similarwww.147ttt.com

REVIEWCurrentprogressininnovativeengineeredantibodiesWilliamR.
Strohl&BiStroBiotechConsulting,Bridgewater,NJ08807,USA&Correspondence:wrstrohl@gmail.
com(W.
R.
Strohl)ReceivedMay12,2017AcceptedJuly8,2017ABSTRACTAsofMay1,2017,74antibody-basedmoleculeshavebeenapprovedbyaregulatoryauthorityinamajormarket.
Additionally,thereare70and575antibody-basedmoleculesinphaseIIIandphaseI/IIclinicaltrials,respectively.
Thesetotal719antibody-basedclinicalstagemoleculesinclude493nakedIgGs,87antibody-drugconjugates,61bispecicantibodies,37totalFcfusionproteins,17radioimmunoglobulins,13antibodyfragments,and11immunocytokines.
Newusesfortheseantibodiesarebeingdiscoveredeachyear.
Foroncol-ogy,manyoftheexcitingnewapproachesinvolveanti-bodymodulationofT-cells.
Thereareover80antibodiesinclinicaltrialstargetingTcellcheckpoints,26T-cell-redirectedbispecicantibodies,and145chimericanti-genreceptor(CAR)cell-basedcandidates(allcurrentlyinphaseIorIIclinicaltrials),totalingmorethan250Tcellinteractingclinicalstageantibody-basedcandi-dates.
Finally,signicantprogresshasbeenmaderecentlyonroutesofdelivery,includingdeliveryofproteinsacrosstheblood-brainbarrier,oraldeliverytothegut,deliverytothecellularcytosol,andgene-andviral-baseddeliveryofantibodies.
Thus,therearecur-rentlyatleast864antibody-basedclinicalstagemole-culesorcells,withincrediblediversityinhowtheyareconstructedandwhatactivitiestheyimpart.
Thesearefollowedbyanextwaveofnovelmolecules,approa-ches,andnewmethodsandroutesofdelivery,demon-stratingthattheeldofantibody-basedbiologicsisveryinnovativeanddiverseinitsapproachestofullltheirpromisetotreatunmetmedicalneeds.
KEYWORDSantibodyclinicalcandidates,engineeredantibodies,chimericantigenreceptorsINTRODUCTIONThisyear,2017,marksthe20thanniversaryoftheapprovalbytheUnitedStatesFoodandDrugAdministration(USFDA)ofRituxan(rituximab)andZenapax(daclizumab),fortreatmentofBcellmalignanciesandforusetosuppressorganrejectioninrenaltransplants,respectively(Table1).
WhiletwoantibodieshadpreviouslybeenapprovedbytheFDA(Table1),theapprovalofRituxanandZenapaxin1997wasawatershedmomentinthehistoryofmonoclonalantibody(mAb)therapeutics.
Thereasonsareverydifferentforeachmolecule.
Rituxanhasbecomebothahugemedicalandcommercialsuccess,withindicationsinBcellmalignanciesaswellasinthetreatmentofrheumatoidarthritis(RA)(Storz,2014).
Rituxaniscurrentlythefourthbest-sellinginnovativedrugofanykindwith2016worldwidesalesof$8,354MM(Table2),about85%ofthosesalescomingincancerindicationsandtheother15%fromsalesfortreatmentofRA(LaMeriePublishing,2017).
IncludingRituxan,sevenofthetoptensellinginnovativedrugsintheworldin2016wereproteins,sixofwhichwereantibody-relatedmolecules(Table2).
Zenapax,ontheotherhand,wasthersthumanizedantibodytobeFDAapprovedbutitneverachievedsignicantcommercialsuccessandwaseventuallywithdrawnfromthemarketin2009.
Daclizumab,however,hasbeenapprovedrecentlyunderthetradenameZinbrytafortreatmentofrelapsingformsofmultiplescle-rosis(MS).
Todate,74unique,innovativeantibodiesandFcfusionproteinshavebeenapprovedfortreatmentofdiseasesinatleastonemajormarket(i.
e.
,US,EU,Japan)(Table1).
Ofthese,sevenhavebeenwithdrawnfrommarketingeitherduetolackofefcacy,poortoxicitytoefcacyproles,orlackofmarketinterest(Table1).
Ofthe74approvedanti-body-basedmolecules,vecontaincompletelymurineTheAuthor(s)2017.
ThisarticleisanopenaccesspublicationProteinCellDOI10.
1007/s13238-017-0457-8Protein&CellProtein&CellTable1.
InnovativemonoclonalantibodiesandfusionproteinsapprovedformarketinginEuropeanUnion,UnitedStates,orJapan*UStradename(Genericname)CompanyApprovaldate(US)**Moleculartarget***Majorindication(s)ProteinformatSourceofvariablesequences**1.
OrthocloneOKT3(Muromonab-CD3)OrthoBiotech(J&J)06/19/86;withdrawn2011CD3E(CD3)OTRMurineIgG2aMousehybridoma2.
ReoPro(Abciximab)Centocor(nowJ&J)/Lilly12/22/94ITGA2B/ITGB3(gPIIb/IIIa)CVDChimericFAbfragmentMousehybridoma3.
Rituxan(Rituximab)Biogen/Idec/Genentech11/26/97MS4A1(CD20)NHL,RAChimericIgG1Mousehybridoma4.
Zenapax(Daclizumab)Biogen/Abbott(PDL/Roche)12/10/97;withdrawn2009IL2RA(IL-2Rα;CD25)OTRHumanizedIgG1Mousehybridoma5.
Remicade(Iniximab)Centocor(nowJ&J)8/24/98TNF(TNF-α)CRD,RAChimericIgG1Mousehybridoma6.
Synagis(Palivizumab)MedImmune06/19/98RSVF-proteinRSVinfectionHumanizedIgG1Mousehybridoma7.
Herceptin(Trastuzumab)Genentech09/25/98ERBB2(HER2)BreastcancerHumanizedIgG1Mousehybridoma8.
Enbrel(Etanercept)Immunex(nowAmgen)11/02/98TNF(TNF-α)RAP75-TNFR-FcfusionFcfusion9.
Simulect(Basiliximab)Novartis12/05/98IL2RA(IL-2Rα;CD25)OTRChimericIgG1Mousehybridoma10.
Mylotarg(Gemtuzumabozogamicin)Wyeth(nowPzer)05/17/00;withdrawn2010CD33LeukemiaHumanizedIgG4-ADC****Mousehybridoma11.
Campath-1H(Alemtuzumab)Genzyme05/07/01;withdrawn2012CD52LeukemiaHumanizedIgG1Rathybridoma12.
Zevalin(Ibritumomabtiuxetan)Biogen/Idec02/19/2002MS4A1(CD20)NHLMurineIgG1radio-conjugate(Y-90,In-111)Mousehybridoma13.
Humira(Adalimumab)CAT,Abbott12/31/02TNF(TNF-α)RA,CRDHumanIgG1Humanantibodyphagelibrary14.
Amevive(Alefacept)Biogen01/30/03CD2PsoriasisCD58(LFA-3)-FcfusionFcfusion15.
Xolair(Omalizumab)Genentech06/20/03IGES(IgE)AsthmaHumanizedIgG1Mousehybridoma16.
Bexxar(Tositumomab-I131)Corixa06/27/03;withdrawn2014MS4A1(CD20)NHLMurineIgG2aradio-conjugate(I-131)MousehybridomaREVIEWWilliamR.
StrohlTheAuthor(s)2017.
ThisarticleisanopenaccesspublicationProtein&CellTable1continuedUStradename(Genericname)CompanyApprovaldate(US)**Moleculartarget***Majorindication(s)ProteinformatSourceofvariablesequences**17.
Raptiva(Efalizumab)Genentech10/27/03;withdrawn2009ITGAL(CD11A)PsoriasisHumanizedIgG1Mousehybridoma18.
Erbitux(Cetuximab)ImClone/BMS02/12/04EGFRCRCChimericIgG1Mousehybridoma19.
Avastin(Bevacizumab)Genentech02/26/04VEGFACRCHumanizedIgG1Mousehybridoma20.
Tysabri(Natalizumab)Biogen/Elan11/23/04ITGA4(α4integrin)MSHumanizedIgG4Hybridoma21.
Orencia(Abatacept)BMS12/23/05CD80/CD86RACTLA4-FcfusionFcfusion22.
Lucentis(Ranibizumab)GenentechNovartis06/30/06VEGFAWetAMDHumanizedFabfragmentHybridoma23.
Vectibix(Panitumumab)Amgen09/27/06EGFRColorectalcancerHumanIgG2TGXenomouse24.
Soliris(Eculizumab)AlexionPharma03/16/07C5PNHHumanizedhybridengineeredIgG2/4Mousehybridoma25.
Arcalyst(Rilonacept)Regeneron02/27/08IL1A(IL-1α),IL1B(IL-1β),IL1RN(IL-1RA)CAPS,MWSIL-1R&IL-1AP-in-lineFcfusionFcfusion26.
Nplate(Romiplostim)Amgen08/22/08MPL(TPO-R)Thrombo-cytopeniaFc-peptidefusion("peptibody")Peptidephagelibrary27.
Simponi(Golimumab)Centocor/J&J04/23/09TNF(TNF-α)RAHumanIgG1HuMAbTGmouse28.
Stelara(Ustikinumab)Centocor/J&J09/25/09IL12B(p40subunitofIL-12andIL-23)PsoriasisHumanIgG1HuMAbTGmouse29.
Removab(Catumaxomab)Fresenius/TrionEUonly4/23/09;withdrawn2017EPCAM,CD3EMalignantascitesRatIgG2b-mouseIgG2ahybridbispecicIgGMouseandrathybridomas30.
Cimzia(Certolizumabpegol)UCB/Schwartz05/14/09TNF(TNF-α)RAPEGylatedhumanizedFAbfragmentMousehybridoma31.
Ilaris(Canakinumab)Novartis06/19/09IL1B(IL-1β)CAPSHumanIgG1HuMAbTGmouse32.
Arzerra(Ofatumumab)GenMab/Novartis#10/26/09MS4A1(CD20)CLLHumanIgG1HuMAbTGmouse30.
Actemra(Tocilizumab)Roche/Chugai/Genentech01/09/10IL6R(CD126)MCD;RAHumanizedIgG1Hybridoma31Prolia/Xgeva(Denosumab)Amgen/GSK06/01/10TNFSF11(RANK-ligand)Osteoporosis,BonecancerHumanIgG2TGXenomouseInnovativeantibodiesREVIEWTheAuthor(s)2017.
ThisarticleisanopenaccesspublicationProtein&CellTable1continuedUStradename(Genericname)CompanyApprovaldate(US)**Moleculartarget***Majorindication(s)ProteinformatSourceofvariablesequences**35.
Benlysta(Belimumab)GSK/HGS03/09/11TNFSF13B(solubleBLyS)SLEHumanIgG1Humanantibodyphagelibrary36.
Yervoy(Ipilimumab)Medarex/BMS03/25/11CTLA4MelanomaHumanIgG1HuMAbTGmouse37.
Nuloji(Belatacept)BMS06/16/11CD80/CD86OTRCTLA-4FcfusionFcfusion38.
ADCETRIS(Brentuximabvedotin)SeattleGenetics/Takeda/Millenium08/19/11TNFRSF8(CD30)Hodgkin'slymphomaChimericIgG1ADC****Mousehybridoma39a.
EYLEA(aibercept)Bayer-Schering/Regeneron11/18/11VEGFAWetAMDVEGF-R-FcfusionFcfusion40.
POTELIGEO(Mogamulizumab)KyowaHakkoKirinJapanonly03/30/12CCR4ATLHumanizedIgG1-AfucosylatedglycanMousehybridoma41.
Perjeta(Pertuzumab)Genentech06/08/12ERBB2(HER2)BreastcancerHumanizedIgG1Mousehybridoma(39b).
ZALTRAP(ziv-aibercept)Sano/Regeneron08/03/12VEGFAMCRCVEGFR-FcfusionproteinTrapFcfusion42.
Abthrax(Raxibacumab)GSK;HumanGenomeSciences12/14/12BacillusanthracisPAtoxinAnthraxbiodefenseHumanIgG1Humanantibodyphagelibrary43.
Kadcyla(trastuzumabemtansine)Roche/Genentech02/23/13ERBB2(HER2)BreastcancerHumanizedIgGADC****Mousehybridoma44.
Gazyva(obinutuzumab)Roche/Genentech/Biogen11/01/13MS4A1(CD20)CLLHumanizedIgG1-lowfucoseMousehybridoma45.
Alprolix(Eftrenonacogalfa)Biogen-IDEC/Biovitrum03/28/14FactorsubstituteHemophiliaBMonomericFactorIXFcusionproteinFcfusion46.
Cyramza(Ramucirumab)Lilly/Dyax04/22/14KDR(VEGFR-2)GastriccancerHumanIgG1Humanantibodyphagelibrary47.
Sylvant(Siltuximab)JanssenR&D/J&J04/23/14IL6MCDChimericIgG1Mousehybridoma48.
Entyvio(vedolizumab)Takeda/Millenium05/20/14ITGA4/ITGB7(α4β7integrin)CRDHumanizedIgG1Mousehybridoma49.
Eloctate(Efmoroctocogalfa)BiogenIdec/SOBI06/06/14FactorsubstituteHemophiliaAMonomericFcdomain-deletedF-VIIIfusionFcfusion50.
Keytruda(pembrolizumab)Merck09/04/14PDCD1(PD-1)MelanomaHumanizedIgG4Mousehybridoma51.
Trulicity(dulaglutide)EliLilly09/18/14GLP1R(agonist)Type2diabetesGLP-1–FcfusionFcfusionREVIEWWilliamR.
StrohlTheAuthor(s)2017.
ThisarticleisanopenaccesspublicationProtein&CellTable1continuedUStradename(Genericname)CompanyApprovaldate(US)**Moleculartarget***Majorindication(s)ProteinformatSourceofvariablesequences**(11).
Lemtrada(alemtuzumab)Genzyme(Sanosubsidiary)11/14/14CD52MSHumanizedIgG1Rathybridoma52.
Blincyto(blinatumomab)Amgen(Micromet)12/03/14CD19,CD3EB-cellALLBiTEMousehybridoma53.
Opdivo(nivolumab)BMS12/22/14PDCD1(PD-1)MelanomaHumanIgG4HuMAbTGmouse54.
Cosentyx(secukinumab)Novartis01/21/15IL17APlaquepsoriasisHumanIgG1HuMAbTGmouse55.
Unituxin(dinutuximab)UnitedTechnologies/NCI03/10/15GD2NeuroblastomaChimericIgG1Mouse56.
Praluent(alirocumab)Sano/Regeneron07/24/15PCSK9HighcholesterolHumanIgG1VelocImmuneTGmouse57.
Repatha(evolocumab)Amgen(AstellasinJapan)08/27/15PCSK9HighcholesterolHumanIgG1TGXenomouse58.
Praxbind(idarucizumab)BoerhingerIngelheim10/16/15DabigatranDrugReversalHumanizedFabfragmentMousehybridoma59.
