Therapeutic complement inhibition in complement-mediated hemolytic anemias: Past, present and future

ContentslistsavailableatScienceDirect

Seminars

in

Immunology

jo u r n al h om ep a g e :w w w . e l s e v i e r . c o m / l o c a t e / y s m i m

Review

Therapeutic

complement

inhibition

in

complement-mediated

hemolytic

anemias:

Past,

present

and

future

Antonio

M.

Risitano

,

Serena

Marotta

Hematology,DepartmentofClinicalMedicineandSurgery;FedericoIIUniversity,Naples,Italy

a

r

t

i

c

l

e

i

n

f

o

Received7March2016

Receivedinrevisedform24April2016 Accepted2May2016

Availableonline23June2016 Keywords: PNH aHUS CAD Complementtherapeutics C5 Eculizumab C3 Compstatin

a

b

s

t

r

a

c

t

Theintroductionintheclinicofanti-complementagentsrepresentedamajorachievementwhichgave tophysiciansanoveletiologictreatmentfordifferenthumandiseases.Indeed,thefirstanti-complement agenteculizumabhaschangedthetreatmentparadigmofparoxysmalnocturnalhemoglobinuria(PNH), dramaticallyimpactingitssevereclinicalcourse.Inaddition,eculizumabisthefirstagentapprovedfor atypicalHemolyticUremicSyndrome(aHUS),alife-threateninginheritedthromboticmicroangiopathy. Nevertheless,suchremarkablemilestoneinmedicinehasbroughttotheforeadditionalchallengesfor thescientificcommunity.Indeed,thelistofcomplement-mediatedanemiasisnotlimitedtoPNHand aHUS,andotherhumandiseasescanbeconsideredforanti-complementtreatment.Theyincludeother thromboticmicroangiopathies,aswellassomeantibody-mediatedhemolyticanemias.Furthermore, morethantenyearsofexperiencewitheculizumabledtoabetterunderstandingoftheindividualsteps ofthecomplementcascadeinvolvedinthepathophysiologyofdifferenthumandiseases.Basedonthis, newunmetclinicalneedsareemerging;anumberofdifferentstrategiesarecurrentlyunderdevelopment toimprovecurrentanti-complementtreatment,tryingtoaddressthesespecificclinicalneeds.They include:(i)alternativeanti-C5agents,whichmayimprovetheheavinessofeculizumabtreatment;(ii) broad-spectrumanti-C3agents,whichmayimprovetheefficacyofanti-C5treatmentbyintercepting thecomplementcascadeupstream(i.e.,preventingC3-mediatedextravascularhemolysisinPNH);(iii) targetedinhibitorsofselectivecomplementactivatingpathways,whichmaypreventearlypathogenic eventsofspecifichumandiseases(e.g.,anti-classicalpathwayforantibody-mediatedanemias,or anti-alternativepathwayforPNHandaHUS).Herewebrieflysummarizethestatusofartofcurrentand futurecomplementinhibitionfordifferentcomplement-mediatedanemias,tryingtoidentifythemost promisingapproachesforeachindividualdisease.

©2016ElsevierLtd.Allrightsreserved.

Contents

1. Introduction...224

2. Thehistoryofcomplementinhibitioninhemolyticanemias...224

2.1. Eculizumabandparoxysmalnocturnalhemoglobinuria...224

2.1.1. Long-termimpactofanti-complementtreatmentinPNH...225

2.2. Eculizumabandatypicalhemolyticuremicsyndrome ... 226

3. Thepresentofcomplementinhibition:unmetmedicalneeds...226

3.1. PNH:hematologicalresponsetoeculizumabandC3mediatedextravascularhemolysis ... 226

3.2. Otherthromboticmicroangiopathies...227

3.2.1. Shiga-toxinproducingE.colihemolyticuremicsyndrome...227

3.2.2. Thromboticthrombocytopenicpurpura...227

3.2.3. Transplant-associatedthromboticmicroangiopathy...227

∗ Correspondingauthorat:HeadofBoneMarrowTransplantationClinicalUnit, Hematology,DepartmentofClinicalMedicineandSurgery,FedericoIIUniversityof Naples,ViaPansini5-80131,Naples,Italy.

E-mailaddress:amrisita@unina.it(A.M.Risitano). http://dx.doi.org/10.1016/j.smim.2016.05.001 1044-5323/©2016ElsevierLtd.Allrightsreserved.

3.3. Antibody-mediatedhemolyticanemias...228

3.3.1. Coldagglutininedisease...228

3.3.2. Paroxysmalcoldhemoglobinuria...228

3.3.3. Autoimmunehemolyticanemias...228

3.4. Rationalefordevelopingnovelanti-complementagents...229

4. Thefutureofcomplementinhibition...229

4.1. Novelinhibitorsofterminaleffectorcomplement...229

4.1.1. Antibody-basedC5inhibitors...230

4.1.2. Smallinterferinganti-C5RNA...230

4.1.3. Coversin...230

4.1.4. Cyclomimetics ... 231

4.1.5. LastgenerationstrategiesofC5inhibition...231

4.2. Inhibitorsofearlycomplementactivation:C3inhibitors...231

4.2.1. Compstatinanditsderivatives ... 231

4.3. Agentsinterferingwithinitialcomplementactivation...233

4.3.1. Selectiveinhibitorsofthealternativepathway...233

4.3.2. Selectiveinhibitorsoftheclassicalpathway...233

4.3.3. Selectiveinhibitorsofthelectin/mannosepathway...233

4.3.4. Inhibitorsbasedonendogenousregulatorsofcomplementactivation...233

5. Concludingremarks...235

References...235

1. Introduction

The complement system is a key component of the innate immunitywhichisfinelyregulatedinhumans.Asfortheadaptive immunity,thephysiologic roleof complement includes protec-tionfrom foreign dangers, mostly infectious agents, aswell as fromself-triggers, like damaged tissues [1,2]. The complement systemalsorepresentsa broadeffector mechanismwhich may playaroleinseveralhumandiseases(e.g.,paroxysmalnocturnal hemoglobinuria [PNH], hemolytic-uremicsyndrome [HUS], kid-neydisorders,age-relatedmaculardegeneration)andconditions (e.g.,sepsis,ischemia/reperfusioninjury,allograftrejection)[2–4]. Thesediseasesmayaffectbasicallyallhumanorgansorsystems; herewefocusondisorderscharacterizedbyacommon hemato-logicalpresentation,whichis hemolysis.Hemolyticanemiasare aheterogeneousgroupofdisorderswhichmayhavecompletely differentcauses;nevertheless,thecomplementsystemhasbeen implicatedaspossiblepathogenicmechanisminmany ofthem. However,sincethepossibleinvolvementofcomplement encom-passesdiseaseswhichtraditionallyhavebeenconsideredlargely independent,asystematicclassificationofcomplement-mediated hemolyticanemiaismissing.Atentativeclassification(seeTable1) maydiscriminatebetweenformscausedbyaprimaryimpairment ofendogenouscomplementregulation(primaryforms),as com-paredwithformscharacterizedbyhyperactivationofcomplement secondarytootherpathogenicevents(secondaryforms). Some-timesthisdistinctionisnoteasy,becauseprimaryandsecondary complement derangements may lead to similar disorders (see forinstancethebroadchapterofthromboticmicroangiopathies, TMA),aswellasprimarydysregulationmayworkasa permis-siveenvironmentwherefurthersecondaryeventsareneededfor thedevelopmentofthedisease.Primaryformsincludethemost typicalcomplement-mediatedhemolyticanemia– namelyPNH –aswellasinheriteddiseasessuchasatypicalHUS(aHUS)and arare congenitaldeficiencyofCD59.While inPNH the impair-ment of complement regulation is restricted toaffected blood cells,eventuallyaccountingforthetypicalhemolysis(seebelow), inaHUSsuchimpairmentissystemic,mostlyinthefluidphase, and it results in possible microangiopathy (aHUS can be con-sideredaprimary,inheritedmicroangiopathy).Secondary forms canbedividedintwosubgroupswithdifferentpathophysiology, accordingtotheeventtriggeringcomplement:(i)auto-immune antibody-mediatedhemolyticanemia(AIHA),and(ii)secondary

thromboticmicroangiopathies.Antibody-mediatedhemolytic ane-mia include cold agglutinine disease (CAD), cold paroxysmal hemoglobinuria (CPH) and otherwarm ormixed auto-immune hemolyticanemias; theseconditionsdifferfor theintrinsic fea-turesofthepathogenicimmunoglobulin(e.g.,antigenspecificity, thermal range and mostly capability of activating the comple-mentcascade),whicheventuallyaccountforthecontributionof thecomplement systemtothemechanismsofhemolysis.TMAs are even more heterogeneous, and include thetypical form of HUS(drivenbybacterialtoxins activatingcomplement),aswell asthrombotic thrombocytopenic purpura (TTP)and transplant-associatedmicroangiopathies(TA-TMA),twoconditionswherethe pathogenicroleofcomplementhasnotyetbeenelucidated.

Here we briefly review the use of therapeutic complement inhibitionincomplement-mediatedanemias,aimingtohighlight how clinicalinterventionscontribute toelucidate complement-mediatedpathophysiology. Based onthesefinding wewillalso reviewthenovelstrategiesofcomplementmodulationswhichare currentlyunderdevelopment,thateventuallyaimtoimprovethe treatmentofdifferentcomplement-mediatedanemias.

2. Thehistoryofcomplementinhibitioninhemolytic anemias

Eculizumab (Soliris®, Alexion) [5] is the first complement inhibitor approvedfor clinical usein humans,initially for PNH and subsequently for aHUS. The experience with this anti-C5 humanizedmonoclonalantibody(mAb),whichinterceptsthe com-plementcascadeatthelevelofitsterminaleffectorpathway,is extremelyinformative.

2.1. Eculizumabandparoxysmalnocturnalhemoglobinuria PNH is a rare hematological disease characterized by three majorclinicalmanifestations:complement-mediated intravascu-larhemolysis,bonemarrowfailureandpropensitytothrombosis [6–8].ThecauseofPNHisaninactivatingsomaticmutationina genecalledphosphatidyl-inositolglycanclassA(PIG-A)[9,10],which eventuallydisablesthebiosynthesisofthe glycosyl-phosphatidyl-inositol(GPI)anchor.Sincethemutationoccursinahematopoietic stemcells,allbloodprogenycellscarrythesameaberrant pheno-typecharacterizedbythelackfromthecellsurfaceofallGPI-linked proteins,includingthetwomajorendogenouscomplement

regula-Table1

Classificationofhemolyticanemiainvolvingacomplement-mediatedpathophysiology.

