1

Antibody-TargetedNanoparticlesforCancerTreatmentThomasCarter1,PaulMulholland1,2,KerryChester1AbstractNanoparticlesarediverseandversatilewithphysicalpropertiesthatcanbe

employedforuseincancermedicine.Targetingnanoparticlesusingantibodies

andantibodyfragmentscouldovercomesomeofthelimitationsseenwith

currenttargetedtherapies.Thisreviewwilldiscusstheroleofantibody-targeted

nanoparticlesinthetreatmentofcancer:asdeliveryvehicles,targeted

theranosticagentsandintheevolvingfieldofcancerhyperthermia.

Keywords:Antibodies,nanoparticles,targeting,cancertreatment,clinicaltranslation,theranostics,hyperthermia1.0IntroductionAsourunderstandingofcancerbiologyhasevolved,sotoohastheappreciation

thatantibodytargetedcancertreatmentofferssignificantadvantagesover

conventionaltherapy.Anumberofstrategieshavebeensuccessfulintheclinic:

mostnotablytheuseofmonoclonalantibodies(mAbs)totargetcancer-specific

antigens[1],delivercytotoxicchemotherapyintheformofantibody-drug

conjugates(ADCs)[2],releasebrakesontheimmunesystem[3]andtorecruit

cytotoxicTcells[4].However,thesuccessesofthesetargetedtherapiesarenot

withoutdrawbacks,includingdose-limitingtoxicities.

Advancesinnanotechnologyhaveenabledthecreationofavastarrayofdevices,

collectivelyreferredtoasnanoparticles(NPs),whichpossessuniquephysical,

chemicalandbiologicalproperties.Theuseofantibodiestotargetthesenano-

sizeddrugdeliveryvehiclesoffersnewpotentialthatcanbeharnessedforusein

cancermedicine[5].Indeed,asmallnumberoftargetedNPshavebeenalready

beensuccessfullytranslatedintoearlyphaseclinicaltrialsusingtargeting

ligands[6-8].Antibodies(Abs)havebecomethemostwidelystudiedofthese

targetingligands[9],duetotheirhighspecificityandthevitalroletheyplayin

moderncancertherapeutics.

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ThemainchallengeforAb-targetedNPsisensuringthataswellas

demonstratingefficacytheNPhasanadequateplasmahalf-lifeandcan

specificallytargetcancercellswithatolerablesideeffectprofile.Thereare

controversiesoverwhichNPplatformscanbestoffertheseproperties,with

unansweredquestionsconcerningcellularinteractionanduptake,potential

toxicitiesandhowNPpropertiesaffectbehaviourbothin-vitroandin-vivo[10].A

robustunderstandingofthephysicalpropertiesofNPsisrequiredtoguide

researchintothecellularandphysiologicalinteractionsofNPs,generating

clinical-gradeproductsthatcouldbecomethenextgenerationofcancer

treatments.

ThisreviewaimstodefineandoutlinethedifferentNPplatformsthatare

currentlyusedinpre-clinicalandtranslationalcancermedicineandthe

challengesfacedintheAb-functionalisationofNPs.Researchregardingthe

variousapproachesandadvantagesofferedbyAb-targetedNPsincancer

therapywillalsobediscussed.

1.1.NanoparticlesNPsaredefinedbytheCommissionoftheEuropeanUnionasanynaturally

occurringormanufacturedparticulatematerialeitherunbound,asanaggregate

oragglomerate,whereatleasthalfoftheparticleshaveoneormoreexternal

dimensionsbetween1and100nminsize[11].However,thereremainsno

formalconsensusonthedefinition[12]andwhilstNPsareoftendefinedbytheir

sizeitisgenerallyconsideredimportanttoalsodefinethembypropertiesnot

sharedwithotherparticleswiththesamechemicalcomposition[13].For

examplehighmobilityinthefreestate,largesurfaceareatovolumeratioand,in

somecases,thenanosize-dependentexhibitionofquantumeffectswhichcontrol

physicalpropertiesandNPbehaviour[14].

Thiswideanddiverserangeoffunctionalpropertiesisfacilitatedbythevariety

ofmaterialsavailabletosynthesiseNPs.Theresultingversatilityoffersattractive

translationalpotentialforanumberofbiomedicalapplicationssuchastargeted

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deliveryoftreatment,innovativeimagingtechniquesandnoveltherapeuticsfor

hyperthermia.Table1showsthefavourablephysicalcharacteristicsoftheNP

treatmentplatformsdiscussedinthisreview.Giventhesecharacteristics,itisnot

surprisingthatoverthelastdecadenanoparticleshavereceivedincreasing

attentionfortheirexcitingpotentialinboththediagnosisandtreatmentof

cancer.

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Nanoparticle Size FavourablePhysicalCharacteristics

NATURALPOLYMERS*(Albumin)[15] 50-300nm

Biocompatible,biodegradable,non-toxicandnon-immunogenic.Straightforwardtocross-linkandchemicallymodify,drugsreadilyincorporatedintoalbuminpolymermatrix.

SYNTHETICPOLYMERS*(Poly-lacticoglycolicacid(PLGA)[16] 50-300nm

FDAapprovedsyntheticmaterialthatisnon-toxicandundergoeshydrolysisin-vivotoproducebiodegradablemetabolites.Drugseasilyincorporatedintothematrix.

LIPOSOMES*[17] 20–1000nm

Wellestablishedascancertherapeutics,canencapsulatehydrophilicandlipophillicdrugs,soluble,lowuptakebymacrophages,favourablestabilityin-vivo,andascolloids,protectdrugsfrombreakdown.

MICELLES*[18] 10-100nm

Uniquecore-shellarchitecture,hydrophobiccoreactsasnaturalcarrierenvironment,hydrophilicshellenablesstabilityinaqueoussolution,structuralmodificationscanfurtheraugmenttumourcelluptake

DENDRIMERS*[19,20] 5–20nm

Symmetricalbranchedpolymericmacromoleculeswithacentralcorealloweitherencapsulationorconjugationoftherapeuticagent.Self-assembling,polyvalent,chemicallystable,non-toxicandsoluble.

IRON-OXIDENANOPARTICLES*(Superparamagneticiron-oxidenanoparticles(SPIONs))[21] 10-100nm

Biocompatibleandbiodegradable,establishedclinicaluseasmagneticresonanceimaging(MRI)contrastagents,controllablebyanexternallyappliedmagneticfield,diverseformulationsallowfine-tuningofphysicochemicalproperties

SILICA(PorousSilicaNanoparticles(pSiNPs))[22] 50-1000nm

Goodchemicalandthermalstability,largesurfaceareaandporevolume.Canencapsulatelargeamountsofbioactivemoleculeandpromotecontrolleddrugrelease.Alsoofferssimplesurfacefunctionalisation

GOLDNANOPARTICLES*(Goldnanoparticles(AuNPs),Goldnanorods)[23] 1-100nm

Intenselightabsorptionandhighphotothermalconversionrate,andeaseofsynthesisinavarietyofshapesandsizes.Goodbiocompatibility,colloidalstabilityandsimpleligandconjugationchemistry.