Strensiq(Asfotasealfa)Alexion(fromEnobia)10/23/15FactorsubstituteHypophos-phatasiaTNSALP-Fcfusion-peptideFcfusion60.
Nucala(Mepolizumab)GSK11/06/15IL5COPDHumanizedIgG1Mousehybridoma61.
Darzalex(daratumumab)JanssenR&D(J&J)/Genmab11/16/15CD38MMHumanIgG1HuMAbTGmouse62.
Portrazza(necitumumab)Lilly/ImClone/Dyax11/24/15EGFRSquamousNSCLCHumanIgG1Humanantibodyphagelibrary63.
Empliciti(elotuzumab)BMS/Abbvie(fromPDL)11/30/15SLAMF7MMHumanizedIgGMousehybridoma64.
Anthim(obiltoxaximab)ElusysTherapeutics03/21/16BacillusanthracisPAtoxinAnthrax-biodefenseChimericIgGMousehybridoma65.
Taltz(Ixekizumab)EliLilly03/22/16IL17APsoriasis;PsAHumanizedIgG4Mousehybridoma66.
Cinqair(Reslizumab)TevaCeption/Cephalon03/23/16IL5EosinophilicasthmaHumanizedIgG4Rathybridoma67.
Tecentriq(Atezolizumab)Roche/Genentech05/18/16CD274(PD-L1,B7-H1)BladdercancerHumanizedIgG1Mousehybridoma(4).
Zinbryta(Daclizumab)Biogen/Abbott(PDL/Roche)May2016IL2RA(IL-2Rα;CD25)RR-MSHumanizedIgG1MousehybridomaInnovativeantibodiesREVIEWTheAuthor(s)2017.
ThisarticleisanopenaccesspublicationProtein&CellTable1continuedUStradename(Genericname)CompanyApprovaldate(US)**Moleculartarget***Majorindication(s)ProteinformatSourceofvariablesequences**68.
Lartruvo(Olaratumab)Lilly/ImClone10/19/16PDGFRASofttissuesarcomaHumanIgG1UltimAbTGmouse69.
Zinplava(Bezlotoxumab)Medarex/MBL/Merck10/22/16ClostridiumdifcileBtoxinCDADHumanIgG1HuMAbTGmouse70.
Siliq(Brodalumab)Valeant/AstraZeneca02/15/17IL17RAPsoriasisHumanIgGTGXenomouse71.
Bavencio(Avelumab)Pzer/MerckKGaA(EMDSerono)/Dyax3/23/17CD274(PD-L1,B7-H1)MerkelcellcarcinomaHumanIgG1Humanantibodyphagelibrary72.
Dupixent(Dupilumab)Regeneron/Sano3/28/17IL4RAtopicdermatitisHumanIgG4S/PVelocImmuneTGmouse73.
Ocrevus(Ocrelizumab)Roche/Biogen3/28/17MS4A1(CD20)Primary,progressingMSHumanizedIgG1Mousehybridoma74.
Imnzi(Durvalumab)AstraZeneca(MedImmune)/Celgene5/1/17CD274(PD-L1,B7-H1)MetastaticurothelialcarcinomaHumanIgG1TGXenomouseAbbreviations:ADC,antibody-drugconjugate;AMD,Age-relatedmaculardegeneration;ATL,adultT-cellleukemia/lymphoma;BiTE,bispecicTcellengager;BlyS,Blymphocytestimulator;C5,complementcomponentC5;CAPS,Cropyrin-associatedperiodicsyndrome;CCR4,C-Cmotifreceptor-4;CD,clusterofdifferentiation;CDAD,Clostridiumdifcile-associateddisease;CLL,chroniclymphocyticleukemia;COPD,chronicobstructivepulmonarydisease;CRC,colorectalcancer;CRD,Crohn'sDisease;CTLA4,cytotoxicT-lymphocyteassociatedprotein-4;CVD,cardiovasculardisease;EGFR,epidermalgrowthfactorreceptor;ERBB2,erb-b2receptortyrosinekinase2;F-VIII,FactorVIII;Fab,fragment,antigen-binding;Fc,fragment,crystallizable;GD2,disialoganglioside-2;GLP-1R,glucagon-likepeptide-1receptor;I-131,Iodine-131(radioactive);HER2,humanepidermalgrowthfactorreceptor-2;Ig,immunoglobulin;IL,interleukin;KDR,kinaseinsertdomainreceptor;LFA,lymphocyte-associatedantigen;MCD,multicentricCastleman'sdisease;MCRC,metastaticcolorectalcancer;MM,multiplemyeloma;MPL,myeloproliferativeleukemiavirusoncogene;MS,multiplesclerosis;MWS,Muckle-Wellssyndrome;ND,notdisclosed;NHL,non-Hodgkinlymphoma;NSCLC,non-smallcelllungcancer;OTR,organtransplantrejection;PA,protectiveantigen;PCSK9,Proproteinconvertasesubtilisin/kexintype9;PDCD1,programmedcelldeath1;PDGFR,platelet-derivedgrowthfactorreceptor;PD-L1,programmedcelldeathproteinligand-1;PEG,poly-ethylene-glycol;PNH,paroxysmalnocturnalhemoglobinuria;PsA,psoriaticarthritis;RA,rheumatoidarthritis;RANK,receptoractivatorofnuclearfactorkappa-B;RR-MS,relapsing-remittingmultiplesclerosis;RSV,respiratorysyncytialvirus;SC,subcutaneous;SLAMF7,signalinglymphocyticactivationmoleculefamilymember7;SLE,systemiclupuserythematosus;S/P,mutationsinhingeofIgG4;TG,transgenic(humanized);TNALP,tissue-nonspecicalkalinephosphatase;TNF,tissuenecrosisfactor;TPO-R,thrombopoietinreceptor;VEGF,vascularendothelialgrowthfactor.
*DataobtainedfromPrescribingInformationreleasedbythemanufacturers,Companywebsites,AdisInsights,andBiStroBiotechConsultingdatabaseonclinicalstagebiologics.
**USFDAapprovaldatesunlessotherwisestated.
***NamesgivenasHUGOGeneNomenclatureCommittee(HGNC)names(Grayetal.
,2015)followedbycommonlyusednamesinparentheses.
****Conjugates:Mylotarg,calicheamicin;Adcetris,monomethylauristatinE(MMAE);Kadcyla,maytansanoidDM-1.
#Currentlynotbeingmarketed;clinicaltrialsinMSsuggestaprobablerelaunchinanewtherapeuticareasoon.
REVIEWWilliamR.
StrohlTheAuthor(s)2017.
ThisarticleisanopenaccesspublicationProtein&CellTable2.
Toptenbest-sellinginnovativedrugsworldwidein2016*#Drug(genericname)ClassMoleculartarget**CompanyPrimaryindications(abbreviated)2015worldwidesales2016worldwidesalesPercentchange1Humira(adalimumab)mAbTNF(TNF-α)AbbvieRA,psoriasis,IBD,others$14,012M$16,078M+14.
7%2Harvoni(ledipasvir/sofosbuvir)SMHCVNS5Bpolymerase,NS5AGileadSciencesHCVinfection$13,864M$9,081M34.
5%3Enbrel(etanercept)FcfusionTNF(TNF-α)Amgen,PzerRA,psoriasis,others$8,697M$8,874M+2.
0%4Rituxan(rituximab)mAbMS4A1(CD20)Roche,BiogenBcellmalignancies,RA$8,354M$8,583M+2.
7%5Remicade(iniximab)mAbTNF(TNF-α)J&J,MerckRA,psoriasis,IBD,others$8,760M$6,561M10.
6%6Revlimid(lenalidomide)SMCRBN(E3ligasecereblon),IKZF1,IKZF3CelgeneMultiplemyeloma$5,801M$6,974M+20.
2%7Avastin(bevacizumab)mAbVEGFRocheMCRC,MRCC,others$6,654M$6,752M+1.
5%8Herceptin(trastuzumab)mAbERBB2(HER2)RocheHER2+breastcancer,gastriccancer,others$6,509M$6,751M+3.
7%9Lantus(insulinglargine)ProteinINSR(insulinreceptor)SanoT1D,T2D$6,770M$6,054M10.
6%10Prevnar13(pneumococcal13-valentconjugatevaccine;CRM197)Vaccine(conjugated)PneumococcalpolysaccharidesPzerPneumoniaprophylaxis$6,245M$5,718M8.
4%Abbreviations:CRM197,non-toxicmutantformofdiphtheriatoxin;mAb,monoclonalantibody;SM,smallmolecule;J&J,Johnson&Johnson;RA,rheumatoidarthritis;IBD,intestinalboweldisease;HCV,hepatitisCvirus;MCRC,metastaticcolorectalcancer;MRCC,metastaticrenalcellcarcinoma;HER2,humanepidermalgrowthfactorreceptor-2;T1D,type1diabetes;T2D,type2diabetes.
*DataabstractedfromLaMerie,2017.
**NamesgivenasHUGOGeneNomenclatureCommittee(HGNC)names(Grayetal.
,2015)followedbycommonlyusednamesinparentheses.
InnovativeantibodiesREVIEWTheAuthor(s)2017.
ThisarticleisanopenaccesspublicationProtein&Cellsequences,ninearemouse-humanchimericantibodies,26arehumanized,23arehumanantibodies,and11areFcfusions(Table1).
Ofthe23fullyhumanantibodies,17arederivedfromtransgenic"humanized"miceandsixarederivedfromhumanantibodyphagedisplaylibraries(Table1).
EightoftheFcfusionsareFc-proteinfusions,twoareFc-peptidefusions,andoneisanFc-proteinfusionwithatissue-targetingpeptidefusedtoit.
Currently,thereare70phaseIIIclinicalstagecandidates,aswellas575knownphaseIorphaseIIantibody-basedclinicalcandidates(Table3).
Thus,asofMay1,2017,thereareatleast719knownantibodyandFcfusionproteinclin-ical-stagecandidates(Table3).
Ofthese,493are"naked"IgGs,13are"naked"antibodyfragments(inbothcases,"naked"referstoantibodiesthatarenotantibody-drugcon-jugates[ADCs],bispecicantibodies,radioimmunothera-peutics,orimmunocytokines),87areADCs,61arebispecicantibodies,37areFcfusionproteins,17areconjugatedwithradioisotopeseitherastherapeuticsorimagingagents,and11areimmunocytokines(Table3andFig.
1).
Itisnotablethat,withtheexceptionofFcfusionproteins,mostofthenon-"naked"antibodiesareskewedtowardsthephaseI/IIclinicalstages,likelyduetothemorerecentdevelopmentofthevariousinnovativetechnologiesincorporatedintothosemolecules(Table3).
Inadditiontotheseproteinantibody-derivedclinicalstagemolecules,thereare145documentedphaseIorIIclinicalstagechimericantigenreceptor(CAR)-Tcellornaturalkiller(NK)cellcandidatesthatincorporateantibodiesastheirCARs(Table3andFig.
1).
Thus,thereareatleast864proteinandcellbasedantibody-derivedconstructseitherapprovedformedicaluseorbeingevaluatedfortheirsafetyandefcacyinclinicaltrials.
ANTIBODYTARGETSThe864uniqueantibody-basedmolecules/cellsindevel-opmentorapprovedfortherapeuticuse(Table3)target328uniqueantigens(Table4).
Becauseseveraltargetsareimportantformultiplediseaseareas(e.
g.
,vascularendothelialgrowthfactor[VEGF]asasignicanttargetinbothoncologyandophthalmologyindications),therearemoreuseslistedthanantibodies.
Thus,864uniquemole-culesareusedin884differentmajortherapeuticareaindi-cations(Table4),andthe328uniquetargetsaredistributedamongst351majoruses(Table5).
About62%oftheseproteinandrecombinantcell-basedcandidatesaredirectedagainsttargetsinoncology(Table4).
Notsurprisingly,all145ofthecurrentCAR-TandCAR-NKcandidatesareinclinicalevaluationforcancerTable3.
Currentstatusofinnovativeantibody,Fcfusionprotein,andchimericantigenreceptor(CAR)drugcandidates*AntibodyformatStageofdevelopmentTotalsPhaseI/IIPhaseIIIApprovedformarketingatsomepoint**NakedIgG305152493Nakedantibodyfragments72413Immunocytokines92011Fcfusionproteins2331137Bispecicantibodies581261IgG-like(41)(1)(1)(43)Fragment-based(14)(0)(1)(15)Nanoparticle***(03)(0)(0)(03)Antibody-drugconjugates#759387Radioimmunoglobulins132217Antibodiesonly5757074719TorNKcellsexpressingCARantibodies14500145Totals7207074864Abbreviations:IgG,immunoglobulinG;CAR,chimericantigenreceptor.
*FromBiStroBiotechConsultingdatabaseonclinicalstagebiologics.
DatabaselockforthesedatawasApril30,2017.
**InnovativeantibodiesandFcfusionproteinsapprovedformarketinginamajormarket(US,EU,Japan).
Five(Raptiva,2009;Mylotarg,2010;OrthocloneOKT3,2011;Bexxar,2014;Removab,2017)havebeenwithdrawnfrommarketing,andtwootherswerewithdrawnandsubsequentlywerere-approvedfornewindicationsunderdifferenttradenames.
***BispecicEGFRxEscherichiacoliO-polysaccharidetandemsinglechain,Fragmentvariable(scFv)antibodiesthattargetminicell-derivednanoparticlestotumors.
#The87antibody-drugconjugatesarecomprisedof68smallmoleculecytotoxicdrugs,10proteins,and9notdescribed.
REVIEWWilliamR.
StrohlTheAuthor(s)2017.
ThisarticleisanopenaccesspublicationProtein&Cellindications.
Thereare,however,preclinicaleffortstogener-ateCAR-Tcellsagainstvirusesandvirus-infectedcelltar-gets(Sahuetal.
,2013;Liuetal.
,2015;Haleetal.
,2017),sothismaychangeinthenearfuture.
Another19%oftheclinicalcandidatesaredirectedagainsttargetsintheimmunologytherapeuticarea(includingautoimmuneandasthma,butexcludingMS)(Table4).
Theremainingca.
19%ofantibody-basedproteinsaredividedamongstotherther-apeuticareas,includingcardiovascularandmetabolism,neurobiology,boneandmuscledisorders,blooddisorders,andinfectiousdiseases.
Ofthe351differentusesfortargets,222(63%)aresingle-passmembraneboundproteinsorcell-boundproteins(e.
g.
,ERBB2[erb-b2receptortyrosinekinase2;akaHer2],EGFR[epithelialgrowthfactorreceptor],ERBB3[erb-b3receptortyrosinekinase3;akaHer3],MS4A1[CD20]).
Another12(3.
4%)areG-coupledproteinreceptors(GPCRs;e.
g.
,CCR4[C-Cmotifchemokinereceptor4],CCR5[C-Cmotifchemokinereceptor5],CXCR4[C-X-Cmotifchemokinereceptor4])orothermulti-pass(e.
g.
,CD47,STEAP[six-transmembraneepithelialantigenofthepros-tate]familymembers)cellsurfacetargets.
Additionally,102(29%)aresolubletargets(e.
g.