Disease Mainpathogenicmechanism Complement pathway(s)involved

Therapeutic complementinhibition Primary

ParoxysmalNocturnalHemoglobinuria LackofCD55andCD59on affectedbloodcells

Alternative; theoreticallyalsoCCP andCLP Eculizumab:approved; Novelagentsin investigation(Table2) CongenitalCD59deficiency[198,199] LackofCD59onbloodcells

andallothertissues(e.g., CNS)

Unknown (alternative?)

Eculizumab:onecase reported[200] ThromboticMicroAngiopathies AtypicalHemolyticUremicSyndrome Systemiccomplement

dysregulation(different genesinvolved); endotheliumaskeytarget tissue?

Alternative Eculizumab:approved

Secondary

Ab-mediatedhemolyticanemias ColdAgglutininDisease Coldantibodyfixingthe complement(mostlyEVH, IVHpossibleaswell)

Classical (±alternative?)

Eculizumab:ongoing trial;[90]

TNT009(Table2) ParoxysmalColdHemoglobinuria Donath-Landsteiner

antibody,fixing complement(IVH)

Classical (±alternative?) OtherAuto-ImmuneHemolyticAnemias Warmantibodies(EVH,

mostlythroughFc receptor)

Classical (±alternative?) ThromboticMicroAngiopathies HemolyticUremicSyndrome Endothelialdamage,Shiga

toxin

Alternative? Eculizumab:small series[55–57] ThromboticThrombocytopenicPurpura Hypercoagulation(ULVWF,

ADAMTS13)

Coagulationcascade (±alternative?) Transplant-AssociatedMicroangiopathy Endothelialdamage,

unknown(iatrogenic? Autoimmune?)

Alternative? Eculizumab:small series[80–83]

IVH:intravascularhemolysis;EVH:extravascularhemolysis.

torsCD55andCD59.CD55(alsonamedDecayAcceleratingFactor, DAF)is a regulator ofearlycomplement activation [11], which works inhibitingthe C3convertaseformation (both C3bBband C4b2a),andalsofavoring itsdecay[12].CD59rathermodulates theterminaleffectorcomplement;[13]indeed,itinteractswith C8and C9preventingtheincorporation ofC9 ontothe C5b-C8 complex,therebydisablingtheassemblyoftheMembraneAttack Complex(MAC)[14].Giventhaterythrocytesdonotexpress Mem-braneCofactorProtein(MCP,alsoknownasCD46),irrespectiveof thepresenceofComplementReceptor1(CR1,alsoknownasCD35) CD55andCD59arethekeymoleculeswhichprotectthesecells fromharmfulcomplementactivation.Indeed,theconcomitantlack ofthesetwomoleculesrendersPNHerythrocytesextremely sus-ceptibletocomplementactivation,eventothelow-ratecontinuous spontaneousactivationduetotheC3tick-over,accountingforthe chronicintravascularhemolysiswhichisthehallmarkofPNH.

Eculizumab[5]isananti-C5humanizedmAbwhichprevents itscleavagebytheC5convertase,therebyinhibiting the termi-nalcomplementeffectorpathway;indeed,inabsenceofC5bthe assemblyofthemembrane attackcomplex(MAC)is hampered. Eculizumabwasinitiallyinvestigatedintwolargemulti-national phase III studies, which in 2007 eventually led to the mar-keting authorization for the treatment of PNH [15,16]. In the firstdouble-blind,placebo-controlled,randomizedtrial(TRIUMPH) recruiting86transfusion-dependentPNHpatients,eculizumabled toadramaticreductionofintravascularhemolysis,resultinginthe abolishmentofredblood celltransfusioninhalfofthepatients [15].Asubsequentopen-labelphaseIIItrial(SHEPHERD)confirmed theseexcellentresultsin abroader populationof PNH patients [15].Boththesestudiesdemonstratedanexcellentsafety with-out clinically meaningful side effects; notably, themost feared concernabout possible increased infectious risk was ruled out [15,16],asalsoconfirmedinthefollowingopen-labelextension study[17].Nevertheless,anti-meningococcalvaccinationisneeded forallpatientsreceivingeculizumab;insomeCountries(e.g.,UK

and France) long-termcontinuous pharmacological prophylaxis isalsorecommended.Furthermore,adequateclinicalmonitoring (andtreatment)isappropriateincaseofanyinfectionoccurring duringeculizumabtreatment.Theextensionstudy,inadditionto thesustainedeffectinpreventingintravascularhemolysis,forthe firsttime demonstratedthateculizumab treatmentsignificantly reducestheriskofthromboemboliccomplications[17],likelyasa consequenceofreducedintravascularhemolysis(e.g.,nitricoxide consumptions[18],pro-thromboticmicro-vesicles)orbecauseofa directeffectonPNHplatelets[19,20].Thus,eculizumabisthe cur-rentgoldstandardforhemolyticanemiainPNHpatients,aswellas forthromboembolicPNH;anti-complementtreatmenthasno ben-efitonpossibleconcomitantbonemarrowfailure,andthusshould notbegiventopatientswithaplasticanemiaasmajorpresentation ofPNH.

2.1.1. Long-termimpactofanti-complementtreatmentinPNH Irrespectiveofthehematologicalbenefit,theeffectofterminal complement blockade by eculizumab was excellent in abolish-ing allclinical symptomsof thedisease, and in preventingthe most feared complication,namelythrombosis [15–17]. Aslong as our experiencewith eculizumab extends, we learn that the relevanceofcomplementinhibitioninPNHexceedsasimple sup-portivecare,becauseitseemstoimpactevenonlong-termsurvival of PNHpatients. Indeed,a firstreport fromKellyetal. showed that 5-year overall survival in a cohort of 79 PNH patients on eculizumabwasashighas95%[21],whichexceedsthatseenina smallcohortof30untreatedpatients[21]orfrompreviousnatural historydata[22–24].Morerecently,Loschietal.haveconducted alargeretrospectivecomparisonbetween123patientsreceiving eculizumaband191historicalcontrolsenrolledintheirNational registry,matchedforentrycriteria(i.e.,hemolyticand/or throm-boembolic PNH) [25]. The authors have found that the overall survivalofpatientsoneculizumabwassignificantlybetterthanthat ofuntreatedpatients(92%vs68%at6years),possiblyduetoalower

incidenceofthromboembolicevents(4%vs27%).Therewasno dif-ferenceintermsofriskofdevelopingahematologicalmalignancy (5%),whileaplasticanemiaseemstobemorefrequentinuntreated patients(1%vs10%,butthisfindingneedstobeconfirmed).Thisis thestrongestevidencesupportingthelong-termbeneficialeffect ofanti-complementtreatmentinPNH,whichdefinitely demon-stratesthateculizumabalteredthenaturalcourseofthedisease eventuallyaccountingforaremarkablesurvivalbenefit.Basedon thesedataonemustconcludethat,irrespectiveoftheinitialfear, therapeuticcomplementinhibitionhasbeenprovenextremelysafe andeffectiveinPNH.Giventhisdramaticimpactondiseasecourse, theobviousnextstepinPNHtreatmentistoextendthistherapy toallaffectedpatients,andpossiblyinvestigatingwhether alter-nativestrategiesofcomplementinhibitionmayfurtherimprove theseexcellentresults.

2.2. Eculizumabandatypicalhemolyticuremicsyndrome

Hemolytic Uremic Syndrome (HUS) is a rare disease which encompasseshematologyand nephrology;[26–28]indeed, it is characterizedbyTMAwhichresultsinmechanicalhemolytic ane-mia,plateletconsumption and end-stage renaldisease[26–28]. Thepathophysiologyofthediseaseimpliesanendothelialdamage whicheventuallypromotesplateletactivationandadhesion,with subsequentdevelopmentofmicrothrombi[28].Accordingtothe triggeringcause,twodifferentformsofHUScanbedistinguished: (i)typical,sporadicHUS(usuallycausedbyShiga-toxinproducing EscherichiaColi[E.Coli],STEC-HUS),and(ii)atypicalHUS(aHUS), whichiscausedbyageneticimpairmentofcomplementregulation. Mutationsindifferentcomplement-relatedgeneshavebeen asso-ciatedwithaHUS,suchascomplementfactorH(CFH),complement FactorI(CFI),ComplementFactorB(CFB),MCP,thrombomodulin andC3[29–32].Indeed,thiswell-provencomplement-mediated pathophysiology[27,28] raised the clinicalinvestigation of the anti-C5mAbeculizumabinthisdisorder.Theclinicalefficacyof eculizumabinaHUShasbeendemonstratedintwodistinctphase IIprospectivetrialswhichincludedtwodistinctpatient popula-tions:(i)patientsresistanttoplasmaexchange(PE)and/orplasma infusion(PI),and(ii)patientsrequiringpersistentPE/PI[33].The firsttrialenrolled17aHUSpatientswiththrombocytopenia,and includedasefficacyendpointtheincreaseinplateletcount,as sur-rogatebiomarkerofceasedTMA.Fifteenofthe17patients(88%) achievedaTMA-freestatus,withonly2patientsrequiringfurther PEtherapy.Allthe17patientsachievedanimprovementinplatelet countafter26weeksofeculizumabtreatment,and88%ofthem (100%ofthe15whoremainedontreatment)alsoreachednormal hematologicalvalues.Morethanhalfofthepatientsimprovedin theirrenalfunction,asmeasuredbycreatinineandGFR(among the5patientsondialysis,4didnotrequirefurtherprocedures) [33].Thesecondtrialenrolled20aHUSpatientsonchronicPE/PI, andincludedasprimaryendpointTMA-freestatus(definedasno plateletdecreaseandnorequirementofPE/PIordialysis).Sixteen ofthe20patients(80%)achievedtheprimaryendpoint(andalso normalizedtheirblood counts and LDH),while 4 patientswho alsoachieveddiscontinuationofPE/PIcouldnotbeclassifiedas TMA-freebecause ofpersistent mild thrombocytopenia [33]. In contrasttotheprevioustrial,theimprovementofrenalfunction wasachievedin onlya thirdof thepatients,and afteralonger exposuretoeculizumab[33].Thesedatasuggestthateculizumab effectivelyinterfereswiththeunderlyingcomplement-mediated pathophysiologyofaHUS,asconfirmedbyitsquickeffectonblood counts;nevertheless,incaseofend-stageorgandamageresulting fromalong-lastingpreviousinjury,thefunctionalrecoveryisnot assuredandmayrequirelongertreatmentperiods.Basedonthese data,eculizumabisnowEMAandFDAapprovedandrecommended foraHUS,evenbeforethatacausativemutationcanbefound,once

otherTMAshavebeenclinicallyruledout[28,34,35]. Neverthe-less,theclinicaluseofeculizumabinaHUSstillcarriesanumberof unansweredquestions,mostlyconcerningpatientsselectionand timinganddurationofthetreatment(anddosagesaswell,since forunclearreasonstherecommendeddosesarehigherthanthose commonlyusedinPNH)[36].

