Seminars in Hematology ] (2018) ]]]–]]]

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Seminars in Hematology

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Review

Expanding Complement Therapeutics for the Treatment of ParoxysmalNocturnal Hemoglobinuria

Dimitrios C. Mastellos, PhDa, Edimara S. Reis, PhDb, Despina Yancopoulou, PhDc,Antonio M. Risitano, MD, PhDd, John D. Lambrisb,⁎

a Division of Biodiagnostic Sciences and Technologies, INRASTES, National Center for Scientific Research “Demokritos”, Athens, Greeceb Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PAc Amyndas Pharmaceuticals, Glyfada, Athens, Greeced Hematology and Bone Marrow Transplant Unit, Department of Clinical Medicine and Surgery, Federico II University of Naples, Naples, Italy

a r t i c l e i n f o

Keywords:Complement therapeuticsPNHC3 inhibitorsExtravascular hemolysisCompstatinsClinical trials

doi.org/10.1053/j.seminhematol.2018.02.00263/$/& 2018 Elsevier Inc. All rights reserved.

esponding author. John D. Lambris, Departmdicine, Perelman School of Medicine, Univers19104.ail address: lambris@pennmedicine.upenn.edu

a b s t r a c t

Paroxysmal nocturnal hemoglobinuria (PNH) is widely regarded as an archetypal complement-mediateddisorder that has propelled complement drug discovery in recent decades. Its pathology is driven bychronic complement dysregulation resulting from the lack of the glycosyl phosphatidyl inositol-linkedregulators DAF and CD59 on susceptible erythrocytes. This complement imbalance fuels persistent C3activation on affected erythrocytes, which culminates in chronic complement-mediated intravascularhemolysis. The clinical application of eculizumab, a humanized anti-C5 antibody that blocks terminalpathway activation, has led to drastic improvement of therapeutic outcomes but has also unveiledhitherto elusive pathogenic mechanisms that are now known to contribute to the clinical burden of asignificant proportion of patients with PNH. These emerging clinical needs have sparked a trueresurgence of complement therapeutics that offer the promise of even more effective, disease-tailoredtherapies for PNH. Here, we review the current state of complement therapeutics with a focus on theclinical development of C3-targeted and alternative pathway-directed drug candidates for the treatmentof PNH. We also discuss the relative advantages and benefits offered by each complement-targetingapproach, including translational considerations that might leverage a more comprehensive clinicalintervention for PNH.

& 2018 Elsevier Inc. All rights reserved.

Everything changes and nothing stands still

Heraclitus (c. 535-c. 475 BC)

Introduction

Pathophysiology and Clinical Landscape of Paroxysmal NocturnalHemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is a chronic,debilitating hematological disorder characterized by the clonalexpansion of hematopoietic stem cells (HSCs) and their progeny,mature blood cells, which carry an acquired somatic mutation inthe phosphatidyl-inositol glycan class A (PIG-A) gene [1]. PIG-A

ent of Pathology and Labora-ity of Pennsylvania, Philadel-

(J.D. Lambris).

codes for an enzyme that is essential for the biosynthesis of theglycosyl phosphatidyl inositol (GPI) anchor, a protein modificationallowing the attachment of proteins to the cell membrane [2]. Thepreferential expansion of these PIG-A mutated HSCs leads to therelease of red blood cells into the circulation that lack, amongother GPI-anchored proteins, the two key complement regulatorsCD55 and CD59 [3,4]. As a result of this deficiency, PNH eryth-rocytes are incapable of withstanding physiologic complementactivation (ie, due to spontaneous C3 tick-over or bystanderactivation) and undergo persistent C3 opsonization and terminalpathway activation that culminate in membrane attack complex(MAC)-mediated intravascular hemolysis. In fact, complement-mediated hemolytic anemia is one of the three cardinal featuresof PNH, along with bone marrow failure and thrombophilia [4–7].

Several studies have indicated that PIG-A inactivation is likelyinsufficient to trigger the disease process, indicating that addi-tional mechanisms are causally involved in driving PNH patho-genesis [8–10]. Both preclinical data and clinical observations haveprovided a concrete framework for adopting the “dual pathophysi-ology” hypothesis for PNH [8,11,12]. According to this hypothesis,

D.C. Mastellos et al. / Seminars in Hematology ] (2018) ]]]–]]]2

an (auto)-immune attack against normal hematopoiesis, similar tothat seen in aplastic anemia, eventually results in the relativeexpansion of PIG-A mutated HSCs within the bone marrow [12].An autoimmune basis for PNH pathophysiology is supported byboth clinical and experimental observations, including the well-known clinical overlap between PNH and aplastic anemia [13] andthe presence of GPI-specific autoreactive T-cells that selectivelytarget normal HSCs for immune destruction. This aberrant T-cellrepertoire apparently spares the PIG-A mutated hematopoieticprogenitors that ultimately expand as a result of this selectiveimmune pressure [14–16]. Whereas mounting evidence lendsfurther credence to this hypothesis the precise molecular mecha-nisms underlying this “immune escape” of PNH cells still remainill-defined.

Intravascular hemolysis is the most typical manifestation of thedisease, affecting to a variable extent all patients with clinical PNH[4,5,17,18]. The second defining clinical feature is cytopenia that ismostly secondary to the underlying bone marrow disorder, whichis embedded in the dual pathophysiology of PNH [8]. The thirdtypical manifestation of PNH is thrombophilia, with thromboem-bolic complications being the main cause of mortality amongpatients with PNH [19–21].

