. The C-terminal REDL motif helps the 37 kDa PEfragmentstobetransportedfromtheTGNtotheendoplasmicreticulum(ER)bybinding to the KDEL-receptors [273], which are normally responsible forrecyclingKDEL-bearingproteinsfromTGNtoER[274]. Inthelipid-dependentsorting pathway, PE still binds to CD91 on the cell surface, however, it isinternalized with the help of detergent-resistant microdomains (DRM) viacaveolin-mediatedinternalization[275].PEthenundergoesthesameprocessesin the endosomes as in the KDEL-dependent pathway,where it is transportedfrom late endosomes to the TGN in a Rab9 dependent manner but issubsequentlytransportedtotheERinaRab6dependentmanner[275].ThetwopathwaysofPEtransportationconverge intheERandbothexploit thecellularER-associated protein degradation pathway to translocate PE to the cytosol[276].Asdescribedearlier,theenzymaticdomainofPEtransfersanADPtothediphthamide residue on eEF2 and protein synthesis in the cell stops. TheintoxicationprocessofPEonlytakes30-90minfrombindingonthecellsurfaceuntilPEhasreachedthecytosol[277].

Figure 8. The internalization pathways of Pseudomonas exotoxin A. Adopted from PhilippWolf,etal.[278].

In order to use PE for therapeutic purposes, full length PE was initiallychemically conjugated to mAbs or receptor ligands [279], [280]. Later,recombinantlyexpressedreceptors’ligandsandvariablefragmentsofantibodiesreplaced the Ia domain of PE resulting in fusion ITs, which were more

Immunotoxins

35

homogeneousandmorecellspecific[281],[282].ThemostoftenusedtruncatedPEtoxinusedinITsisPE38(Mw38kDa).ComparedtonativePE,PE38lacksthenative CD91 binding domain Ia (Δ1–250) and part of Ib (Δ365–380) [283].Several scFv ITs containing PE38 have entered clinical trials, including ITstargeting CD22 [284], mesothelin [285] and CD25 [286]. However, a largeportionofpatientsquicklydevelopedanti-PEantibodiesduringtheclinicaltrials.Whilethisproblemwaslesssevereforpatientswithhematologicaltumors,duetoacompromisedimmunesystem[250].

Figure9.NativePseudomonasexotoxinA(PE)andvariantscommonlyusedinimmunotoxins.PEhasthreedomains,domainI,IIandIII.DomainIcanbefurtherseparatedintodomainIaand Ib. Four disulfide bridges are marked with brown lines. The furin cleavage site isindicatedwithanarrow.PE38 is truncatedversionofPE, lackingmostofdomain I,aminoacids1-250and368-380.PE38X8isaversionofPE38wheremouseB-cellepitopeshavebeenremoved by the followingmutations: R313A, Q332S, R432G, R467A, R490A, R513A, E548SandK590S.Themutationsaremarkedwithorangelines.LysosomeresistantPE(PE-LR)wasobtainedbyremovingaminoacids1-273and285-394.PE25wasobtainedbyintroducing10mutations,R427A,D463A,R467A,R490A,R505A,F443A,L477H,R494A,R538A,andL552E,toPE-LR.

Itwasfoundthat2-5cyclesoftreatmentwithPE-basedITsareneededtoreachamajorclinicalresponseincludingsomecompleteremissions[250].Therefore,toallow multiple cycles of treatment before anti-PE antibodies emergence, theimmunogenicityofPEtoxinhastobedecreased.Onewaytoallowmorecyclesoftreatment is to administer PE-based ITs together with immune suppressivedrugs. Another way is to reduce the immunogenicity by protein engineeringstrategies. PEGylation is a common strategy to reduce immunogenicity by‘masking’ immunogenic epitopes [287]. It increases the size of the IT andprolonged half-life in circulation aswell [287]. Although PEGylation strategies

1 252/253364/365

404/405 613

Ia II Ib III

Native Pseudomonas exotoxin A (PE)

Ia

△(1-273) △(285-394)

Ia

△(1-250) △(368-380)

Ia

△(1-273) △(285-394)

Ia

△(1-250) △(368-380)

PE38

PE38X8

PE-LR

PE25

Immunotoxins

36

showed promising results [287], [288],more efforts were put into identifyingandremovingB-andTcellepitopesinPE.Atfirst,mouseepitopesinPE38wereidentifiedbycharacterizinganti-PE38antibodiesisolatedfrommiceimmunizedwithPE38ITs[289].Mutationsofeightresidueswithlargesidechainstoalanine,glycineandserineontheidentifiedB-cellepitopesresultedintheconsiderablyless immunogenic version PE38X8 with retained cytotoxicity [290]. However,mouseB-cellepitopescannotbefullytranslatedintohumanB-cellepitopes.Atthe same time, it was found that domain II of PE is susceptible to proteasedigestion in the lysosome. A version of PE, lacking domain II but keeping thefurin cleavage site (PE-LR (lysosome resistant)), were therefore designed andwas found to have a similar cytotoxicity compared to PE38, while it had anincreased tolerability in mice [291]. The removal of domain II also removedseveral B- and T-cell epitopes, and as a consequence, PE-LR showed reducedimmunogenicityinanimalstudies[291],[292].InordertoidentifyandremovehumanB-cell epitopes, Liu et al. displayedFvs ofB-cells frompatients treatedwith the immunotoxins, SS1P (anti-mesothelin PE38 immunotoxin) and HA22(anti-CD22 PE38 immunotoxin), on phages [293]. By incubating the phageparticleswithapoolofPE38variants,eachhavinganalaninesubstitutionoftheresidueswith largebulkysidechains, sixB-cellepitopeswere identified [293].The new immunotoxin HA22-LR-LO10, lacking domain II and bearing 7 pointmutations(R427A,R458A,D463A,R467A,R490A,R505AandR538A)indomainIII, was found to have low immunogenicity [293] in humans. Besides B-cellepitopes, elimination of T-cell epitopes was also carried out to reduceimmunogenicity.TheT-cellepitopeswereidentifiedbystimulatingPBMCsfrom50donorswithanaïve immunerepertoire,representingpeople inthewesternworld, with 15-meric peptides spanning PE38 followed by detection of IL-2secretion [294]. Eight epitopes were identified, one of which in domain II isimmuno-dominant, in people with a variety of HLA alleles [294], [295]. TheidentifiedepitopeswereeliminatedbydeletingdomainIIandbyintroducingsixmutations (R427A, F443A, L477H, R494A, R505A, and L552E) in domain III[295]. The immunotoxins withmutated/deleted T-cell epitopes, LMB-T18 andLMB-T20, showed greatly reduced immunogenicity compared with theirparental immunotoxins [295], [296].Thereare twopositions(R427andR505)shared by bothB- andT-cell epitopes. The combined removal of T- andB-cellepitopescreatedPE25,whichlacksdomainIIexceptforafurincleavagesite,andbears10mutationsindomainIII(R427A,D463A,R467A,R490A,R505A,F443A,L477H, R494A, R538A, and L552E) [297]. PE25 was found to have greatlyreducedimmunogenicity,goodstabilityandwaswelltoleratedinanimals[297].

4.3 Alternativescaffoldstoxinsfusions

Alternative scaffolds proteins are good alternatives as targeting moieties inimmunotoxins.Alternativescaffoldsimmunotoxinscanusuallybeexpressedinasolubleformwithhighyieldinbacteria.Thesmallersizeofalternativescaffoldsalsohelpstoreducethesizeoftheimmunotoxins,whichmaytranslatetobettertumor penetration. There are not many reports of immunotoxins based onalternative scaffolds published yet, andmost of themuseDARPins or affibodymoleculesforcelltargeting.