3. Thepresentofcomplementinhibition:unmetmedical needs

Theintroduction ofeculizumab represented a majorstep in medicine,sinceforthefirsttimeclinicianswhereableto inter-fere withcomplement asthepathogenicmechanism of several diseases.Nevertheless,theavailabilityofaneffectivetherapythen raisesadditionalmedicalneeds,whichincludethepossible exten-sionofitsusetootherconditions,aswellasthepossibilitytofurther improvethestandardtreatment.Indeed,differentunmetclinical needsmaybeidentifiedinthecontextofcomplement-mediated hemolyticanemias.

3.1. PNH:hematologicalresponsetoeculizumabandC3 mediatedextravascularhemolysis

Aftermore than 10 years of experience witheculizumab in PNH,itbecameevidentthatnotallPNHpatientshavethesame benefitfromanti-complementtreatment,atleastintermsof hema-tologicalresponse.Indeed,about25–40%ofpatientscontinueto requireregularredbloodcelltransfusions,andasimilar propor-tionexhibitspersistent mild tomoderateanemia [15,16,37,38]. Such suboptimalhematological responsetoeculizumabmaybe explainedbythreedifferentcauses.

iThemostintuitive causeis apartialeffectof eculizumab on intravascularhemolysis,whichcanbetrackbypersistently ele-vatedLDHlevels.Intrinsicresistancetoeculizumabhasbeen reportedinafewJapanesepatientscarryingarareC5 polymor-phismwhichhampersthebindingbetweenC5andeculizumab; [39]however,thisisextremelyrareandlikelyrestrictedto spe-cificethnicities,sincealltheotherPNHpatientsshowadequate controlofintravascularhemolysisduringeculizumabtreatment. Nevertheless,eveninsomepatientswithwildtypeC5residual intravascularhemolysiscanbedemonstratedinsome circum-stances,referredtoasbreakthrough.About10–15%ofpatients exhibita“pharmacokineticbreakthrough”,whichusuallyoccurs recurrently1–2daysbeforethenextdosingofeculizumab,and maybenefitfromincreaseddoseofeculizumaborfromreduced dosinginterval[40].Inaddition,irrespectiveofeculizumab lev-els,intravascularhemolysismayreappearinconcomitancewith infectiousepisodes,duetoexaggeratedsystemiccomplement activation;thisphenomenon isknownas“pharmacodynamic breakthrough”, and it is usually self-limiting (evenif severe hemolysismayrequireimmediateredbloodcelltransfusions); iiAnothercommoncauseofpartialhematological benefit dur-ingeculizumabtreatmentisinadequateerythropoiesisdueto aconcomitantbonemarrowfailuresyndrome.Assaidabove, aplasticanemiaisthethirdtypicalpresentationofPNH,which maydevelopanytimeduringthediseasecourse,evenin con-comitance with hemolysis and thrombophilia. PNH patients harboringabonemarrowfailurecanbeeasilyidentifiedbylow orinadequate(forindividualHb levels)reticulocytecountin absenceofsignsorsymptomsofintravascularhemolysis.Since thepathophysiologyof bonemarrowfailureinPNH isbased onaTcellimmune-mediatedresponse[6],anti-complement treatmenthasnoroleinthiscondition,andratheritshouldbe avoided.Indeed,patientswithclinicallyrelevantbonemarrow

failureinthecontextofPNHshouldbetreatedasthosewith idio-pathicacquiredaplasticanemia.Thetwostandard treatment optionsarehematopoieticstemcelltransplantation(HSCT)or immunosuppression[6],thislattereveninconcomitancewith eculizumabifclinicallyappropriate;[41]thechoiceshouldbe basedonpatient’sageandavailabilityofaHLA-identicaldonor; iiiThethirdandmostcommoncauseofresidualanemiainPNH patientsoneculizumabhasbeenidentifiedbyourgroupin2006 andreferred asC3-mediatedextravascularhemolysis[37].In contrasttothetwopreviouscauses,thisphenomenonisrather commonanddetectableinallPNHpatientsoneculizumab,since itresultsdirectlyfromthemechanismofactionofeculizumab. Indeed,eculizumabisexpectedtoinhibittheterminaleffector complementatthelevelofC5,servingasasurrogateofCD59 inpreventingtheMACformation.Hovewer,eculizumabcannot functionallyreplacethelackofCD55,andthusearly comple-mentactivationremainsuncontrolledonPNHerythrocytes.As aconsequence,PNHerythrocytesdonotsuccumbbecauseofthe MAC-mediatedintravascularhemolysis,butremainexposedto C3activation,eventuallyaccumulatingontheirsurfaceC3and itssplitfragments.Withtheirprogressivedeposition,C3 frag-mentsworkasopsoninsforprofessionalphagocytesintheliver andinthespleen,whichharborC3-specificreceptors[42].Thus, inadiseasetypicallycharacterizedbyintravascularhemolysis, eculizumabmayresultinchronicextravascularhemolysisdueto theselectivedestructionofPNHerythrocyteswhichhavebeen exposed to threshold complement activation. Assaid above, thisineluctablebiologicalphenomenonbecomesclinically rel-evant only in aboutone third of patients, for still unknown reasons[37,38,43–45].Polymorphichypo-functionalvariantsof CR1 havebeenfoundassociated witha much higherchance ofclinicallymeaningfulC3-mediatedextravascularhemolysis [46],provingtheconceptthatgeneticvariantsofanyothergene modulatingthecomplementcascademightcontributetothis phenomenon.However,thefullreasonsaccountingforthis het-erogeneityamongpatientsneedtobefurtherelucidated.

Atthemoment,C3-mediatedextravascularhemolysisremainsa medicalconditionlackinganyappropriatetreatment.Indeed,while thereisnodoubtthatsteroidsshouldnotberecommended[47],a possibleeffectivestrategyissplenectomy;[48]however splenec-tomymaybeconsideredonlyinselectedpatients,becauseitmaybe associatedwithintra-orperi-operatorythromboticcomplications andlateinfectiouscomplications[49].Thus,C3-mediated extravas-cularhemolysisrepresentstodaythemosturgentunmetclinical needin hemolyticPNH;becauseof theobviousinvolvement of physiologicC3tick-over,whichinabsenceofCD55leadsto uncon-trolledactivationofthecomplementalternativepathway(CAP), inhibitorsinterferingwithC3orwithearlyphasesoftheCAPare consideredapossibletherapeuticstrategy.

3.2. Otherthromboticmicroangiopathies

Thepossiblebenefitoftherapeuticcomplementinhibitionhas beenhypothesizedinsomethromboticmicroangiopathiesother thanaHUSbasedonthepossibleinvolvementofcomplementin theirpathophysiology(seeTable1).

3.2.1. Shiga-toxinproducingE.colihemolyticuremicsyndrome Shiga-toxin producing E. Coli HUS (STEC-HUS) is the typi-cal,acquired formof HUS which usuallyoccurs acutelyafter a gastrointestinal infection [50]. Clinically, STEC-HUS is indistin-guishablefromaHUS,withthecommontriadofacuterenalfailure, hemolyticanemiaandthrombocytopenia,evenifusuallyithasa self-limitingcourse,withslowrecoveryeveninabsenceof treat-ment[50].In contrasttoaHUS,thediseaseis clearlyassociated

withacolitiscausedbybacteriaproducingST,whicheventually leads toendothelial damage and microthrombi formation [51]. The involvement of the complement systemin its pathophysi-ology is confirmed by thedetection of serum-soluble MAC, C3 and CFBbreakdown fragments(suggestinga majorrole for the alternativepathway astriggeringevent)[52,53]. Sincetheonly availabletreatmentisplasmaexchange[54],thepossiblebenefit ofpharmacologiccomplementinhibitionhasbeenhypothesized, similarlytothekindreddiseaseaHUS.Unfortunately,theeffects ofeculizumabinSTEC-HUShavenotbeensystematically investi-gatedinprospectivetrials;afewpatientswithseverepresentation withneurologicalinvolvementshowedanexcellentoutcomeafter eculizumabtreatment[55].Largerseriesweretreatedduringthe outbreakofE.ColiO104inEuropein2001,withconflictingresults [56,57].Indeed,thedissectionbetweentheeffectofeculizumab andspontaneousrecoveryremainhard,evenifthefast normal-izationofdiseasebiomarkersafterearlytreatmentconfirmsthat eculizumabeventuallydisablethekeypathogenicmechanismof microangiopathy even in the STEC-induced form of HUS [57]. Thus, therapeutic complement inhibition seems appropriate in STEC-HUS,especiallyincaseswithacomplicateddiseasecourse; nevertheless,prospectivetrialsremaindesirableforeculizumabor anyotherinhibitortargetingthealternativepathway.