,TNF[tumornecrosisfactor-alpha,TNF-α],IL6[interleukin-6,IL-6],VEGFA[vascularendothelialgrowthfactorA]),and15(4.
3%)areinfectiousdiseasetargets(e.
g.
,respiratorysyncytialvirus[RSV]-Fprotein,Bacillusanthracisprotectiveantigen[PA]toxincomponent,inuenzahemagglutinin2[HA2;stalkportion],humanimmunodeciencyvirus[HIV]envelopproteingp120)(Table5).
Cellsurfacetargetsinoncologytendtofallintothreecategories.
Therstcategory,whichincludesabout90receptors(e.
g.
,CD19,CD20,EPCAM[epithelialcelladhe-sionmolecule,EpCAM],CEACAM5[carcinoembryonicABCKJIHGMLFED"knob""hole"1.
Antibody2.
Linker3.
Naturalproduct-basedcytotoxinTcellCD28CD247(CD3ζ)TNRSF9TNFRSF4(4-1BB)(OX40)orHNHNH2NHONHNNNHNHHNHOOOOOOOOOOFigure1.
Cartoonsofmoleculesandconstructsdiscussed.
(A)IgGmonoclonalantibody;(B)FAbfragment;(C)Singlechainfragment,variable(scFv);(D)HeterodimericIgG-basedbivalent,bispecicantibody;(E)scFv-basedbispecicantibodysuchasaBiTE("bispecicT-cellengager");(F)IgG-scFv-basedtetravalent,bispecicantibody;(G)TetravalentscFv-basedantibodycalledTandAb;(H)IgG-basedImmunocytokine(cytokineisdenotedbygreenoval);(I)TandemscFv-immunocytokine(cytokineisdenotedbygreenoval);(J)Fc-peptidefusion(peptidesdenotedbysquiggledlines);(K)Fc-proteinfusion(proteindenotedbygrayoval);(L)Antibodydrugconjugatewiththreeparts(antibody,linker,cytotoxicdrug);(M)Chimericantigenreceptor(CAR)-Tbasedantibody(scFvsonsurfaceofrecombinantTcell;examplesofintracellulardomainsnotedinbox).
InnovativeantibodiesREVIEWTheAuthor(s)2017.
ThisarticleisanopenaccesspublicationProtein&Cellantigenrelatedcelladhesionmolecule5],MUC1[mucin1,cellsurfaceassociated]),areessentially"postaladdresses"towhichkillingmechanismscanbetargeteddirectly.
Thesekillingmechanismscaninclude,eitherindividuallyorincombinations,antibody-dependentcellularcytotoxicity(ADCC)(Ochoaetal.
,2017),antibody-dependentcellularphagocytosis(ADCP)(Shietal.
,2015),complement-de-pendentcytotoxicity(CDC)(TaylorandLindorfer,2016),antibody-drugconjugates(ADC)(TsuchikamaandAn,2016;Becketal.
,2017),antibody-inducedapoptosis(Sunetal.
,2017;Wangetal.
,2017),antibody-induced,non-apoptoticprogrammedcelldeath(Alduaijetal.
,2011),bispecicantibody-redirectedkillerTorNKcells(LumandThakur,2011;Sattaetal.
,2013;Suzukietal.
,2015),orCAR-T/CAR-NKcells(RuellaandGill,2015;RuellaandJune,2016;Smithetal.
,2016).
Thesecondgroup,whichoverlapswiththerstgroup,arereceptorswhichmaybetargetedtoblockligandbindingandsignaltransduction(Esparis-Ogandoetal.
,2016;ZhangandZhang,2016).
Thenalcategoryarecheckpointmodulators,eithertoblockTcellinhibitorypath-waysortodirectlystimulateTorNKcellsormacrophages.
Thereareabout20T-cellrelatedoncologytargetsinthiscategory.
Ofthe328uniquetargetsforantibody-baseddrugcan-didates,themostwidelytargetedantigenisCD19,whichisrecognizedby64clinicalcandidates,53ofwhichareCARs(Table6).
ThesecondmosttargetedproteinisCD3E,foundin32clinicalstageorapprovedmolecules,ofwhich26areTcell-redirectingbispecicantibodycandidates(Table6).
Thus,thetwotoptargets,CD19andCD3E,areresponsiblefortheengineeredretargetingofTcells,eitherasCAR-Tcells(RuellaandGill,2015;RuellaandJune,2016;Smithetal.
,2016)orT-cellredirectingbispecicantibodies(LumandThakur,2011;Sattaetal.
,2013;Suzukietal.
,2015),tokillcancercells.
Ofthenon-T-cellrelatedtargets,thepro-teinscurrentlymostwidelytargetedareERBB2(HER2),EGFR,MS4A1(CD20),CD22,PDCD1(PD-1),MSLN(me-sothelin),andERBB3(Her3),allforcancerindications.
TheTh17cytokine,IL17A,towhich14antibody-relatedbiologicsaredirected,iscurrentlythetopnon-oncologytarget(Table6).
Thereare382uniquemoleculesorrecombinantCARsdirectedagainstthetop29targetsshowninTable6,representingabout44%ofalloftheclinicalstageorapprovedantibody-basedmolecules/cells;theremaining482(56%)candidatestargettheremaining299uniquetargets.
Table4.
Therapeuticareastargetedbyinnovativeantibodies,Fcfusionproteins,andCARsinclinicaldevelopment*TherapeuticareaMajorindicationsforantibodiesinphaseofdevelopmentTotalsPhasesIandIIPhaseIIIMarketedOncology(antibodiesandFcfusionproteins)3463033409(46%)Oncology(CAR-TandCAR-NKclinicalcandidatesincorporatingantibodies)14500145(16%)Inammationandautoimmunediseases1321525172(19%)Ophthalmology162220(2.
3%)Infectiousdiseases286438(4.
3%)Neurobiologydiseases203326(2.
9%)Cardiovascularandmetabolicdiseases230528(3.
2%)Blooddiseases125421(2.
4%)Pain3609(1.
0%)Boneandmusclediseases74213(1.
5%)Otherornotdisclosed2103(na)Totalnumberofusesineachtherapeuticarea7347278(allaremAbs/Fcfusionproteins)884totalusesintherapeuticareasTotalnumberofuniquetargets(alltherapeuticareas)–––328uniquetargetsNumberofprogramspertarget–––Average2.
7clinicalprograms/targetAbbreviations:CAR,chimericantigenreceptor;NK,naturalkiller;mAbs,monoclonalantibodies;Fc,fragment,crystallizable.
*DatabaselockforthesedatawasApril30,2017;BiStroBiotechConsultingLLCdatabase.
Thetotalnumberoftherapeuticareaindicationsisgreaterthanthenumberofmoleculesbecausesometargetedantibodieshavebeenusedwidelyindifferentindications(e.
g.
,anti-vascularendothelialgrowthfactor[VEGF]antibodiesusedinbothoncologyandophthalmologyindications).
REVIEWWilliamR.
StrohlTheAuthor(s)2017.
ThisarticleisanopenaccesspublicationProtein&CellThe74approvedmAbsandFcfusionproteinsaredirectedagainst39uniquetargets,withTNF(TNF-α)andMS4A1(CD20)beingthemostwidelytargeted,withveantibody-basedmoleculeseach(Table1).
ThevemostvaluabletargetsforapprovedmAbsandFcfusionproteinsareTNF(TNF-α),VEGF,ERBB2(HER2),MS4A1(CD20),andPDCD1(PD-1)(Table7).
Antibodiesagainsttherstfourofthesetargetswereapprovedmorethantenyearsago,sothemarketvaluehasbuiltupovertime.
Remarkably,how-ever,theanti-PD-1antibodies,KeytrudaandOpdivo,wereapproved2014,makingPDCD1(PD-1)averyfastrisingtargetofvalue(Table7).
Thetoptenantibody-basedtherapeutictargets(Table7)comprise85%ofthevalueofthetotal39targets,withtheanti-TNFmoleculesleadingthewaywithamarketshareof36%(Table7).
Basedon2016salesgures,recombinantproteinscomprisedsevenofthetop10bestsellingdrugsworldwide(Table2).
Ofthesesevenproteins,ve(Humira,Rituxan,Remicade,Avastin,Herceptin)aremAbsandone(Enbrel)isanFcfusionprotein(Table2).
Finally,sinceJanuary2014(thepast3.
3years),antibodiesandFcfusionproteinshavecomprised24%(29/121)ofinnovativeUnitedStatesFoodandDrugAdministration(USFDA)drugapprovals(Fig.
2).
Thisrepresentsthegreatestpercentageeversincethebeginningoftheantibodyera.
Thus,itisclearthatmAbsandFcfusionproteinsaremakinganenormousimpactonthepharmaceuticalindustry,bothasnovelapproachestotreatdifcultdiseasesandmeetunmetmedicalneeds,aswellasprovidinganexcitingnewgrowthareafortheindustry.
BRIEFOVERVIEWOFANTIBODYENGINEERINGHumanIgGshavebeenengineeredinamultitudeofwaystogeneratedifferenteffects(StrohlandStrohl,2012),asshowninTable8.
Intheearlierdaysofantibodyengineering,thefocuswasonmanipulationofthevariableregionstohumanizeandafnity-matureantibodies,ortogeneratedif-ferenttypesofantibodyfragmentssuchasscFvs(Birdetal.
,1988;Hustonetal.
,1988),diabodies(Holligeretal.
,1993),PEGylatedFabs(Choyetal.
,2002),TandAbs(Kipriyanovetal.
,1999),anddomainantibodies(Wardetal.
,1989).
Thenextwaveofantibodyengineeringfocusedmoreonthegenerationandapplicationof"tforpurpose"antibodies(Strohl,2011)withtunedFcfunctionssuchasincreasedADCC,ADCP,andCDC(StrohlandStrohl,2012;BrezskiandGeorgiou,2016;SondermannandSzymkowski,2016;BarnhartandQuigley,2017),ormutedorsilencedFcfunc-tions(Labrijnetal.
,2008;Vafaetal.
,2013;Schlothaueretal.
,2016;Loetal.
,2017;Borroketal.
,2017).
Thesefunctionshavebeenapproachedusingbothglyco-engi-neeringstrategiessuchaslowornofucosylationforhigherFcγRIIIabindingandincreasedADCC(Shieldsetal.
,2002;Ferraraetal.
,2006;Malphettesetal.
,2010;Golayetal.
,2013),higherlevelsofsialylationfordampenedimmuneresponses(AnthonyandRavetch,2010),ornon-Table5.
DistributionoftargetsforantibodiesandFcfusionproteinsbymajorindicationsintherapeuticareasandlocation*TherapeuticareaAntibodiesbindingtotargettypesTotalsCell-boundtargets**SolubletargetsGPCRsormulti-passreceptorsoncellsInfectiousagentsandtoxinsOncology1432651175Inammationandautoimmunediseases(includingasthma,butexcludingMS)52421095Ophthalmologicaldiseases17008Infectiousdiseases20111infectiousagents;3toxins17NeurobiologydiseasesincludingMS750012Cardiovascularandmetabolism914014Painandmigraine32106Bloodhomeostasis3170020Boneandmuscle22004Totalsandpercentoftotal222(63%)102(29%)12(3.
4%)15(4.
3%)351Abbreviations:Fc,fragmentcrystallizable;MS,multiplesclerosis;GPCR,G-proteincoupledreceptor.
*Thesenumbersadduptomorethanthe328uniquetargetsnotedinTable4becauseseveraltargetshavemajorindicationsinmultipletherapeuticareas(e.
g.
,anti-vascularendothelialgrowthfactor[VEGF]antibodieswithmajorindicationsinbothoncologyandophthalmology).
**Mostlysingle-passmembranetargets,eitherasmonomericcell-boundproteins,homodimericreceptors,orheterodimericreceptors.
InnovativeantibodiesREVIEWTheAuthor(s)2017.
ThisarticleisanopenaccesspublicationProtein&CellglycosylatedantibodiesforpartlysubduedFceffectorfunc-tion(Walkeretal.
,1989;Nessporetal.
,2012).
TheseandmoreFceffectormodulationscanalsobegeneratedwithaminoacidchangesinthelowerhingeandFc(Shieldsetal.
,2001;Lazaretal.
,2006;Strohl,2009;StrohlandStrohl,2012;Vafaetal.
,2013;BrezskiandGeorgiou,2016;Son-dermannandSzymkowski,2016;BarnhartandQuigley,2017).
Therearecurrentlythreeapprovedantibody-basedmoleculeswithmodiedFcfunctionality.
TheseincludetheCTLA4-Fcfusionproteins,abatacept(Orencia)andbelat-acept(Nujolix),bothofwhichhavemodiedhingestoreduceFcfunctionality(Davisetal.
,2007),andthehumanizedanti-C5mAb,eculizumab(Soliris),whichhasanIgG2/IgG4hybridFctoreduceFcfunctionality(Rotheretal.
,2007).
AllthreeofthesemoleculesbindtoimmuneTable6.
ToptargetsbasedonnumberofmoleculesdevelopedtowardsthemTarget(aloneorinbispecicpairing)TherapeuticareaPhaseofdevelopmentPhaseI/IIPhaseIIIApprovedTotalCD19ONCYYAAAABBB(53T)YB64CD3EIMM,ONC,CVMYYYYM(24B)-YBB32ERBB2(HER2)ONCYYYAAABBBBBBBTTTTTTTYYYA24EGFRONCYYYATBBBBBBBBBYYYYY19MS4A1(CD20)ONCYYACCBBTTYYYYYRR16IL17AIMMYYYYYYBBBBB-YYY14CD22ONCABTTTTTTTYAAR-13ERBB3(HER3)ONCYYYYYYYYABBBY-13PDCD1(PD-1)ONCYYYYYYYYYF-YY12MSLN(Mesothelin)ONCYAAATTTTTTTT12APP(Amyloid-β)NSYYYYYYYYFYYY-12VEGFAONC,OPHTYYBBBBBYYYF11GD2gangliosideONCBCTTTTTTTT-Y11TNF(TNF-α)IMMYYYBBF-YYYYF11CD274(PD-L1)ONCYYYYYYBYYY10IL3RA(CD123)ONCYYABBBTTT9CD33ONCYABRTTTAA9MET(cMet)ONCYYYYABBTY-9TNFRSF4(OX40;agonist)ONCYYYYYYYF8IL6ONCYYYYF-YYY8GPC3(Glypican-3)ONCYBTTTTTT8TNFRSF8(CD30)ONCBTTTTTT-A8CEAONCBBBCRRTT8TNFRSF18(GITR;agonist)ONCYYYYYYY7EGFR-variantIII(EGFRvIII)ONCYAATTTT--7CD40(antagonist)ONC,IMMYYYYYYY7ANGPT2ONC,OPHTYYYYBBB7IL13IMMYYYYBYY-7FOLH1(PSMA)ONCAABBTTT7Abbreviations:A,antibodydrugconjugate(ADC);ANGPT2,angiopoietin2;B,bispecicantibody;C,immunocytokine;F,Fcfusionprotein;R,radioimmunoconjugate;IMM,immunology;ONC,oncology;OPHT,ophthalmology;NS,neurosciences;CEA,carcinoembryonicantigen;CVM,cardiovascular/metabolism;FOLH1,folatehydrolase1;GITR,glucocorticoid-inducedTNFRfamilyrelatedgene;PSMA,prostatespecicmembraneantigen;T,CAR-T,TCR-T,orCAR-NKcells;Y,nakedIgGorantibodyfragment;otherabbreviationsareasinTable1.