Immunotoxins

37

Ananti-HER2DARPinwasfusedtoPE40,consistingofdomainII,domainIbanddomain III, with a mouse IgG3 hinge region linker [298]. The DARPin-PE40fusiontoxin,targetingHER2,couldbeproducedwithahighyieldinE.coli(160mg/L culture medium in shake flasks) and could selectively kill HER2-overexpressingtumorcellsinvitroandinvivo[298].Ananti-EpCAM-PE38fusiontoxinshowedsimilaranti-tumoractivityonEpCAM-overexpressing tumorcellsin vitro and in vivo [299]. The anti-EpCAM-PE38 was also site-specificallyPEGylated on PE38 resulting in a prodrug-like fusion toxin [300]. When theprodrug-like fusion toxin was treated with a particular protease, cleaving thelinkerbetweenPEGandanti-EpCAM-PE38, the cytotoxicityof thedePEGylatedfusion toxin was increased by a 1000-fold. PEGylation also increased thehydrodynamicradius,whichgavea10-foldincreaseinserumhalf-life[300].Anti-HER2affibodymolecules(ZHER2)andPE38fusiontoxins,denotedaffitoxins,havebeeninvestigatedinseveralstudies.AZHER2-PE38affitoxinshowedpotentcytotoxicity on HER2-overexpressing tumor cell lines while up to 1000-foldlowercytotoxicityonaHER2-lowexpressingcelllineinvitro[301].Inaninvivostudy,theZHER2-PE38affitoxinshowedapotentanti-tumoreffectonthreeHER2-overexpressing xenografted tumors bydelaying tumor growth, and in the bestcasekeepingtumorlessthan5%comparedtobeforetreatmentattheendofthe80 days study [302]. However, the half-life of the affitoxin was onlyapproximately15min,andsignificantamountsofanti-affitoxinantibodiesweredetectedafter the thirddose [302]. Since thehalf-life of the affitoxinwasveryshort, the efficacy of such affitoxinsmight be improved if its serumhalf-life isprolonged.Ihaveinvestigatedthe invitroactivityand invivobiodistributionofananti-HER2affitoxinextendedwithanABDbetweentheaffibodymoleculeandPE38X8toincreaseserumhalf-lifeinpaperIandII.LaterGuoetal.fusedanABDto theN-terminalof theanti-HER2affitoxin to increase thehalf-life [303].ABDfusionincreasedthehalf-lifeoftheanti-HER2affitoxinfrom13.5minto330min.Although theABD fused affitoxin had a 2.5-fold lower cytotoxicity invitro, theprolonged half-life was translated to better efficacy in mice with xenograftedtumorswithoutincreasingformationofanti-PEantibodies[303].Guoetal.alsofused a version of PEwith deleted human B-cell epitopes [293] to ZHER2. Thisdeimmunized anti-HER2 affitoxin had significantly higher tolerability in mice,and could be given at doses up to 10 mg/kg. Formation of anti-affitoxinantibodies was also significantly lower [304]. In summary, prolonged half-lifeandlowerimmunogenicityweretranslatedtoahigheranti-tumoractivityinthexenografttumormodelsused.

Presentinvestigation

38

Chapter5

5 PresentinvestigationAntibodieshavebecomeoneofthemainclassesofmoleculesfortargetedcancertherapyduringthelastdecade,eitheractingontheirownorinconjugationwithcytotoxicpayloads.Alternativescaffoldaffinityproteinshaverecentlyemergedascompetingaswellascomplementingagentstoantibodies.Thestudiesinthisthesis focus on construction and evaluation of two alternative scaffold affinityproteins,affibodymoleculesandADAPTs,inconjugationwithcytotoxicproteinsandsmallmoleculardrugsfortargetedcancertherapy.

AffibodymoleculesandADAPTsthemselvesusuallydonotpossesspotentanti-tumor cytotoxic effects. Arming them with an effector molecule, such as acytotoxic protein or small molecule drug, is an attractive way to produce atumor-targeted therapeutic agent. HER2 was chosen as target in the studiescomprising this thesis, since it is an established tumor marker. Cytotoxicpayloads involved in this thesis include Pseudomonas exotoxin A (PE), PE38,PE38X8, PE25 (Fig. 9) and mc-DM1. Prolonged serum half-life is generallyrecognizedasadesiredproperty.Wehaveusedanalbumin-bindingdomaintoprolongtheserumhalf-lifeinallthestudiesinthisthesis.InpaperI,Idevelopedand characterized anti-HER2 affibody molecule (ZHER2), PE38X8 and ABDcontaining fusion toxins for killing of HER2 overexpressing tumor cells. ThebiodistributionofthefusiontoxinsdesignedinpaperIwasstudiedinpaperII.InpaperIII,wedesignedandevaluatedHER2targetingfusiontoxinsconsistingofADAPT6, an ABD and PE25. In paper IV, several different versions of ZHER2-containingtargetingdomainswereconjugatedwithmc-DM1andtheantitumoractivitywasinvestigatedinvitroandinvivo.


39

5.1 PaperI-Target-specificcytotoxiceffectsonHER2-expressingcellsbythetripartitefusiontoxinZHER2:2891-ABD-PE38X8,includingatargetingaffibodymoleculeandahalf-lifeextensiondomain

Immunotoxins usually consist of antibody fragments, such as Fabs or scFvs,coupledwithcytotoxicproteins,suchasPseudomonasExotoxinAordiphtheriatoxin.Antibodyfragmentsratherthanfullantibodiesareusedinimmunotoxins,mainly due to their smaller sizes and easier expression in bacterial systems,which is generally resistant to the cytotoxic action of immunotoxins. Affibodymoleculesarealternativescaffoldaffinityproteinswithaverysmallsize(7kDa)that gives high expression yield in E. coli. Therefore they are interestingalternatives to antibody fragments. Previously, a fusion toxin consisting of ananti-HER2 affibody molecule (ZHER2:342) and PE38, a truncated version ofPseudomonas Exotoxin A, was presented and named an affitoxin [301], whichwas created for treatment of HER2-overexpressing tumors The affitoxindisplayed very potent and selective anti-tumor activity towards HER2-overexpressingtumorcellsinvitro.However,aninvivopharmacokineticstudyoftheaffitoxin indicated that itshalf-lifewasonlyaround8min [302],whichwethoughtmightlimititsefficacy.Inthispaper,wehaveconstructedseveralZHER2-PEfusiontoxinsbasedonZHER2-PE38(Figure10).Thenewfusiontoxinsdifferfromthepreviousoneinseveralaspects. In order to prolong the half-life of the affitoxin, an albumin bindingdomain (ABD) was added. The purification tag was changed from the hexa-histidine-tag toa tagwith theaminoacidsequenceHEHEHE,whichpreviouslyresulted in a decreased liveruptakeof a radiolabeled affibodymolecule [305].Theanti-HER2affibodymoleculewaschangedfromZHER2:342toZHER2:02891,whichisabackboneoptimizedversion[140].TherewereeightmutationsintroducedinPE38,resultinginavariantdenotedPE38X8,whichhaspreviouslybeenshowntogiveadiminishedimmuneresponseinmice[290].


40

Figure 10. Construction and initial characterization of the fusion toxins. (A) Schematicrepresentationsofthegenesencodingthefusionproteinsareshown.Restrictionendonucleasesitesusedduringconstructionandlinkersconnectingthethreedomains,withtheaminoacidsequence(S4G)3,areindicatedonZHER2:2891-ABD-PE38X8.(B)ThepurifiedfusiontoxinswereanalyzedbySDS-PAGEona10%gel.NumberstotheleftindicatethemolecularweightofthemarkerproteinsinkDa.1,ZHER2:342-PE38;2,ZHER2:2891-PE38X8;3,ZHER2:2891-ABD-PE38X8;4,ZTaq-PE38X8;5,ZTaq-ABD-PE38X8.(C)The chromatograms recorded during gel filtration of the fusion toxins are displayed. (D)Thespectraofalkylatedproteinsrecordedduringmassspectrometryanalysis.