3.2.2. Thromboticthrombocytopenicpurpura

Thromboticthrombocytopenicpurpura (TTP)isanotherTMA clearlydistinctfromHUS,becauseplateletadhesion/aggregation isduetotheexcessofultralargevonWillebrandfactor(ULVWF) multimericstrings[58].Itisnowwellestablishedthatthediseaseis duetoadeficiencyofADAMTS-13(eitherconstitutionaloracquired [59],e.g.,duetoneutralizingantibodies[60]),ametallo-protease which is responsible for the physiologic processing of ULVWF [61,62].Eveniftheroleofthecoagulationcascaderemainspivotal, quiterecentlynewevidencesemergedsuggestingtheinvolvement of thecomplement systemin the pathophysiologyof TTP[63]. Indeed,C3andC5b-C9canbedetectedontissuebiopsiesobtained fromTTPpatientsoreven in thebloodstream; [64,65]looking attheinter-playbetweenULVWWandthecomplementsystem, ithasbeendemonstratedthatULVWFmaydirectlyactivatethe alternativepathwayonceboundtoendothelialcells[66].Notably, thecross-talkbetweencomplementandcoagulationalsoincludes CFH,whichenhancesADAMTS-13mediatedULVWFcleavage;[67] indeed,inamousemodelusingaknock-outadamts-13the inhibi-tionofCFHbyneutralizingantibodiesisneededtodeveloptheTTP phenotype[68].AllthesedatadescribeascenariowhereULVWF multimericstringsmayactivatethecomplementalternative path-wayatathresholdwhichmayexceedendogenouscomplement regulators(such asCFH),eventually leadingtoendothelial(and bloodcells?)complement-mediateddamage,withsubsequent for-mation of microthrombi [63]. Thus, even if likely it cannot be resolutiveinallpatients,therapeuticcomplementinhibitionmight beclinicallyusefulalsoinsomeTTPcases[69].

3.2.3. Transplant-associatedthromboticmicroangiopathy

Transplant-associatedTMA(TA-TMA)isarareformof microan-giopathyspecificallyoccurringinthecontextofHSCT[70].Asfor alltheotherTMAs,thekeyclinicalfeaturesarehemolyticanemia, thrombocytopeniaandrenalfailure;indeed,thediagnosisisbased onlaboratorycriteriawhichareassociatedwiththeseconditions (i.e.,redcellfragmentation,anemia,increasedLDH,reduced hap-toglobinandthrombocytopenia)[71,72].IncontrasttootherTMAs, thepathophysiologyofTA-TMAhasnotbeenfullyelucidated,even ifadamageoftheendotheliumseemstoplaya majorrole,and theassociationwithdrugs(e.g.,calcineurineinhibitors),immune reactions(e.g.,clinicalgraft vshostdiseaseorsubtleallogeneic immuneresponse)orinfectiouscomplicationsiswellestablished

[70].Evenifthekidneyisthetypicallyaffectedorgan,ithasbeen demonstratedthattheendothelialdamageissystemicanditcan affectotherbodyorgans orsystems,suchasthelungs[73],the gastro-intestinaltract[74],theheartorthecentralnervous sys-tem[75].Moreinteresting, evidencesofcomplement activation havebeenfoundinTA-TMApatients, andelevatedsoluble C5b-C9hasalsobeenindicatedasaprognosticmarkerinthiscondition [76].Themechanismoftheinvolvementofcomplementhasbeen investigatedmoreindetail,lookinginparticulartothe alterna-tivepathway;afunctionalimpairmentofendogenouscomplement regulation,eitherinheritedoracquired,wasfoundin many TA-TMA patients. Indeed, neutralizing anti-CFH antibodies can be detectedinsomepatients;[77]furthermore,arecentstudy demon-stratedthatmorethanhalfofTA-TMApatientshaveatleastone germline mutation/polymorphism in some complement-related genes (e.g., CFH, CFB, CFI, CFD, CD55, CD46, C3, C5, ADAMTS-13,andCFH-relatedgenesCFHR1-5)[78].Thus,similarlytoTTP, TA-TMAseemsassociatedwithexaggeratedcomplement activa-tion(mostlythroughthealternativepathway,possiblydrivenby differenttriggers)whicheventuallyexceedstheregulatory capa-bilityofendogenousmodulators(possiblyfunctionallyimpaired becauseofconstitutionaloracquiredfactors).Thus,a predispos-ingfunctionalderangementofcomplementregulationmayemerge inpresence of HSCT-associatedevents, finallyleading toa sys-temic complement-mediated endothelial damage withpossible end-stage organ disease[77,79]. Because of theseevidences of acomplement-mediated pathophysiology, eculizumabhasbeen investigatedinTA-TMA,withpromisingresultsfromatleasttwo groups.Indeed,afterafirstindexcase[80]deFontbruneetal.have reportedaseriesof12TA-TMApatientstreatedwitheculizumab, showinghematologicalresponsein50%ofcases,with33%overall survival[81].Thesedataareinagreementwiththepediatric expe-riencereportedbyJodeleetal.,whichinitiallyshowedaresponse rateof66%(4of6 treatedpatients)[82].Amore recentreport fromthesameCincinnatigrouponacohortof18patients demon-strateda response rateof61%, withanoverall survivalof 56%, whichwas farbetterascompared withhistorical controls(9%) [83].Notably,inthis studythepre-treatmentextentof comple-mentactivation(basedonC5b-C9plasmalevels)correlatedwith fastereculizumabclearance,whichmayresultinsub-therapeutic plasmalevelsofeculizumab(measuredashemolyticcomplement activity)andlack ofclinicalresponse[83].Thus, thesystematic investigationof anti-complementtreatmentfor TA-TMAandits clinicaloptimizationrepresentsanotherunmetneedinthefield ofcomplement-mediatedanemias.

3.3. Antibody-mediatedhemolyticanemias

Complementisinvolvedalsointhepathogenicmechanismsof mostautoimmunehemolyticanemias,withadifferential contribu-tionaccordingtothespecificcausingantibodies(Ab)(Table1).The possiblyuseoftherapeuticcomplementinhibitioninthesedisease remainsanopenissuewhichiscurrentlyunderinvestigation. 3.3.1. Coldagglutininedisease

Coldagglutininedisease(CAD)isanautoimmuneanemiadue toauto-Abof IgMtypewhich bindtoerythrocytes atlow tem-peratures and cause their agglutination [84,85]. These cold Ab arespecificforsomeerythrocyteantigens(usuallyI,morerarely i)[85]andareabletoactivatethecomplementcascade[84,85]. Thepeculiarityoftheseauto-Abistheirthermalamplitude,which accountsfor theirbinding to erythrocytesurface in the cooler acralcirculation;then,subsequentcomplementactivationoccurs inthewarmercentralcirculation,throughthecomplement classi-calpathway(CCP).TherearedifferentformsofCAD:thetypical primary form is a clonal lymphoproliferative disorder with an

indolent course,characterized bychronic or sub-acute anemia. In contrast,secondaryforms usually occurin thecontext ofan infectiousdisease(e.g.,MycoplasmaPneumoniae,mononucleosis) orassociatedtootherlymphoidmalignancies,andtendstohavea self-limitingcourse[86,87].ThepathophysiologyofCAD,atleastof itshemolyticpresentation,includessurfacecomplementactivation triggeredbyerythrocyte-boundIgM;thismayleadtoMAC assem-blyandsubsequentintravascularhemolysis,butmorefrequently itsimplycauseC3-fragmentopsonizationwhicheventuallycause boosted erythrocyteclearance viatheFc- and the C3-receptors onprofessionalphagocytes[86,87].Thus,theuseofcomplement inhibitorshasbeenpostulatedforCAD,andremarkable therapeu-tic benefit hasbeen demonstrated in a few patientswho have receivedeculizumab [88,89]. A prospectivephase II,open-label study witheculizumab (registered at http://clinicaltrials.gov as NCT01303952)is currentlyongoing, and preliminarydata have justbeenpresented[90].Thestudyincluded13CADpatientswith LDHlevel≥2xuppernormallimit,andshowedanaverage reduc-tionofLDH level of56%(this wastheprimaryendpointofthe study),withreductionoftransfusionalneedin9of10 transfusion-dependentpatients[90].Evenifthetreatmentwitheculizumab wassafeandwelltolerated,theclinicalbenefitwaslimited,likely becauseoftheheterogeneityofthepatientpopulationandoftheir pathogenicmechanisms.Indeed,eculizumabisexpectedtocontrol onlyMAC-mediatedintravascularhemolysis,whichinthemajority ofthepatientscontributesonlypartiallytotheanemia.Sinceitis wellestablishedthatFc-andC3-mediatedextravascular hemoly-sisisthekeypathogenicphenomenoninCAD[86,87],therapeutic complementinhibitionremainsanintriguingtreatmentoptionfor CADpatients,butstrategiesalternativetoeculizumabandanti-C5 agentsmaybeinvestigated(seebelow).

3.3.2. Paroxysmalcoldhemoglobinuria

Paroxysmalcoldhemoglobinuria(PCH)isaveryrare autoim-munehemolyticanemiawhichiscausedabiphasicAb(alsoknow as the Donath-Landsteiner hemolysin) targeting the P antigen of erythrocytes [91]. Similarly to CAD, the Donath-Landsteiner hemolysin binds to erythrocytes at the lower temperature (in contrast to most Ig causing AIHA) in the peripheral blood circulation, and eventually, once it reaches warmer parts of the body, it activates complement leading to MAC-mediated intravascular hemolysis [92]. However, in contrast to CADthe Donath-Landsteinerhemolysin doesnot induceagglutination of red blood cells. PCH is usually polyclonal, and it occurs in the contextofdifferentinfectiousdiseases,suchasmeasles,mumps, influenza,adenovirus,chickenpox,cytomegalovirus,Epstein-Barr virus, syphilis (this wasby farthe mostcommon causein the pastcentury), Haemophilusinfluenzae and Mycoplasma pneumo-nia.PCHusuallypresentswithacuteintravascularhemolysiscrisis, includingsymptoms likehemoglobinuria, chills, rigors, myalgia and nausea; however thediseasecourseis almost always self-limiting and transient,and etiologic treatment arenot needed. However,incase ofseverecrises(whichmayalsoleadtoacute renalfailure)thiswouldbeanotherconditionwhichmightbenefit fromanti-complementtreatment.Atthemoment,theonly anec-doticcasetreatedwitheculizumabwasnotveryencouraging,likely becauseofitsconfoundingassociationwithmultiplemyeloma[93]. 3.3.3. Autoimmunehemolyticanemias

ThemajorityofAIHAareduetowarmAb,ortoAbofmixed type;theseIgG-typeAbleadtoerythrocytedestructiontypically inthereticulo-endothelialcellsofthespleenandtheliver,viathe Fcreceptor[85].Inanycase,evenwarmormixed-typeAbmay fixcomplement,whichmayeventuallycontributetothe mech-anismofhemolysis(mostlybyC3opsonization,morerarelyby MAC-mediatedintravascularhemolysis)[85,92].Thetreatmentof

Classical pathway Lectin pathway Alternative pathway C1q C1r C1s C2 C4 C4b2a C3 MBL MASPs C3bBb C3(H₂O)Bb C3 hydrolysis fB fI P (tick-over) Immune complexes Bacterial LPS

and membranes

C4b2aC3b C3bBbC3b C6 MAC C7 C8 C9 C3 convertases C5 convertases Lytic complex C3b C3a C5 C5b C5a

Novel anti-C5 agents:

• mAbs(LFG316, Mubodina)

• Coversin

• Small peptides (RA101348)

• Aptamersand SOMAmers

• Affibodies

• siRNAs

Broad C3 inhibitors:

• Compstatin and analogs Proximal complement inhibitors (alternative pathway-specific):

• Complement Receptor 1 (CR1)-based proteins*

• Complement Factor H (CF H)-based proteins (TT30, mini-CFH)

• Complement Factor B (CFB) inhibitors

• Complement Factor D (CFD) inhibitors

• Properdin (P) inhibitors

*: may also modulate other complement pathways

Amplification loop

Fig.1. Complementsystemanditstargetedmodulation.