*Wherepossible,namesgivenasHUGOGeneNomenclatureCommittee(HGNC)names(Grayetal.
,2015)followedbycommonlyusednamesinparentheses.
REVIEWWilliamR.
StrohlTheAuthor(s)2017.
ThisarticleisanopenaccesspublicationProtein&CellsystemcomponentsandthemutedFcdesignwasintendedtoincreasethesafetymargin.
Additionally,twoglyco-engineeredantibodieswithimprovedADCCactivitieshavebeenapprovedinatleastonemajormarket.
Therst,mogamulizumab(Poteligeo),isanafucosylatedanti-CCR4mAbapprovedinJapanforadultT-cellleukemia/lymphomathatisproducedbyacelllinewithamutationintheFUT8(α-1,6-fucosyltransferase)gene(PotelligenttechnologyfromBioWa;Yamane-Ohnukietal.
,2004;Kandaetal.
,2006;Malphettesetal.
,2010).
Thesecond,obinutuzumab(Gazyva),ananti-MS4A1(CD20)mAbwithlowfucosecontent,hasbeenapprovedfortreat-mentofchroniclymphocyticleukemia(CLL)(Golayetal,2013).
Thelowfucoseofobinutuzumabisduetotheadditiontotheproducingcelllineofaβ-1,4-N-acetylglucosaminyl-transferaseIII(GntIII)genewhichaddsthebisectingN-acetylglucosamine(GlcNAc)thatinterfereswithfucosylation(GlycartGlycoMabtechnologyacquiredbyRochein2005)(Ferraraetal.
,2006).
Moreover,therearefourglycoengineered(lowornofucose)andoneaglycosyl-IgGphaseIIIclinicalcandidatesinthelatestageclinicalpipeline.
CurrentlytherearenoapprovedIgGswithaminoacid-modiedincreasedFceffectorfunction,althoughtherearetwosuchFc-modied,increasedFceffectorfunctionIgGsinlatestageclinicaltrials,theanti-CD19mAb,Mor208(Mor-phosys,Xencor),inphaseII/IIIclinicaltrialsfortreatmentofBcellmalignancies(NCT02763319),andtheanti-ERBB2(HER2)mAb,margetuximab(Merck,Macrogenics),inphaseIIIclinicaltrialsforbreastcancer(NCT02492711).
Moreover,therehavebeenmanyeffortstomodulateaspectsofIgGbiologybeyondjustincreasingordecreas-ingFceffectorfunction(Table8).
Therstoftheseismod-ulationofhalf-lifeviamodiedinteractionoftheFcwiththerecyclingreceptorFcRn(RoopenianandAkilesh,2007;Table7.
MostvaluabletargetsforMabsandFcfusionproteinsasoffull-year2016#Target*NumberofdrugsTherapeuticareaExampledrugsFirstapprovaloftargetTotalvalue2016**Percentoftotalvalue1TNF(TNF-α)5InammationandautoimmunityHumira,Enbrel,Remicade1998$38.
7B36%2VEGF3Cancer,ophthalmologyAvastin,Eylea,Lucentis2004$15.
3B14%3ERBB2(HER2)3CancerHerceptin,Perjeta,Kadcyla1998$9.
5B9%4MS4A1(CD20)3CancerRituxan,Gazyva1997$7.
5B7%5PDCD1(PD-1)2CancerOpdivo,Keytruda2014$6.
0B5.
6%6IL12B(p40subunitofIL-12andIL-23)1InammationandautoimmunityStelara2009$3.
2B(3.
23)3%7TNFSF11(RANK-ligand)1Osteoporosis,cancerProlia/Xgeva2010$3.
2B(3.
16)3%8C51BloodhomeostasisSolira2007$2.
8B3%9EGFR3CancerErbitux,Vectibix,Portrazza2004$2.
4B2.
2%10IGES(IgE)1AsthmaXolair2003$2.
3B2.
2%Total–576differentdiseaseareas–1997–2014$90.
9B***85%Totalmarketvalueforallinnovatorantibodiesin2016$106.
9BAbbreviations:CD,clusterofdifferentiation;EGFR,epidermalgrowthfactorreceptor;HER2,humanepidermalgrowthfactorreceptor-2;Ig,immunoglobulin;IL,interleukin;PD-1,programmedcelldeathprotein-1;RANK,receptoractivatorofnuclearfactorkappa-B;TNF,tissuenecrosisfactor;VEGF,vascularendothelialgrowthfactor.
*NamesgivenasHUGOGeneNomenclatureCommittee(HGNC)names(Grayetal.
,2015)followedbycommonlyusednamesinparentheses.
**Roundedtoonedecimalpoint.
DataabstractedfromLaMerie,2017.
***$90.
9Bof$106.
9Bis85%oftotalmAbandFcfusionproteinvaluein2016(10of69totalactivelymarketedantibody-basedproducts).
InnovativeantibodiesREVIEWTheAuthor(s)2017.
ThisarticleisanopenaccesspublicationProtein&CellBakeretal.
,2009).
Themostimportantofthesemodica-tionshasbeenthe"YTE"mutation(Dall'Aquaetal.
,2002;Dall'Aquaetal.
,2006)fromMedImmune(AstraZeneca),whichhasbeenincorporatedintoafewearlystagecandi-datesatthispoint(Robbieetal.
,2013).
Otherhalf-lifeextensionmutationsoftheFcalsohavebeenmade,includingXencor'sXtendtechnology(Zalevskyetal.
,2010),whichhasbeenincorporatedintoatleasttwoearlystageclinicalcandidates,Alexion'santi-C5mAb,ALXN5500,andtheNationalInstitutesofHealth's(NIH's)anti-CD4mAb,VRC01LS.
SeveralotherIgGengineeringtechnologieshavebeenreportedthathavethepotentialtomodulatethecapabilitiesofexistingandfutureclinicalcandidates(Table8).
Theseincludeproteaseactivated"probody"IgGsfortumor-local-izedactivity(Ersteretal.
,2012;Desnoyersetal.
,2013;PoluandLowman,2014),protease-resistantIgGsthatarestableinthetumormicro-environment(Kinderetal.
,2013),hexamericantibodieswithhighC1qbindingandconcomitantCDCeffectorfunction(Cooketal.
,2016;deJongetal.
,2016),pH-dependentbinding(Igawaetal.
,2010a;Cha-parro-Rogersetal.
,2012;Devanaboyinaetal.
,2013)andsweepingantibodies(Igawaetal.
,2013)thatimproveupontheeliminationproleforsolubleantigens(Igawaetal.
,2016),engineeringvariableregionsforimprovedsolubilityanddevelopability(Clarketal.
,2014;Seeligeretal.
,2015),modulationofthepIorchargeoftheantibodyvariablesequencesforimprovedhalf-life(Igawaetal.
,2010b;Lietal.
,2014;Datta-Mannanetal.
,2015)and/orseparationandpurication(Sampeietal.
,2013),andmutationofpro-teinAbindingsiteforimprovedpuricationofaheterobis-pecicIgG(Tustianetal.
,2016).
AnotherareaofantibodyengineeringthatisstartingtoseesignicantactivityistheengineeringofIgMsastherapeutics,especiallywherehighavidityeffectsaredesired(Chromikovaetal.
,2015;Wangetal.
,2017b).
Inarecentexample,ananti-TNFRSF10B(DR5)IgMdemonstrated10-foldgreateravidityand1000-foldgreaterkillingeffectthanasimilarIgG(Wangetal.
,2017b).
ANTIBODYDRUGCONJUGATES(ADCS)ADCstargetacytotoxicdrugtoatumortokillcancercellswhileloweringthesystemicexposureoftheactivemoiety,withthegoalofincreasingthesizeoftheefcacy/toxicitywindowofhighlytoxicanti-tumordrugs(StrohlandStrohl,2012;TsuchikamaandAn,2016;Becketal.
,2017).
ADCs1997199819992000200120022003200420052006200720082009201020112012201320142015201620175101520253035404550NMEsandantibodiesapprovedbyUS-FDA172136202218242621303927414522243212229106242127353039219Year532520332245211255135665214236666211127923189112817161519151319312321SmallmoleculeNMEsapprovedbyFDAAntibodiesandFc-fusionproteinsapprovedbyFDABLAbiologicsapprovedbytheFDAThrough5/1/1751Figure2.
Smallmoleculedrugs(bluelines),biologicsincludingvaccines(greenlines),andmonoclonalantibodies/Fcfusionproteins(redlines)approvedbytheUnitedStatesFoodandDrugAdministrationfrom1997toMay1,2017.
ThisinformationwassourcedandextractedfromtheUSFDAwebsite(https://www.
fda.
gov/drugs/developmentapprovalprocess/druginnovation/ucm537040.
htm).
REVIEWWilliamR.
StrohlTheAuthor(s)2017.
ThisarticleisanopenaccesspublicationProtein&CellTable8.
ExamplesofantibodyengineeringandkeyearlydatesforthevarioustechnologiesdevelopedTypeofengineeringKeyearlydateNotesorcommentsExamplereferencesChimerization1984MousevariablesequencesfusedtohumanconstantsequencesMorrisonetal.
,1984Humanization1986MouseCDRsinhumanframeworksJonesetal.
,1986;Queenetal.
,1989GenerationofscFvs1989FvdomainsfusedwithlinkerBirdetal.
,1988;Hustonetal.
,1988Fcfusionproteins1989IgGFcfusedwithpeptidesorproteinsCaponetal.
,1989Afnitymaturation1990–1992ImprovementinbindingtotargetHawkinsetal.
,1992IsotypeswitchingformodiedFcfunctionality1990–1993ChangeinFcactivityGreenwoodetal.
,1993AglycosylIgG1993N297xmutationtogenerateaglycosylatedIgGtoreduceFcγRactivityBoltetal.
,1993;Nessporetal.
,2012HeterodimericFcengineeringtomakebispecics1996Knobs-into-holeswasrstheterodimericFcplatformRidgewayetal.
,1996SilencedFcactivity1997IgGσandotherplatforms;AbataceptandEculizumabrstclinicalcandidatestoincorporateMuelleretal.
,1997;Vafaetal.
,2013Glyco-engineeredforincreasedADCC1999IncreasedbindingtohumanFcγRIIIatoincreaseADCC;Poteligent,GlycoMax;MogamulizumabandObinutuzumabrstclinicalcandidatestoincorporateUmanaetal.
,1999;Shieldsetal.
,2002ModicationofproteinAbindingforpurication2000FirstengineeringtomodulatepuricationTustianetal.
,2016Antibody-cytokinefusions2001CytokinefusedtotargetingIgGorscFvPenichetandMorrison,2001;Halinetal.
,2002SequencemodicationforincreasedFcactivity2001IncreasedbindingtomultipleFcγRstoincreaseADCC,CDC,and/orADCPShieldsetal.
,2001;Lazaretal.
,2006Longerhalf-life2002ModicationofFcsequencestoimprovepH-dependentbindingtoFcRn;"YTE"mostwidelyrecognizedhalf-lifeextensionmodicationsDall'Aquaetal.
,2002;Dall'Aquaetal.
,2006Targetingpeptide2004RGDtargetingofIgG;AsfotasealfarstclinicalcandidattoincorporateLietal.
,2004pHdependentbindingtoantigen2010ImprovedremovalofsolubleantigenswhilerecyclingantibodyIgawaetal.
,2010a;Chaparro-Rogersetal.
,2012;Devanaboyinaetal.
,2013ModicationofpIinvariableregionsforlongerhalf-life2010Engineeringvariablesequencestoimprovehalf-lifeIgawaetal.
,2010bProtease-activated"probody"IgGfortumorlocalizedactivity2012LackofbindingactivityuntilactivatedbyproteolyticcleavageErsteretal.
,2012;Devanaboyinaetal.
,2013ClinicalcandidatesusingIgG-mediatedtranscytosis2012,2014Anti-insulinIgG-enzymefusionfornextgenerationenzymereplacementsforCNSERTsBoadoetal.
,2012;Boadoetal.
,2014Protease-resistantIgGs2013IgGresistanttomicrobialandtumor-elicitedproteasessuchasMMP9Kinderetal.
,2013InnovativeantibodiesREVIEWTheAuthor(s)2017.
ThisarticleisanopenaccesspublicationProtein&Cellconsistofthreecomponents,thetargetingantibody,thecytotoxicpayload,andthelinkerthatcouplesthosetwocomponentstogether(Fig.
1).
WiththosethreecomponentscomeveconsiderationsforthedesignandconstructionofanADC:First,thetargetingantibodymustbindtoaproteinthatisfoundeitherexclusivelyoncancercellsorsignicantlyoverexpressedoncancercellsascomparedwithexpressiononnormaltissues.
ThebesttargetsforADCsmaybeoncofetalantigensortargetsthatmaybeoverexpressedincancercellsbutpresentinnormaltissuesatlowcopynumberorintissuesinwhichthetoxicityistolerable.
Thecellsurfaceproteinsmostwidelytargetedwithclinicalstage(orapproved)ADCscurrentlyareHer2(veADCstargeting),CD19(fourADCstargeting),CD22(threeADCstargeting),andmesothelin(3ADCstargeting).
CEA-CAM5,EGFR(wild-type),EGFR(variantIII),CD33,andCD70eachhavetwoclinicalstageADCstargetingthem.
ThepropertiesofgoodADCtargets,aswellasdescriptionsofcandidateADCtargets,havebeenreviewed(Teicher,2009;StrohlandStrohl,2012).
AninterestingstrategybeingemployedbyCytomXtoincreasethetumorspecicityoftheirADCsistheuseofpro-antibodiesthatpossessapeptidesequencecoveringtheparatope,preventingbindingtotheirtargetuntilitreachesthetumormicroenvironment(TME).
OnceintheTME,theparatope-shieldingpeptideiscleavedbymatrixmetalloproteinases(MMPs),whichareinhighconcentrationsinmostTMEs,allowingtheantibodytobindtotargetsinthatlocalenvironment(Desnoyersetal.
,2013;PoluandLowman,2014).
Second,theADC-directingantibodymustberapidlyinternalizeduponligationtoitstargetedreceptor.
Antibodiesthatbindcellsurfacereceptorsmayormaynotinternalizerapidly,sowhenisolatingtheantibody,incorporationofinternalizationscreensintothediscoveryprocessiscritical(Pouletal.
,2000;Zhouetal.
,2010).
Third,theidentity,number,andtypeoflinkerattachmentsitesisacriticalissue.
InrstgenerationADCs,thelinkersweretypi-callyattachedtothe-amineoflysineresidues(TsuchikamaandAn,2016;Becketal.
,2017).
Giventhatthereareabout80lysineresiduesinatypicalIgG,tenofwhichcanbeaccessedforchemicalcoupling(TsuchikamaandAn,2016),theresultsofsuchconjugationsarehighlyheterogeneous.