CARBONNANOPARTICLES(Carbonnanotubes(CNTs))[24] 1-10nm

Highcarryingcapacity,andhighpropensitytotraversecellmembranesEasilychemicallymodifiedorfunctionalizedthroughformationofstablecovalentbonds.

QUANTUMDOTS(QDs)[25] 2-10nm

Broadabsorptionspectraandhighfluorescencequantumyield,highphotostability.Possibilityofmultiplexing(differentcoloursofQDsusedwithinoneassaywithasingleexcitationsource).CanbecombinedwithotherNPplatformsforcancertheranostics.

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Table1.PhysicalcharacteristicsofthecommonlyusedNPplatformsexploitedforclinicalapplications.NPslabelledwithanasterix(*)havebeenusedintheclinicforthediagnosisortreatmentofcancer[26].1.2.TargetingNanoparticlesforCancerTreatmentTheabnormalhyperpermeablevasculatureandimpairedlymphaticdrainage

foundwithintumours[27]canleadtopassiveaccumulationofNPswithin

malignanttissues.Thisprocessisknownasenhancedpermeabilityandretention

(EPR).EPRhasbeendemonstratedextensivelyinpre-clinicalresearchandinthe

clinicalsettingusingliposomaldrugssuchaspegylatedliposomaldoxorubicin

(PLD).ThesuccessofPLDliesinencapsulatingthetoxicanthracycline

doxorubicinwithin100nmliposomestolimitdruguptakeintosensitivehealthy

tissuessuchasthemyocardiumtherebyavoidingtheseveredose-limiting

cardiotoxicitycommonlyseenwithfreedoxorubicin[28].Oncelocalisedwithin

thetumour,liposomebreakdownreleasesthedoxorubicin,causinglocaltumour

celldeath.

WhilstEPRcanincreaseoveralltumouruptakeofNPs,specifictargetingofNPs

tocancercellscanbebestachievedthroughtheadditionofatargetingmoiety

[29],anumberofwhichhaveshownpromiseincludingproteins,nucleicacids

andsmallmolecules(Figure1).

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Figure1.SchematicshowingthemostcommonlyusedligandstoactivelytargetNPsincludingDNAaptamers(A),antibodies;asbothwholeimmunoglobulinGmolecules(IgGs)(B)andfragmentssuchassinglechainvariablefragments(scFv)(C),non-immunoglobulinproteinssuchastransferrin(D)andsmallmoleculessuchasfolicacid(E).Figureadaptedfrom[30].Antibodies,themostwidelyusedligandsforNPtargeting,recogniseavastarray

ofantigens,duetotheiruniquespecificityconferredbythecomplementarity

determiningregions(CDRs)(Figure2).Antibodiescanbereadilygeneratedas

intactIgGsorasfragmentssuchassinglechainvariablefragments(scFvs)

(Figure2)indefined,recombinantform[31,32].Antibodytargetingappearsto

becomplementarytopassiveintratumouralaccumulationmediatedbyEPR[33-

35].Ab-NPsalsoshowimprovedefficacyandcytotoxicitycomparedwithnon-

targetedNPs[36].

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Figure2.ImmunoglobulinG(IgG)Structure:Theheavychain(blue)ismadeupofonevariableregion(VH)andthreeconstantregions(CH1,CH2,CH3).Thelightchain(green)hasonevariable(VL)andoneconstant(CL)region.Oneachvariablechaintherearethreecomplementaritydeterminingregions(CDRs),whichbestowthevastvariabilityinspecificantigenbindingcapacity.VHandVLtogetherwithCH1andCLareknownastheFab(fragment,antigenbinding),andtheremainderofthemoleculeisknownastheFcregion(fragment,crystallisable),whichconfersbiologicalactivityandhalf-life.Recombinantantibodyfragmentsincludesinglechainvariablefragments(scFvs),whicharemadeupoftheVHandVLsegmentsjoinedusingapeptidelinker.2.0Antibody-Nanoparticlebio-conjugationThechallengesinestablishingconjugationstrategiesthatpreservethe

functionalityofboththeantibodyandNPduringtheconjugationprocesscanbe

broadlydividedintothreemaincategories;controllability,stabilityand

reliability.Controllabilityrelatestothenumberofantibodiesconjugatedtoeach

NPandtheorientationofthisinteraction,bothofwhichcangreatlyaffectthe

subsequentfunctionalabilityofanAb-NPconjugate[37].Whilstitremains

difficulttopre-determinethenumberofAbsconjugatedtoeachNP,itispossible

tofractionatefunctionalisedNPsbasedonthenumberoflinkermoleculesonthe

NPsurface[38].Sophisticatedtechniqueshavealsobeendevelopedtocontrol

antibodyorientation[39,40],resultinginpreservationofAbbioactivity[41].

ThesenewtechniqueshavebeenreportedtoachieveAbbioactivityashighas

88%ofthenakedantibody[41,42].

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ItiscruciallyimportanttomaintainstabilityoftheNPandAbthroughoutthe

conjugationprocessandtoensurethatthefinalconjugatehasacceptable

stabilityin-vitroandin-vivo.Harshreactionconditionsshouldbeavoided,as

thesecantriggerantibodyunfoldingwhichimpairsantigenbinding[43].Itis

alsonecessarythattheattachmentofAbtoNPsurfacedoesnotleadtounfolding

fromunfavourableinteractionsoftheproteinsidechainswiththeNPsurface

[44].Thechoiceofconjugationstrategyhasalsobeenshowntoaffectlong-term

stability.Forexample,whilstthehighlyspecificinteractionbetweenstreptavidin

andbiotinadaptormolecules(engineeredontothenanoparticlesurfaceand

antibodyrespectively)successfullygenerateAb-NPconjugatescapableof

targetingtumourcellsin-vitro[45],theseconjugatesdidnotdemonstrate

acceptablelong-termcolloidalstability.However,byaddingareactivethiol

grouptotheAbandcovalentlycross-linkingitviaabifunctionallinkermolecule

tothenanoparticlesurfacehigherbindingefficiencyandimprovedstabilitywas

achievedwithequalin-vitrotargetingefficacy[46].

Reliable,reproducibleandwellvalidatedconjugationproceduresareessentialto

enableupscaledevelopmentforuseinclinicalapplications[47]andtodate,this

hasbeenmostsuccessfullyachievedbydirectcovalentconjugationvialinker

molecules.Establishedcovalentconjugationmethodsincludecarbodiimide

coupling,maleimidecouplingandclickchemistry(coppercatalysedalkyne-azide

cycloadditionreactions)[48],asshowninfigure3.InthecaseofAuNPsgold-

sulphurlinkagecanbeexploited,utilisingthehighaffinitywithwhichthiol

modifiedligandsbindtogoldsurfaces[49].Theadvantagesanddisadvantagesof

conjugationstrategiesaresummarisedintable2,andhavebeenreviewedin

depthbyMontenegroet.al.[50].