Overviewofthecomplementcascade,includingallmainfunctionalcomponentsandphysiologicregulators.Thethreeactivatingpathways(alternative,classical,and mannose/lectin)areindividuallydepicted,togetherwiththealternativepathwayamplificationloop.Candidateinhibitorsaregroupedaccordingtotheirspecifictarget;their modulatoryeffectsareindicatedbyredlinesinterceptingspecificstepsofthecomplementcascade.Seealsomaintextforamoredetaileddescription.

AIHAincludesseveraloptions,rangingfromsteroidstorituximab andotherimmunosuppressiveagents,orevensplenectomy[94]. Nevertheless,therapeuticcomplementinhibitionmightbeuseful inspecificcircumstances,suchasfulminantcasesof intravascu-lar hemolysis or refractory diseases, even if systematic studies arelacking.Uptodate,justafewanecdoticcasesofsevere, life-threteningAIHAhavebeen reported,which benefitedfromthe useof eculizumab[95],orfroma C1-esteraseinhibitor concen-trate[96,97].Futurestudiesareneededtoassessthepossibleuse oftheseorothercomplementinhibitorsinthecontextofAIHA.

3.4. Rationalefordevelopingnovelanti-complementagents More than a decade of therapeutic complement inhibition demonstratedthatthisapproachisfeasible,safe,andpotentially effectivein differentcomplement-mediated diseases. Neverthe-less,asdiscussedabovenewmedicalneedsemerge,andthenext challengeistoaddresstheseunmetneedsbyimprovingourcurrent complement therapeutics. Indeed, novelinsights in the patho-physiology of different diseases are providing the rationale for developingalternativestrategies ofcomplementinhibition.This isespeciallytrueforPNH,where itbecameobviousthat target-ingC3orearlierstepsofthecomplementcascademightresultin improvedefficacy;however,thelistofdiseaseswhichmay ben-efitfromanti-complementtherapiesisgrowing,andforsomeof themtargetsalternativetoC5maybeconsideredmechanistically moreappropriate(e.g.,CCPinhibitorsforAb-mediateddiseases). Finally,atalmost10yearsfortheapprovalofeculizumab,themain limitation of anti-complement treatment remains its restricted

access: now that the clinical benefit has been largely demon-strated,it appearsunethicalthat abroaderworldwideaccessis hamperedbytheexaggerated cost of thecurrent treatment.All togetherthesearguments,hopefullymorethanfinancialinterest of pharmaceuticalcompanies,should drivethedevelopmentof additionalcomplementmodulators,which mayexploitdifferent strategiespossiblytailoredforindividualdiseases.

4. Thefutureofcomplementinhibition

Aftertheexcellentresultswiththefirstcomplementinhibitor eculizumab,thereisnowasecondgenerationofcomplement mod-ulatorswhicharestartingtheirpreclinicalorclinicaldevelopment [98,99].Table1includesthemostrelevantcompounds,whichare groupedaccordingtotheirspecifictargetsinthecomplement cas-cade(seealsoFig.1);roughly,novelcomplementinhibitorscan be dividedinto inhibitors of theterminal complement effector pathway(i.e.,targetingC5ordownstream complement compo-nents), inhibitorsofearlycomplement activation(i.e.,targeting thekeymoleculeC3)andinhibitors/modulatorsofinitial comple-mentactivation,whichmayalsoworkselectivelyonthedifferent complementactivatingpathways.Fig.2summarisesthestatusof developmentofthemost promisingagents, someofwhomare alreadyinclinicalinvestigationinhealthyindividualsorpatients. 4.1. Novelinhibitorsofterminaleffectorcomplement

Theinhibitionoftheterminalcomplementeffectorpathwayhas beenprovensafeandeffectiveinhumans.Differentstrategiesof

Preclinical: laboratory Preclinical: animals PhaseI: healthysubjects PhaseI: PNH PhaseII Phase II PNH Phase III Approved TT30 APL-2 AMY-101 Coversin ALN-CC5 ALXN1210 AchillionCompdA TNT009 ALXN5500 LFG316 SOBI002 Eculizumab

Anti

-C3

Anti-C5

Anti-CC

P

Anti-CA

P

Novartis anti-FD

Novelmedanti-P Mirococept CDX-1135 C1-INHs Lampalizumab AMY-201

Fig.2.Novelcomplementtherapeuticstowardstheirclinicaltranslation.

Graphicaloverviewofthestatusofartofclinicaltranslationformostrelevantnovelcandidatecomplementtherapeutics(modifiedfromMorganandHarris)[202].Thecolors ofthetargetindicatethephaseofdevelopment(frompreclinicalworktomarketingauthorization):inwhitepreclinicalwork(laboratoryworkoranimalmodels);inlight bluephaseItrials(inhealthyindividualsorinPNH);inredphaseIItrials(inoterhindicationorinPNH);inyellowphaseIIItrialormarketingauthorization.Thetargetis dividedin4quadrantswhichindicatethefourmostrelevantclassesofcounds(basedontheirtargets):upperleft,inhibitorsoftheclassicalcomplementpathway(CCP); lowerleft,inhibitorsoftheclassicalalternativepathway(CAP);upperright,inhibitorsofthecomplementcomponent5(C5);lowerright,inhibitorsofthecomplement component3(C3).Eacharrowindicatesaspecificagentwithitsstageofdevelopment(seemainmanuscriptfordetaileddescription).

C5inhibitorsarecurrentlyunderdevelopment,aimingtoimprove currentanti-C5therapy.Whilethemostobviousneedistotreat patientscarryingtheC5polymorphismaffectingeculizumab bind-ing[39],thesestrategiesmayalsoaimtodevelopagentswithlonger half-life,oradministeredbysubcutaneousinjections,eventually addressingpatients’ desiderataconcerningpossiblereduction of frequenthospitalizations.

4.1.1. Antibody-basedC5inhibitors

Atleast four novelanti-C5 mAb antibodies are currently in clinicalorpreclinicaldevelopment.Indeed,Alexionisdeveloping twoanti-C5mAbwithlongerhalf-life(ALXN1210andALXN5500, thislatterforsubcutaneoususe),whichareexpectedtoreproduce theefficacyofeculizumabwithabettercomplianceforpatients. The first of these compounds, ALXN1210, is currently under investigationinaphaseIItrialrecruitingPNHpatientswithout pre-viousexposuretoanti-complementtherapy(NCT02605993)[100]. NovartisandMorphosysaredevelopingafullyhuman combinato-rialantibodylibraryanti-C5mAbnamedLFG316;thisagentwas initiallydevelopedinalocalformulationforage-relatedmacular degeneration(AMD)andotherophthalmologicdiseases.LFG316is nowinphaseIIassystemicintravenoustherapyinopthalmology (NCT01624636);[101]aproofofconceptstudyinuntreatedPNH patientshasjustbeenlaunched(NCT02534909),anditwillrecruit patientsinJapan(includingsubjectswiththeC5polymorphism) andEasternEurope[102].Anotheranti-C5strategyisbasedona “minibody”,whichisanengineeredAbfragmentincludingonlythe C5-specificvariableregionsofitsparentalanti-C5mAb[103,104]. TheminibodyMubodina®(AdiennePharma&Biotech)invitro

pre-ventsC5cleavageandconsequentiallytheformationofthelytic MAC[105],andithasbeengrantedwiththeorphandrug designa-tionforsomekidneydiseasesbybothFDAandEMA.Inadditionto thesetwomolecules,otheranti-C5Ab(includingbiosimilars)are inpreclinicaldevelopmentbydifferentpharmaceuticalcompanies. 4.1.2. Smallinterferinganti-C5RNA

AnovelstrategyofC5inhibitionwasexploitedbyAlnylamusing aGalNAcconjugatedsmall-interferingRNAduplexspecificforC5 (ALNCC5) [106]. In animals, subcutaneous injection of ALNCC5 resultedinefficientsilencingofC5productionfromtheliver,with robust(>95%)complementinhibitionlastingmorethan2weeks [107].ThesedatawereconfirmedinaphaseI/IItrialinhealthy indi-viduals[108,109],whichdemonstratedthatALNCC5wassafein32 subjectswhoreceivedeithersingleormultipleascendingdoseof thecompound.TheknockdownofserumC5wasashighas99%and aftermultipledoseslasteduptoonemonth;complement inhibi-tionwas>95%[108],whichhopefullywillbeclinicallysufficientfor differentcomplement-mediatedhemolyticanemias.Thistrialalso includedathirdarmrecruitinguntreatedPNHpatients,whichhas starteditsenrollment.

4.1.3. Coversin

Anotheranti-C5 candidate agent is coversin (also knownas OmCI,AkariTherapeutics),asmall(16kDa)proteinofthelipocalin familyisolatedfromthetickOrnithodorosmoubata[110].Coversin bindstoC5preventingitscleavagebytheC5convertases[111,112]. Invitro,coversinwasabletopreventhemolysisofPNHerythrocytes [113,114],eveninpatientscarryingtheC5polymorphism[115].