Current Treatment Paradigm: Targeting C5

The treatment of PNH has dramatically changed since theintroduction of the first clinically approved complement C5 inhibitor,eculizumab (trade name Soliris, Alexion). Eculizumab is a recombi-nant humanized monoclonal antibody that selectively targets theterminal complement component C5, preventing its cleavage intoC5a and C5b and the assembly of the pore-forming MAC [22].Notably, the discovery of eculizumab was spearheaded by studiesdating back to the late 1980s, when the first monoclonal antibody(mAb) against murine C5 (BB5.1) was generated [23]. Followingproof-of-concept studies of therapeutic C5 inhibition in rodentmodels [24], Alexion developed a primate and human C5-specificmAb and, finally, the humanized antibody eculizumab [25,26].

The efficacy of eculizumab in controlling intravascular hemol-ysis in patients with PNH was consolidated in 2 large multicentertrials [27,28], which recorded improved clinical responses(ie, reduced transfusion needs, hemoglobin stabilization, and theresolution of all hemolysis-related symptoms). Sustained inhib-ition of the terminal complement pathway and abrogation ofsubsequent intravascular hemolysis may result in transfusion-independence in about half of the treated patients, with somepatients also achieving a substantial increase in their hemoglobinlevels. Furthermore, eculizumab seems to reduce the risk ofthromboembolic events [29], possibly resulting in improvedlong-term survival [30]. Despite its profound clinical gains inPNH, anti-C5 therapy entails high annual costs for treatment andrequires bimonthly intravenous infusions in a hospital setting inmost countries, thereby limiting patient compliance and interfer-ing with the patient’s social activity and productivity at work.

Emerging Clinical Needs in the Era of Anticomplement Therapy

The advent of anti-C5 therapy has brought clinical benefit to~70% of patients with PNH [31]; however, a significant proportionof patients receiving anti-C5 are either suboptimal responders orunresponsive to therapy and still require blood transfusions totreat residual anemia [32–35].

An insufficient response to anti-C5 therapy may be attributedto several factors. Bone marrow failure is the most obvious reasonbehind a poor hematological response; however, this conditioncannot be treated with complement inhibition and may requirealternative measures (ie, either bone marrow transplantation or

immunosuppression) [6]. “Breakthrough” intravascular hemolysishas been described in about 10%-15% of patients with PNH,potentially requiring an increased dosage of eculizumab (pharma-cokinetic breakthrough); in addition, some in vitro observationssuggest that, regardless of its levels, eculizumab may fail tocompletely prevent intravascular hemolysis in circumstances thatevoke massive complement activation (“pharmacodynamic break-through”) [36]. A pharmacodynamic breakthrough appears tocorrespond clinically to hemolytic paroxysms observed at the timeof infections in patients with PNH under eculizumab treatment.This insufficient response to eculizumab may be attributed to aforceful alternative pathway (AP)-driven amplification of C3bdeposition at high surface densities that curtails the full inhibitoryeffect of anti-C5 agents [37].

More importantly, anti-C5 treatment has unmasked a novelpathogenic mechanism that partly explains the limited hemato-logic benefit afforded to some patients with PNH by C5 blockade.Persistent C3 opsonization of surviving PNH RBCs coupled touncontrollable AP amplification, resulting from the geneticabsence of GPI-linked CD55, may lead to C3-mediated extrava-scular hemolysis and consequent anemia, which exacerbate thepatients’ long-term clinical course [32,33]. Complement regulationremains impaired on PNH erythrocytes as a result of the lack ofCD55; more importantly, deregulated C3 fragment deposition onPNH cells promotes the phagocytic engulfment of C3-tagged PNHerythrocytes by hepatosplenic macrophages. It is now well estab-lished that extravascular hemolysis fueled by persistent AP dysre-gulation on PNH cells is the most important cause of theinsufficient response to eculizumab [35,38]. Remarkably, thismechanism does not apply only to the one-third of PNH patientswho remain transfusion-dependent on eculizumab; indeed, C3opsonization and subsequent extravascular hemolysis can bedocumented with variable clinical signs in all patients with PNHon eculizumab [34,35]. Recent studies have provided mechanisticinsight into the basis for C3-mediated extravascular hemolysis,showing that C3dg-opsonized RBCs from eculizumab-treatedpatients with PNH are recognized and efficiently phagocytozedby macrophages or monocytes in vitro [39].

A genetic basis for the refractory phenotype of certain PNHpatients receiving anti-C5 treatment has been identified, withcomplement gene polymorphic variants (ie, C5 and CR1) impli-cated as genetic modifiers that skew therapy responses, forexample, by modifying recognition of eculizumab’s epitope onC5 or altering the inherent capacity of PNH cells to withstand C3activation [40,41].

Overall, even though the clinical experience gained so far witheculizumab has consolidated its clinical efficacy for patients withPNH, it has also revealed previously elusive pathogenic mecha-nisms that cannot be addressed by the standard treatment. Theseclinical observations have accentuated the need for developinganticomplement agents that can afford broader therapeuticefficacy by intercepting multiple disease-exacerbating processes.This effort may yield significant clinical gains with profoundsocioeconomic effect, if coupled with drug optimization strategiesthat can enhance patient compliance (ie, via alternative dosingroutes), and offer wider access to a more affordable therapeuticoption.