All the fusion toxins designed were purified to homogeneity after sequentialimmobilized metal-ion affinity chromatography (IMAC), anion exchangechromatography and finally size-exclusion chromatography (SEC) and showedthecorrectmolecularweightsmeasuredbymassspectrometry(Figure10).Thefunction of ZHER2 and ABD were investigated by biosensor analysis. Theequilibrium dissociation constant (KD) of ZHER2:2891-PE38X8 and ZHER2:2891-ABD-PE38X8towardtheextracellulardomainofHER2wereboth5nM.AninterestingfindingisthattheaffinityofZHER2:2891-ABD-PE38X8toHER2was12nMKD,afteritwaspre-incubatedwithhumanserumalbumin(HSA).Thisdemonstratedthatthe interaction between ZHER2:2891-ABD-PE38X8 and HER2 was only slightlyimpairedwhenitisboundtoHSAinthecirculation.Theequilibriumdissociationconstants of the interaction between affitoxins and HER2 was dramaticallyweakerthanthatofZHER2:342(KD=22pM)andZHER2:2891(KD=60pM)[306][140].ThefusionofPE38andABDprobablyincreasedsterichindranceandthereforedecreasedtheaffinity.TheaffinityreductioninpresenceofHSAmightalsodueto increased steric hindrance by bulkyHSA. The anti-Taq polymerase affibodymolecule (ZTaq)was used as negative control, and the two ZTaq PE38X8 fusiontoxinsdidnotbindtoHER2atanyoftheconcentrationstested.ABDinthefusiontoxinsboundtoHSAandmouseserumalbumin(MSA),butnottobovineserumalbumin(BSA),asexpected[175].


41

Figure 11. Analysis of relative HER2 expression on SKOV-3, SKBR-3 and A549 cells anddetermination of the cytotoxicity of the fusion toxins. (A, C and E) The relative expressionlevels of HER2 on SKOV-3, SKBR-3 and A549 cells, respectively, were determined by flowcytometry.Bluelinesindicatespectraafterincubationwithprimaryandsecondaryantibody,red lines indicate control spectra recorded after incubation with secondary antibody onlyand green lines indicate control spectra recorded of the cellswithout incubationwith anyantibody.ThecytotoxicitieswereanalyzedbyincubatingserialdilutionsofthefusiontoxinswithSKOV-3,SKBR-3andA549cells.(B,DandF)Cellviabilityareplottedonthey-axisasafunction of fusion toxin concentration on the x-axis. The viabilities of cells cultivated ingrowthmediawithoutadditionofanytoxinweresetto100%foreachpanel.Eachpointwasmeasuredintriplicatesandtheerror-barscorrespondto1SD.ZHER2:342-PE38(●);ZHER2:2891-PE38X8(n);ZHER2:2891-ABD-PE38(u);ZTaq-PE38X8(▼);ZTaq-ABD-PE38X8(▲).

Thecytotoxicityof the fusion toxinswasmeasured invitroonSKBR-3,SKOV-3andA549celllinesexpressingdifferentamountsofHER2.TheHER2expressionlevelsof these threecell linesweremeasuredby flowcytometry.BothSKOV-3and SKBR-3 cell lines had high HER2 expression and A549 had low HER2expression (Figure 11). The ZHER2 affitoxinsweremuchmore potent than ZTaqfusiontoxinsonallthecelllines,demonstratingover1000-folddifference(Table1).Inaddition,ZHER2affitoxinsweremorepotentonSKBR-3andSKOV-3thanontheA549cell line,which indicates thatHER2expression level isadeterminingfactorofthecytotoxicity.AmongthethreeZHER2affitoxins,theorderofpotencywas ZHER2:2891-ABD-PE38X8< ZHER2:2891-PE38X8< ZHER2:342-PE38 on all the celllinestested(Table1).However,ZHER2:2891-ABD-PE38X8wasstillverypotentwithapicomolar IC50value.The reasons forweaker cytotoxicitymightbeaweakeraffinityincombinationwiththeeightmutationsonPE38.


42

ThepotencyofZHER2:2891-ABD-PE38X8wasalsodemonstratedbyacontacttimeexperiment.WeincubatedSKOV-3cellswithZHER2:2891-ABD-PE38X8fordifferenttimesataconcentrationthatwouldkillcloseto100%ofthecells(500pM).A15minincubationtimecaused50%celldeath.Table1. Cytotoxicityoffusiontoxinsondifferenttumorcelllines.

Cellline

HER2expression

level

IC50(pM)

ZHER2:342-PE38

ZHER2:2891-PE38X8

ZHER2:2891-ABD-PE38X8 ZTaq-PE38X8

ZTaq-ABD-PE38X8

SKBR-3 High0.16(0.12-0.23)a

2.1(1.5-2.9) 5.6(4.0-7.8)

22,000(14,000-32,000)

1,400(900-2,100)

SKOV-3 High5.1(4.0-6.4)

6.9(5.5-8.8) 25(20-32)

24,000(19,000-30,000)

19,000(15,000-24,000)

A549 Moderate 50(35-71) 160(110-230)

300(210-430)

32,000(22,000-45,000)

37,000(26,000-52,000)

aRange(95%confidenceinterval).

The HER2-specific cytotoxicity was further investigated with two blockingexperiments. In one experiment, the cytotoxicity of ZHER2:2891-ABD-PE38X8 onSKOV-3cellscouldbeblockedbyanexcessamountofZHER2:342,whichconfirmedtheHER2-specificcytotoxicity(Figure12).ZHER2:342bindstothesameepitopeasZHER2:2891 on HER2 [140]. In another experiment, SKOV-3 cells were pre-incubatedwithdifferentexcessamountsofZHER2:342toblockavailablereceptorsbefore incubationwithZHER2:2891-ABD-PE38X8.Theresults showed thataround100- to 1000-fold excess of ZHER2:342 could fully block the cytotoxicity ofZHER2:2891-ABD-PE38X8(Figure12).


43

Figure12.Analysisof toxinspecificity.SKOV-3cellswere incubatedwithdifferentproteinsfor72hfollowedbydeterminationofcellviability.Theviabilityofcellscultivatedingrowthmediumwithoutadditionofanytoxinwassetto100%foreachpanel.(A)Fromlefttoright,incubation with 25 pM ZHER2:2891-ABD-PE38X8; 100 nM transferrin; 100 nM ZHER2:342; cellswere pre-incubated with 100 nM transferrin for 5 min followed by addition of 25 pMZHER2:2891-ABD-PE38X8;cellswerepre-incubatedwith100nMZHER2:342for5minfollowedbyaddition of 25 pM ZHER2:2891-ABD-PE38X8. (B) Cells were pre-incubated with an excess ofZHER2:342for5minfollowedbyadditionof500pMZHER2:2891-ABD-PE38X8.Numbersbelowthepanel correspond to the differentmolar ratios between ZHER2:342and ZHER2:2891-ABDPE38X8.Each data-point corresponds to the average measured viability of three independentexperiments.Theerror-barsindicate1SD.

Inconclusion,atripartitefusiontoxinbasedonadeimmunizedversionofPE38was successfully constructed. Although ZHER2:2891-ABD-PE38X8 had lowercytotoxicity compared to the original fusion toxin, it will be interesting toinvestigatefurthertocharacterizeitsextendedhalf-life,lowimmunogenicityandpossiblelowliveruptakefeaturesinvivo.


44

5.2 PaperII-InfluenceofmoleculardesignonbiodistributionandtargetingpropertiesofanAffibody-fusedHER2-recognisinganticancertoxin

In paper I, we have shown that the affibody fusion toxins had potent specificcytotoxicity to HER2 overexpressing tumor cells in vitro. However, the in vivoefficacy of a drug is influenced by many factors, of which one is itsbiodistribution. Normally, a good anti-tumor therapeutic agent shouldaccumulate at a sufficiently high concentration in the tumor,while having lowaccumulationinhealthytissue,whichistranslatedtohighdrugavailabilityandminimaltoxicitytohealthytissues.Thedrugshouldalsohavelongenoughhalf-life incirculationso that frequentdrugadministrationcanbeavoided,and thebloodlevelwillbemoreeven.The aim of this paper was to study the influence on biodistribution of newfeatures introducedbymolecular design to an affibody-PE38 fusion toxin. Thestudy was carried out by comparing the biodistribution of H6-ZHER2:342-PE38,(HE)3-ZHER2:2891-PE38X8 and (HE)3-ZHER2:2891-ABD-PE38X8 to determine theinfluence of the purification tag and the ABD on biodistribution. These fusiontoxinswerepurifiedtohomogeneitythesamewayasinpaperI.Theywerethenradiolabeledwith111Inandusedtoperformthefollowingexperiments.