InaphaseIstudyinhealthyvolunteerssubcutaneousinjectionof coversinwassafeapparentlywithoutevidenceof immunogene-ity,andshowedexcellentpharmacokineticandpharmacodynamic profiles;[114]aclinicaltrialinPNHisreadytogo(NCT02591862) [116].

4.1.4. Cyclomimetics

Rapharma is developing CyclomimeticsTM_{, a class of small,}

cyclic,peptide-likepolymerswithbackboneandside-chain mod-ifications,producedbyribosomalsynthesisofunnaturalpeptides [117].Cyclomimeticshavebeneficialpropertiesascomparedwith naturalpeptides,includingalowriskofimmunogenicity(dueto poorMHCpresentation)andincreasedcellpermeability,stability, potency,andbioavailability(duetothestructuralmodifications). Theleadanti-C5macrocyclicpeptideRA101348showedexcellent inhibitionofhemolysisinaninvitromodelofPNH[118,119],and aclinicaltranslationprogramforPNHhasbeenannounced. 4.1.5. LastgenerationstrategiesofC5inhibition

The spectrum of C5 inhibitors also includes several newer classesofcompoundsexploitingnoveltechnologieswhichallow thedesignofbettertarget-specifictherapies.Aptamersarelarge oligonucleicorpeptidecompoundscreatedbyusinglargerandom sequencepools,fromwheretarget-specificmoleculesareselected [120]. ARC 1905 (ZimuraTM_{, OphthotechCorp. Princeton, NJ)is}

a PEGylated, stabilized oligonucleic aptamertargeting C5 [121] whichiscurrentlyinphaseIIclinicalinvestigationfor ophthalmo-logicdiseases(AMD),atthemomentforlocaluse(NCT00950638) [122].Afurtherevolutionofaptamersexploitsasystematic evo-lutionofligandsbyexponentialenrichment(SELEX);SOMAmers® (SlowOff-rateModifiedAptamers)arethesenovelclassof com-poundswithamorefavorablePK/PDprofile;SOMAmersspecific for different key components of the complement cascade (C5, C3,CFDandCFB)arecurrentlyunderpreclinicaldevelopmentby SomaLogic(Colorado,USA)[123],andcanbeconsideredfor thera-peuticapplication.Anothertechnologyaimingtoidentifytargeted inhibitorsiscurrentlyexploitedbytheSwedishcompanyAffibody, thatisdevelopingsmallantibodymimeticproteins(about6kDa) bya combinatorial proteinengineering approach; asmall non-immunoglobulinproteinwithhighaffinitybindingforC5hasbeen described[124].Morerecently,SwedishOrphanBiovitrumhas cre-atedaC5-specificaffibodyfusedtoanalbumin-bindingdomain (SOBI002;12kDa);SOBI002bindshumanC5withlow-nanomolar affinity(KD∼1nM),anddemonstratedexcellentbioavailabilityin non-humanprimates,withexcellentterminalhalf-life(>2weeks) duetoitsalbumin-bindingmoiety[125].SOBI002hasbeenshown effectiveinpreclinicalmodelsofcomplement(C5)-mediated dis-eases,anditisnowunderinvestigationinaphaseIstudyevaluating safety,tolerabilityandPK/PDinhealthyvolunteers(NCT02083666) [126]. However,the clinicaldevelopmentof thefirst candidate SOBI002hasbeendiscontinueddue totransientadverse events duringthehealthyvolunteerphaseIstudy.

4.2. Inhibitorsofearlycomplementactivation:C3inhibitors Theclinicalexperiencewithanti-C5treatmentinPNHledtothe conclusionthat,atleastinthisdisease,theearlyphasesof comple-mentactivationmaystillaccountforclinicalconsequences,suchas C3-mediatedextravascularhemolysis.Thus,anidealcomplement modulator should intercept the complement cascade upstream (seeFig.1), preventingtheearlyphases ofcomplement activa-tion(e.g.,continuousspontaneousC3tick-over)[127]andmostly defusingtheamplificationmechanisms(i.e.,theCAPamplification loop) which maymagnify initial complement activation occur-ring through anyinitiating pathway.C3 cleavage into C3a and C3bby C3convertases isthe key eventof complement

activa-tion;thus,C3isanobvioustargetforinhibitingearlycomplement activation.A systemic blockade ofC3 activationby mAb (simi-lartotheanti-C5eculizumab)iscomplicatedbytheabundance (∼1mg/mL)ofC3;furthermore,ablockadealong allthe activa-tionpathwaysmaytheoreticallybeassociatedwithincreasedrisk ofinfectiousandautoimmunecomplications.Adifferentapproach triedtotargetselectivelyonlyactivatedC3fragments(C3b/iC3b) rather than nativeC3.Indeed, ananti-C3b/iC3b mAb (3E7,and itsde-immunizedderivativeH17;EluSysTherapeutics),hasbeen testedinPNHinvitro,showingacompleteabrogationof hemoly-sisaswellasofC3depositiononsurvivingPNHerythrocytes[128]. Theseanti-C3mAbworkasselectiveinhibitorsoftheCAP(see para-graph4.3.1),andmaybeofinterestinPNH,ifadequatelymodified. Indeed,completeanti-C3AbcannotbeusedinPNH,becausethey wouldserveasadditionalopsoninsonPNHerythrocyteseventually worseningratherthanreducingphagocyte-mediated extravascu-larhemolysis.Morerecently,engineeredderivativesofH17have beendescribed,withaFabfragmentofH17thatwasinvestigated inananimalmodelofrenaldisorders[129]andmightbecomeof interestevenforcomplement-mediatedanemias.Nevertheless,the mostpromisingapproachtointerceptC3fortherapeutic applica-tionisaclassofsmallcompoundsrelatedtoamoleculenamed compstatin.

4.2.1. Compstatinanditsderivatives

Compstatin is a 13-residue disulfide-bridged peptide which bindstohumanandnon-humanprimate(NHP)nativeC3,andtoits activefragmentC3b[130],preventingthecleavageofC3intoC3b, anddisablingtheincorporationofC3btoformC3/C5convertases [131].Thus,compstatinisabroadC3inhibitorwhichcompletely abrogatescomplementactivationalongallthethreecomplement pathways (CCP, CAP and complement lectin/mannosepathway [CLP]), including thekey CAP-mediated amplification loop (see Fig.1)[132].Thepreclinicaldevelopmentofcompstatinhasbeen pioneeredbyProf.Lambrisabouttwentyyearsago,anditledtothe generationofseveralcompstatinderivativeswhicharecurrently intheirpreclinicalorclinicaldevelopment[133].Indeed,thefirst therapeuticcompstatinanalog4(1MeW)wasinvestigatedinphase IclinicaltrialsforAMDwithpositiveresults[134].Thismolecule wasthenfurtherdevelopedbyPotentiaPharmaceuticalswiththe nameofPOT-4,anditisnowinvestigatedinotherconditionsby ApellisPharmaceuticals,underthenameofAPL-1[133].A second-generationofcompstatin derivativesisnowgrowing, aimingto improvePKandPDprofilesforsystemictherapeuticapplication [135–137].Thesecompounds,delelopedbyAmyndas Pharmaceu-ticals,are considereda verypromising strategy toimprove the efficacyofcurrentanti-complementtreatment,especiallyinPNH. Indeed,theAmyndasleadanalogAMY-101(alsoknownasCp40) [133]wasextensivelyinvestigated inanin vitromodel ofPNH [138].Cp40/AMY-101(anditsN-terminallyPEGylatedderivative PEG-Cp40)completelyabrogatedMAC-mediatedhemolysisofPNH erythrocytes, consistently with a complete complement inhibi-tionoftheterminalcomplement effectormechanisms; [138]in addition,asanticipatedbytheirupstreameffectoncomplement activation, Cp40/AMY-101 also prevented the depositionof C3 opsoninsonPNHerythrocytes [138].Thesedatawerealso con-firmed by another group who has tested different compstatin derivativesindevelopmentatApellisPharmaceuticals(APL-1and APL-2)[139],andledtoclinicaltranslationplanforPNH.These findings demonstratethatthecompstatin familyprevents early complement activationin PNH, eventually predicting a clinical effectonbothMAC-mediatedintravascularandonC3-mediated extravascular hemolysisof PNH erythocytes.Amyndas Pharma-ceuticalshascompletedpre-clinicalstudiesofitscompoundsin non-human primates,showing that unmodified Cp40/AMY-101 hasanexcellentbioavailabilityafterrepeatedsubcutaneous

injec-A.M. Risitano, S. Marotta / Seminars in Immunology 28 (2016) 223–240 Table2

CandidatecomplementinhibitorsindevelopmentforPNHandothercomplement-mediateddiseases.

Target Name Company Classofmolecule Statusofdevelopment Reff.