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Figure3.Establishedmethodsforthecovalentconjugationofantibodiesto

nanoparticles:(A)carbodiimidecoupling,(B)maleimidecouplingand(C)

Coppercatalysed‘click’cycloadditionreaction[48].

ConjugationStrategy

Advantages Disadvantages

PhysicalAdsorption(ionic,electrostatic,van-der-Waals)

• Simpleandstraightforward• UnnecessarytomodifyeithertheAbortheNP

• Electrostaticattractioncanorientatetheantibodies‘endon’preservingbindingability

• Reversible• HydrophobicinteractioncancausedenaturationofAb

• ElectrostaticattractionisweakandpHdependent

• Competitivedisplacementbyserumproteinscanoccur

CovalentConjugation(includingvialinkermolecules)

• Higherstabilityandimprovedreproducibility

• ModificationstoAbusuallynotrequired

• Orientedbindingpossible• Useoflinker-moleculecanavoidhostilereactionconditions

• Possibletocontrolvalency

• Reactionconditionsmayleadtoproteinunfolding/reduction

• Conditionscanaffectantigenbindingcapacity

• Choiceofbindingmoietiesorlinkercansignificantlyaffectfunction

Useofadaptormolecules(biotin/streptavidin)

• Usuallyorientatedbinding• Canresistharshreactionconditions

• Difficulttocontrolvalency• Expensivetechnique• Reversibleattachment

BispecificAntibodyConjugation[51]

• Noneedfordisruptivechemicalconjugation

• MinimalimpactuponNPstability

• Longtermstabilityunknown• Onlyoneantigenbindingsiteavailable

Table2.SomeadvantagesanddisadvantagesofAb-NPbio-conjugationstrategies.Tableadaptedfrom[50,51].

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3.0ClinicalApplicationsofNanoparticlesforCancerTherapyAnumberofuntargetedNPshaveenteredinearlyphaseclinicalcancertrials

[26].Theseincludecyclodextrin-polymercamptothecin-containingNPsfor

advancedovariancancer[52],micellesdeliveringpaclitaxeltopatientswith

advancedgastriccancer[53]andAuNPsdeliveringrecombinanthumanTNF-

alphatopatientswithadvancedsolidtumours[54].SuperparamagneticIron-

oxideNPs(SPIONs)havefoundclinicalapprovalforuseascontrastagentsin

cancerimaging[55,56]andhavealsobeensuccessfulinearlyphasetrialsin

cancerhyperthermia[57,58].SomeuntargetedNPshavebeenfullyapprovedfor

clinicaluseascancertherapeutics[26]includingtheliposomaldrugPLD[59],

andnanoparticlealbumin-boundpaclitaxel(nab-paclitaxel)[60].

Ab-targetedNPsintheclinicareshowingearlypromisebuthavebeensofar

beenrestrictedtoliposomes.Inonetrial,ascFvagainsttransferrinreceptorwas

usedtotargetliposomesbearingp53DNAinpatientswithavarietyofadvanced

solidtumours[61].Doserelatedaccumulationofthetransgenewasobservedin

tumoursbutnotinnormalskintissue.Furthermore,7outof11patientshad

stablediseaseafter6weeksoftreatmentwithtolerablesideeffects[61].PLDhas

alsobeentargetedtotheFDA-approvedchimericanti-epidermalgrowthfactor

receptor(EGFR)mAbcetuximabinaphaseIclinicaltrialwithsomeevidenceof

clinicalefficacy.Furthermore,thesideeffectsseenwerelessseverethanwould

beexpectedwitheitherfreedoxorubicinorcetuximab[6].Themostcommon

dose-limitingtoxicitywasmyelosuppression,whichcouldbemanaged

prophylacticallywithgranulocyte-colonystimulatingfactor(G-CSF)[6].

TargetedPLDhasalsobeeninvestigatedintheclinicusingananti-human

epidermalgrowthfactorreceptor2(HER-2)scFv[7].Theseearlyclinicaltrials

areencouragingandfurthertranslationofAb-targetedNPsisunderpinnedbya

numberofexcitingpreclinicalstudies.Theongoingpre-clinicaldevelopmentof

Ab-targetedNPsfordeliveryofcytotoxicdrugs,radiotherapyandnucleicacids

andtheirroleincancertheranosticsandcancerhyperthermiaisdiscussed

below.

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4.0PreclinicalDevelopmentofantibody-targetednanoparticlesforcancertherapy4.1DeliveryofcytotoxicagentsAbtargetedNPdrugdeliveryoffersanumberofbenefitsoversystemic

administrationoffreedrugs.Theseincludeimprovedintratumouraldrug

distribution,controlledreleaseofdrugswithinthetumourmicroenvironment,

superiorefficacyandmoretolerablesideeffects.Althoughantibody-targeted

liposomes(immunoliposomes)remainintheleadfordeliveryofcytotoxicagents

[62],otherinnovativeapproachesareclosebehindusingwholeAbsaswellasAb

fragments.Thisisillustratedbythevarietyofdrug-loadedNPsinpreclinical

development(Table3).

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Nanoparticle Antibody/Fragment

MolecularTarget

CytotoxicAgent

Stage Cancer Reference

PEGylatedPLGAPolymer

mAb DR-5 Camptothecin In-vivo Colorectal [63]

Liposome mAb GD2 Etoposide In-vitro Mixed(GD2expressing)

[64]

Lipid-PolymerHybrid

mAb EGFR Adriamycin In-vivo HepatocellularCarcinoma(HCC)

[65,66]

PLGAPolymer mAb EGFR Paclitaxel In-vivo Lung [36,67]PorousSilicaNPs(pSiNPs)

mAb p75(NTR),EGFR,CD20

Camptothecin In-vitro Glioblastoma,Neuroblastoma,B-celllymphoma

[68]

Polymericnano-micelles

mAb HIF-1α Paclitaxel In-vitro Gastric [69]

CarbonNanotube(CNT)

mAb CEA Doxorubicin In-vitro Colon [70]

AuNPs mAb EGFR Gemcitabine In-vitro Pancreatic [71]PLGA/MMTPolymer

mAb HER2 Paclitaxel In-vitro Breast [72]

SPIONs mAb HER2 Paclitaxel/Rapamicin

In-vitro Breast [73]

Liposome mAb 2C5 Doxorubicin In-vivo Glioma,Lung [74,75]PLAPolymer mAb SM5-1

bindingprotein

5-FU In-vivo HCC [76]

Lipid-PolymerHybrid(PLGA)

Half-Ab(hAb)

CEA Paclitaxel In-vitro Pancreatic [77]

Iron-Oxide(SPION) ScFv Endoglin Docetaxel In-vitro Ovarian [66]PEGylatedPolymer ScFv CD44v6 Arsenic

trioxideIn-vivo Pancreatic [67]

PLGAPolymer ScFv SM5-1bindingprotein

Paclitaxel In-vitro HCC [78]

Liposome ScFv HER-2 Doxorubicin PhaseIClinicalTrial

Breast [7]