Novel Therapeutic Avenues for Tackling Complement Dysregulation inPNH

The need for broadly inhibitory complement therapeutics thatcan further improve hematological responses in PNH has mate-rialized into a creative toolbox of diversified complement-target-ing approaches and drug candidates (Fig.) [38,42,43]. Being theobvious target for tackling intravascular hemolysis, C5 inhibition

Fig. Schematic overview of the complement activation cascade illustrating targets for therapeutic modulation in PNH. Complement activation via any of the 3 pathways(CP ¼ classical, AP ¼ alternative, or LP ¼ lectin pathway) triggers a cascade of proteolytic reactions that converge at the cleavage of the central component, C3, by short-lived enzymatic complexes, termed C3 convertases of the CP (C4b2b) or AP (C3bBb). In the absence of the GPI-linked AP regulator DAF/CD55, PNH erythrocytes undergouncontrollable C3 opsonization that leads to C3b deposition at high surface densities. This AP-amplified opsonic turnover triggers C5 convertase activity that leads to MACassembly on target cells. In the absence of CD59, regulation of MAC formation on PNH erythrocytes is impaired and consequently this persistent C3 opsonization culminatesin chronic, MAC-mediated intravascular hemolysis, one of the cardinal clinical manifestations of PNH. In the era of eculizumab treatment, C3-mediated extravascularhemolysis of C3-opsonized PNH cells in the hepatosplenic compartment has emerged as a significant unmet clinical need for PNH patients who are suboptimal responders toanti-C5 therapy. Furthermore, a fraction of PNH patients may show incomplete response to eculizumab due to residual intravascular hemolysis which is attributed topharmacodynamic or pharmacologic breakthrough during inhibitor dosing. These unmet needs have sparked the development of next-generation complement therapeuticsthat can modulate C3 or C5 activation and also target AP convertase activity (shown in callout boxes). In principle, C5 blockage results in complete abrogation of intravascularhemolysis without however affecting upstream AP dysregulation. Conceivably, the application of different anti-C5 agents (or their combination) may offer deeper terminalpathway inhibition and/or improved patient compliance through alternative dosing routes. Upstream complement intervention at the level of C3 is anticipated to affordbroader inhibition of complement responses that drive PNH pathology. C3 inhibition can abrogate both intravascular and extravascular C3-mediated hemolysis while AP-targeted inhibitors can also afford broader coverage by controlling AP amplification and downstream effector generation.

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has retained the lion’s share in the pipelines of pharmaceuticalcompanies with at least six anti-C5 mAbs in preclinical/clinicaldevelopment, a small-interfering RNA (siRNA), and two smallmolecule-based approaches also being evaluated (Table) [38,42].A second and highly promising approach for clinical interventionaims at blocking the central hub of the cascade, C3, with broadlyinhibiting peptidic drugs that protect the native protein fromconvertase-mediated activation, thereby abolishing all down-stream effector functions. A third promising approach targetsthe AP, the pathway fueling chronic pathology and residualextravascular hemolysis, with inhibitors that either dismantle theAP C3 convertase (C3bBb) or block the function of individualAP components (factors B and D, or properdin) [38,42,44].This class of therapeutics includes mAbs, small-moleculeinhibitors, as well as engineered fusion proteins encompassingthe regulatory activity of endogenous AP regulators, such asFactor H (Table) [45].

Alternative Anti-C5 Agents

The ongoing clinical experience with eculizumab has bolsteredconfidence in complement therapeutics that target C5 [42]. How-ever, the identification of patient subgroups that fail to optimallyrespond to eculizumab because of inherited genetic polymor-phisms, and the partial hematologic responses observed in certaineculizumab-treated patients as a result of breakthrough hemolysishave accentuated the need for alternative anti-C5 therapeutics thatcan circumvent such obstacles [35,40]. In this respect, a fullyhuman anti-C5 antibody that binds to a different epitope on C5than eculizumab (tesidolumab, LFG-316, Novartis) is currently inclinical evaluation as a treatment option for patients with PNHrefractory to eculizumab therapy [46]. In an effort to improve thebioavailability and plasma residence of anti-C5 agents, Roche, incollaboration with Chugai, has developed a humanized anti-C5recycling antibody (SKY59/RO7112689) with improved circulatory

TableComplement therapeutics in various stages of development for PNH and other hematological disorders.

Complementtherapeutic

Entity Target Mechanism of action Mode ofadministration

Pharmaceuticalsponsor

Stage ofdevelopment

Eculizumab(Soliris)

mAb C5 Inhibition of C5 activation/blockage of C5convertase activity

IV infusions Alexion Pharmaceuticals In the clinic

ALXN1210/ravulizumab

mAb C5 C5 inhibition/same epitope as ecu Bimonthly IVinfusions

Alexion Pharmaceuticals Phase II/III

ABP959 mAb C5 Inhibition of C5 activation/biosimilar of ecu IV infusions Amgen Phase IIISKY59/RO7112689

mAb C5 Inhibition of C5 activation/different epitopefrom ecu

IV and SCinjections

Hoffmann-La Roche Phase I/II

LFG-316/tesidolumab

mAb C5 Inhibition of C5 activation/different epitopefrom ecu

IV infusions Novartis Phase II

REGN3918 mAb C5 n.a. n.a. Regeneron Phase IMubodina minibody (Fab

based)C5 C5 inhibition/ different epitope from ecu n.a. Adienne Preclinical stage