Cell specific binding was confirmed by blocking the interaction betweenradiolabeled fusion toxins and SKOV-3 cells with non-labeled ZHER2. The cell-associated radioactivity was significantly reduced when the cells were pre-incubatedwithanexcessamountofZHER2.All fusiontoxinsdemonstratedrapidbindingtoHER2overexpressingcells,reachingaplateauat1-2hafteraddition(Figure13).Aninterestingfindingisthatboth(HE)3-ZHER2:2891-ABD-PE38X8and(HE)3-ZHER2:2891-PE38X8 showed a much faster internalization rate than H6-ZHER2:342-PE38; 65-70% compared to <30% cell-associated radioactivity wasinternalized after 2 h incubation (Figure 13). Part of the explanation may beattributed to the N-terminal modification, which consisted of replacing thehexahistidine-tagwith a (HE)3-tag. And the eightmutations on PE38may alsoplayarole.Regardlessoftheunderlyingreasons,ahigherinternalization(bothrateandtotalamount)maybetranslatedtobetterefficacy.Theaffinity(KD)of

Figure13.Cellularprocessingof111In-labeledH6-ZHER2:342-PE38(A),(HE)3-ZHER2:2891-PE38X8(B)and(HE)3-ZHER2:2891-ABD-PE38X8(C)bySKOV-3cellsduringcontinuousincubation.Dataarepresentedasanaveragevaluefrom3samples±SD.


45

111In labeled(HE)3-ZHER2:2891-ABD-PE38X8and(HE)3-ZHER2:2891-PE38X8toHER2receptorsonSKOV-3 cellswas in the lownanomolar range,measuredby real-timeradiotracer-receptorinteractiondetection,andwassimilartothemeasuredaffinityinpaperI(Figure14).Thedatawerefittedtoa1:2modelratherthan1:1,which is likely caused by the influence of co-expression of other HER familyreceptors on cell surface. It was found earlier that binding of ZHER2 to HER2-expressing cell lines invitrowas influencedby interactionofHER2withotherHER2 family receptors [307]. ZHER2 might have a weaker interaction with asubsetofheterodimerizedHER2.

Figure 14. Representative LigandTracer sensorgrams (binding of 111In-(HE)3-ZHER2-ABD-PE38X8(A)and111In-(HE)3-ZHER2-PE38X8(B)toSKOV-3cells).

The substitution of the H6-tag with the more hydrophilic (HE)3-tag waspreviously shown to reduce liver uptake of a ZHER2 affibody molecule [305].Therefore, we compared the biodistribution of H6-ZHER2-PE38 and (HE)3-ZHER2:2891-PE38X8 to determine if liver uptake was affected similarly in thecontext of a fusion toxin. The (HE)3-tag in combination with the eight pointmutations in PE38 in 111In-(HE)3-ZHER2:2891-PE38X8 reduced liver uptake byalmost20%at4hafter injection (Figure15).However, the liveraccumulationwasstillhighcomparedtoanaffibodymonomeralone[305].ThisindicatesthatPE38 is the major part responsible for high liver uptake in the fusion toxin,which is supported by similar results on other PE based immunotoxins [308].The reduction in hepatic uptake of the (HE)3-tag containing construct wasaccompaniedbyasignificantlyincreasedrenaluptake,whichislikelyduetothattheradiolabelledaffitoxinisreabsorbedbytheproximaltubularcellsafter it islost to primary urine in the kidneys, where it will be prone to enzymaticdegradation[308][309].


46

Figure 15. Comparative biodistribution of 111In-H6-ZHER2-PE38 and 111In-(HE)3-ZHER2-PE38X8 in female NMRI mice, 4 h after injection. The uptake values are presented as anaveragevaluefrom4animals±SD.

SinceH6-ZHER2:342-PE38hadveryshorthalf-life(~8min)[302],weincludedanABD to prolong the half-life of the fusion toxin to increase the efficacy. Thecomparison of 111In-(HE)3-ZHER2:2891-PE38X8 and 111In-(HE)3-ZHER2:2891-ABD-PE38X8pharmacokineticsshowedthat theadditionofanABD increasedbloodretentionby28fold(seetableinpaperII).Asurprisingfindingwasthatitwasstillclearedmuchfaster thanpreviouslystudiedABD-fusedAffibodymolecules[310], [311]. This could be explained by the high liver uptake of PE38 basedfusiontoxinsmentionedearlier.Asaresultofthelongerretentioninblood,111In-(HE)3-ZHER2:2891-ABD-PE38X8 showed a >5-fold reduced renal accumulation incomparison with 111In-(HE)3-ZHER2:2891-PE38X8. The higher renal excretion of111In-(HE)3-ZHER2:2891-PE38X8may explain the significantly lower accumulationofthisconstructintheliver.

Wealsostudiedthebiodistributionof111In-(HE)3-ZHER2:2891-ABD-PE38X8inmicewithxenograftedSKOV-3tumors.Thetumoruptake(5.5±1%ID/gat24hafterinjection) was significantly (p<0.05) higher than the corresponding non-targeting control construct (Figure 16), but it was lower than its parentalAffibodyZHER2:2891(11±4%ID/gat24hafter injection)[312].ThisphenomenonwasalsoobservedbyZilienskietal.[302]whostudiedthebiodistributionofH6-ZHER2-PE38 and ZHER2 in mice with BT-474 xenografts. They attributed theunevendistributionofH6-ZHER2:342-PE38toitslargersizecomparedtoZHER2:342.


47

Figure 16. Comparative biodistribution of 111In-labelled ZHER2:2891-ABD-PE38X8 andZTaq-ABD-PE38X8 in female BALB/c nu/nu mice bearing HER2 expressing SKOV-3xenografts.Dataarepresentedasanaveragevaluefrom4animals±SD.

In order to find out the maximum tolerated dose of the novel 111In-(HE)3-ZHER2:2891-ABD-PE38X8, three doses (0.55, 0.275 and 0.1375 mg/kg) wereinjected 5 times to female BALB/c mice (n=6) and their body weight wasmonitored (Figure 17). Themice in the highest dose (0.55mg/kg) group hadsignificant loss of averageweight (onemousewas removedbecauseof criticalweight loss) but recovered at the end of the experiment, indicating that 0.55mg/kgwasabovethelimitoftolerabledose.Whenmicewereinjectedwiththelower doses (0.275 and 0.1375 mg/kg), equivalent doses providing a cleartherapeutic effect in the study reportedbyZielinski and co-workers [302], themice did not experience significant weight loss during the study and did notshowanyothersignsoftoxicity.

Figure17.ChangeinweightovertimeinfemaleBALB/cmiceinjectedwithdifferentdosesof(HE)3-ZHER2:2891-ABD-P38X8 as indicated by arrows. Data are presented as an averagevaluefrom6animals±SD.

ThispaperrevealedtheinfluenceofABDfusionandN-terminalmodificationonthebiodistributionofanaffitoxin.The(HE)3-tagdecreasedhepaticuptakeoftheaffitoxincomparedto theH6-taggedaffitoxin.However, thehepaticuptakewasstill more than 5 times higher than the uptake of non-toxin fused affibodymolecules [305], indicating that the liver uptake of the affitoxin was mainlydrivenbythePEpart.TheadditionofanABDsignificantlyprolongedthehalf-lifeof theaffitoxinand loweredthekidneyuptake. It is interestingto investigate ifthesechangesalsoincreasethetherapeuticefficacyoftheaffitoxin.Inastudyby


48

Guoandtheco-workers[303],theinvivoefficacyofasimilarfusiontoxin,ABD-ZHER2-PE38 were found to have an increased anti-tumor efficacy in mice withxenografted tumors comparedwith a construct without ABD addition. Similarresultswerealsoobservedforanti-mesothelinimmunotoxins[313].