Liposome ScFv c-Met Doxorubicin In-vivo Lung [79]PLGA Fab HER2 Pseudomonas

ExotoxinA(PE38KDEL)

In-vivo Breast [80]

Liposome Fab EGFR Doxorubicin PhaseIClinicalTrial

Mixed(EGFRexpressing)TumourTypes

[6,71]

Table3.Drug-loadedNPstargetedusingantibodiesorantibodyfragments.WholeAbs:ThemajorityofwholeAbtargetedNPshavebeendirectedtoEGFRor

HER2;bothofwhichhaveillustratedtheprospectofNP-mediatedintracellular

drugdeliverythroughreceptormediatedinternalisation.Oneexampleused

cetuximabtotargetgemcitabine-loadedAuNPs[71].Intheseexperiments,the

authorsdemonstratedspecifictargetingoftheNPstoEGFRexpressing

pancreaticcelllinesin-vitroandadditionalantibody-mediatedcytotoxicity

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comparedtonon-targetedcounterparts[71].TargetingEGFRhasalsoshownin-

vivoefficacyinarangeofmodels.Forexample,indeliveryofpaclitaxel-loaded

polymericNPstolungtumours[36],camptothecinloadedporoussilicaNPstoa

rangeofEGFRover-expressingtumoursincludingglioblastoma[68]and

adriamycinencapsulatedpolymer-lipidNPstohepatocellularcarcinoma[65].In

thisfinalexample,therewasalsoevidencesuggestingthatboththeantibodyand

adriamycinwerecontributingtotheobservedcytotoxicity[65].HER2targeting

usingtrastuzumabhasbeenshowntobeeffectiveusingpaclitaxelandrapamicin

partitionedwithinpolymercoatedSPIONs;In-vitroefficacywasover7times

higherthanthenon-targetedcounterparts[73].Inanotherstudy,HER2-targeted

polymericNPsdeliveringpaclitaxelshowedsustaineddrugreleasein-vitro[72].

PolymericNPshavealsobeensuccessfullytargetedtothepro-apoptoticcell

surfacedeath-receptor,DR5[63].Intheseexperiments,theDR5-targeted

polymericNPswereshowntoinitiateapoptosisin-vitroand,whencaptothecin

wasencapsulated,theNPwasabletoovercometheresistancecommonlyseen

withsingleagentanti-DR5therapyin-vivo.Inanotherexample,hypoxia

induciblefactor1α(HIF-1α),anextracellularproteinoverexpressedina

numberofhumancancersinresponsetolocalhypoxia,wassuccessfullytargeted

withanti-HIF-1αAbfunctionalisedpaclitaxelloadedpolymericNPs[69].The

NPswereshowntoselectivelyinternaliseincellsoverexpressingHIF-1αand

paclitaxelmediatedcytotoxicitywasshowntobespecifictowardsHIF-1α

expressingcells.Othertargetsinvestigatedforcontrolleddeliveryofdrug-loaded

NPsincludethecellsurfaceglycoproteinandtumourmarkercarcinoembryonic

antigen(CEA)andthep75neurotrophinreceptor(NTR)[68].NTRisamember

ofthetumournecrosisfactor(TNF)superfamily,whichhasaroleincelldeath

andisoverexpressedinanumberofmalignanciesincludingsarcomaand

malignantmelanoma[81].Drug-loadedNPshavealsobeentargetedusingSM5-

1,amouse-humanchimericantibodywhichishighlyspecifictotheSM5-1

bindingprotein,atargetknowntobeoverexpressedinanumberofcancers

includinghepatocellularcarcinoma(HCC),melanomaandbreastcancer.Binding

oftheSM5-1Abtoitstargetproteininhibitscellgrowthandinducesapoptosisin

cancercellsinacaspasedependentmanner[82].PolymericNPsloadedwith5-

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Flurouracil(5-FU)havebeensuccessfullytargetedusinghumanisedSM5-1inits

wholeAbform,demonstratingsustaineddrugreleaseandfavourableanti-

tumouractivityagainstsubcutaneousandorthotopicHCCxenografts[76].

PLDhasbeenshowntoexhibitenhancedtumourretention,Abdependent

endocytosisandincreasedcytotoxicitywhentargetedusingtheanti-nucleosome

mAb2C5inorthotopicgliomatumoursin-vivo[74].The2C5Abspecifically

recognisesextracellularandtumour-cellboundnucleosomesthatarisefrom

apoptotictumourcellsin-vivo[75].Evenwhenusedatsubtherapeuticquantities,

2C5isaneffectivetumourtargetingmoietyforPLD,alsoshowingefficacy

againstprimaryandmetastaticlungtumoursinmice[75].Centralnervous

system(CNS)tumourspresentauniquechallengetotreatmentduetothe

presenceofthebloodbrainbarrier(BBB);auniquephysiologicalbarrierwhich

functionstoprotectthebrainbutalsolimitsdrugdeliverytotheCNS[83].To

facilitatemovementpasttheBBB,polymeric,2C5-targetedNPshavealsobeen

co-targetedusingananti-transferrinreceptor(TfR)Ab.In-vivointratumoural

localisationwasfoundtobesignificantlyhigherforthedualtargetedNPthan

thatseenforNPstargetedwitheachantibodyalone[84].Morerecently,

transferrinreceptorbispecificantibodyplatformshavebeenshownto

successfullycrossandtargetpasttheBBB[85],andthesebispecificAbscould

equipdrugloadedNPstomoreeffectivelytargetCNStumours.These

experimentssuggestthatAbtargetedNPscouldofferanovelsolutiontodeliver

treatmentpasttheBBB.

AbFragments:Abfragmentsareconsideredtomakeattractivetargetingagents

thankstoasmallersizeandpotentiallyreducedimmunogenicitycomparedwith

wholeAbs[86].Themostcommonlyusedfragments,scFvs,arereadily

generatedusingrecombinantantibodytechnologyandhavebeensuccessfully

appliedinthefollowingexamples.ScFvshavebeensuccessfullyexploitedto

targetarsenicloadedpolymericnanoparticlestargetedtoCD44v6,a

transmembraneglycoproteinoverexpressedonpancreaticadenocarcinoma

cells.Accumulationandretentionwithinthetumourwassignificantlyhigherfor

CD44v6-targetedNPsthanforthenon-targetedcontrols.Furthermore,the

targetedNPsinhibitedtumourgrowthsignificantlymorethanthenon-targeted

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NPsorfreearsenic[67].Inanotherexample,phagedisplaytechnologywasused

toobtainacell-internalisingscFvtotargetc-Met;atyrosinekinasereceptor

expressedonbothtumourandendothelialcells[79].Whenthisanti-c-MetscFv

wasusedtotargetPLDtolungtumours,significantdisruptionofthetumour

vasculaturewasobservedinadditiontodirecttumourcellcytotoxicity[79].