Coversin (OmCI) Recombinantprotein

C5 Inhibition of C5 activation SC injections Akari Therapeutics Phase II

RA101495 Peptide C5 Allosteric inhibition of C5 activation Daily SCinjections

Ra Pharmaceuticals Phase II

Cemdisiran(ALN-CC5)

siRNA C5 Silencing of hepatic C5 production SC injections Alnylam Phase II

AMY-101 Peptide C3 C3 inhibition/ blockage of C3 convertaseactivity

Daily SCinjections

AmyndasPharmaceuticals

Phase I completed/Phase IIannounced

APL-2 PEGylatedpeptide

C3 C3 inhibition/ blockage of C3 convertaseactivity

Daily SCinjections

Apellis Pharmaceuticals Phase Ib/II, (Phase IIIannounced)

TT30 (CR2/FΗ) Recombinantprotein

AP C3convertase

Surface-directed inhibition of AP SC and IVinfusions

Alexion Pharmaceuticals Phase I (terminated)

mini-FH/AMY-201

Recombinantprotein

AP C3convertase

Surface-directed inhibition of AP n.a. AmyndasPharmaceuticals

preclinical stage

LNP023 Small molecule Factor B Inhibition of AP C3 convertase formation orally Novartis Phase IIACH-4471/ACH-0144471

Small molecule Factor D Inhibition of AP C3 convertase formation orally AchillionPharmaceuticals

Phase II

TNT009/BIVV009

mAb C1s CP inhibition/inhibition of C1s proteaseactivity

IV infusions True North Therapeutics/Bioverativ

Phase Ib (CAD patients)

AP ¼ alternative pathway; CP ¼ classical pathway; CAD ¼ cold agglutinin disease; IV ¼ intravenous; SC ¼ subcutaneous; n.a. ¼ not available.

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residence, thanks to a combination of pH-dependent C5 bindingand enhanced recycling via the neonatal Fc receptor [47]. Extend-ing the toolbox of antibodies targeting C5 in sites distant from theeculizumab epitope, Adienne has developed a fully human (Fabbased) minibody against C5 (Mubodina) that is still listed as aproduct in the preclinical discovery phase [48]. On the otherhand, Regeneron has recently registered an anti-C5 antibody(REGN3918) into phase 1 trials for PNH without disclosing anyinformation on the exact nature or properties of this agent [49].Anticipating the expiration of the patent on eculizumab, othercompanies such as Amgen have focused on developing biosimilarsof eculizumab. The anti-C5 Mab ABP959 has been evaluated inPhase I trials [50], and a phase III trial in Europe is currentlyunderway to assess its safety and efficacy in comparison toeculizumab [51]. In addition to antibody-based therapeutics, thetoolbox of complement C5 inhibitors has embraced diverse andmechanistically subtle approaches exemplified by peptidic inhib-itors, aptamers, recombinant proteins, and siRNA therapeutics[42]. These alternative agents might help circumvent the genet-ically driven resistance to eculizumab therapy and might also offeradvantages for lower production costs and enhanced pharmaco-kinetic profiles, including the potential for oral administration.

Coversin (OmCI, Akari Therapeutics) is a tick-derived 16-kDaprotein that blocks C5 activation and also inhibits leukotriene B4activity [52]. Coversin has shown preclinical efficacy in in vitroPNH models [53] and has reached Phase II clinical trials, deliveredas a subcutaneous injection in untreated PNH patients orpoor responders to eculizumab [54]. Although the potentialimmunogenicity of this protein may raise concerns in chronicintervention protocols, no neutralizing antibodies have beenreported so far in ongoing trials, with coversin showing biological

efficacy and promise as an alternative PNH treatment that couldalso enable self-administration.

RA101495 (RA Pharma) is a synthetic macrocyclic peptide thatbinds C5 with high affinity and allosterically inhibits its conver-tase-mediated cleavage [55]. Daily subcutaneous administration ofRA101495 in healthy individuals (Phase I trials) achieved sustainedsuppression of complement activity [56], and this peptide-basedanti-C5 agent is currently being evaluated in two Phase II studiesin naive and eculizumab-treated patients with PNH, respectively[57,58]. These ongoing studies will discern whether RA101495 canprovide therapeutic benefit as a monotherapy or as an add-ontherapy in patients with PNH with insufficient response toeculizumab.

Expanding the C5-targeted therapeutic arsenal, Alnylam hasdeveloped a silencing approach for shutting down hepatic C5production. Cemdisiran (ALNCC5) is a siRNA therapeutic that canachieve almost complete inhibition of C5 synthesis by hepatocytesin animal models [59]. Phase 1/2 trials of cemdisiran as a mono-therapy in PNH did not appear to reach full efficacy, indicatinga potential contribution of locally produced C5 to diseasepathogenesis [60] and the need of complete (499%) C5 inhib-ition to achieve therapeutic effect. In view of these results,cemdisiran is likely envisioned as a potential add-on to eculizu-mab that could improve hematological responses in certain poorresponders [61].