C5 ALXN1210 Alexion Monoclonalantibody Active.Clinical(PhaseII,PNH) [100]

C5 ALXN5500 Alexion Monoclonalantibody Active.Preclinical(PNH,unknown)

C5 LFG316 Novartis/Morphosys Monoclonalantibody Active.Clinical(PhaseII,AMDandPNH) [101,102]

C5 Mubodina® _{Adienne Monoclonalantibody(minibody) Active.Preclinical(non-PNH,TMAs) [103–105]}

C5 Anti-C5siRNA Alnylam Si-RNA Active.Preclinical(non-PNHandPNH);clinical

(PhaseI,healthyvolunteerandPNH)

[107–109]

C5 Coversin(OmCI) Akari Smallanimalprotein(recombinant) Active.Preclinical(PNH);clinical(PhaseI,

healthyvolunteersandPNH)

[114,116]

C5 RA101348 Rapharma Smallmolecule(unnaturalpeptide) Active.Preclinical(unknown);clinical(PNH,

planned)

[117–119]

C5 ARC1005 NovoNordisk Aptamers Active.Preclinical(non-PNH);clinical(PhaseI,

AMD)

[121,122]

C5 SOMAmers SomaLogic Aptamers(SELEX) Active.Preclinical(non-PNH) [121,123]

C5 SOBI002 SwedishOrphanBiovitrum(Affibody) Affibody(fusedwithalbumin-bindingdomain) Notactive.Preclinical(non-PNH);clinical

(PhaseI,healthyvolunteers)

[121,125,126]

C3(C3b/iC3b) H17 EluSysTherapeutics Monoclonalantibody Notactive.Preclinical(PNHandnon-PNH) [128,129]

C3/C3b 4(1MeW)/POT-4 Potentia Compstatinfamily Notactive.Preclinical(non-PNH);clinical

(PhaseIandII,AMD)

[134]

C3/C3b Cp40/AMY-101,PEG-Cp40 Amyndas Compstatinfamily Active.Preclinical(PNHandnon-PNH);clinical

(PhaseI,healthyvolunteersandPNH,planned)

[138]

C3/C3b 4(1MeW)/APL-1,APL-2 Apellis Compstatinfamily Active.Preclinical(PNHandnon-PNH);clinical

(PhaseI,healthyvolunteersandPNH)

[139–141]

CFB TA106 AlexionPharmaceuticals Monoclonalantibody Notactive.Preclinical(unknown) [99]

CFD Lampalizumab(FCFD4514S) Genentech/Roche Monoclonalantibody Active.Preclinical(non-PNH);clinical(PhaseII,

AMD)

[143]

CFB Anti-FBsiRNA Alnylam Si-RNA Notactive.Preclinical(non-PNH) [107]

CFBandCFD SOMAmers SomaLogic Aptamers(SELEX) Notactive.Preclinical(non-PNH) [121]

CFBandCFD NA-04-WV99; JD-43-RB65 Novartis Smallmolecules(chemicals) Active.Preclinical(non-PNH) [148–150]

CFBandCFD ACH-3856ACH-4100ACH-4471 Achillion Smallmolecules(chemicals) Active.Preclinical(PNH);clinical(PhaseI,

healthyvolunteersandPNH,planned)

[151–153]

Properdin NM9401 Novelmed Monoclonalantibody(andmAbderivatives) Notactive.Preclinical(unknown) [145,146]

C1s TNT003 TrueNorthTherapeutics Monoclonalantibody Active.Preclinical(CAD);clinical(PhaseI,

healthyvolunteersandCAD)

[154–156]

MASP-1,MASP-2,MASP-3 OMS721(anti-MASP-2) Omeros Monoclonalantibody Active.Preclinical(PNHandnon-PNH);clinical

(TMAsandkidneydisorders)

[160–162]

C1r/C1s Cinryze® _{Shire Humanpurifiedprotein(C1-INH) Active.Clinical(approvedforHereditary}

Angio-Edema)

[165,166]

CAPC3convertase TT30(CR2/CFH) Alexion CFH-basedprotein Active.Preclinical(PNHandnon-PNH);clinical

(PhaseI,PNH)

[172–174]

CAPC3convertase Mini-FH Amyndas CFH-basedprotein Active.Preclinical(PNHandnon-PNH) [175]

CAPC3convertase Mini-FH n.a. CFH-basedprotein Notactive.Preclinical(non-PNH) [177]

CAPC3convertase CRIg/CFH n.a. CFH-basedprotein Notactive.Preclinical [179]

CAPandCPC3convertase sCR1(CDX-1135) Celldex CR1-basedprotein Notactive.Preclinical(non-PNH);clinical

(PhaseI,DDD)

[186–188]

CAPandCCPC3convertase Mirococept(APT070) n.a. CR1-basedprotein Active.Preclinical(non-PNH);clinical(Phase

III,renaltransplant)

[190–193]

tions(theterminalhalf-lifeisestimatedinatleast12–24h),with sustainedpharmacologicallevelswhichallowa dailyorbi-daily treatmentschedule[138].ThisexcellentplasmastabilityandPK profileisevenimprovedwiththelong-lastingderivativePEG-Cp40, which harbored a half-life of almost a week [138]. The poten-tialtherapeutic benefitofCp40/AMY-101overstandard anti-C5 treatmenthasbeenrecognizedbymedicinesagenciesfromboth Europe(EMA)andUS(FDA),whorecentlygrantedOrphanDrug designationtoAMY-101forthetreatmentofPNH.Amyndas Phar-maceuticals hasalready started a clinical translation plan with humanstudiesin healthyvolunteersandthen PNHpatients;at themoment,theselectedcompoundistheunmodified Cp40/AMY-101.Cp40/AMY-101waspreferredforitsshorterhalf-lifethatmay allowaquickerrestorationofcomplementactivityincaseof infec-tiouscomplications orofothersideeffects, toruleoutpossible safetyconcernsofsystemicC3inhibition.Assaidabove,different compstatinanalogsareinclinicaldevelopmentatApellis Pharma-ceuticals;afirstphaseIstudyaddingAPL-2(along-lastingversion ofPOT-4)ontop oneculizumabtreatmenthasrecentlystarted (NCT02264639)[140],whereasanotherphaseIinuntreatedPNH patientshasbeenrecentlylaunched(NCT02588833)[141]. 4.3. Agentsinterferingwithinitialcomplementactivation

ThekeyeventincomplementactivationisC3cleavagebyC3 convertasesgenerated alongone ofthethree different comple-ment activatingpathways CCP,CAPand CLP; allthese events eventuallyendintotheactivationoftheharmfulterminal effec-tormechanisms.Sinceeachcomplementpathwayexploitsspecific complementcomponents,inadditiontotargetingC3directlythere isalsothepossibilitytopreventC3activationactingupstream,at thelevelofinitialpathway-specificeventsthatlead toC3 acti-vation(seeTable2andFig.1).Thisstrategymaybeparticularly usefulin diseases where a specificcomplement pathway hasa dominantpathogenicrole,asforinstancetheCAPinPNH,orthe CCPinantibody-mediatedhemolyticanemias.Belowwedescribe differentstrategies tointerceptthecomplement cascade atthe levelofitsinitialevents,upstreamC3activation.Notably,ithas tobehighlightedthatboththeCCPandtheCLPexploittheCAP toamplifytheirinitialresponse;thus,anymodulationoftheCAP wouldineluctablyaffectalsotheproperfunctioningofCCPandCLP. 4.3.1. Selectiveinhibitorsofthealternativepathway

Complement factor B(CFB)and complement factor D(CFD) are the key molecules of the activation along the CAP, which alsoincludesproperdin.CFBbindstoC3(H2O)generatedfromthe

C3tick-over(spontaneoushydrolysisofitsthioesterbond)[127], becoming availablefor cleavage byCFD. Indeed,CFD generates Ba(thenon-catalyticchain)and Bb,theactivecatalytic subunit whichtogetherwithC3(H2O)(orwithC3b)constitutestheactive

C3convertase C3(H2O)Bb (or C3bBb)[142].These CAPC3

con-vertasesarestabilizedbyproperdin,andthenconvertedintoC5 convertasebytheadditionofafurtherC3bmolecule.Thus,one may try to modulate the CAP by targeting any of these com-plementcomponents.Ab-basedstrategies areavailablefor CFB, withtheanti-CFBFabfragmentTA106indevelopmentatAlexion [99,143].Genentech/Rocheisratherdevelopingananti-CFDmAb fragment(FCFD4514S,alsoknownaslampalizumab),whichis cur-rentlyinphaseIIIforophthalmologicdiseases(NCT02247479and NCT02247531)[144,145]. Finally, Novelmedis developing anti-complementagentsbasedonhumanizedanti-properdinmAband anti-properdinantigen-bindingportions[146,147].However,the mostpromisingapproach tointerceptearlyCAPcomponentsis basedonsmallcompoundswhichpreventtheinteractionbetween CFBandCFD.Indeed,smallanti-CFBandanti-CFDinhibitorsare inpreclinicaldevelopmentinNovartis[148,149].Preliminarydata

usingdifferentanti-CFDagentsinPNHinvitrohavebeenrecently presented,showingthatCFDinhibitorspreventbothlysisandC3 opsonizationofPNHerythrocytes[150].DifferentCFDinhibitors arealsoindevelopmentatAchillion(ACH-3856,ACH-4100, ACH-4471);againthesecompoundsexhibitedaselectiveinhibitionof theCAP,withfullpreventionof hemolysisin surrogatemodels ofPNH[151,152],aswellasusingPNHerythrocytes[153].These compoundsseemtohavesomeoralbioavailabilityinNHP,evenif co-administrationofritonavirwasrequiredtosustain pharmaco-logicallevels[150,151].Otherneweranti-CFBstrategiesexploiting eithersmall-interferingRNA(Alnylam[107])orSOMAmers (Soma-Logic,[121])havebeenannounced.

4.3.2. Selectiveinhibitorsoftheclassicalpathway

TheactivationoftheCCPrequirestheactivationofthetetramer C1r2s2,whichistriggeredbyC1qonceithasboundto

immune-complexes(antigen-Ab).Then,theactivatedC1ssubunitscleave C4 into C4a and C4b, which eventually bind to C2 promoting itscleavagebyC1s;theresultingC4b2bcomplexconstitutesthe CCPC3convertase,whichcangenerateC3bmoleculeseventually enabling theCAP-mediatedamplification loop.The key compo-nentsoftheCCPmaybetargetedforaselectivemodulationofthis complementpathway,whichplaysamajorroleinAb-mediated hemolyticanemias.TruenorthTherapeuticshasdescribedan anti-C1smAbTNT003(amouseIgG2a)whichbindstoC1sanddisables its catalytic effectonC4. In anin vitro model of CAD,TNT003 has been shown effective in preventing Ab-mediated surface complementactivation,preventingdirectcomplement-mediated hemolysis triggered by the agglutinins, as well as C3 decora-tionoftargeterythrocytes[154].Indeed,similartoCAPinhibitors in in vitro modelsof PNH, by blockingthe initialAb-mediated CCPactivation,TNT003preventsbothC3-mediatedextravascular hemolysis(whichisthedominantmechanismofhemolysisinCAD) aswellaspossibleMAC-mediatedintravascularhemolysis(which is minimalim mostCAD)[154].Thehumanized versionof this anti-C1smAb(TNT009)seemsaverypromisingagentforCADand otherAb-mediatedhemolyticanemias;[155]TNT009isnowina phaseItrialenrollingbothhealthyvolunteersandCADpatients (NCT02502903)[156].