Duetotheirmagneticproperties,ithaslongbeenproposedthatSPIONscouldbe

targetedusinganexternallyappliedmagneticfield[87].However,following

successfulinitialin-vivostudies,problemswithbloodvesselembolisationledto

thedevelopmentofalternativemethodsoftargeting,includingAbs[88].More

recently,thepossibilityofmagnetictargetinghasbeenrevisitedincombination

withanscFvtargetedtoendoglin,acellsurfaceglycoproteinoverexpressedon

ovariancancerendothelialcells[66].ThescFvswereconjugatedtothesurfaceof

ironoxidenanoparticlespre-loadedwithβ-cyclodextrinencapsulateddocetaxel

tocreatedrug-loaded,endoglintargetedSPIONs.In-vitrostudiesshowedthatthe

scFvdirectedspecificbindingontoendoglinpositivecellsandalsothatthe

majorityoftheSPIONswerelocalisedwithinthemagneticfield[66].

ProgresshasalsobeenmadeusingFab-targetedNPs.Forexample,Fab

fragmentsderivedfromahumanisedanti-HER2mAbhavebeensuccessfully

usedtotargetpseudomonasexotoxinA(PE)encapsulatedwithinPLGANPs[80].

PEisapotentimmunotoxinwithsignificanttoxicity,whichhassofarlimitedits

useasananti-cancerdrugdespiteevidenceofimpressiveanti-tumouractivity

[89].Theresultsfromtheseexperimentsdemonstratedimprovedanti-tumour

activityandreducedsystemictoxicitywhencomparedtothenon-encapsulated

anti-HER2-PEAb-drugconjugate(ADC).

4.2DeliveryofradiationtreatmentRadiationistheprimarytreatmentmodalityinthemanagementofmany

cancers.Themainchallengeinradiotherapyishowtoachieveadequate

radiationexposuretothetumourwhilstavoidingdamagetosurrounding

healthyareas,especiallyinsensitivetissuessuchasthebrain.Ab-targetedNPs

haveshownpromiseinaddressingthischallengeforexamplebyfacilitating

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neutroncapturetherapy(NCT);atechniquewhichreliesontheintratumoural

injectionofanon-radioactiveisotope,mostcommonlyBoron-10(10B),which

actsasacaptureagentandreleaseslocalisedionisingradiation[90].In-vitro

studieshavetargeted10B-loadedliposomestoEGFR-expressinggliomacellsin-

vitro[91],raisingthepossibilitythatAb-targetedNPscouldovercomethe

barrierofdirectintratumouralinjectionofcaptureagents.Inasecondexample,

cetuximab-conjugatedSPIONswerefoundtosensitiseradioresistantgliomasto

theeffectsofradiation,possiblythroughproductionofreactiveoxygenspecies

andtheinhibitionofDNAdamagerepair[92].

4.3.Deliveryofnucleicacids

Ab-targetedNPdeliveryvectorssuchaspolymericNPs[93],AuNPs[94]and

liposomesarealsobeginningtofindaroleindeliveryofnucleicacidsforcancer

therapy.ThisprovidesopportunityforthedeliveryofDNAtoinduceexpression

oftherapeutictumoursuppressorgenes,ordeliveryofsmallinterferingRNA

(siRNA)oranti-senseDNAsequenceswhichcandisruptthetranslationof

oncogenes[95].EncapsulationofnucleicacidinNPsaimstoovercomebarriers

suchasphysiologicalinstability,poorintracellulardeliveryandoff-targeteffects

[96].Forexample,inonestudy,polyethyleniminewasusedtocondenseand

compactDNAencodingthep53gene[97]generatingpolymer-DNANPswhich

werethentargetedusingJ591,ananti-PSMAmAb.TheresultantJ591targeted

NPsshowedefficienttransfectioninvariousprostatecancercelllinesin-vitro

andeffectivePSMAspecifictargetingwhentestedin-vivoinprostatecancer

xenografts[97].J591isparticularlyfavourableforclinicaltranslationduetoits

previoususeinaphaseIIclinicaltrial[98].

Fordeliveryofanti-senseDNA,AuNPshavebeencoatedwithahighlyorganised

anti-sensenucleicacidlayerdesignedtobindHER2mRNA.Correspondingsense

DNAsequenceswerefunctionalisedviaclickchemistrytoanti-HER2Absand

hybridisedtotheanti-senseDNAontheNPsurface.TheseNPshadsuperior

uptakeintoHER2positivecellsin-vitrocomparedtonon-targetedcounterparts.

Inaddition,aconcentrationof1nManti-HER2NPswassufficienttocompletely

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blockexpressionofHER2intargetcellswhilstcontrolNPseithercontaining

scrambledanti-sensenucleicacidsorfunctionalisedusinganoff-targetAb

controlwereunabletoinhibitanyexpression[99].Asimilarapproachwas

undertakenusingclickchemistrytoconjugatechemicallystabilisedantisense

nucleicacidsandtrastuzumabtothesurfaceofpolymericmicelles.Thesedual

functionalisedpolymericNPsdemonstratedHER2targeteddeliveryandefficient

geneknockdowninHER2positivecellsin-vitrowhencomparedwithnon-

targeted,non-stabilisedcontrols[100].

Anotherexampleofefficientnucleicaciddeliveryutilisedaliposomal

formulationofantisensenucleicacidsagainstBCL2,anantiapoptoticgene

overexpressedinthemajorityofacuteleukaemias.Inthisstudy,theliposome

wastargetedusingtheanti-CD20mAbrituximab[101].Thetargetedliposomes

demonstratedeffectivereductionsofBcl-2proteinwithincancercells.Further

in-vivostudiesusinghumanBurkitt’slymphomaxenograftsshowedsignificant

reductionsintumourgrowth.Furthertothis,theliposomeswerefoundtobe

stableforover1yearinstoragewhetherasasuspensionorlyophilisedpowder,

highlightingfeasibilityforclinicaltranslation[101].

4.4.Antibody-conjugatednanoparticlesastheranosticagents:

NPsofferanattractiveplatformfordevelopmentofso-calledtheranosticagents,

whichcombinetherapyanddiagnosiswiththeaimofreducingtheneedfor

multi-stepproceduresandavoidingtreatmentdelays[102].Antibodytargeting

hasamajorroletoplayinthisfield(Figure4),andSPIONshavebecomethe

mostadvancedtheranosticNPsusingarangeofinnovativeapproaches.One

successfulexampleofAb-SPIONtheranosisusedthecommercialanti-EGFRmAb

cetuximab,conjugatedtoPEG-polymercoatedSPIONsthroughcarbodiimide

chemistry,totargetEGFRandthemutantvariantEGFRvIIIexpressingorthotopic

gliomatumours.ThemAb-functionalisedSPIONstriggeredapoptosisinhuman

gliomacellsin-vitroandconferredasignificantsurvivaladvantageoverboth

cetuximabandSPIONsalonewheninfusedintoorthotopichumanGBMmodels

[103].Intheseinnovativestudies,thechallengeofcrossingtheBBBtoachieve

18

adequateintratumouraldistributionwasaddressedusingconvection-enhanced

delivery(CED).PotentinhibitionofEGFRphosphorylationandincreasedlevels

ofcaspasecleavagewereobservedalongwithconsistentcontrastenhancement

oftheSPIONsonT2-weightedMR.Inadditiontothispromisingexperimental

data,toxicityexperimentsinhealthymiceshowednoevidenceofdamageto

normalbraintissues[103].