49

5.3 PaperIII-FusiontoxinsconsistingofaHER2-interactingABD-derivedaffinityprotein(ADAPT)fusedtotruncatedversionsofPseudomonasexotoxinAwithhighpotencyandspecificity.

Thealbuminbindingdomain(ABD)maynotonlyserveasa fusionpartner forhalf-life extension as in paper I and II, it can also be used as an alternativescaffoldproteinwhereitsspecificityisredirectedtotargetsotherthanalbumin.ThisclassofalternativescaffoldproteinsisnamedABD-derivedaffinityproteins(ADAPTs).Theyconsistofonly46aminoacids,andarethusevensmallerthanaffibodymolecules(58aminoacids).ADAPTstargetingHER2withhighaffinityhave been generated [178]. A variant of and anti-HER2 ADAPT (denotedADAPT6), binds toHER2withhighpicomolar affinity (KD=0.5nM)andhasnoaffinitytoalbumin.IthasdemonstratedhighcontrastinaHER2-xenografttumorimagingstudy[180].EncouragedbytheresultsobtainedwiththePseudomonasexotoxinAaffitoxinsfortargeteddeliveryoftoxicactioninpaperIandII,webelievethatADAPT6canalso direct toxins to tumor cells. we therefore constructed several differentADAPT-PE immunotoxins in this study. As mentioned in paper I, one of thebiggestproblemsofPE-basedimmunotoxins is the immunogenicity,preventingmultiple cycles of treatment. In paper I and II, we used PE38X8which was aversionwhere themouseB-cell epitopeshadbeen removed. In this paper,weusedaminimizedandhumanB-andT-cellepitoperemovedversionofPE,PE25(Mw~25kDa)[297].

In this study, we constructed three ADAPT6 Pseudomonas exotoxin Aimmunotoxins, namely ADAPT6-PE25, ADAPT6-ABD-PE25, and ADAPT6-ABD-PE38X8, and a negative control immunotoxin, ZTaq-ABD-PE25 (Figure 18A). Toourknowledge,ADAPT6-PE25isoneofthesmallestimmunotoxinsthathasbeencreated. The small sizemight be beneficial in the sense of tumor penetration,howevertheserumhalf-lifewaspredictedtobeveryshort.WiththeadditionofABD, ADAPT6-ABD-PE25 was predicted to have an increase in half-life. Thepurpose of constructing ADAPT6-ABD-PE38X8 is to compare the properties ofPE25andPE38X8bothinvitroandinlaterstudiesthebiodistributionandanti-tumor activitywill also be compared. ZTaq-ABD-PE25 is not a perfect negativecontrol, since ZTaq is slightly bigger than ADAPT6. However, both ZTaq andADAPT6adoptasimilarthree-α-helicalbundlestructureandareofsimilarsize.All the ADAPT6 immunotoxins were expressed in E. coli and purified tohomogeneitybyIMACfollowedbyanionexchangechromatographyforADAPT6-PE25, or HSA-based affinity chromatography followed by anion exchangechromatographyfortheotherconstructs(Figure18B).Analyticalsizeexclusionchromatograms indicated no formation of aggregates for any of theimmunotoxins(Figure18C).

TheaffinitiesofthepurifiedADAPT6immunotoxinsforHER2weremeasuredbybiosensoranalysis inaBiacorewithachipwithimmobilizedHER2,followingasimilarprocedureaswasdescribedinpaperI.Theaffinity(KD)ofADAPT6-PE25,ADAPT6-ABD-PE25,andADAPT6-ABD-PE38X8toHER2were10,15and26nM,respectively.Similartoaffibody-basedimmunotoxins,theseimmunotoxinshada


50

decreased affinity compared to the ADAPT6 monomer (KD 0.5 nM) [178] forHER2. The affinities were affected to a less extent in the case of ADAPT6immunotoxinscomparedtotheaffibodyimmunotoxins investigatedinpapersIand II. Itwasalso found that thebigger the fusionpartnersare, themore theyhave a negative influence on affinity. The sizes of the immunotoxins and thevalues of KD followed the same order, ADAPT6-PE25<ADAPT6-ABD-PE25<ADAPT6-ABD-PE38X8.

Figure 18. Construction, purification and initial biochemical characterization of the fusiontoxins.A)Schematicrepresentationofthefusiontoxins.B)Thepurifiedtoxinswereanalyzedbyseparationona10%SDS-PAGEgel.Numberstotheleftarethemolecularweightofthemarkerproteins(kDa) in laneM.Lane1,ADAPT6-PE25; lane2,ADAPT6-ABD-PE25; lane3,ADAPT6-ABD-PE38X8; lane 4, ZTaq-ABD-PE25. C)Overlay of chromatograms obtained afteranalytical size-exclusion chromatography of the fusion toxins. The numbers above theoverlayedchromatogramsarethemolecularweights(kDa)ofproteinstandards.

TheaffinitiesofADAPT6immunotoxinstoHSAandMSAwerealsomeasuredbybiosensoranalysisonachipwithimmobilizedHSAandMSA.BothADAPT6-ABD-PE25 and ADAPT6-ABD-PE38X8 had nanomolar range KD affinity to HSA andMSA,wheretheaffinitiestoHSAwereseveralfoldshigherthanthattoMSA.Thisresult confirmed that ABD was functional, although the affinities weredramaticallylowerthantheaffinityofABDaloneforthesamemolecules(KD=50fM)[175].ThereasoncouldbesterichindranceformedbyADAPT6andPE.

Table2.CytotoxicityofADAPTfusiontoxinsondifferentcelllines

Cellline

HER2expression

IC50(pM)

ADAPT6-PE25

ADAPT6-ABD-PE25

ADAPT6-ABD-PE38X8

ZTaq-ABD-PE3825

SKBR-3 +++ 47 85 34 7600

AU565 +++ 46 130 130 100,000

SKOV-3 +++ 140 310 180 N.D.a

A549 + 5,500 18,000 3,400 100,000aN.D.notdetermined

ADAPT PE25

PE25ABD

Z PE25ABD

A

B

PE38X8ABD

M 1 2 3 49766

45

30

20.1

14.4

C

1.0 1.5 2.00

5

10

Elution volume (ml)

A28

0nm

(mA

U)

ZTaq-ABD-PE25

ADAPT6-PE25

ADAPT6-ABD-PE38X8ADAPT6-ABD-PE25

75 29 13.7

6

ADAPT6

ADAPT6

Taq

Figure 1


51

WefurtherdeterminedthecytotoxicitiesofalltheimmunotoxinsonbothHER2high(SKBR-3,AU565andSKOV-3)and low(A549)expressingtumorcell lines.The ADAPT6 immunotoxins demonstrated picomolar IC50 values on SKBR-3,AU565 and SKOV-3 cells (Figure 19 and Table 2). For SKBR-3 cells the IC50-valuesrangedfrom34to85pM,forAU565theyrangedfrom46to128pMandforSKOV-3 theyranged from138 to310pM.The IC50valuesof thenon-targetcontrol,ZTaq-ABD-PE25,werefoundtobebetween7.6nMtomorethan100nMfor the same cell lines. The difference in cytotoxicity between the ADAPT6immunotoxinsand theZTaq immunotoxinwas two to theeordersofmagnitudefor the three cell lines, indicatingawide therapeuticwindow.All fusion toxinsdemonstrated substantiallyweaker cytotoxic potential forA549 cellswith lowHER2 expression (Figure 19) compared to the cell lines with high HER2expression. The IC50-value for A549 ranged from 3 to 15 nM for the ADAPT6containingfusiontoxinsandwasmorethan100nMforthecontrol.Thisresultsupportedthewidetherapeuticwindowfromanotherangle.Oneof thevariants,ADAPT6-ABD-PE25,waschosenfor further investigationofthespecificity.Theexperimentsetupwasbasically thesameas that inpaper I.Theresultsshowedthatonlyanexcessamountof freeADAPT6ratherthantheirrelevant transferrin control protein could block the cytotoxicity of ADAPT6-ABD-PE25.AndtheblockingeffectsweredependentontheconcentrationoffreeADAPT6,whereanincreaserescuedcellviability.