Evenwithoptimization,conjugationtolysinesresultsinadrugtoantibodyratio(DAR)ofabout2–4,witharangeof0–7(Lazaretal.
,2005;TsuchikamaandAn,2016;Becketal.
,2017).
TherearemultiplechallengeswithheterogeneousADCsincludinganalyticalchallenges,batch-to-batchconsis-tencies,thestabilityoftheADC,andthepotentialforvariablepharmacokineticsifconjugationsitesinsomeantibodiesinterferewithnormalFcRn-mediatedrecycling(Becketal.
,2017).
Sitespecicconjugation,whichhasbeenachievedthroughavarietyofmethodsandcanresultinverytightDARsandincreasedhomogeneity(Junutulaetal.
,2008;Panowskietal.
,2014;Perezetal.
,2014;Beerlietal.
,2015;Ihospiceetal.
,2015;Siegmundetal.
,2016;Thompsonetal.
,2016;TsuchikamaandAn,2016;Becketal.
,2017),appearstobeasignicantadvancement.
Newapproachesusingextensionsequences,suchasdevelopedbyMersana,canachieveadrug/antibodyratioof20(Yurkovetskiyetal.
,2015).
Fourth,thestabilityofthelinkercanhaveahugeinu-enceontheefcacyandtoxicityoftheADC.
Intheory,amorestablelinkerwhichisonlydegradedwithinthelyso-someshouldhavethebestsafetyprole.
Unfortunately,itisnotthatsimple,astherearecasesinwhichhighlystablelinkersresultedinsafetyissues.
Someofthesemaybeduetomannosereceptor,orpotentiallyalsoFcγR-me-diatedbindingandinternalizationofADCs,whichcouldresultin"off-target"toxicityissues(GorovitsandKrinos-Kiorotti,2013;Becketal.
,2017).
Finally,notallcancercellswithinatumoraretargetantigen-positive(Singhetal.
,2016),thusallowingpotentialTable8continuedTypeofengineeringKeyearlydateNotesorcommentsExamplereferencesModicationofpIinvariableregionsforeasierpurication2013EngineeringvariablesequencestoimprovepuricationSampeietal.
,2013Sweepingantibodies2013HighlyactiveremovalofsolubleantigenswhilerecyclingantibodyIgawaetal.
,2013;Igawaetal.
,2016Antibodyengineeringforimprovedmanufacturability2014ModicationofvariablesequencestoimprovesolubilityanddecreaseaggregationClarketal.
,2014;Seeligeretal.
,2015IntracellulardeliveryofIgG2014BioactiveIgGescapesendosometobindtocytosolictargetChoietal.
,2014;Kimetal.
,2016HexamericIgGformation2016HexamerizationofIgGsoncellsurfaceswithhighlyimprovedC1qbinding;CDCCooketal.
,2016;deJongetal.
,2016Abbreviations:BBB,bloodbrainbarrier;CDC,complement-dependentcytotoxicity;CDRs,complementaritydeterminingregions;CNS,centralnervoussystem;ERT,enzymereplacementtherapy.
REVIEWWilliamR.
StrohlTheAuthor(s)2017.
ThisarticleisanopenaccesspublicationProtein&Cellescapeoftheantigen-negativecellsfromtargetedtherapies.
Ithasbeendemonstratedthatmembranepermeabilityofthecytotoxinisacriticalfactorforpotentialbystanderactivity(Lietal.
,2016).
Thus,designoffutureADCswillneedtotakethechemistryoftheresultantADCintoaccounttooptimizebystandereffectandefcacy.
Therecurrentlyare87clinicalstageADCs,includingthreeapprovedADCs,nineinphaseIIIdevelopment,andanother75inphaseI/IIclinicaldevelopment.
ThethreeapprovedADCsincludeMylotarg(2000,withdrawnin2010),theCD30-targetingAdcetris,andtheERBB2(Her2)-targetingKadcyla.
These87clinicalstageADCmoleculesaredirectedagainstatleast53differentknowntargets,althoughafewhavenotbeendisclosed,sotheactualnumbermaybehigher.
ThemosttargetedcellsurfacereceptorscurrentlyareERBB2andCD19(4ADCsagainsteach),andCD33,CD22,andMSLN(mesothelin)(3ADCsagainsteach).
Thereare16knowndifferentclassesofdrugsincorpo-ratedintoclinicalstageADCs,11ofwhicharesmallmole-culeclassesandveofwhichareprotein-based.
ThemostwidelyuseddrugclassincorporatedintoclinicalstageADCsaretheauristatins(employed31times),followedbythemaytansanoids(in16ADCs),andbenzodiazepines(usedin9ADCs)(Table9).
Ofthebiologics,PseudomonasexotoxinPE38isincorporatedintofourADCs(Table9).
EventhoughthreeADCshavebeenapprovedforthera-peuticuse,thistechnologyisstillrelativelyearlyinthedevelopmentalcycleandmanyofthe"rules"foroptimizedADCsarestillbeingsortedout(DrakeandRabuka,2015;Becketal.
,2017).
Moredetailsonthedesignandcon-structionofADCscanbefoundinTsuchikamaandAn(2016)andinBecketal.
(2017).
FCFUSIONSFcfusionsarefusionsoftheIgGFcdomainwitheitheraproteinorpeptide.
Intheory,thefusioncanbetoeithertheC-orN-terminusoftheFc,butmostFcfusionsonthemarketandinclinicaldevelopmenttodayareN-terminalfusions.
TheprimaryreasonforgeneratingFcfusionsistoextendthehalf-lifeofpharmacologicallyrelevantproteinorpeptidebyusingtheFcRn-mediatedrecyclingoftheFc(StrohlandStrohl,2012;Strohl,2015).
Currently,11FcfusionproteinsTable9.
Classesofdrugscurrentlybeingemployedinantibodydrugconjugatecandidates*ClassofdrugDrugtypeNumberofADCsperphaseTotalPhaseI/IIPhaseIIIApprovedatsomepointforMarketing*AuristatinsSMnaturalproduct-derived291131MaytansanoidsSMnaturalproduct-derived141116Benzodiazepines**SMnaturalproduct-derived8109PseudomonasaeruginosaexotoxinPE38Proteintoxin-based2204Calicheamicin***SMnaturalproduct-derived1113DiphtheriatoxinProteintoxin-based2002Irinotecans(SN38)SMnaturalproduct-derived1102DuocarmycinSMnaturalproduct-derived2002ExatecanSMnaturalproduct-derived2002StaphylococcusaureusenterotoxinA/E-120Proteintoxin-based0101DoxorubicinSMnaturalproduct-derived1001TubulysinSMnaturalproduct-derived1001AntibacterialantibioticSM1001ShigatoxinProteintoxin-based1001RicinProteintoxin-based1001UreaseEnzyme1001NotdisclosedorunknownNA9009Totals768387*FromBiStroBiotechConsultingLLCdatabaseonclinicalstagebiologics.
DatabaselockforthesedatawasApril30,2017.
**Includingbothpyrrolobenzodiazepinesandindolobenzodiazepines.
***Mylotarg,whichcontainedacalicheamicinADC,waswithdrawnfrommarketingin2010.
InnovativeantibodiesREVIEWTheAuthor(s)2017.
ThisarticleisanopenaccesspublicationProtein&Cellhavebeenapprovedfortherapeuticuse(Table1),threeareinphaseIIIclinicaltrials,and23arebeingevaluatedinearlierstageclinicaltrials(Table3).
ManyoftheearlierFcfusionsgenerallywereconstructedusingreceptorexodo-mainsinimmunepathways(e.
g.
,TNFRSF18[p75],CD58[LFA3],CTLA4,IL1R1[IL-1receptor])fusedtotheFctomodulatetheimmunesystem,eitherbyblockingsolublecytokinesorbybindingtocells.
MorerecentFcfusionpro-teinshavebecomemorediverse(Strohl,2015),withthepharmacologicallyactive"headgroups"beingbloodfactors,suchasF9(FactorIX)andF8(FactorVIII),peptidessuchasGCG(GLP-1)andaTHPO(thrombopoietin)analogue,andanenzyme,suchasthetissuenon-specicalkalinephos-phatase(TNSALP;Millanetal.
,2008)inasfotasealfa(Strensiq)(Hofmanetal.
,2016).
IMMUNOCYTOKINESCertainhumancytokinessuchasIL2havebeenapproved(marketedname,Proleukin)forsystemicdeliveryanduseinseverediseasessuchasmetastaticmelanomaandmetastaticrenalcellcarcinoma(Dutcher,2002).
SystemicdeliveryoftheTcell-activatingcytokine,IL2,however,bringswithitthepotentialforadverseevents.
Theconceptofusingantibodiestotargetcytokinestoeithertumorsortospecictissuescameintofruitionaroundtheturnofthecentury(PenichetandMorrison,2001;Halinetal.
,2002).
Sincethattime,therehasbeenanefforttotargetIL2,orothercytokinessuchasIL12andTNF,tothetumormicroenvironment,wherethedesiredactivitycantakeplacewithreducedadversesystemiceffects(NeriandSondel,2016).
Thisapproachhasbeenactualizedbythefusionofcytokinestoantibodiestomakeimmunocytokinesthatmaytargetvas-culatureassociatedwithtumors(Pascheetal.
,2012;Hem-merliandNeri,2014),tumorcellsurfaceantigens(Kleinetal.
,2017),ortargetsthatwouldassistinaccumulationininamedjoints(Hughesetal.
,2014).
Immunocytokinescomeintwomajorformats,cytokine-scFv(orotherfragment)fusionswhichhaveashortcirculatinghalf-lifeandcytokine-IgGfusions,whichretainalonghalf-life(NeriandSondel2016).
Thereareatleast11immunocytokinescurrentlybeingevaluatedinclinicaltrials.
TwooftheseareDarleukin(-bronectinextradomainB[EDB]-targetingscFvL19-IL2fusion)andFibromun(EDB-targetingscFvL19-TNFfusion),whicharebothinphaseIIIpivotalclinicaltrialsascombinationtherapyformalignantmelanoma(NCT02938299).
Otherclinicalstageimmunocytokinesincludeexam-plessuchasDekavil(bronectinextradomainA[EDA]targetingscFvF8-IL-10fusioninphaseIIfortreatmentofRA[NCT02270632]),Teleleukin(tenascinCalternativesplicevariantEDA1-targetingscFvF16-IL-2fusioninphaseIfortreatmentofacutemyeloidleukemia[AML;NCT02957032]),RG7461FAP(broblast-activationprotein)-IL2fusioninphaseIfortreatmentofsolidtumors[NCT02627274]),andcergutuzumabamunaleukin,ananti-CEA(carcinoembryonicantigen-IgGfusedwithIL2,currentlyinphaseIclinicaltrials(NCT02350673)fortreatmentofsolidtumors.
CHECKPOINTMODULATORSAntibody-directedmodulationofimmunecellcheckpointreceptorshasbecomeoneofthemostexcitingandimpor-tantnewareasinantibodytherapeuticsoverthepastfewyears.
MosteffortshavebeenfocusedonTcellcheckpointmodulation,butthereisincreasinginterestinBcell,NKcell,andmyeloidcellcheckpointmodulationaswell.
Tcellactivationisregulatedbyaseriesofthreesignals.
TherstsignalisprovidedbytheinteractionoftheTcellreceptor(TCR)withmajorhistocompatibilitycomplex(MHC,HLA)classI(forCD8Tcells)orMHC(HLA)classII(forCD4Tcells)onantigenpresentingcells(APCs).
Thesecondarysignalisprovidedthroughoneofseveralcheckpointrecep-tors(Table10),whichcaneitherprovideacostimulatorysignaltoactivatetheTcells,orablockingsignaltodampenTcellresponse(Topalianetal.
,2015).
Thethirdsignalcomesfromtheproductionofeitherpro-inammatory,Tcell-activatingcytokinesoranti-inammatorycytokinesthatwouldacttoreduceTcellresponse(Chikumaetal.
,2017;Schirdewahnetal.
,2017).
CancercellscanexpressligandsforTcellinhibitoryreceptorssuchasPDCD1(PD-1)(ligandisCD274[PD-L1]),CTLA-4(ligandsareCD80andCD86),andHAVCR2(akaTIM3)(ligandreportedtobeGAL9)toinhibitTcellactivationandcytolyticTcellresponses.
LigationofthesereceptorscanleadtoTcellanergyorexhaustion,resultingintheinabilityoftheimmunesystemtokillcancercells.
InhibitionoftheblockingresponsestoTcellactivationusinganti-PDCD1,anti-CTLA4,oranti-CD274antibodieshasprovenclinicallytoresultinimprovedresponsesforasubsetofpatientswithmetastaticmelanoma,NSCLC,andpotentiallyotherformsofcancer(AchkarandTarhini,2017;Kimetal.
,2017).
Additionally,effortsareongoingtousecombinationsofanti-PD1andanti-CTLA4antibodiestoincreasetheper-centageofpatientsexperiencingdurableresponses,i.
e.
,"raisingthetailofthesurvivalcurve"(Harrisetal.
,2016).
Alternatively,severalclinicalcandidatesareagonistsofT-cellactivatingreceptorssuchasTNFRSF4(OX40),CD40,TNFRSF9(CD137,4-1BB),TNFRSF18(GITR),ICOS(CD278),CD27,orCD28tostimulateTcellresponses(Antoniaetal.
,2016;Table10).
Additionally,Tcellcheckpointpathwaysarepotentiallyimportantininfectiousdiseases,inwhichTcellexhaustionhaltsTcellsfromeliminatingviralandbacterialpathogens(DyckandMills,2017).
Finally,antibodyinterventioninTcellcheckpointpathwaysmayplayaroleinautoimmunedis-eases,whereblockingtheactivatingsignalsorincreasingtheblockingsignalsmayresultinloweringtheTcellacti-vationresponse(vanderVlistetal.
,2016).
FivemAbsandtwoFcfusionproteinsthattargetTcell/APCcheckpointshavebeenapproved(Table10).
TwomoreTcellcheckpointinhibitorantibodiesarecurrentlyinphaseIIIREVIEWWilliamR.
StrohlTheAuthor(s)2017.
ThisarticleisanopenaccesspublicationProtein&Cellclinicaltrialsand77areinphaseI/IIclinicaltrials,covering19differentTcellcheckpointtargets.
Someofthesecheckpointtargetsarebeingtestedinbothimmuneandoncologyrelateddiseases.
Forexample,CD28,CD40,andTNFRSF4(OX40)antagonistsareinearlystageclinicaltri-alsfortreatmentofvariousimmunedisorders,whereasCD28,CD40,andTNFRSF4(OX40)agonistsareinearlystageclinicaltrialsforvariouscancerindications(Table10).
Checkpointligandsexpressedoncancercellsalsoarepotentiallyexcellenttargets,bothbecausetheycanblocktheinhibitorycheckpointinteractionaswellastargetingtheligand-expressingcancercellswithFc-activeantibodies.