Capitalizing on the clinical success of eculizumab and in aneffort to extend its inhibitory potency across less frequent dosingintervals, Alexion has developed a next-generation, long-actingmAb (ravulizumab and ALXN1210) that shares the same epitopeon C5 with eculizumab [62]. Targeted mutagenesis has extendedthe antibody’s plasma residence through pH-dependent lysosomal

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release and increased FcRn-mediated recycling [62]. This antibodyhas shown efficacy in 2 Phase I/II trials, delivering sustained C5inhibition that resulted in normalization of key hematologicalmarkers of disease [63]. ALXN1210 is being further evaluated in2 large Phase III trials that will ascertain its safety and biologicalefficacy as a therapy for naive patients with PNH or as a switchtherapy for patients already on eculizumab [64,65]. This new anti-C5 antibody is anticipated to provide a more patient-complianttreatment option for PNH, extending the dosing window ofeculizumab from 2-8 weeks.

Targeting the Core of the Cascade: C3 Inhibitors

C3 serves as the nodal point of all complement activationpathways [66]. Its activation can trigger a plethora of inflammatoryand immunostimulatory processes, with detrimental consequen-ces for the host when regulatory control in the circulation or onhost cell surfaces is compromised [66,67]. Although C5 blockageabrogates MAC formation thereby preventing intravascular hemol-ysis, it does not interfere with upstream AP amplification, which isself-perpetuated on PNH cells because of the absence of DAF[68,69]. This process fuels persistent C3 opsonization on PNH cellsand culminates in the extravascular phagocytic clearance of C3-osponized PNH cells in the hepatosplenic compartment [39]. Inview of this pathogenic mechanism, C3 interception has emergedas a promising and perhaps superior approach for therapeuticintervention in PNH. In principle, agents targeting C3 offer theunique benefit of concomitantly abrogating both intravascularhemolysis and extravascular C3-mediated clearance of PNH RBCs[68]. Hence, this approach may afford greater hematologic benefitto patients with PNH than the currently approved treatment.

Compstatin AnalogsSince the discovery of the parental compound, compstatin, in

the mid 1990s [70], this family of small-sized, peptidic comple-ment inhibitors has grown to include a series of highly potent andselective C3 inhibitors that have propelled the clinical advance-ment of C3-based therapeutics for PNH and other complement-mediated diseases [71,72]. The discovery, molecular character-ization, structure-guided optimization, and most of the preclinicaldevelopment of these inhibitors were achieved through purelyacademic endeavors coordinated by the senior author of this paperat the University of Pennsylvania [71,73]. All next-generationcompstatin analogs have been built on the scaffold of the originalcompstatin, a cyclic 13-aminoacid peptide with strong affinity andselectivity for human and nonhuman primate (NHP) C3 [71]. Themost recent analogs have incorporated unnatural aminoacidsto increase the inhibitory potency, and possess an extendedN-terminus that can afford additional contacts with C3, thusfurther improving their binding affinity [73]. Compstatin analogsbind to a shallow pocket formed between the MG4 and MG5domains of the β chain of C3 and impair the binding of the nativeprotein to assembled C3 convertases, regardless of the initiatingcomplement pathway [74]. In this way, compstatin-based drugsprevent propagation and amplification of complement responsesand blunt effector generation via any of the three activationpathways (CP, LP, or AP) (Fig.).

The third-generation compstatin analog Cp40 (clinicaly devel-oped as AMY-101, Amyndas Pharmaceuticals) has demonstratedsustained C3 inhibition and consistent therapeutic efficacy in anumber of in vitro, ex vivo, and in vivo (NHP) models of comple-ment-mediated activation [71]. The pharmacological studies con-ducted with Cp40/AMY-101 include: (1) an ex vivo model of PNH(discussed below); (2) an in vitro model of C3 glomerulopathy inwhich Cp40 effectively restored complement regulation and

ameliorated key pathological drivers that promote kidney injuryand inflammation [75]; (3) an in vivo NHP model of hemodialysis-induced inflammation in which two discrete Cp40 treatmentregimens have led to complete inhibition of hemodialysis-inducedcomplement activation [76]; (4) inhibition of heme-induced com-plement activation and malarial inflammation [77]; (5) early organprotection and improved clinical outcome in an NHP model oftrauma-induced hemorrhagic shock [78]; and (6) inducible andnatural NHP models of periodontitis, in which local application ofAMY-101 inhibited complement activation, attenuated clinicalindices of periodontal inflammation and, more importantly,decreased inflammatory bone loss [79].

C3-based inhibition has shown clinical promise as a newtreatment option for PNH. The Cp40 analog abrogated MAC-mediated hemolysis of PNH RBCs and completely prevented C3deposition on surviving RBCs from PNH patients receiving anti-C5therapy (eculizumab) [80]. In this ex vivo PNH model, fresh RBCsobtained from patients with PNH were exposed to complementactivation, paralleling—or even exceeding—physiological levels ofcomplement activation (ie, the continuous low-level activation dueto spontaneous hydrolysis of C3, also known as C3 “tick-over”)occurring in humans. AMY-101 completely abrogated hemolysiswith an efficacy better than that observed with eculizumab undersimilar conditions; moreover, and in contrast to eculizumab, C3opsonization on RBCs was completely abolished [80]. PK or PDstudies of Cp40 in nonhuman primates revealed favorable drugelimination profiles, safety, and inhibitory efficacy after repeatedsubcutaneous administration, making this compound (AMY-101/Cp40) amenable to chronic application [68,73]. With a targetaffinity in the subnanomolar range, almost 6000-fold greater thatthe first-generation compstatin, and an extended plasma half-lifeof more than 50 hour, Cp40 is well suited for prolonged ther-apeutic application [42,71]. Notably, this pharmacokinetic profileobviates the need for further peptide modifications of Cp40, suchas PEGylation, that could entail the risk of eliciting adverseevents related to PEG-specific immune responses over extendeddosing periods. Furthermore, the pharmacokinetic behavior ofCp40 potentially enables better control in a clinical setting(ie, interruption of therapy and swift recovery of complementactivity in the case of adverse events) and may have importantimplications for tissue distribution, dosing, and administration in adisease-tailored context. Given that the production costs ofunmodified Cp40 are expected to be lower than those ofPEGylated peptides, this drug candidate is well-poised for pavingthe way to a broadly efficacious and affordable C3-targetedtherapy for PNH.