Figure 19.Determination of the in vitro cytotoxicity of the fusion toxins. The cytotoxicitieswere determined by incubating serial dilutions of the fusion toxins with SKBR-3, AU565,SKOV-3andA549cells.(A,B,CandD)Cellviabilityisplottedonthey-axisasafunctionoffusion toxin concentrationon thex-axis.Theviabilityof cells cultivated ingrowthmediumwithoutadditionofanytoxinwassetto100%foreachpanel.Thedatawasfittedwithalog(inhibitor) vs. response (four parameters) function. Each data point corresponds to theaverageoffourindependentexperimentsandtheerror-barscorrespondto1SD.

10-14 10-12 10-10 10-8 10-60

50

100

Concentration (M)

Rel

ativ

e vi

abilit

y (%

)

SK-BR-3

10-14 10-12 10-10 10-8 10-60

50

100

Concentration (M)

Rel

ativ

e vi

abilit

y (%

)

AU565

10-12 10-10 10-8 10-60

50

100

Concentration (M)

Rel

ativ

e vi

abilit

y (%

)

SK-OV-3

10-12 10-10 10-8 10-60

50

100

Concentration (M)

Rel

ativ

e vi

abilit

y (%

)

A549

ADAPT6-ABD-PE25

ADAPT6-ABD-PE38X8

ADAPT6-PE25

ZTaq-ABD-PE25

A B

C D

Figure 4


52

Inconclusion,thenovelalternativescaffoldaffinityproteinADAPTcanbeusedas a targeting moiety in immunotoxins. ADAPT Pseudomonas exotoxin Aimmunotoxins constructed in this paper had potent and specific anti-tumoractivityinvitro.


53

5.4 PaperIV-Affibody-derivedDrugConjugates:PotentCytotoxicDrugsForTreatmentOfHER2Over-ExpressingTumors

Antibodydrugconjugates(ADCs)arepromisingtherapeutics,sincetheydelivera higher cytotoxicity compared to naked antibodies and may sometimesovercomeresistancetoantibodytherapies.SeveralADCshavebeenapprovedbythe FDA and the EMA, and several more are under clinical and pre-clinicaldevelopment.Wehypothesized that affibodymolecules can alsobe conjugatedwithcytotoxicdrugs for targetedcancer therapy.Affibodymoleculesmayhaveadvantages over antibodies in terms of site-specific conjugation. Affibodymoleculesdonotcontainanycysteines,which isamajorreactionsite fordrugconjugation in ADCs. One or several cysteine residues could therefore beintroducedinaffibody-basedconstructsforsite-specificandhomogeneousdrugconjugation. It is also easy to separate affibody-drug conjugates from non-conjugatedaffibodymoleculesaftertheconjugationreaction,duetothefastandeasy refolding of affibody molecules. Harsher separation methods, such asreverse phase high performance liquid chromatography (RP-HPLC), can beemployedforpurificationoftheconjugatestohomogeneity.

Figure20.Illustrationofthemechanismofactionoftheaffibody-mc-DM1conjugates.

Inthisstudy,wegeneticallyintroducedacysteineresidueforconjugation,attheC-terminal end of several variants of ZHER2 molecules, including a ZHER2-Cysmonomer, a ZHER2 dimer linkedwith a G4S linker ((ZHER2)2-Cys), a ZHER2 dimerfollowedwithanABDmolecule((ZHER2)2-ABD-Cys)and(ZTaq)2-ABD-Cysasnon-specificcontrol(Figure21).ThecytotoxicpayloadDM1wasalreadycoupledtoamaleimidocaproyl(mc)linker,whichmayreactwiththesulfhydrylgrouponthecysteines, forming a thioether bond at pH 6.5 to 7.5. Themc-DM1 conjugatedaffibodymoleculescouldbeeasilyseparatedfromnon-conjugatedaffibodyandunreactedmc-DM1usingRP-HPLC.Theconjugation ratesvariedbetween40%and 90%. The molecular weights of the purified affibody molecule mc-DM1conjugateswereverifiedwithMALDI-TOF-MS.


54

Figure 21. Production and initial characterization of the affibody-mc-DM1 conjugates. (A)Schematicrepresentationsof theproteinsareshown.Alldomains inmulti-domainproteinswereconnectedby theaminoacidsG4S,but isonly shown for (ZHER2:2891)2-Cys. (B)ThepurifiedproteinswereanalyzedbySDS-PAGEunderreducingconditions.Numberstotheleftindicatemolecularweight (kDa) of themarker in laneM. (C) Schematic representation ofmc-DM1conjugatedtotheC-terminalcysteineofanaffibody.(D)ConjugatesafterfinalRP-HPLC purificationwere analyzed by SDS-PAGEunder reducing conditions.Numbers to theleftindicatemolecularweight(kDa)ofthemarkerinlaneM.

ToverifythefunctionofZHER2afterRP-HPLCpurification,thepurifiedconjugateswereanalyzedbyareal-timebiosensor.Theequilibriumdissociationconstants(KD)ofZHER2-mc-DM1and indoacetamide(IAA)alkylatedZHER2-Cyswere foundtobesimilar(112and137pM,respectively)indicatingthatmc-DM1conjugationdidnotaffecttheaffinityofZHER2toHER2.Forthebivalent(ZHER2)2-ABD-mc-DM1,the kinetic data were fitted using 1:1 interaction model and the acquiredapparent KD was 200 pM for the HER2 immobilized surface. The result thusshowed a strong interaction of (ZHER2)2-ABD-mc-DM1 with HER2, despite mc-DM1 conjugation. The interaction of (ZHER2)2-ABD-mc-DM1 with differentalbumins was analyzed on a chip with immobilized serum albumin of human(HSA),mouse(MSA)andbovine(BSA)origin.Asexpected, theconjugatecouldinteractwithHSAandMSA,butnotBSA[175].

The in vitro cytotoxicity of affibody molecule mc-DM1 conjugates wasinvestigated by incubating tumor cell lines, having different levels of HER2expression,withtheconjugates.Wealsoperformedahead-to-headcomparisonwiththeFDAapproveddrugtrastuzumab-DM1inthisstudy.Sinceouraffibody-mc-DM1conjugatesonlyhaveonemoleculeofDM1peraffibodymoleculewhileT-DM1hasonaverage3.5DM1pertrastuzumabmolecule,IC50valuesofT-DM1were translated to IC50 per DM1molecule. The T-DM1 IC50 values mentionedbelow are all translated values. All the HER2-targeting affibody-mc-DM1conjugatesshowedstrongcytotoxicitytotheHER2overexpressingbreasttumorcelllineSK-BR-3,withvaluesrangingfrom0.2to0.5nM,similartoT-DM1(IC50=


55

0.2nM)(Figure22).Thenon-targetingcontrolhadanIC50over50nM,indicatingat least a two orders of magnitude therapeutic window. All the conjugatesshowed similar cytotoxicity for the breast tumor cell line AU565. On anotherHER2-overexpressingovariantumorcelllineSK-OV-3,theanti-HER2conjugatesalso had strong cytotoxic effects, with IC50 values ranging from 1.9 to 7.8 nM,whichareslightlyhigherthanT-DM1(IC50=0.5nM).Thetransitionsof thekillcurves of the affibody-mc-DM1 conjugates were broader compared totrastuzumab-DM1.Previously,trastuzumab-DM1alsoshoweddifferencesinthetransitionofkillcurvesondifferentcelllines[195].ItisnoteworthythattheIAAalkylated(ZHER2)2-ABDitselfshowedgrowthinhibitionoftheSKBR-3andAU565cell lines,while had a growthpromotion effect on SKOV-3 cells, at the highestconcentrations.ThisphenomenahasalsobeenobservedearlierfordimericZHER2on SKOV-3 cells [314]. The specificity of the anti-HER2 affibody-mc-DM1conjugates were demonstrated by rescuing SKBR-3 cells from ZHER2-mc-DM1actionbyanincreasingamountoffreeZHER2.PreincubationwithequalamountofZHER2before incubationwithZHER2-mc-DM1,demonstrated that50%ofSKOV-3cellscouldberescuedfromZHER2-mc-DM1.