Forthisapproach,therearenowthreeapprovedanti-PD-L1antibodiesandanotherseveninclinicaltrials,aswellasthreeclinicalstageanti-CD70(CD27ligand)mAbsandoneCD70-targetingCAR-TcellproductinphaseIclinicaltrials.
,aswellasfouranti-CD276(B7H3)antibodiesarecurrentlyinphaseIclinicaltrials.
BcelltransitionalcheckpointsarecenteredaroundBcellhomeostasisandthechoiceofwhethertheBcellshouldmatureorproceedtoapoptosis.
ThisprocessensuresthatBcellsexpressingautoreactiveimmunoglobulinsarepurged(Cancroetal.
,2009).
KeyregulatorsofBcellmaturationthatfunctioninBcellcheckpointsareTNFSF13B(solubleBLyS,ligandsBlymphocytestimulator;alsoknownasBcellacti-vatingfactor[BAFF])andTNFSF13(APRIL,aproliferation-Table10.
mAbsandFcfusionproteinsdirectedtowardsimmunomodulationandcheckpointtargets*Target***ActivityTherapeuticareaPhaseofdevelopmentTotalnumberofcandidatesPhaseI/IIPhaseIIIApprovedCD80/CD86AntagonistIMM0022CTLA4AntagonistONC1113PDCD1(PD-1)AntagonistONC130215CD274(PD-L1,B7-H1)AntagonistONC61310PDCD1LG2(PD-L2)AntagonistONC1001CD28AntagonistIMM3004totalAgonistONC100TNFRSF4(OX40)AntagonistONC20010totalAgonistONC800TNFSF4(OX40ligand,CD252)AntagonistONC0**000CD40AntagonistIMM70013totalAgonistONC600CD40LG(CD154;CD40ligand)AntagonistONC2002ICOS(CD278)AntagonistONC1003totalAgonistONC200ICOSLG(ICOS-ligand;B7RP-1;CD275)AntagonistIMM1001TNFRSF18(GITR)AgonistONC7007HAVCR2(TIM3)AntagonistONC2002TNFRSF9(CD137,4-1BB)AgonistONC2002LAG3(CD223)AntagonistONC3003VSIR(VISTA)AntagonistONC1001TIGITAntagonistONC2002CD47AntagonistONC4004CD27AgonistONC1001Totals––762785*Abbreviations:IMM,immunology;ONC,oncology.
**Knownpreclinicalprogramsthatshouldprogresstoclinicaltrialsbyendof2017.
***NamesgivenasHUGOGeneNomenclatureCommittee(HGNC)names(Grayetal.
,2015)followedbycommonlyusednamesinparentheses.
InnovativeantibodiesREVIEWTheAuthor(s)2017.
ThisarticleisanopenaccesspublicationProtein&Cellinducingligand).
TNFSF13BcanbindtheTNFSF13Breceptor(BR3;alsoknownasBAFF-R)topromoteBcellsurvival,andbothTNFSF13BandTNFSF13canbindTNFRSF13B(transmembraneactivator-1andcalciummodulatorandcyclophilinligand-interactor,TACI)andTNFRSF17(Bcellmaturationantigen,BCMA),bothofwhichresultinIgclassswitchingandTcell-dependentresponses(Cancroetal.
,2009).
OverexpressionofTNFSF13Bcanleadtoautoimmuneconsequences,suchassystemlupuserythematosus(SLE)orSjgren'ssyndrome(Cancroetal.
,2009).
OneBcellcheckpointinhibitor(anti-TNFSF13BmAb,Benlysta)isapproved,twomorearecurrentlyinphaseIIIclinicaltrials,andthreeareinphaseI/IIclinicaltrials,alltargetingtheBcellactivatingfactorregulatorypathway.
AnotherapproachthathasgainedinterestinveryrecentyearsistheimmunomodulationofNKcells.
NKcells,aswellasCD8Tcells,expressaseriesofinhibitoryreceptorsincludingKLRC1-formA(NKG2A),TIGIT,CD96,andKIRfamilymembers(Carotta,2016).
Asanimmunedefensemechanism,tumorcellsexpressligandstobindtothesereceptorstoinhibitunwantedactivationofNKcells.
Cur-rentlytherearesixantibodiesinphaseI/IIclinicaltrialsbindingthesetargetstoremovethebrakeonNKcellactivation.
Finally,anothercheckpointthatregulatestheactivityofmacrophagesandtheirphagocytosisoftargetcellsistheCD47/SIRPA(signalregulatoryproteinalpha)andCALR(calreticulin)/LRP1pathway.
TheCD47/SIRPAligationisoftenreferredtoasthe"don'teatme"signal,whereasCALR/LRP1ligationisknownasthe"eatme"signal(McCrackenetal.
,2015).
BlockingofCD47byantibodiesorFcfusionproteinscanleadtoanimbalanceandapro-"eatme"response(McCrackenetal.
,2015).
Currently,fouranti-CD47antibodiesorFcfusionproteinsarebeingevaluatedinclinicaltrialsfortreatmentofcancer(Table10).
ANTIBODYMIXTURESOneapproachthathasgainedinterestinrecentyearsisthecombinationormixtureofantibodies,usuallyagainstasingletarget,includedintoasingledosage(RajuandStrohl,2013;Carvalhoetal.
,2016).
Thusfar,antibodymixturesarebeingusedmostlyforoncologyandinfectiousdiseaseindications.
TheDanishbiotechnologycompany,Symphogen,hasledthisspace,withfourantibodymixturescurrentlybeingtestedinclinicaltrials.
TheseincludeSYM004,amixtureoftwoanti-EGFRmAbs,SYM013,amixtureofsixantibodiesagainsttheERBB(Erb-b2receptortyrosinekinase)familyofreceptors(Ellebaeketal.
,2016),SYM015,amixtureoftwoantibodiestargetingMET(cMET),andSYM009,anundis-closedmixtureofantibodiespartneredwithGenentechforaninfectiousdiseasetarget.
Atleastnineotherantibodymixturesarebeingevaluatedinclinicaltrials,allofwhichareagainstinfectiousdiseasestargetssuchasEbolavirus,botulinumtoxin,andotherviruses.
Oneveryinterestingnewapproachinthisareathatcouldseesignicantlygreaterupsideinthecomingyearsisthegenerationoffullyhumanantibodymixtures,orpolyclonalmixtures,intransgenic(tg)cattle(Matsushitaetal.
,2014,2015).
Thesemay,iffoundsafeandefcacious,atleastpartiallyreplace"specic"intravenousimmunoglobulin(IVIG),whichisIgGpuriedfromindividualswhohavebeenvaccinatedorfromconvalescingpatientswhohavepro-ducedIgGsagainstaspecictarget(Llewelynetal.
,1992;Mireetal.
,2016).
Theupsideoftgcattle-producedhumanIgGsissupply,consistencyacrosslots,andtheabilitytovaccinatethecowswithantigensnotavailableforhumanvaccinationduetoregulatoryandsafetyconsiderations.
Onesuchpolyclonalmixturefromtgcattlealreadybeingevalu-atedinclinicaltrialsisSAB-301(SABTherapeutics),apolyclonalmixtureofhumanIgGstargetingmiddleeastrespiratory(MERS)virus(NCT02788188;Lukeetal.
,2016).
BISPECIFICANTIBODIESBispecicantibodies,rstconceptualizedin1983(MilsteinandCuello,1983),areantibodiesthatcanbindtwodifferentantigenssimultaneously.
Therearevefundamentalgroupsofbispecicantibodyformats:(i)asymmetricbivalent,bis-pecicIgG-likeantibodieswithheterodimericheavychains(HCs)(Ridgewayetal.
,1996;Merchantetal.
,1998;Guna-sekaranetal.
,2010;Stropetal.
,2012;Kleinetal.
,2012;Labrijnetal.
,2013VonKreudensteinetal.
,2013;BrinkmannandKontermann,2017);(ii)tetravalentmultispecicanti-bodiesthatarecomprisedofIgGs,withadditionalbindingdomains,e.
g.
,scFvs,Fvs,VHHdomains,ornon-antibodybindingscaffoldssuchasfynomers(Bracketal.
,2014;Silaccietal.
,2016),fusedtoeithertheN-orC-terminiofeithertheheavyorlightchains(LCs)(ColomaandMorrison,1997);(iii)engineeredbindingdomainswithinthenormalIgGstructure,suchasthe"two-in-one"bispecicapproachfromGenentech(Bostrometal.
,2009;EigenbrotandFuh,2013)andtheF-STARapproachofdesigningnovelsecondbindingsiteswithintheCH3domain(Leungetal.
,2015),(iv)engi-neeredantibodyfragmentslinkedbyshortpeptidelinkerswhichcanbemadeintobivalent,trivalent,ortetravalentformatsaddressingtwotothreetargets(Macketal.
,1995;HolligerandWinter,1997;Kipriyanovetal.
,1999;Reuschetal.
,2015;Eganetal.
,2016).
ThesemaybefusedtoanFcdomainorotherhalf-lifeextendingmolecule(Liuetal.
,2017);and(v)IgGsthatarechemicallycoupledtogenerateIgG-IgGconjugates(e.
g.
,Brennanetal.
,1985;Garridoetal.
,1990).
ExamplesofthesevebasicformatsareshowninFig.
3.
Manyvariationsonthesecentralthemeshavebeenreviewedmultipletimes(Kontermann,2012;Spiessetal.
,2015;KontermannandBrinkmann,2015;Haetal.
,2016;BrinkmannandKontermann,2017).
Overthepastdecadetherehasbeenaliteralexplosionofnovelbispecicantibodytechnologies,approaches,andclinicalcandidates.
Todaythereareatleast61bispecicorbifunctionalantibodiesinclinicaltrialsthataremadefromatREVIEWWilliamR.
StrohlTheAuthor(s)2017.
ThisarticleisanopenaccesspublicationProtein&Cellleast24differentbispecicplatformtechnologies(Table11).
TheseincludetenasymmetricalIgG-basedplatforms(17bispecicantibodies),veappendedIgGplatforms(17bis-pecicantibodies),asingleplatformforchemicallycoupledIgGs(fourbispecicantibodies),eightfragment-basedplat-forms(22bispecicantibodies),andoneIgG-basedbis-pecicgeneratedwithanunknownplatform(Table11).
Twobispecicantibodieshavethusfarbeenapprovedformed-icaluse,bothintheeldofoncology.
Therstbispecicantibodyofanykindtobeapprovedwascatumaxomab(Removab),abivalent,trifunctional,hybridmouseIgG2a–ratIgG2bantibodytargetingCD3EwithonearmandEPCAMwiththeother.
Catumaxomab,approvedin2009(onlyintheEuropeanUnion)fortreatmentofmalignantascites,wasgeneratedbythethree-wayfusionofamouseB-cell,aratB-cell,andamyelomacelltoformaquadromacellline(Triomabtechnology)(Zeidleretal.
,1999).
Thesecondbispecicantibodytobeapprovedwastheanti-CD3Exanti-CD19"BispecicTCellEngager"(BiTE)MT-103,constructedbylinkingtwoscFvswithaveresidue(G4S)1linker(Macketal.
,1995).
ThisBiTE,nowknownasblinatumomab(tradename,Blincyto),wasapprovedin2014fortreatmentofB-cellacutelymphoblasticleukemia(ALL).
The61currentclinicalstagebispecicantibodiesareusedforavarietyofdifferentpurposes.
Forexample,10ofthembindtwosolubleantigenssuchasIL13andIL4(e.
g.
,SAR156597;NCT02345070),ninebindtworeceptorsonthe"knob""hole"α-CD3ABTriomabKIHDVD2-in-1MATBiTEDuobodylgG-scFvfusionbAdnaTydobonaNChemicalDARTCOOHNH22HNHOOClinkerMAbtyrinCDEα-EpCAMMouselgG2algG2bRatS-SFigure3.
Fivebasictypesofbispecicantibodies.
(A)Bivalent,bispecicIgG-likeantibodieswithheteromericheavychains(e.
g.
,Triomab,knobs-into-holes(KIH),Duobody,etc);(B)TetravalentmultispecicantibodiescomprisedofIgGswithotherbindingdomainsfusedtoeithertheN-orC-terminiofeithertheheavyorlightchains(e.
g.
,dualvariabledomain[DVD],IgG-scFvfusion,Mabtyrin(IgGwithnon-antibodybindingscaffold"centyrin"fusedtoC-terminalendofheavychains);(C)IgGstowhichadditionalantigencombiningsiteshavebeenaddedwithinthestructure(e.
g.
,two-in-oneantibodies,MAT"ModularAntibodyTechnology"platformfromF-Star);(D)Engineeredantibodyfragmentslinkedbyshortpeptidelinkerswhichcanbemadeintobivalent,trivalent,ortetravalentformatsaddressingtwotothreetargets(e.
g.
,bispecicT-cellengager(BiTE),Nanobodyplatform,dual-afnityre-targeting(DART)antibodies,"tandemantibody"structures(TandAbs));(E)ChemicallycoupledIgGs.
InnovativeantibodiesREVIEWTheAuthor(s)2017.
ThisarticleisanopenaccesspublicationProtein&CellTable11.
SummaryofbispecicantibodyplatformscurrentlyrepresentedbyclinicalcandidateantibodiesBispecicantibodyplatformSubgroupGroupinFig.
3LightchainsolutionNumberofclinicalcandidateantibodiesImmunecellredirectedcandidatesMostadvancedcandidateCompanyorinstituteReferenceforplatformRat/mouseTriomabAsymmetricASpecies-specicLCs11ApprovedinEUFresenius,TrionZeidleretal.
,1999ART-IgAsymmetricACLC1(+1IgG-scFv*)0Phase3ChugaiSampeietal.
,2013Knobs-into-holesAsymmetricACLC,CFS51Phase2GenentechRidgewayetal.
,1996;Merchantetal.
,1998DuobodyAsymmetricAPost-productionassembly31Phase1GenmabLabrijnetal.
,2013BiclonicsAsymmetricACLC21Phase1MerusThrosbyetal.
,2016BiMabAsymmetricACLC10Phase1Oncomedhttp://drugspider.
com/drug/navicixizumabAzymetric(ZW1)AsymmetricALCmutations10Phase1ZymeworksVonKreudensteinetal.
,2013XmabHAAsymmetricAFabplusscFv11Phase1XencorMooreetal.
,2011BEATAsymmetricAFabplusscFv11Phase1GlenmarkMorettietal.
,2013ProteinAdifferentialAsymmetricACLC11Phase1RegeneronSmithetal.
,2015;Tustianetal.
,2016IgG-scFvAppendedIgGBNA6*1*Phase2EliLilly,Merrimack,andothersColomaandMorrison,1997DVD-IgandDVD-Ig-likeAppendedIgGBNA30Phase2AbbvieWuetal.
,2007IgG-FabAppendedIgGBCFS21Phase1RocheKleinetal.
,2016IgG-peptideAppendedIgGBNA30Phase1MedimmuneKonkaretal.
,2016IgG-fusionproteinorenzymeAppendedIgGBNA30Phase1Roche,ArmagenBoadoetal.
,2007;2012;2014ChemicallycoupledIgGsChemicallycoupledIgGsENA44Phase2BarbaraAnnKarmanosCancerInstBrennanetal.