After receiving orphan drug designation for C3G and PNH[81,82], the Cp40-based drug candidate AMY-101 (Amyndas)entered clinical development in 2017. An open-label first in-human clinical trial assesed the safety, tolerability, and both PKand PD profile of AMY-101 after a single ascending dose andmultiple doses (MD) administered systemically in healthy volun-teers, using both IV and SC dosing routes [83]. According topreliminary results released by Amyndas, AMY-101 dosing dis-played safety and a PK profile that can support further clinicaldevelopment with sustained C3 inhibition through SC dosingevery 48 hours [84]. Amyndas has announced plans for Phase IIstudies of AMY-101 in both untreated patients with PNH andpatients with poor clinical response to eculizumab, as well as inABO-incompatible kidney transplantation, periodontitis, and C3glomeruolopathy [84]. In addition, fourth-generation compstatinanalogs are in preclinical development for various indications.

Further credence to the clinical translatability of peptidic C3inhibitors has been provided by the clinical development of thesecond-generation compstatin analog 4(1MeW)7W/POT-4, whichwas licensed to Apellis by the University of Pennsylvania. This drug

D.C. Mastellos et al. / Seminars in Hematology ] (2018) ]]]–]]]6

candidate has been developed as a long-acting, PEGylated deriv-ative for several indications, including PNH and age-relatedmacular degeneration (AMD) (APL-2, Apellis Pharmaceuticals).APL-2 is currently being evaluated in 2 Phase Ib trials, both as amonotherapy and “add-on” therapy in PNH patients who are poorresponders to eculizumab [85,86]. According to preliminary resultsreleased by Apellis, daily SC dosing of APL-2 has resulted inimprovement of key markers of hematologic response (eg, LDLand Hb levels), both in naive patients with PNH and in poorresponders to eculizumab [87]. Although more concrete data forpatient follow-up and optimum choice of therapy (add-on vsmonotherapy) have yet to be provided, Apellis has announcedplans for advancing this inhibitor to Phase III trials for PNH [87].Notably, C3 inhibitors have also shown promise as a treatmentoption for geographic atrophy (GA), an advanced form of AMD[71]. APL-2 met the primary endpoint of lesion reduction in a largemulticenter Phase II study following a monthly intravitreal dosingscheme in patients with GA [88]. Taken together, these studiesattest to the initial safety and feasibility of clinical C3-basedintervention, paving the way to more comprehensive, affordable,and patient-compliant complement therapies.

Targeted AP Inhibitors for PNH

Surface-Directed Engineered RegulatorsThe cardinal role of chronic AP dysregulation in PNH patho-

genesis has galvanized efforts to develop targeted AP inhibitorsthat can intercept convertase activity in close proximity to comple-ment-opsonized surfaces [45]. In this respect, several groups havedesigned chimeric regulator of complement activation (RCA)-typeinhibitors exploiting the capacity of endogenous regulators (ie,Factor H), to destabilize C3 convertases and degrade C3b [45].Indeed, the opsonin-targeted FH derivative TT30 (Taligen orAlexion) was one of the first engineered AP regulators to reachclinical trials for PNH in the posteculizumab era [89]. TT30 consistsof the complement regulatory domains of FH (CCPs 1-5) fused tothe iC3b/C3dg binding region of complement receptor 2 (CR2)(CCPs 1-4) [90]. Although TT30 demonstrated complete abrogationof intravascular hemolysis and C3 opsonization of PNH cellsin vitro and showed pharmacological activity in a Phase I trial inuntreated PNH patients, its clinical program was terminated forundisclosed reasons, possibly including the very short half-life ofthis chimeric protein in circulation [91].

Embracing the same concept of surface-directed AP modula-tion, research efforts have yielded a miniaturized form of humanFH (mini-FH/AMY-201, Amyndas) engineered to exert its inhibitoryactivity on opsonized cells after systemic administration [92].Mini-FH consists of the regulatory and surface-recognizing seg-ments of FH connected together by a peptide linker. Despite asignificant reduction in size, this engineered regulator retains ahigh or even superior binding affinity for C3-derived opsonicfragments and it has shown therapeutic efficacy abrogating bothintravascular hemolysis and C3 opsonization in PNH models [93].