Targetingchemotherapyincombinationwithimagingagentsdemonstrates

anothertheranosticapproachthathasbeenexemplifiedusinghybrid

superparamagneticiron-platinumNPs(SIPPs)targetedtotheprostatecancer

targetPMSA.TheSIPPswereencapsulatedwithpaclitaxelinsidephospholipid

micellesandthenconjugatedusingstreptavidin/biotinadaptormoleculesto

anti-PMSAAbs.Theresultinghybrid-NPswereshowntotargetPMSApositive

prostatecells,inducecytotoxicitysimilartofreepaclitaxelin-vitroandfunction

asMRIcontrastagentsin-vivo[104].

Iron-oxidemagneticnano-crystals(MNCs)havealsobeeninvestigatedfor

theranosticpotential.TheseparticlesretaintheimagingabilitiesofSPIONsbut

aresmalleranddonothavesurfacecoatings.Assuch,theyhavetheadvantageof

beingreadilyincorporatedintothestructureoforganicpolymericNPsto

generatemultifunctionalNPs.Inoneexample,doxorubicinandMNCloaded

PLGApolymernanoparticleswereconjugatedviacarbodiimidecouplingtothe

anti-HER2mAbtrastuzumabformingNPswiththepotentialtobothdetectand

treatmalignantbreastcancercells.In-vitroresultswiththeseNPsdemonstrated

excellentefficacyasMRprobeswithspecificityforHER2positivecells.Aswellas

this,theNPsshowedsustainedreleaseofdoxorubicin[105].IronoxideMNCs

havealsobeensuccessfullyemployedastheimagingcomponentofdocetaxel

loadedPLGANPstargetedusingascFvagainstprostatestemcellantigen(PSCA).

PSCApositivecellsincubatedwiththetargetedNPsappeareddarkeronT2-

weightedMRimagingcomparedwithnon-targetedequivalents[106].Unusually

inthisstudy,drugreleaseshowedinitialrapiddesorptionofdrugfromthe

particlesurface,followedbymoresustainedreleaseduringdegradationofthe

polymermatrix.In-vitroefficacystudiesindicatedthatthedocetaxel-loadedNPs

19

wereeffectiveatalowerdosethanfreedocetaxel.PolymericPLGAnanoparticles

havealsobeenusedtoprovideavehiclefortheranosisbycombining

doxorubicinwithindocyaninegreen(ICG);aheat-generatingnear-infrared(NIR)

dye[107].Whenconjugatedtoanti-HER-2Absusingcarbodiimidechemistry,

theNPsdemonstratedtargetedcelluptake.Furthermore,NIRlaserexcitationof

theICGreleasedcytotoxicheatin-vitro.Theseresultsshowcaseasingle

theranosticNPwithtriplefunctionsin(i)imaging,(ii)deliveryofchemotherapy,

and(iii)inductionoflocalhyperthermia[107].

TheranosticAb-targetedlipidbasedNPshavealsobeencreatedusingquantum

dots(QDs)astheimagingcomponent,rapamycinfortherapyandtrastuzumab

fortargeting.TheresultingNPstargetedandimagedHER2positivecellsin-vitro,

showninboth2Dand3Dmodelsusingconfocalmicroscopy.Inthisstudy,lipid-

encapsulationaloneappearedtoimparta2foldincreaseineffectivenessover

freerapamycin,whilsttheadditionofaHER2targetingAbimpartedafurther5

foldincreaseinefficacyinHER2positivecells[108].

Multi-walledcarbonnanotubes(CNTs)offernewpotentialascancer

theranostics,duetotheirhighcarryingcapacityandhyperechogenicity

(increasedultrasoundcontrast).Preliminaryresultsinvestigatingthe

theranosticapplicationofCNTsconjugatedwithAbagainstPSCAhavebeen

encouraging.WhenfunctionalisedwithAbs,theCNTsshowedefficacyas

contrastagentsandenhanceduptakeintoPSCA-expressingcells.Moreover,after

loadingwithdoxorubicin,theAb-functionalisedCNTsaccumulatedwithin

tumourtissuesandinhibitedtumourgrowthin-vivo[109].

20

Figure4.Schematicillustratingthe4majorcomponentsofanAb-targetedtheranosticNP:(1)Deliveryvehicle,(2)Imagingagent,(3)Therapeuticcomponentand(4)TargetingAb.Figureadaptedfrom[110].4.5.CancerhyperthermiaAlmost50yearsagoitwasshownthattemperaturesofjust42°Ccouldinduce

cancercelldamagewithinarelativelyshorttimeframewhilstnon-malignant

cellswereabletowithstandthisriseintemperature[111].Morerecentwork

hasshownthatthehypoxictumourmicroenvironmentandtheincreasedmitotic

rateofmalignantcellspredisposemalignantcellstotemperaturesensitivity

[112],andthathyperthermiatreatmentshowssynergismwithconventional

therapies[113].Whilstpotentiallyaneffectivetreatment,themajorchallenge

fortherapeuticapplicationofhyperthermiaisrestrictingtreatmenttodiseased

areasandavoidingdamagetohealthytissues.Heat-generatingNPsofferan

elegantsolutiontothisproblem,particularlyiftheNPscanbelocalisedwithin

tumourspriortoheatinduction.Localisationhasbeenachievedbydirect

injectionintotumourtissue,butincreasinglyantibodyfunctionalisationisbeing

utilisedtotargetNPstothetissueofinterest(Table5),asillustratedwiththe

examplesbelow.

21

Nanoparticle Externalactivatingfield

Antibodytarget

Setting CancerTreated

Reference

SuperparamagneticIron-oxide

nanoparticles(SPIONs)

Alternatingmagneticfield(AMF)

MembraneProtein

In-vivo Breast [114,115]

GoldNanoparticles(AuNPs)

Laserlight EGFR In-vitro OralSquamousCellcarcinoma

[116]

Mucin-7 In-vitro UrothelialCells [117]Near-Infrared(NIR)light

TROP-2 In-vitro CervicalCancer [118]EGFR In-vitro OralSquamous

Cellcarcinoma[119]

Short-waveradiofrequency(RF)energy

EGFR In-vivo Pancreatic [120,121]

CarbonNanotubes(CNTs)

NIRlight CD133 In-vitro Glioblastoma [122]HER-2&IGF1R

In-vitro Breast [123]

CD22/CD25 In-vitro BurkittsLymphoma

[124]

Table4.Ab-targetedNPsemployedforcancerhyperthermia.