4.3.3. Selectiveinhibitorsofthelectin/mannosepathway

The CLP hasrecently emerged as a distinct pathway which mayplayaroleincomplementhomeostasisandpossiblyinsome humandiseases.Indeed,duetoitscross-talkwiththeother com-plementpathwaysCCP and CAP,componentsof theCLPmight serve as a therapeutic target. The key proteins of theCLP are themannose-bindinglectin-associatedserineproteases(MASPs, whicharesimilartoC1randC1s),whichactbycleavingC4[157]. MASP-2isthemoleculeinvolvedintheactivationoftheCCP,which thusplayamajorroleinthewell-establishedtypicalCLP[157].In contrast,ithasbeenreportedthatMASP-3maybecrucialforthe properfunctioningoftheCAPbyactivatingCFD[158],evenifthis rolemaybesomehowredundant[159].Sinceinhibitorsof indi-vidualMASPshavebeendescribedbyOmeros[160],theseagents mightbeofinterestforpossibleselectivetherapeuticinterception ofindividualcomplementpathways.Indeed,ananti-MASP-2mAb (OMS721)iscurrentlyinphaseIIforTMAs[161]andrenaldisorders [162].

4.3.4. Inhibitorsbasedonendogenousregulatorsofcomplement activation

In physiologic conditions, the complement system is finely tunedbyseveralcomponentsactingeitherassurfaceproteinsorin thefluidphase.Indeed,theimpairmentoftheseregulatory mecha-nismsiscrucialindifferenthumandisease,suchasthelackofCD55 andCD59inPNH,orcomplement-genemutationsinaHUS.Given

thepleiotropiceffectsoftheseregulatorsofcomplementactivation (RCAs),someofthemmaybeinsomewayexploitedevenfor thera-peuticstrategies.Threedistinctapproachescanbeidentifiedwhich arebasedonthekeycomplementregulatorsC1esteraseinhibitor, CFHandCR1.

4.3.4.1. C1esterase inhibitor. C1esterase inhibitor(C1-INH) is a serineprotease inhibitor (SERPIN) which binds to C1r and C1s disablingtheircatalyticeffectonC4andC2;C1-INHalsohavea similareffectonMASPs,whichmakesitabi-functionalinhibitor ofCCPandCLPatthesametime.ThekeyroleofC1-INHiswell known,sinceitsconstitutionaldeficiencyordysfunctionresultsin thelife-threateningdiseasehereditaryangio-edema(HAE).Indeed, in additiontomodulate thecomplement system,C1-INH plays a major role in regulating bradykinin production by kallicrein anditssubstratehighmolecularweightkininogen[163,164].HAE is characterized by uncontrolledbradykinin generation leading to increased vascular permeability, eventually associated with catastrophic episodes of subcutaneous and submucosal edema whichmay havefatal outcome;thenatural history of HAEhas been changed by the availability of different C1-INH analogs [165].ThefirstC1-INHapprovedforclinicaluse(second comple-mentinhibitor ingeneral,aftereculizumab)is namedCinryze® (ViroPharma/Baxter),which isa humanplasma-derivedprotein preparedbynanofiltration[166].Morerecently,otherthree for-mulationsofC1-INHbecameavailableintheclinic;thefirsttwo, likeCinryze, areobtainedfromhumanplasma(theirnamesare BerinertandCetor[165]),whereasthelastoneisarecombinant C1-INHwhichhasbeenmadeavailablebySalixPharmaceuticals. AlltheseC1-INHsareapprovedfor thetreatmentof HAEin US andEurope;morerecently,theirusehasbeenhypothesizedeven forcomplement-mediatedhemolyticanemias.SinceC1-INHsare selectiveforCCPandCLP,themostappropriatesettingwouldbe Ab-mediatedhemolyticanemias,evenifthisclinicalapplicationis challengingbecauseveryhighdosesarerequired[96,97].In con-trast,evenifsomeinvestigatorshaverecentlyreportedthatCinryze maypreventbothhemolysisandC3decorationofPNHRBCsinvitro [167],theuseofC1-INHinPNHseemsnotappropriate.

4.3.4.2. CFH-based inhibitors. CFHhasa pleomorphicrole inthe regulationoftheCAPbecauseitactsbothpreventingthe forma-tionoftheCAPC3convertaseandacceleratingitsdecay;[168]in addition,itisalsorequiredfortheinactivationofarmedC3bintoits inactivatedformiC3bbyCFI[169].Whiletheuseofrecombinant CFHhasbeenhypothesized(butformulationsarestilllacking),the mostintriguingapproachesaimtogenerateengineered recombi-nantproteinswhichexploitthefunctionalcomplementregulatory domain of CFH. This approach was pioneered by Prof. Holers, whoinitiallydevelopedfusionproteinscombiningtheinhibitory domainof CFH withother protein domains which may recog-nizesitesofcomplementactivation[170].TheleadmoleculeTT30 (AlexionPharmaceuticals)isa65kDaconsistingofthe iC3b/C3dg-bindingdomainofcomplementreceptor2(CR2)fusedwiththe functional domain of CFH; [171] this engineered protein was designedwiththeaimofdeliveryinhibitoryCFHatsitesof com-plementactivation,identifiedbythepresenceofiC3b/C3dg.TT30 hasbeentestedinvitroinPNH,demonstratingacomplete inhi-bitionofMAC-mediatedintravascularhemolysis,aswellasafull preventionofC3fragmentdepositiononsurvivingPNH erythro-cytes [172]. The inhibitory effectof TT30 was dose-dependent, andrequiredanefficientmembrane-targetingonerythrocyte sur-face;indeed,ananti-CR2mAbimpairingthebindingofTT30to C3resultedinpartialreversionofitsinhibitoryeffect[172].These observationsanticipate thatTT30 shouldinhibitMAC-mediated intravascular hemolysis typical of PNH, and may also prevent C3-mediatedextravascularhemolysiseventuallyemergingduring

anti-C5therapy.Asingleascendingdose,phaseIstudywithTT30in untreatedPNHpatientshasjustbeencompleted(NCT01335165); [173]preliminaryresultsshowedthatTT30wassafeandwell toler-ated.InitialPKandPDdatademonstratethatTT30treatmentmay resultinpharmacologicallyrelevantCAPinhibitioninPNH,as sup-portedbyLDHdecrease;nevertheless,furtherclinicaldevelopment ishamperedbytheshorthalf-lifeofthiscompound[174].A differ-entapproachaimingtoimprovesurfacetargetingofCFHexploits anengineeredminiaturizedversionofCFH,namedmini-FH; differ-entversionsofmini-FHhavebeendescribed[175–177],allaiming tomaximizetheCAPinhibitoryeffectofCFH(C3convertasedecay andco-factoractivities)atsitesofcomplementactivation[175]. Mini-FH (Amyndas, AMY-201) is a small (43kDa) protein con-sisting oftheregulatorycomplement controlprotein(CCP) 1–4 domainsofCFHattachedtoitsCCP19–20domains(which har-bora C3binding-site)[175].Onceinvestigatedin PNH invitro, resultswereoverlapping tothose observedwithTT30:mini-FH effectivelyinhibitedintravascularhemolysisofPNHerythrocytes, aswellastheiropsonizationbyC3fragments[175].Apparently, completeCAPinhibition wasachievedwithmini-FHat concen-trationabout10-foldlowerthanthoseseenwithTT30;[175]this wasconfirmedinaformalcomparisonofmini-FHwithanother versionofaCFH-CR2fusionprotein[178].Allthesemini-FH ver-sions,aswellasanotherCFH-basedproteinfusingCFHwiththe complementreceptoroftheimmunoglobulinsuperfamily(CRIg) asthetargetingmodule[179],remainexcellentcandidateagents tobedevelopedforPNH.Tocompletethelistofcomplement thera-peuticsconceptuallyexploitingCFH-basedmodulation,adifferent strategywasrecentlyinvestigatedbyAmyndas,which is devel-opingCFH-bindingpeptides(AMY-301);indeed,ifappropriately targeted onspecific tissues which are undergoing complement activation,thesepeptidesmaybeabletorecruitendogenousCFH preventingtissue-specificcomplement-mediateddamage[180]. 4.3.4.3. CR1-basedinhibitors. CR1isanotherRCAwhichworks sim-ilarlytoCFH asCFIco-factor and regulatingC3/C5convertases; [181–183]however,in contrasttoCFH,CR1exerts itseffecton alltheCAP,CCPandCLP,becauseitbinds alsotoC3bandC4b includedintheconvertasesoftheCCP(andoftheCLP)[184,185]. ThatCR1playsamajorroleinendogenouscomplementregulation is confirmedalso byclinical observations in PNH, since a low-expressionpolymorphismofCR1isassociatedwithalowerchance ofgoodclinicalresponse toeculizumab [46].Furthermore,PNH erythrocytescarryingthehypomorphicCR1alleleharborafaster andincreasedsurfacedepositionof C3fragments onceexposed tocomplementactivationinvitro;[46]thefactthatthiseffectis morepronouncedinpatientshomozygousforthepolymorphism (ascomparedtotheheterozygousones)clearlysupportsthatthe amountofsurface-bound CR1is essential tomodulate comple-mentactivation,especiallywhenothercomplementregulatorsare lackingsuchasonPNHbloodcells[46].

AsforCFH,differentstrategiestodevelopCR1-basedinhibitors havebeencarriedout.AsolubleformofCR1(sCR1,TP10)named CDX-1135abrogatingallthecomplement pathwayswas devel-opedbyCelldex.CDX-1135hasbeenshownsafeinmorethan500 patientsenrolledindifferentclinicaltrials,withpharmacological effectivelevelsachievedwithoutrelevantsideeffects.Preclinical datafromdensedepositdisease(DDD,aC3-mediated glomeru-lopathy)haveshownexcellentefficacyofCDX-1135inamouse model [186], and some clinicalbenefit has been reported in a short-termcompassionatetherapyinachild[187].However,its furtherclinical developmentis limited toa pilot trialfor DDD that hasbeenstarted and terminated (NCT01791686);[188]at themomentitisnotcleariffurtherclinicalplansexistfor CDX-1135.AnothercandidateCR1-basedproteinismirococept(APT070) [189],anengineeredmoleculeconsistingofthefirstthreeshort