Figure22.Analysisofthecytotoxicityoftheaffibodymc-DM1conjugates.(A)Dilutionseriesof the affibody-mc-DM1 conjugates, (ZHER2:2891)2-ABD-IAA and trastuzumab emtansinewereincubatedwiththecell lines indicatedabovethecurves.Theconcentrationrangesfortheconstructswere:100nMto2pM(SKBR-3),100nMto0.6pM(AU565),1µMto30pM(SKOV-3),250nMto0.2pM(MCF7).RelativeviabilityofthecellsisplottedontheY-axisasafunctionofthecompoundconcentrationontheX-axis.Therelativeviabilityofcellscultivatedin medium only was used as reference (100%). Each data point is the average of fourindividualexperimentsandtheerrorbarsinthefigurescorrespondto1SD.

Thepromisinginvitroresultsmotivatedustoinvestigatetheanti-tumoreffectofthe affibodymc-DM1 conjugates in vivo. (ZHER2)2-ABD-mc-DM1was chosen forthe invivo study because prolonged half-life byABD is probably beneficial fortherapeutic efficacy. The dimeric ZHER2might also have a faster internalizationratebydimerizationofHER2,whichmaytriggerinternalization[314].(ZHER2)2-ABD-mc-DM1wasradiolabeledwith111Intobeabletotraceitduringtheinvivostudy. The 111In labeled (ZHER2)2-ABD-mc-DM1 was found to still retain theinteractionwithHER2onSKOV-3cells.Theinternalizationof111In-(ZHER2)2-ABD-


56

mc-DM1 was also determined to be moderate, with 32% of cell associatedradioactivityinternalized4hpostincubation.Tostudy thebiodistributionof 111In-(ZHER2)2-ABD-mc-DM1, itwas injected intoNMRImice and radioactivity uptake in each organwasmeasured 4 h p.i.. IAAalkylated (ZHER2)2 was used as a control. Similar to other ABD conjugatedaffibody molecules, (ZHER2)2-ABD-mc-DM1 had significantly longer half-life incirculation (t1/2 14 h) and significantly reduced kidney uptake (84±7 vs.194±18%ID/g, 4 p.i.). However, the half-life was relatively short and hepaticuptake was significantly higher in comparison with other ABD-conjugatedaffibodymolecules [311].This indicatedapossible increased liver clearanceoftheconjugate,whichmightbeduetothehydrophobicpropertiesofmc-DM1.

The 111In-(ZHER2)2-ABD-mc-DM1 demonstrated HER2-specificity in mice withxenografted SKOV-3 (HER2 positive) or RAMOS (HER2 negative) tumors.ObtainedSPECT/CTimagesrevealedthattheuptakeoftheconjugateinSKOV-3tumorswas14timeshigherthanthatintheRAMOStumor.Toinvestigatethetherapyeffectof(ZHER2:2891)2-ABD-mc-DM1,micewithSKOV-3tumorswere treatedby5weekly injectionsof twodosesof the conjugate (8.5and 4.2 mg/kg). The survival of mice in both treatment groups increasedsignificantly(p<0.02)comparedtothePBScontrolgroup,withamediansurvivalof46d(4.2mg/kg),37.5d(8.5mg/kg)and25d(PBScontrol).Theweightsofthemice were not affected by the injections of the conjugate, indicating goodtolerance.Histologicalexaminationofexecratoryorgans,liverandkidney,whichhad the highest uptake of the conjugate among the organs, did not show anymorphological differences or signs of injury between treated and non-treatedgroups.Inanothertherapystudy,wecomparedtheanti-tumoreffectof(ZHER2)2-ABD-mc-DM1 to non-targeting control (ZTaq)2-ABD-mc-DM1 and non-toxiccontrol IAAalkylated (ZHER2)2-ABD,withadoseof8.5mg/kg for all constructs(Figure23).Themediansurvivalinthe(ZHER2:2891)2-ABD-mc-DM1treatedgroupwas58d, significantly longer than the (ZHER2:2891)2-ABD-IAAgroup (21d), PBSgroup(35d)and(ZTaq)2-ABD-mc-DM1group(26d). Interestingly, (ZHER2:2891)2-ABD-IAAdecreased the survivalofmice compared toPBSand (ZTaq)2-ABD-mc-DM1treatments. Itwaspossiblycausedbystimulationof tumorgrowthbythedimericaffibodythatwefoundintheinvitroexperimentsandthatwasfoundbyEkerljungetal.[314],[315].


57

Figure 23. Specificity of treatment with (ZHER2:2891)2-ABD-mc-DM1. (A) Survival ofBALB/cnude mice (n=6-8)bearings.c. HER2-expressing SKOV-3 tumors on the abdomen (138±46mm3)afterreceiving6 intravenous injectionsof8.5mg/kg(ZHER2:2891)2-ABD-mc-DM1(red),8.3mg/kg (ZTaq)2-ABD-mc-DM1 (black), 8.1mg/kg (ZHER2:2891)2-ABD-mc-IAA (blue) or PBS(green)everyweek(1injectionperweek).(B)Representationoftheaveragebodyweightasanindicationoftoxicityduringtreatment.Miceweightsweredeterminedtwiceweeklyandvaluesarepresentedasaverageweightof6-8animals±SD.Arrowsindicatetheinjections.

In conclusion, we created affibody drug conjugates that could target HER2-overexpressing tumors. The drug was well tolerated by mice and couldsignificantlyprolongsurvivalofmicebearingHER2over-expressingtumors.Theaffibodydrugconjugateholdspromiseforfuturetherapyinhumans.


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5.5 Conclusionremakesandfutureperspectives

The studies presented in this thesis have been devoted to using affibodymolecules and ADAPTs as the targeting moieties in immunotoxins and drugconjugatestodevelopcompoundssuitablefortargetedtumortherapies.Boththeaffibody molecules and the ADAPT in this thesis target HER2, which isoverexpressed in around 20-30% of breast tumor and is strongly related torecurrenceandpoorprognosis. It is thereforean importantbiomarkerandisaclinicallyvalidatedtargetfortargetedtumortherapy.InpaperIandII,weinvestigatedtheanti-tumoractivityofanti-HER2affibodyPE38X8affitoxins.Wealsoinvestigatedtheinfluenceofa(HE)3-purificationtaginsteadofaH6-tagandanABDontheinvitroandinvivoproperties.TheaffitoxinscanbeeasilyexpressedinE.coliinsolubleform.Anti-HER2affitoxins,withorwithoutanABD,hadhighlypotentantitumoractivitytowardsHER2overexpressingtumorcellswhiletheyweremuchlesspotenttowardsHER2low-expressingtumorcells.Tofurtherincreasethespecificityoftheaffitoxins,bi-specificdimericaffibodymolecules,suchasZHER2-ZHER3,targetingtwotumorantigenssimultaneouslymaypossiblybeusedsothatpreferentiallythetumorcellsexpressingbothtumorantigensmayreceivealethaldoseoftoxin,comparedtonormalcellsexpressingonlyoneofthetumorantigens.Bi-specificaffitoxinsmayhaveincreasedinternalizationrateaswell.Anactivationmechanismmaypossiblyalsobeintroducedtoincreasethespecificity.Forexample,aprodrugformofaffibodymolecules(pro-affibody)canbeusedinsteadofcommonaffibodymolecules.Thepro-affibodyconsistsacommonaffibodymoleculeandapeptidemaskingtheparatopeofthecommonaffibody,whicharelinkedtogetherwithapeptidelinkercontainingasubstratesequenceforcancer-associatedprotease[316].Thebindingactivityofthepro-affibodyisblockeduntilitreachesthetumortissuewherethecancer-associatedproteaseisoverexpressed.ThebiodistributionresultsshowedthattheadditionofanABDledtoappreciableextensionoftimeincirculationandseveral-foldreductionofrenaluptake.Thesubstitutionofthehexa-histidinetagwiththe(HE)3tagresultedindecreasedliveruptake.However,themaindriverofliveruptakewasPE38X8,sinceliveruptakeoftheaffitoxinwereseveralfoldshighercomparedtoaffibodymoleculewithoutPE38X8fusion.Inordertofurtherimprovethebiodistributionprofileoftheaffitoxin,apossibilityistoincreasethehydrophilicityoftheaffitoxinbye.g.PEGylationorintroducinghydrophilicaminoacidpointmutationsinPE38X8.