SPIONs:ArethemostclinicallyadvancedNPsforheattreatmentduetotheir

historyofuseasapprovedMRIcontrastagents[55]andinpioneeringclinical

trials(untargeted)forheattreatmentofglioblastoma[57].Whenexposedtoan

alternatingmagneticfield(AMF)SPIONsconvertmagneticenergytothermal

energythroughBrownianandNeelrelaxation.SuperparamagneticNPs,unlike

largerferri-orferromagneticNPs,donotretainmagnetismwhenthefieldis

removedandcangenerateheatatlowermagneticfieldamplitudes,makingthem

moreattractiveforbiomedicalapplications[125,126].

AntibodytargetingofSPIONsforhyperthermiawasfirstachievedbyconjugating

an111InradiolabelledmAbagainstanintegralmembraneglycoprotein(highly

expressedonanumberofhumancancers)to20nmdextran-coatediron-oxide

nanoparticles[115].FollowingintravenousinjectionoftheNPsintoinathymic

micebearingHBT3477(breast)xenografttumours,micewereexposedto

variouslevelsofexternalAMF.Electronmicrographstakentwodaysfollowing

AMFtherapyshowedtumourcellnecrosisatallstrengthsofAMFapplied.

22

TumoursdidnotrespondtoeitherSPIONsorAMFalone,andtoxicitywasonly

seenatthehigheststrengthofAMFapplied.Subsequentstatisticallyvalidated

resultsshowedthattherapeuticresponsesweregeneratedwithoutnormal

tissuetoxicityandthattheheatingdosecorrelatedwellwithresponse[114].

Despitein-vivosuccess,subsequentstudieshavemetwithchallengesduetothe

rapidclearanceofSPIONsfromcirculationviathereticuloendothelialsystem

(RES)wheninjectedintravenously[127].Whilstthischaracteristiclendsitself

welltoSPIONuseascontrastagents,itisdetrimentaltoachievingsufficient

intratumouralSPIONconcentrationstogeneratetherapeuticheating.Anumber

ofsolutionshavebeenproposedtoovercomethis,includingtheuseofblocking

agentstoprolongSPIONcirculatorytime[128],tailoringtheNPsurface

chemistriesorthetargetingmoietiestooptimisecellularinteractionsofSPIONs

[41,129]ordirectintratumouralinjectionwithtargetingantibodiesfunctioning

toretainSPIONsattheinjectionsite.

AuNPs:Ab-targetedAuNPshavealsobeenexploitedasvectorsforphotothermal

therapy(PTT).Thisprocessreliesuponthegenerationofvibrationalheatenergy

followingtheexcitationofphotosensitisersthroughtheabsorptionofspecific

wavelengthsoflight;mostcommonlyinthenear-infrared(NIR)range.AuNPs

stronglyabsorbandscatterlight;thisabsorptioncanbetunedbymodifications

inthesizeandshapeoftheNPsinadditiontotheincorporationofother

materialssuchassilica[116,119].ThelightabsorbedbyAuNPsconvertsreadily

andrapidlyintoheatandthis,alongwiththeestablishedbiocompatibilitymake

AuNPsattractivephotothermalagents[116].ToavoidaccumulationofAuNPsin

healthytissues,anumberofpassiveandactivetargetingstrategieshavebeen

testedtotargetthemtotumourcells.ThesestrategiesincludePEGylation,

liposomeencapsulationandantibodyconjugation[130].Thiol-terminatedPEG

derivativesarecommonlyusedtocoatthesurfaceofAuNPsactingtoimprove

colloidalstability,avoidtheRESuptakeofAuNPsandprovidea‘linker’to

conjugateAuNPstobiomoleculessuchasantibodies[131].RecentAb-targets

investigatedincludetrophoblastcellsurfaceantigen2(TROP2),a

transmembraneglycoproteinoverexpressedinanumberofepithelialcancers

andassociatedwithpoorprognosisincervicalcancer[118].Inthisstudy,hollow

23

goldnanosphereswereconjugatedtoanti-TROP2mAbsusingathiol-terminated

PEGlinker,generatingfunctionalisedNPsabletoreducecellviability

significantlymorethannon-targetedcontrolsfollowingexposuretoNIRlaser

[118].Inanotherstudy,goldnanospheresweretargetedtourothelialcancer

cellsusingantibodiesagainstMucin-7;acommonlyusedtargetinbladdercancer

[117],andactivatedusinggreenlaserlight.

AswellasabsorbingNIRorvisiblelight,Ab-targetedAuNPsalsoreleaseheat

followingabsorptionofshort-waveradiofrequency(RF)energy;behavingas

targetedradiofrequencyablation(RFA)agents.Short-waveRFfieldshavethe

advantageofpenetratingdeeperintotissuesthanNIRlight.Whentheanti-EGFR

antibodycetuximabwasconjugatedto20nmgoldnanorods,selectivetargeting

wasseenin-vitroand,followingexposuretoshort-waveRFfields,necrotic

cellularinjurywasachieved;similartotheeffectsofinvasiveRFAintheclinic

[120].WhenthesameNPsweretestedin-vivo,significantcelldeathwas

observed36hoursfollowingtreatmentofEGFRamplifiedpancreaticxenograft

tumours,withnodamageinselectedhealthytissuesincludingtheliver[121].

CNTs:NIRactivatedAb-targetedCNTshavealsobeenemployedtoinducePTT

in-vitro[132].ExamplesoftargetsinvestigatedincludeHER2andIGF1R,to

targetCNTstobreastcancercells[123],CD22andCD25totargetBurkitts

lymphoma[124]andCD133totargetglioma-likestemcellsinthetreatmentof

glioblastoma[122].Whilsttheseearlystudiesshowpromise,CNTsaccumulate

intheliver,arenon-biodegradable,andshowpoorsolubilitywithatendencyto

aggregate.Thesechallengesoftoxicitylimitthecurrentclinicaltranslationof

CNTs,andarebeingaddressed[133].

5.0Conclusions

ThisreviewhasillustratedthewaysinwhichthediversityandversatilityofNPs

canbefurtherexploitedbyAbtargeting.Awiderangeofpre-clinicalresearch

hasdemonstratedthatAb-targetedNPscanbeusedtodeliverycytotoxic

chemotherapydirectlytocancercells,resultinginefficacioustreatmentwith

24

reducedsideeffectsprofilescomparedtofreedrugs.Breakthroughshavealso

beenmadeinutilisingAb-NPstoaugmentradiotherapyandaidincancergene

therapy.CancertheranosticshasevolvedinparalleltoNPs,andanumberof

innovativeapproacheshaveshowcasedsingleagents,whichcancombinecancer

imagingandtherapy.Finally,theuseofhyperthermiainthetreatmentofcancers

hasbeenmadepossiblethankstotheinherentphysicalpropertiesofSPIONs,

AuNPsandCNTs.

Targetedtherapyremainsattheforefrontoftranslationalcancerresearchand

significantprogresshasbeenmadeintheclinicaltranslationofantibody-

targetedNPbasedcancertreatment,includingphaseIclinicaltrialsusing

antibodytargeteddrug-loadedliposomes.Thesepioneeringstudieshavebuilt

uponprevioussuccessesdevelopingNPs,refiningbio-conjugationstrategiesand

optimisingthebiocompatibilityandbioavailabilityoftheseproducts.