Factor D or Factor B InhibitorsInstead of modulating convertase activity at the C3 breakdown

level, one can effectively block convertase assembly by targetingany of its assembling elements. In this respect, small-moleculedrug discovery platforms that target serine proteases involved inconvertase formation (ie, Factors B and D) have attracted consid-erable attention by pharmaceutical companies [42]. FD is consid-ered an attractive drug target due to its relatively low plasmaconcentration, high specificity, and bottleneck role in AP C3convertase assembly [94]. Novartis has developed potent, orallyavailable, and highly selective small-molecule FD and factor B

inhibitors that have shown efficacy in preventing AP dysregulationon PNH cells in vitro [95,96]. A Phase II study to assess the safety,PK or PD profile, and efficacy of the factor B inhibitor LNP023 asadd-on therapy for patients with insufficient response to eculizu-mab has recently been announced by Novartis (Eudra CT Number2017-000888-33). It remains to be seen whether upstream APmodulation will synergize with anti-C5 therapy in abrogating theresidual intravascular hemolysis observed in these patients.

Achillion has adopted a similar approach introducing orallybioavailable small FD inhibitors that have shown efficacy inmodulating AP activity in preclinical PNH and aHUS models[97]. One of these FD inhibitors, ACH-4471/ACH-0144471 (Achil-lion) has shown efficacy in abrogating intravascular hemolysisand attenuating C3 opsonization in PNH models [97] and hasentered clinical development for PNH as an orally administeredtherapeutic. Following a Phase I study that established its safetyand tolerability in healthy volunteers, ACH-4471 is being eval-uated in 2 ongoing Phase II trials as a single agent for PNHtreatment [98]. Achillion has also announced a Phase II trial toevaluate ACH-4471 in combination with eculizumab (Eudra CTNumber 2016-003526-16), based on in vitro results suggesting apotential synergistic effect with eculizumab in improving clinicalresponses [99]. In view of the recently described FD bypasspathway that enables kallikrein-mediated AP activation in theabsence of FD [100] and the failure of the FD-blocking antibodylampalizumab to meet the primary endpoint in one large PhaseIII trial in patients with GA [101], particular caution should beexercised when projecting the therapeutic outcome of suchinhibitors in pathologies involving aberrant AP activation. Inprinciple, factor D or factor B inhibitors are anticipated to providebroad coverage against both intravascular hemolysis and extrava-scular clearance of C3-opsonized PNH erythrocytes in patientswith PNH. Actual clinical data from ongoing clinical trialsevaluating targeted AP inhibitors in PNH patients are thereforehighly anticipated.

Although inhibitors targeting upstream complement compo-nents, such as C3 or FD or FB, offer the promise of a broadercontrol of complement-mediated anemia and may therefore proveto be efficacious as a monotherapy, there is still a possibility thatsubtotal inhibition of complement activity at the level of twodiscrete components (eg, combined anti-C3 and anti-C5 therapy)would also result in a similar clinical benefit for patients with PNH,maintaining also residual complement activity for pathogenimmune surveillance. This plausible scenario is worth investigat-ing in future clinical trials.

Translational Considerations

Anti-C5 therapy has drastically improved the clinical land-scape of PNH affording significant clinical gains as the firstcomplement-specific treatment approved for this debilitatingdisorder [19,29]. However, the unmasking of previously elusivepathogenic mechanisms that limit the hematologic response ofpatients, the necessity for more patient-compliant therapies andemerging genetic obstacles that limit patients’ responsiveness toeculizumab have all set forth important scientific questionsregarding the optimal choice of therapeutic agents, dosingroutes, and treatment algorithms and whether solid scientificrationale lies behind these emerging therapeutic concepts andtargets [35,38].

New anti-C5 agents are being evaluated with the goal ofachieving a comparable clinical response to that of eculizumab,likely improving issues related to dosing frequency and patientcompliance through self-administration of the drug. For example,the development of long-acting anti-C5 antibodies, such asALXN1210, or other anti-C5 agents delivered via alternative

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systemic routes (eg, SC) (eg, coversin and ALNCC5) might signify amajor leap forward for increasing patient compliance. The possiblecombination of anti-C5 drugs might also offer a more effectivemeans of controlling residual terminal pathway activity in case ofPK or PD breakthrough. To this end, many of these agents are beingclinically evaluated as add-on regimens to eculizumab therapy.Recent studies have shown that in settings of pronounced comple-ment activation, high C3b densities on the target surface can leadto forceful C5 convertase generation in a way that overrides thefull inhibitory effect of anti-C5 agents such as eculizumab [37].Complete abrogation of residual hemolysis can be achieved inthese cases by simultaneous use of C5 inhibitors. This perspectivemight be worth investigating in PNH, given the availability ofmultiple anti-C5 agents in the drug development pipeline [38].Genetic resistance due to C5 polymorphisms located within theeculizumab binding epitope may further limit clinical responses[40]. The availability of new anti-C5 agents targeting sites on C5distal to the epitope of the drug might offer an importantalternative for improving clinical responses.

Undeniably, the main pathogenic mechanism that remainsunaddressed by the standard treatment is C3-mediated extrava-scular hemolysis, which leads to partial hematologic responses in~30% of patients with PNH under eculizumab treatment [33]. Inthis context, the clinical advancement of C3-based or AP-targetedtherapeutics may constitute a major milestone for PNH therapy,since this strategy will likely afford broader clinical gains forabrogating intravascular hemolysis and intercepting residual ane-mia resulting from extravascular clearance of C3-opsonized PNHcells [68]. Of note, peptide-based therapeutics (ie, compstatins)may likely develop into a relatively affordable and broadly acces-sible treatment option, given the projected lower costs for large-scale peptide manufacturing nowadays [71]. The availability oforally active factor B or factor D-targeting agents provides anoption for increasing patient compliance. Furthermore, selectivetargeting of the AP may allow for maintenance of antimicrobialsurveillance in patients with PNH through an intact CP or LP.Conversely, the use of such AP-specific agents might come withthe risk of fueling hemolytic paroxysms through opportunistic,forceful CP or LP activation.