InpaperIII,weusedananti-HER2ADAPTcoupledtoPE25,andperformedasimilarstudytothatinpaperI,toinvestigatethefeasibilityofADAPTimmunotoxins.TheADAPTimmunotoxinswereevensmallercomparedtoaffitoxinsinpaperI.Theanti-HER2ADAPTPE25fusiontoxinsselectivelykilledHER2overexpressingcellswithalargetherapeuticwindow,albeitPE25demonstratedslightlylowerpotencycomparedtoPE38X8.Inconclusion,immunotoxinsconsistingofanaffibodymoleculeorADAPTastargetingmoietyhadhighpotencyandspecificity.Sinceanti-HER2affibodymoleculesandtheADAPTbindtodifferentepitopes,theseimmunotoxinsmaybeusedincombinationinafuturestudy.Theaffinityoftheanti-HER2ADAPTforHER2wassimilartothatofscFvcounterpart,whilemuchweakerthanthatoftheanti-


59

HER2affibodymolecule.However,inthecontextofimmunotoxin,anti-HER2ADAPTimmunotoxinhadonlyabitweakeraffinitytoHER2comparedwithaffibodymoleculeimmunotoxin.Theweakeraffinityresultedinweakercytotoxicity.However,aweakeraffinitymayincreasethetumoruptakeandpenetrationofsolidtumorsbytheimmunotoxins.

Sincethescaffoldofaffibodymoleculesarecysteine-free,wewereabletosite-specificallyconjugatemc-DM1toauniquecysteineintroducedattheC-terminalendofanti-HER2affibodyconstructsfortargeteddeliveryofDM1totumorcells.AllvariantsofZHER2mc-DM1conjugateshadspecificantitumoractivitytowardHER2overexpressingtumorcelllinesandthe(ZHER2)2-ABD-mc-DM1alsodelayedtumorgrowthandprolongedsurvivalofmicewithxenograftedtumors.Inconclusion,affibodydrugconjugatesarepromisingalternativestoantibodydrugconjugates(ADCs),sincetheyhadshownpotentanti-tumoractivitybothinvitroandinvivo.ThehydrophobicnatureofDM1mighthowevercontributetoincreasedunspecificuptakeoftheconjugates.Inaddition,wenoticedthatfreeze-thawoftheaffibodydrugconjugatessometimesledtoaggregationandprecipitation,whichmightbeduetothehydrophobicnatureofDM1.Inthefuture,wewouldliketoincreasethehydrophilicityoftheconjugatesbyeitherintroducingmorehydrophilicaminoacidsorbyPEGylation.Themaleimidocaproyl(mc)linkerwasshownpreviouslylosingpayloadsincirculationviaaretro-Michaeltypereaction,wherepayloadsaretransferredtoothermoleculeshavingSHgroup,suchasserumalbumins.Morestablelinkersorreactionsstabilizingmclinkercanbeappliedinproductionofconjugatesinthefuture.

Analternativetothetargetedtherapyinvestigatedinthisthesisisimmunotherapy,whichisalsoanewtypeofcancertreatmenthavinghighspecificity.ChimericantigenreceptorT-cell(CAR-T)therapyisonerepresentativepossibilityofimmunotherapythathasdrawnattentionrecently.Lastyear,theFDAapprovedthefirst-everCART-celltherapy,tisagenlecleucel(tradenameKymriah®),totreatB-cellprecursoracutelymphobasticleukemiapatientswhohavenotrespondedtostandardtherapyorwhohaverelapsedatleasttwice.CART-celltherapieshavethespecificitiesprovidedbyscFvsinthechimericantigenreceptors(CARs)andcytotoxicitesprovidedbyCD8+T-cells.Inasense,CART-cellsaresimilartoimmunotoxins,withCD8+cellsaseffector“moieties”.Itispossiblethataffibodymoleculesand/orADAPTsareusedinproductionofCART-cellsinthefuture.

Takentogether,thisthesishasshownthepotentialofaffibodymoleculesandADAPTsfortargetedcancertherapy,bydirectingcytotoxicpayloadstotumorcells.

Acknowledgements

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AcknowledgementsTorbjornGräslund,mymainsupervisor,thanksforacceptingmeasaPhDstudenttostudyinKTHandlettingmedotheseexcellentprojects.Thankyouforyourpatientguidanceonresearchandlifeallthroughthesefiveyears.WheneverIhaveaquestionoraproblem,Icanjustknockonyourofficedoorandstartaconversation.Ihavelearnedalotfromthecountlessconversationswehad,whichwillbeapreciousassetformyfuturecareer.

SefanStåhl,myco-supervisor,thanksyouforfantasticadviceonmyresearchprojectsandcommentsonmythesis.Youareaveryniceandkind,andmoreimportantlyahappyprofessor.Per-ÅkeNygren,thanksfortheinspiringconversationswehad.Thecommentsandquestionsyouhadintheseminarsalwaysinspiredmealot.

SophiaHober,thanksforthefantasticcollaborationanddiscussionontheADAPTproject.Youareverykeenonaskingthemostimportantquestionsofaresearchandfindingsolutions.

Uppsalagroup,VladimirTolmachev,AnnaOrlovaandMohamedAltai,thankyouallforthefantasticcollaborationonalltheprojects.Withoutyourwork,myworkwon’tbesomeaningful.JohnLöfblom,thankyoufortakingtimereviewingmythesisandgivingmeexcellentfeedbacks!

JohanSeijsing,youaremyfirstSwedishfriend.ThankyouforshowingandteachingmemanypracticalthingsonPlan3.ThankyouforhelpingmebothonresearchandlifeinSweden.WehadmanyhappyconversationsaboutSwedenandChina.Thankyouforinvitingmetoyourparents’house.Iwon’tforgetthetimespentonthearchipelago.

IfeelluckytositinanofficeB3:1052.Duringtheseyears,manypeoplecomeandgo.Thankstoyouall,includingJohan,Anna-Luisa,Shengze,Jonas,Joel,Marlin,Mona,Jakob,Magda,Aman.

IalsowanttothankallthepeopleonPlan3forcreatingsuchaniceenvironmentandbeinghelpfulallthetime.Ken,Anna-Lusia,Johan,Hanna,Feifan,Shengze,Jonas,Tarek,Sara,Sarah,Rezan,Anne-SophiaS.andT.,Lanlan,Anders,Haozhong,Wenetc.,youallhelpedmeinvariousways.

I’dliketothankallthenewChinesefriendsIgottoknowduringmyPhDstudy.Wereallyhadalotoffuntogether,suchasBBQ,fishing,takingtoursaroundEuropeandplayingMaJiangJ.我觉得我还应该感谢我的发小们，刘琪、王炳科、杨晶、刘奭川、史⼀洋、赵博、马鹏、赵晨、郝⽂佳、李良⼦等等……。能认识你们我很幸运。今天的我有你们每个⼈的影响。愿我们友谊长存！最后，我想感谢我的家⼈。我⼗分感谢爸爸妈妈⼀直以来对我的学业和⽣活的支持。没有你们⽆私的支持，我不可能有今天的成绩。老婆，感谢你⼀直以来对我的科研的支持。尤其要感谢你在读博期间给我们⽣下了可爱的刘瑞涛（Edwin）。他的降⽣给我们带来了很多快乐也让我变得更加成熟。我永远爱你们！

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