6.0FuturePerspectivesFutureresearchonAb-NPswilldependuponreliableandreproduciblebio-

conjugationstrategiesthatcanbescaleduptogoodmanufacturingpractice

(GMP)standardforclinicaltranslation[129].OtherNPssuchasmesoporous

silicaNPs(MSNs)(Table1)couldbeutilisedwhichofferimpressivedrug

carryingcapacity,andcanbeequippedwithtargetingligands[134].Inaddition,

multifunctionalhybridNPscanbegeneratedforuseincombinationtherapy

and/ortheranostics.

Theroleofimmunotherapyinthetreatmentofcancercontinuestoevolve.

WhilsttheuseofAb-targetedNPsincancerimmunotherapyisearlyin

development[135],recentbreakthroughsincludetheuseofPLGANPs

conjugatedtobothprotein-MHCcomplexesandanti-CD28mAbstoactas

syntheticantigenpresentingvehicleswhenadministeredin-vitro[136].Inthe

future,Ab-targetedNPscouldbeusedtoprimetheimmunesystemnotonlyto

recognisetumourantigensbutalsotostimulateanti-tumourimmunity.Whilst

Ab-targetedNPsareonlyjustreachingtheclinicalsetting,theirdiversepotential

25

foruseincancerdiagnosticsandtherapeuticspredictsanimportantroleinthe

futureofcancertreatment.

6.0ExecutiveSummaryIntroduction

• Targetedcancertreatmentofferssignificantadvantagesoverconventionaltherapybutcurrenttargetedtreatmentsareoftenlimitedbysystemictoxicity

• Nanoparticles(NPs)possessuniquephysical,chemicalandbiologicalpropertiesthatcouldovercometheselimitationsandbeharnessedforuseincancermedicine

Nanoparticles• NPsarenaturallyoccurringormanufacturedparticulatematerialeitherunbound,asan

aggregateoragglomeratewithonedimensionbetween1and100nminsize.• NPspossessinterrelatedproperties;highmobilityinthefreestate,largesurfaceareato

volumeratios,andsometimestheexhibitionofquantumeffects.• ThewideanddiverserangeoffunctionalpropertiesexhibitedbyNPsispossiblethanks

totheutilityofavarietyofmaterialstosynthesisethem.Targetingnanoparticlesforcancertreatment

• PassiveaccumulationofNPsoccurswithinmalignanttissuesinaprocessknownasenhancedpermeabilityandretention(EPR).

• SpecifictargetingofNPstocancercellscanbeachievedthroughtheadditionofatargetingmoiety,includingproteins,smallmoleculesandaptamers.

• Antibodies(Abs)arethemostpromisingtargetingligandsandcanbereadilygeneratedasintactIgGmoleculesorasfragments(ScFvs)indefined,recombinantform.

• AntibodytargetingiscomplementarytoEPRandenablesspecificreceptormediatedinternalizationoftheNP.

Antibody-Nanoparticlebio-conjugationstrategies• Challengesinbio-conjugationcanbebroadlydividedintothreemaincategories;(i)

controllability,(ii)stability,and(iii)reliability.• Sophisticatedconjugationtechniquesenablethecontrolofantibodyorientationsto

ensuretargetbindingandpreserveAbbioactivity• Whilstbothbifunctionallinkermoleculesandadaptormoleculescangeneratefunctional

conjugates,covalentlinkermoleculesdemonstratesuperiorlongtermstability.• Establishedcovalentconjugationstrategiesincludecarbodiimidecoupling,maleimide

couplingandclickchemistry(coppercatalyzedalkyne-azidecycloadditionreactions).ClinicalApplicationsofNanoparticlesforCancerTherapy

• NPbaseddrug-deliverysystemssuccessfullytranslatedintoearlyphaseclinicaltrialsincludeliposomes,polymericNPs,dendrimers,micellesandgoldNPs(AuNPs).

• SuperparamagneticIron-oxideNPs(SPIONs)havebeenclinicallyapprovedforuseascontrastagentsincancerimagingandhavebeensuccessfulinearlyphasetrialsforcancerhyperthermia

• Antibody-targetedliposomes(immunoliposomes)havebeenusedinthreeearlyphaseclinicaltrials.

PreclinicalDevelopmentofantibody-targetednanoparticlesforcancertherapy• ChemotherapyloadedAb-NPscanofferimprovedintratumouraldrugdelivery,superior

efficacycomparedwithfreedrugs,andcontrolledreleaseofdrugswithintumourcells.• Targetingpastthebloodbrainbarrier(BBB)totargetcentralnervoussystem(CNS)

tumoursmaybepossiblethroughdualtargetingoftransferrinreceptorandcancerspecifictargets.

• Ab-targetedpolymericNPs,AuNPsandliposomescanbeusedtostabilisenucleicacidsanddeliverthemdirectlytocancercellsforgenetherapy.

• SPIONsbecombinedwithotherNPsincludingpolymersandquantumdotstoproducemulti-functionaltheranosticNPsthatcanimagetumoursanddeliverycytotoxicdrugsincombination

• SPIONs,AuNPsandCNTsareabletogenerateheatfromwithinthetissueofinterestwhenactivatedbyexternallyappliedalternatingmagneticfields(SPIONs)ornear-infraredfields(AuNPs/CNTs).

26

• SPIONshavebeentargetedusingAbsinthepre-clinicalsettingwithsuccesslimitedbyrapiduptakebythereticuloendothelialsystemandclearancein-vivo.Strategiesarebeinginvestigatedtoreducethisuptake.

Conclusions• Ab-targetedNPsofferaversatileplatformforthedevelopmentofthenextgenerationof

cancertherapeutics• Progresshasbeenmadeintheclinicaltranslationofantibody-targetedNPbasedcancer

treatmentsFuturePerspectives

• Reliableandreproduciblebio-conjugationstrategiesthatcanbescaleduptogoodmanufacturingpractice(GMP)standardwillfacilitateclinicaltranslationofAb-targetedNPs.

• Ab-targetedNPsmayhaveafutureroleincancerimmunotherapy,includinginthedevelopmentofsyntheticantigen-presentingvehicles.

Financial&competinginterestsdisclosureTheauthorsaregratefulforthesupportreceivedfromtheSeventhFrameworkProgramme(FP7)forDARPintargetedmagnetichyperthermictherapyforglioblastoma(DartrixECGRANT:278580),andfromtheDepartmentofHealthandCancerResearchUKExperimentalCancerMedicineCentre(ECMC).Theauthorshavenootherrelevantaffiliationsoffinancialinvolvementwithanyorganizationorentitywithfinancialinterestinorfinancialconflictwiththesubjectmatterormaterialsdiscussedinthemanuscriptapartfromthosedisclosed.Thefundershadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscriptNowritingassistancewasutilisedintheproductionofthismanuscript.Bibliography1. JarboeJ,GuptaA,SaifW.Therapeutichumanmonoclonalantibodies

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