Longstanding, yet largely hypothetical, discussions concerningthe safety of C3 therapeutics need to be placed into a clinicallyvalidated context. Although clinical observations from rare cases ofprimary complement deficiencies have indicated an increasedsusceptibility to certain bacterial infections, especially during earlyage, this phenotype appears to subside during adulthood, likelydue to compensatory mechanisms of pathogen immune surveil-lance [102,103]. It should be stressed that complement-basedpharmacologic intervention cannot recapitulate the phenotype ofan inherited deficiency. Importantly, the clinical experience gainedwith eculizumab should similarly guide antimicrobial prophylaxisin the case of C3 intervention. A comprehensive vaccinationprogram directed against highly virulent encapsulated bacteriasuch as meningococci, streptococci, and Haemophilus influenzashould provide ample antimicrobial coverage in prolongedC3-targeted intervention protocols. A cautionary note, however,is necessary regarding the variable bactericidal activity elicitedagainst meningococci in immunized patients, depending on themode of complement inhibition [104]. All in all, one should avoidextrapolating clinical phenotypes from theoretical discussionsabout safety before obtaining actual clinical data from trialsevaluating C3-based inhibitors. Of note, preliminary results fromsmall ongoing trials have not revealed any treatment-relatedadverse events, supporting a favorable safety profile for anti-C3agents. Overall, long-term monitoring of patients with PNH treatedwith anti-C3 agents will ultimately decide the safety and clinicalefficacy of C3 intervention.

Concluding Remarks and Outlook

Undeniably, the clinical approval of eculizumab has drasticallychanged the clinical management of PNH, offering an effectivetherapy that tackles the main clinical hallmark of the disease,intravascular hemolysis. Following this clinical breakthrough, aseries of next-generation complement therapeutics have beenthrust into the limelight of clinical research with the goal ofimproving PNH management. More than 10 drug candidates arecurrently in clinical development for PNH, marking a remarkableresurgence of complement therapeutics. Upstream complementintervention, either at the level of C3 or through targeted APmodulation, offers the promise of broader therapeutic coverageagainst established and emerging pathogenic mechanisms andmay therefore represent a tangible new milestone for furtherimproving the management of patients with PNH. It is foreseeablethat once new complement therapeutics become approved forPNH, they will likely translate into clinical benefit for morecomplement-mediated indications that are currently in need ofan etiologic therapy or lack effective treatment. These indicationsinclude, among others, hematological disorders fueled by auto-immune factors (eg, cold agglutinin disease) a spectrum of rarerenal pathologies driven by AP dysregulation (ie, C3 glomerulop-athy) and transplant or biomaterial-triggered thromboinflamma-tory complications (eg, antibody-mediated allograft rejection).Further raising optimism about this approach, drug leads specif-ically targeting the CP are now in clinical development as anotherviable option for tackling complement-driven pathology in hem-atological diseases. Indeed, an anti-C1s antibody (TNT009/BIVV009, True North Therapeutics/Bioverativ) is currently in PhaseIb trials as a treatment option for patients with cold agglutinindisease, a rare autoimmune hemolytic anemia that lacks approvedtherapy [105].

The integration of solid clinical data from ongoing trials withrefined diagnostic algorithms for monitoring complement activitywill enable a more comprehensive and unbiased evaluation of theclinical efficacy of these inhibitors. The increasing appreciation ofthe impact of patient-specific genetic variance on clinicalresponses to anticomplement agents will enable a more rationalstratification strategy for enrolling those patients in clinical trialswith the greatest chance to benefit from each anticomplementagent. Hopefully the next decade will witness a long-awaitedexpansion of the arsenal of clinically approved complementtherapeutics for the treatment of PNH and other complement-driven diseases.

Disclosure Statement

J.D.L. and A.M.R. are inventors of patents or patent applicationsthat describe the use of complement inhibitors for therapeuticpurposes, some of which are developed by Amyndas Pharmaceut-icals. J.D.L. is the founder of Amyndas Pharmaceuticals, which isdeveloping complement inhibitors, including third-generationcompstatin analogs such as AMY-101, for the treatment of comple-ment-mediated diseases. J.D.L. is also the inventor of the comp-statin technology licensed to Apellis Pharmaceuticals [4(1MeW)7W, also known as POT-4 and APL-1 and PEGylated derivativessuch as APL-2]. A.M.R. has received research support from Alexion,Alnylam, RA Pharma, and Novartis; A.M.R. has received lecture feesand serves as member of an investigator board for Alexion,Novartis and Roche. AMR is involved as an investigator in clinicaltrials evaluating the following agents: TT30, ALXN1210, SKY59,ACH-4471, LNP023, and AMY-101. The rest of the authors have nocompeting interests.

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Acknowledgments

We thank Dr Deborah McClellan for her excellent editorialassistance. The authors’work was supported by grants from the USNational Institutes of Health (AI068730 and AI030040), the USNational Science Foundation (Grant no. 1423304), as well as fromthe Aplastic Anemia and Myelodysplastic Syndrome (AAMDS)International Foundation and the Italian PNH Association (AIEPN).

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