,1985;Garridoetal.
,1990REVIEWWilliamR.
StrohlTheAuthor(s)2017.
ThisarticleisanopenaccesspublicationProtein&CellTable11continuedBispecicantibodyplatformSubgroupGroupinFig.
3LightchainsolutionNumberofclinicalcandidateantibodiesImmunecellredirectedcandidatesMostadvancedcandidateCompanyorinstituteReferenceforplatformBiTEAntibodyfragment-basedDNA55ApprovedinUSandEUAmgen(Micrometacquisition)Macketal.
,1995;Schlerethetal.
,2005;Baeuerleetal.
,2008TandAbAntibodyfragment-basedDNA22Phase2AfmedKipriyanovetal.
,1999TandemscFvAntibodyfragment-basedDNA40Phase2EngeneICandseveralothersMadrenasetal.
,2004Dock-and-LockAntibodyfragment-basedDNA10Phase2ImmunomedicsSharkeyetal.
,2010DARTandDART-FcAntibodyfragment-basedDNA65Phase1MacrogenicsJohnsonetal.
,2010;Rootetal.
,2016Nanobody-basedAntibodyfragment-basedDNA20Phase1AblynxVanHeusdenetal.
,2013TCR-scFvAntibodyfragment-basedDNA11Phase1ImmunocoreBossietal.
,2014ADAPTIRTMAntibodyfragment-basedDNA11Phase1AptevoHernandez-Hoyosetal.
,2016UnknowndesignIgG-based-unk10Phase1BioCad(NCT03103451)Totals24differentknownplatforms–61differentcandidates27–––*OneoftheART-IgmoleculesisconstructedasanIgG-singlescFv.
Abbreviations:ART-Ig,asymmetricre-engineeringtechnology-immunoglobulin;BEAT,bispecicengagementbyantibodiesbasedontheTcellreceptor;BiTE,bispecicTcellengagers;DART,dualafnityretargeting(antibody);DVD-Ig,dualvariabledomain-immunoglobulin;LC,lightchain;CLC,commonlightchain;CFS,cross-Fab(orcross-mAb)sequences;Fab,fragment,antibody;Fc,fragment,crystallizable;scFv,singlechain,fragment,variable;LC,lightchain;NA,notapplicable;scFv,singlechainFragment,variable;TandAb,tandemdiabodies;TCR,Tcellreceptor.
InnovativeantibodiesREVIEWTheAuthor(s)2017.
ThisarticleisanopenaccesspublicationProtein&CellsamecellsurfacesuchasEGFRandMET(e.
g.
,JNJ-61186372;NCT02609776),andfourbindacellsurfacetar-getsuchasDeltaLikeCanonicalNotchLigand4(DLL4)withonecombiningsiteandasolubleligandsuchasVEGFwiththeother(e.
g.
,navicixizumab;NCT02298387).
Twocurrentclinicalstagebispecicantibodiesarebiparatopic,i.
e.
,botharmsbindtothesamereceptor,albeitattwodifferentnon-overlappingepitopes(e.
g.
,ZymeworksZW25,whichbindstwonon-overlappingepitopesofERBB2;NCT02892123).
Themostsignicantuseofbispecicantibodies,how-ever,isforTcellredirection,inwhichonecombiningsiteisdirectedtowardacellsurfacetargetonacancercellandtheothercombiningsitebindsCD3EonTcellstoredirectthosecellstothetargetedcancercell(seebelow).
TCELLREDIRECTIONTwenty-sevenclinicalstagebispecicantibodiesareimmunecellredirectionbispecicantibodies.
OneofthesetargetsFCGR3A(CD16a)forNKcellredirection,whiletheother26bispecicantibodiestargetCD3EonTcellstoredirectthecytotoxicTcells(CTLs)tokillandlysecancercells.
Ofthese,14areconstructedfromantibodyfragments,sevenareasymmetricbispecicIgGs,fourareconjugatedbispecicIgGsthatareusedtoactivateTcellsexcorporally(Brennanetal.
,1985;Garridoetal.
,1990),andtwoarebispecicIgGswithappendeddomains(Table11).
ThetwoappendedIgGsalsoutilizeanasymmetricFcformatsothatonlyoneCD3E-bindingarmispresent.
ItisgenerallyacceptedthatthemostpotentTcellredirectingantibodiesarefragments,withunmodiedBiTEsandDARTs(dual-afnityre-targetingantibodies)demonstratingsub-picomolarIC50valuesforinvitrokillingactivities(Mooreetal.
,2011).
Ofthetwoapprovedantibodies,BlincytoisamouseBiTE,whileRemovabisanasymmetricrat/mouseIgG.
Giventhatbothare"rstgeneration"Tcellredirecting,fullymouseantibodiesforverydifferentindications,itisdifculttosaytodaywhichtypeofplatform(fragmentvs.
IgG-based)willultimatelybethemostefcaciousfortreatmentofdiseases.
ThelargerIgG-basedformsappeartobesignicantlylesspotentbasedoninvitroactivitiesandinvivopreclinicaldosingthanarefragments(unpublisheddata).
Thus,thereisabalancebetweensheerpotency,whichcanbeachievedwithsmallsize,andlonghalf-life,whichtypicallybringswithitgreatersizeandlesspotency.
Additionally,boththesizeofthecellsurfacereceptorofthetargetcellsandtheepitopetowhichtheantibodybindsappeartobecriticalfactorsinpotencyaswell(Bluemeletal.
,2010).
Moreover,thepotencyofTcellredirectedbispecicantibodiesdependsontheafnityofthearmsforeachantigen.
TypicallyinthecaseofbispecicTcellredirectionantibodies,theafnityforthecancercellsurfacetargetismuchhigher(i.
e.
,10-foldormore)thantheafnityfortheCD3EchainonTcells(Zhu-kovskyetal.
,2016).
Insummary,factorsthatmayinuencepotencyinTcellredirectedantibodiesaresizeoftheantibody,sizeofthetargetcellsurfaceprotein,epitopeonthatproteintowhichtheantibodybinds,andafnity.
AnotherareathathasnotyetbeenfullyinvestigatedwithrespecttoTcellredirectionistheroleofFcfunctionality.
TheTriomabplatform,onwhichRemovabisdesigned,hasahighlyactiveFcdomainthatinteractswithhumanFcγRstoincreasetheimmuneresponse(Cheliusetal.
,2010;Hessetal.
,2012).
Ontheotherhand,mostofthecurrentfrag-ment-Fc,asymmetricIgG,orappendedIgGplatformshaveusedmutedorsilencedFcssoasnottooverstimulatetheimmunesystemviainteractionswithmyeloideffectorcells.
EvenwiththeabsenceofFcactivity,manytreatmentswithTcellredirectingbispecicantibodiesareaccompaniedbycytokinestormsthatneedtobeaddressedaspartofthetherapeuticparadigm(Leeetal.
,2016).
Thus,itseemslikelythatmostTcellredirectingantibodiesmadeinthefuturewillcontinuetoavoidFcactivityinanefforttolimitthereleaseofpro-inammatorycytokinesbyTcellsandothereffectorcellsinthetumormicroenvironment.
CAR-TCELLSANDTCR-TCELLSCARsareanti-tumortargetedantibodiesthathavebeenfusedgeneticallytoastalkorlinker,atransmembranedomain,andintracellularTcellactivationdomainsthathavebeenborrowedfromactivationcheckpointreceptorssuchasCD28,TNFRSF9(CD137),and/orTNFRSF4(OX40)(Fig.
1M;Figueroaetal.
,2015;vanderStegenetal.
,2015;RuellaandGill,2015;Smithetal.
,2016;RuellaandJune,2016;LimandJune,2017).
WhiletheconceptofCAR-Tcellshasbeenaroundsincetheearly1990s(Eshharetal.
,1993),theadvancementoftechnologiesrequiredtoturnthisintoaviable"manufacturable"processwasonlyrealizedinrecentyears.
Thus,similartobispecicantibodytechnology,whileconceptuallyold,trulydevelopableCAR-Ttechnologyisstillrelativelyyoungandstilldeveloping(LimandJune,2017).
TherearefundamentallytwotypesofCARs.
Therstisautologous,inwhichapatient'sTcellsarecollectedbyaprocessknownasapheresis,andtheneitherasawholepool,orafractionedpoolofCD8Tcells,CD4TcellsorpossiblybothCD4andCD8Tcells,aretransducedwiththeCARsusingeitherviralvectorssuchasLentivirusortrans-posonssuchasSleepingBeautyorPiggyBack(Figueroaetal.
,2015;LimandJune,2017).
TherecombinantTcells,nowarmedwithCARstargetingatumorexpressedontheirsurface,areactivatedandinfusedbackintothepatientsfromwhichtheywerederivedtokillcancercellsbearingtheantigen(Figueroaetal.
,2015).
ThesecondmajortypeofCARisallogeneic,oruniversal.
An"off-the-shelf"celllineisconstructed,typicallydevoidofMHCclassImolecules(Renetal.
,2017a)andendogenousTcellreceptors(MacLeodetal.
,2017;Renetal.
,2017a)todecreasetheriskofhostvs.
graft(rejection)andgraftvs.
hostdisease(GvHD),respectively.
ThisuniversalTcelllinealsowouldexpressCARsfortreatmentofcancerorpossiblyviralinfections.
Thusfar,thebarrierstogeneratetrulyoffthe-REVIEWWilliamR.
StrohlTheAuthor(s)2017.
ThisarticleisanopenaccesspublicationProtein&Cellshelfallogeneiccelllinesarestillquitehigh,withcontrolofproliferation,continuedactivationofthecellsoncetheyareengrafted,andincorporationofkillswitchesforsafetypur-posesascriticalissuesstilltobeworkedout.
Nevertheless,signicantprogresshasbeenmadeinjustthepastyearsuggestingthatfullymodiedallogeneicCAR-Tcelltherapyisquicklybecomingareality(Renetal.
,2017a,b).
Todate,therearefourgenerationsofautologousCAR-Tcellcon-structs.
Therstgenerationtypicallyconsistedoftheextra-cellular,cancercell-targetingscFvfusedtotheCD8stalkandtransmembranedomainfollowedbyCD247(akaCD3ζ),whichprovidedtheactivationsignal(ParkandBrentjens,2010;Figueroaetal.
,2015;LimandJune,2017).
TherstgenerationCARspossessedamplecytotoxicitybutlackedproliferativeandsurvivalsignals.
Thesecond-generationCARstypicallylinkedtheexodomainscFvtothetrans-membranedomainofCD28,TNFRSF9(CD137,4-1BB),orTNFRSF4(OX40)toprovideaproliferationsignal,followedbyCD247(CD3ζ)toprovidethecytolyticactivationsignal.
ThethirdgenerationCARshavetypicallylinkedthetargetingscFvtotheCD28transmembranedomain,followedbyeithertheTNFRSF9(CD137,4-1BB),orTNFRSF4(OX40)acti-vationdomains,andthenCD247(CD3ζ)(ParkandBrent-jens,2010;Figueroaetal.
,2015;Smithetal.
,2016;LimandJune,2017).
TheseCARscombinedcytolyticactivitywithbothproliferationandsurvivalsignalstoenhanceboththeiractivityandtheirpersistenceinthepatient'sserum.
FourthgenerationCARsaddnewactivitiessuchasasuicidemechanismtokillofftheCARsincasetheybecomeover-proliferative,orutilizeTcellsthathavebeenconditionedtorecognizeviralantigenswhichcanbeusedas"vaccines"toincreasethepersistenceoftheCAR-Tconstruct(Ch-mielewskietal.
,2014;Smithetal.
,2016;LimandJune,2017).
Therearecurrently145differentCARconstructsinclini-caltrials.
Asstatedearlier,alloftheCARcandidatesareinphaseIorIIclinicaltrials.
Almosthalf(72/145)ofthecurrentCARsoriginatedinChina,with67originatingintheUS,and6originatinginEurope.
CARshavebeengeneratedagainst38differenttargets,37ofwhicharecell-surfaceproteinsoncancercellsandone,WT1,anMHC-displayedpeptidetar-getderivedfromanintracellularantigen(Raqetal.
,2017).
Fifty-three(37%)clinicalCARcandidatesaredirectedagainstCD19.
ThenextmosttargetedantigensareGD2andMSLN(mesothelin)(8CARseach),ERBB2(HER2)andCD22(7CARseach),andGPC3(glypican-3)andTNFRSF8(CD30)(6CARseach).
MostofthecurrentclinicalstageCARconstructsareautologousCAR-TconstructsgeneratedfromαβTcells(Table12),butthereareafewexamplesofotherformats,includingearlyformatsofallogeneicCAR-Tcells,autologousCARγδTcells,bothautologousandallo-geneicCAR-NKcells,CAR-NKTcells,andCARsmadefromTCRs(Table12).
ItistooearlytojudgethesuccessoftheCAReld,althoughitisclearthatthisareahasgeneratedanenormousamountofinterest,aswellasfundingwellexceeding$1B.
ItisnoteworthythatNovartisrecently(3/29/17)ledabiologicslicenseapplication(BLA)totheUSFDAfortreatmentofrelapsedandrefractoryB-cellacutelymphoblasticanemia(B-ALL)withCTL019(tisagenlecleucel-T),makingittherstCARconstructtobesubmittedforregulatoryapproval(Kingwell,2017).
Moreover,KitePharmaannouncedshortlythereafter(3/21/17)thattheyhadcompletedtheirrollingBLAsubmissionfortreatmentofnon-Hodgkinlymphoma(NHL)usingKTE-C19(axicabtageneciloleucel).
IfeitherCTL019orKTE-C19is,orbothare,approvedwithinthenextyear,itwillmarkahugemilestoneinthisexcitingneweld.
DELIVERYOFANTIBODIESTONOVELCOMPARTMENTSAnareathathasbeenofinterestformanyyears,buthasprovenchallenging,isthetargetingofantibodiestocom-partmentsintowhichtheydonotnormallygo.
Theseinclude,forexamples,targetingantibodiestothegutviaanoralroute,tothebrainbycrossingtheblood-brainbarrier,ortothecytosolicintracellularcompartment.
Allofthesecom-partmentspresentsignicantchallenges,butinthepastfewyears,signicantstrideshavebeenmadeforallofthem.
Themostadvancedtissue-targetedantibody-basedpro-ductthebone-targetedenzymereplacement-Fcfusion,asfotasealpha(Strensiq),whichwasapprovedbytheUSFDAfortreatmentofhypophosphatasia(Hofmanetal.
,2016).
Asfotasealpha(TNSALP-Fc-deca-aspartatefusionprotein)istargetedtobonewithadeca-aspartatepeptidefusedtotheC-terminusoftheFc(Millanetal.
,2008).
Thesecondareaofantibodytargetingthatisrepresentedbyclinicalcandidatesisbasedontherouteofdeliverytogettheantibodiestothedesiredcompartment.
Atleastthreeorally-delivered,antibody-relatedproteinstargetedtotheintestinaltractarecurrentlybeingevaluatedinclinicaltrials.
TheseincludePRX-106(Protalix),ananti-TNFplantcell-expressedanddeliveredFcfusionproteininphaseIIclinicaltrials(NCT02768974)forthetreatmentofulcerativecolitisTable12.
Chimericantigenreceptor(CAR)-andT-cellreceptor(TCR)-basedimmuno-oncologyclinicalcandidates*TypeNumberAutologousCARαβTcells128AutologousCARγδTcells1AllogeneicCARαβTcells6AutologousCAR-NKcells3AllogeneicCAR-NKcells2AutologousCAR-NKTcells2AutologousrecombinantTCR-Tcells3TotalnumberofCARandCAR-likeclinicalcandidates145*FromBiStroBiotechConsultingLLCdatabaseonclinicalstagebiologics.
DatabaselockforthesedatawasApril30,2017.
InnovativeantibodiesREVIEWTheAuthor(s)2017.
ThisarticleisanopenaccesspublicationProtein&Cell(Ilanetal.
,2017).
TheplantcellsarethoughttoprotecttheFcfusionwhiletraversingthroughthestomach.
Overthelastdecade,themouseanti-CD3EmAb,OKT3,hasbeeneval-uatedinclinicaltrialsfororaldeliverytothegutfortreatmentofnonalcoholicsteatohepatitis(NASH;NCT01205087),withresultssuggestiveofclinicalactivity(Lalazaretal.
,2015).
Asecondanti-CD3EmAbformulatedfororaldeliveryisfor-alumab(NI-0401;NovImmune,TizianaLifeSciences),afullyhumanmAbcurrentlybeingpreparedforphaseIIclinicaltrialsfororaldeliveryforthetreatmentofNASH.
Gettingantibodiestocrosstheblood-brainbarrierhasbeenagoalforwellovertwodecades.
IgGlevelsinthehumanbrainareapproximately0.
1%oftheserumconcen-trationof9–10mg/mL(Abbottetal.
,2010).
Thisdifferentialisduetotheblood-brainbarrier(BBB)whicheffectivelykeepsantibodiesoutofthebrain.
Consideringthewealthofpotentialtargetsforbiologicsinthecentralnervoussystem(CNS),therehasbeenagreatefforttondmechanismstoimprovetheabilitytotranslocatebiologicsintotheCNS.
Inrecentyears,signicantprogresshasbeenmadeingettingantibodiestotraversetheBBB.
Yuetal.
(2011)usedabivalent,bispecicantibodybindingTFRC(transferrinreceptor,CD71)withonearmandBACE1(β-secretase-1)withtheotherarm,todemonstratethatlowafnityantibodiestoTFRCweremoreefcientattranscytosisthanhighafnityantibodies.
Whiletheyonlyachievedabout12-foldhigheraccumulationsofantibodyinthebrainovercontrols,theyclearlydemonstratedanti-BACE1pharmacologicalactivityoftheantibody,provingthattheantibodyhadaccumulatedwithinthebrain(Yuetal.
2011).
Theyalsogeneratedabivalent,bispecicantibodytargetinghumanandnon-hu-manprimate(NHP)TFRCwithonearmandhumanBACE1withtheotherarm(Yuetal.
,2014).
Thebestvariants,whichwerelow-to-moderateafnityantibodiestoTFRC,wereaccumulated15-foldhigherinthebrainthancontrolanti-bodiesandtheydemonstratedinvivopharmacologicalactivityinNHPs(Yuetal.
,2014).
Neiwoehneretal.
(2014)comparedtheefciencyoftranscytosisusingatetravalent,bispecicantibodywithtwoarmseachbindingtoTFRCandAPP(amyloid-beta,Aβ)toatrivalent,bispecicantibodywithonlyonearmbindingTFRC.
TheyfoundthatmonovalentbindingtoTFRCpro-motedefcienttranscytosiswhereasbivalentbindingtoTFRCresultedinshuttlingthecomplextowardslysosomaldegradation.
Theydemonstrateda55-foldimprovementintargetengagementoverthecontrol(Neiwoehneretal.
2014).
Incontrasttothesestudiesinwhichmonovalenttargetingofthetranscytoticreceptorwasoptimal,theanti-TMEM30A(α(2,3)-sialoglycoprotein),llamasingle-domainantibody,FC5(Abulrobetal.
,2005),appearedtobetran-scytosedmoreefcientlyasadimerratherthanamonomer(Farringtonetal.
,2014).
Recently,FC5wasfusedinanscFvformattotheN-terminusoftheHCofananti-GRM(gluta-matemetabotropicreceptor1,mGluR1)antagonistIgGtoshuttleitacrosstheBBB(Websteretal.
,2016),achievingpharmacologicalactivitywitha10-foldenrichmentoftheantibodyinthebrainparenchyma(Websteretal.
,2016).
Thus,itstillappearsthatthereismuchtobelearnedaboutoptimizingantibodiesfortranscytoticdeliveryofproteinstotheCNS.
WilliamPardridgeandhiscolleagueshaveisolatedananti-humanINSR(insulinreceptor)antibodythatcanbetranscytosedbyINSRonendothelialcellsliningthevascu-latureinthebrain(Boadoetal.
,2007).
Theyhaveusedtheanti-INSRantibodyasatranscytoticcarriertomoveenzymesacrosstheBBBforCNSenzymereplacementtherapy(ERT)(Boadoetal.
,2012,2014).
ThesecandidatesareconstructedbyfusionoftheenzymestotheC-terminusoftheBBB-traversinganti-INSRIgG"HIRMAb"(Boadoetal.
,2012,2014).
AGT-181,whichisatetravalent(twoantibodyarmsandtwoenzymes)fusionofananti-INSRantibodyandα-L-iduronidase(ALI)(Boadoetal.
,2012),isbeingevalu-atedinphaseIclinicaltrials(NCT02371226)forthetreat-mentofmucopolysaccharidosisI(MPSI;HurlerSyndrome).
AGT-181wasrecentlydemonstratedtobetakenupbynon-humanprimatebrainat1.
2%ofinjecteddoseascomparedto0%injecteddoseofα-L-iduronidasealone(BoadoandPardridge,2017),demonstratingthepharmacologicalrele-vanceoftheBBB-traversingbispecicantibody.
AGT-182,comprisedofafusionofiduronate2-sulfatase(IDS)totheC-terminioftheanti-INSRHCs(Boadoetal.
,2014),isunderphaseIclinicaltesting(NCT02262338)forthetreatmentofmucopolysaccharidosisII(MPSII;HunterSyndrome).
Thenaldelivery-relatedtechnologythathasgottenveryinterestinginrecentyearsisthedeliveryofmAbstothecytosolofcellsviapinocytosisandendosomalescape(Marschalletal.
,2014;Lnnetal.
,2016;Stewartetal.
,2016;Limetal.
,2017).
Multipleapproacheshavebeentakentogetbiologicallyactiveantibodiesintothecytosolofcells,includingtheuseofcellpenetratingpeptides(Mar-schalletal.
,2014;Lnnetal.
,2016;Limetal.
,2017).
Justrecently,auniqueantibodyhasbeengeneratedforthedeliveryofanIgGtothecytosolofcellsviaendosomalescape(Choietal.
,2014).
Thisantibody,whichhasauniquesequenceinitslightchainvariableregion,hasbeenmaturedtoincreasetheproportionofIgGthatentersthecytoplasm(Kimetal.
,2016).
This,andothercellpenetrationtech-nologies(Marschalletal.
,2014;Lnnetal.
,2016;Limetal.
,2017)bringhopethatoneday,antibodieswillbeusedtotargetcytosolicantigens.
NEWFORMSOFDELIVERYOFANTIBODYGENES(DNA,RNA,AAVS,ONCOLYTICVIRUSES)TraditionalformsofdeliveryformAbsandFcfusionproteinshasbeenviaeitherintravenous(IV)orsubcutaneous(SC)administrationofformulatedproteins.
Generally,highdosemAbsforoncologyindicationsarelimitedtoIVdosing,whereaslowdoseantibodiessuchasadalimumab,goli-mumab,andustekinumabcaneasilybedeliveredinSCdoses.
Additionally,inrecentyearstherehasbeenincreasedinterestinintratumoraldosingofantibodiesandotherREVIEWWilliamR.
StrohlTheAuthor(s)2017.
ThisarticleisanopenaccesspublicationProtein&Cellbiologicsforcertaintypesofcancerwherethetumorismoreaccessible(Zeltsmanetal.
,2016).
AnovelapproachfordeliveringmAbsand/orFcfusionproteinsisviadeliveryofthegeneorgenesthatproducethem,eitherasnakedDNA,RNA,orbyaviral-basedvector.
Thisisnotanentirelynewapproach,sincestudiesweredonearoundtheturnofthecenturyshowingthatRNA(Giraudetal.
,1999)andviral(Lewisetal.
,2002)deliveryofIgGgenescouldresultindemonstrationofinvivoIgGactivity.
Nevertheless,therewasnotmuchinterestuntilthepastfewyears,whenithasbecomeevidentthatvectoredornucleicaciddeliveryofIgGcouldpotentiallybeasignicantnewapproachtodeliverantibodiesfortherapeuticuse.
Oneofthemoreexcitingformsofdeliveryistheintra-muscularinjectionofadeno-associatedviruses(AAVs)encodingantibodies,followedbyyearsofconsistentlyhighexpressionofthoseantibodiesinnon-humanprimates(Fuchsetal.
,2016;Greigetal.
,2016).
ItisimportanttonotethatAAVsexistinthemusclecellsasextrachromosomalelementsanddonotintegrate,whichincreasesthesafetyoftheiruseforlongtermexpressionofantibodiesorotherproteins(Greigetal.
,2016).
Thissuggeststhatsuchanapproachmightbeappropriatefordeliveryofanti-HIVanti-bodiestohelppatientseithertobecomecuredor,minimally,lessreliantonhighlyactiveanti-retroviraltherapy(HAART)(SchneppandJohnson,2014a;FuchsandDesrosiers,2016).
Thereareseveralverypromising,potentanti-HIVantibodiesinclinicaltrialscurrently,someofwhichhavebeenexpressedinvivousinggene-baseddeliveryofanti-bodiesforpotentialtherapeuticuse(SchneppandJohnson,2014b;YangandWang,2014;Fuchsetal.
,2016;FuchsandDesrosiers,2016).
Similarly,butwithadifferenttwist,AAV-deliveredanti-bodiestothenasalpassagesofmicehavedemonstratedexcellentprophylaxisagainstuvirus(Limberisetal.
,2013;Balazsetal.
,2014;Adametal.
,2014).
SincetheseAAVsenterepithelialcellsthataresloughedoffoverseveralmonths,thisprovidesapotentiallysaferoutefordeliveryofprophylacticanti-uantibodiesthatwouldcovertheentireuseason.
ThepotentialsignicanceofthisapproachisthatthereareseveralHA-bindingandneutralizingantibodiesavailablenowthatarenearlyuniversalinuenzavirusinhi-bitors.
Thesecouldpotentiallybeusedinclinicaltrialstodeterminewhetherornotthisprophylactic,pan-inuenzanasaldeliveryapproachmightbefeasible.
Finally,theconceptofusingoncolyticvirusestodeliveranti-tumororcheckpointmodulatingantibodiestoatumorisveryexciting.
Oncolyticviruseshavebeenengineeredforyearstodeliverimmune-modulatingmoleculessuchasCSF2(GM-CSF)totheTME(Bommareddyetal.
,2017),soitmakessensethattheycouldbeengineeredtodeliverTMEmodulatingantibodies(Duetal.
,2014).
Severalrecentexampleshavedemonstratedthepotentialforvarioustypesofoncolyticvirusesexpressingimmunecheckpointinhibitorssuchasanti-PDCD1(PD-1),anti-CD274(PD-L1),andanti-CTLA4(Duetal.
,2014;Kleinpeteretal.
,2016;Tanoueetal.
,2017),aswellasotheranti-tumorantibodies(Adelngeretal.
,2015;Liikanenetal.
,2016;Fajardoetal.
,2017).
SUMMARYOverthepastdecadetherehasbeenasignicantshiftfromdiscoveryanddevelopmentofbasicantibodies,e.
g.
,nakedIgG1isotypeantibodieswithnoadditionalengineeringotherthanperhapshumanizationandafnitymaturation,tomoresophisticatedformsofantibodiesinallkindsofshapesandsizes.
ThesenewerformsincludeFc-modied,glyco-engi-neered,bispecic,drug-conjugated,andcellsurfaceexpressedantibodies(i.
e.
,CARs)asnewweaponstoghtdifculttotreatdiseases.
Wenowseethisdramaticshiftinthetypesandnumbersofmodiedantibodiesnowreachingclinicaltrialstudies.
Thisnewphaseofantibodydrugdis-coveryanddevelopmentrepresentsanexcitingandboldnewerathatshouldseeantibody-basedtherapeuticsexpandingtheirinuenceinmanytypesofdiseases.
InthenextfewyearswewilllikelyseetherstregulatoryapprovalsofCAR-Tbasedantibodiesandimmunocytokines,aswellasapprovalsofadditionalnewbispecicantibodies,newADCs,Fcengineeredantibodies,andglyco-engineeredantibodies.
Additionally,weshouldseenewadvancesintargetingantibodiestotheCNSandintracellularcompart-ments,aswellasnucleicacidorviral-vectoreddelivery.
Whatanexcitingtimetobeanantibodyengineer!
ABBREVIATIONSAAVs,adeno-associatedviruses;ADC,antibody-drugconjugates;ADCC,antibody-dependentcellularcytotoxicity;ADCP,antibody-dependentcellularphagocytosis;ADCs,antibody-drugconjugates;CAR,chimericantigenreceptor;CCR5,C-Cmotifchemokinereceptor;CDC,complement-dependentcytotoxicity;CXCR4,C-X-Cmotifchemokinereceptor4;EGFR,epithelialgrowthfactorreceptor;EpCAM,epithelialcelladhesionmolecule;ERBB2,erb-b2receptortyrosinekinase2;GPCRs,G-proteincoupledreceptors;HIV,humanimmunodeciencyvirus;mAb,monoclonalantibody;MS,multiplesclerosis;NK,naturalkiller;RA,rheumatoidarthritis;RSV,respiratorysyncytialvirus;TNF-α,tumornecrosisfactor-alpha;VEGF,vascularendothelialgrowthfactorCOMPLIANCEWITHETHICALGUIDELINESWilliamRStrohldeclaresthathehasnancialinterestinJohnson&Johnson,forwhomhewasrecentlyanemployee,butnootherpotentialconictsofinterest.
Thisarticledoesnotcontainanystudieswithhumanoranimalsubjectsperformedbytheauthor.
OPENACCESSThisarticleisdistributedunderthetermsoftheCreativeCommonsAttribution4.
0InternationalLicense(http://creativecommons.
org/licenses/by/4.
0/),whichpermitsunrestricteduse,distribution,andInnovativeantibodiesREVIEWTheAuthor(s)2017.
ThisarticleisanopenaccesspublicationProtein&Cellreproductioninanymedium,providedyougiveappropriatecredittotheoriginalauthor(s)andthesource,providealinktotheCreativeCommonslicense,andindicateifchangesweremade.
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