Complement alternative pathway in ANCA-associated vasculitis: Two decades from bench to bedside

Journal Pre-proof Complement alternative pathway in ANCA-associated vasculitis: Two decades from bench to bedside

Benoit Brilland, Anne-Sophie Garnier, Alain Chevailler, Pascale Jeannin, Jean-François Subra, Jean-François Augusto PII:

S1568-9972(19)30234-4

DOI:

https://doi.org/10.1016/j.autrev.2019.102424

Reference:

AUTREV 102424

To appear in:

Autoimmunity Reviews

Received date:

26 June 2019

Accepted date:

30 June 2019

Please cite this article as: B. Brilland, A.-S. Garnier, A. Chevailler, et al., Complement alternative pathway in ANCA-associated vasculitis: Two decades from bench to bedside, Autoimmunity Reviews(2019), https://doi.org/10.1016/j.autrev.2019.102424

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© 2019 Published by Elsevier.

Journal Pre-proof

Complement alternative pathway in ANCA-associated vasculitis: two decades from bench to bedside

Benoit Brilland1,2, Anne-Sophie Garnier1, MD; Alain Chevailler2,3, MD PhD; Pascale Jeannin2,3, PharmD PhD; Jean-François Subra1,2, MD PhD; Jean-François Augusto1,2,‡, MD PhD.

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1- Service de Néphrologie-Dialyse-Transplantation, Université d’Angers, CHU Angers, Angers, France. 2- CRCINA, INSERM, Université de Nantes, Université d'Angers, Angers, France.

Corresponding author:

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Professor Jean-François Augusto, MD, PhD

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‡

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3- Laboratoire d’Immunologie, Université d’Angers, CHU Angers, Angers, France.

Service de Néphrologie Dialyse Transplantation

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CHU Angers, 4 rue Larrey, 49033 Angers CEDEX 09, France.

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[email protected]

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Declarations of interest: none.

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Abstract word count: 152. Text word count (including abstract): 6220. Tables: 1 Figures: 3

Journal Pre-proof Highlights -

Murine and human data support a critical role of complement alternative pathway (cAP) in AAV pathogenesis.

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cAP activation leads to the generation of the anaphylatoxin C5a resulting in neutrophils priming and chemotaxis in an auto-amplification loop manner.

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C5aR blockade suppresses glomerular lesions in murine AAV models.

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CCX168 (avocapan), a C5aR antagonist, has proven its safety and efficiency in phase I and

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phase II trials conducted in AAV patients. CCX168 is currently being compared to steroids in a phase III trial for remission induction in

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AAV patients.

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Abstract

Anti-neutrophil cytoplasmic autoantibodies (ANCA)-associated vasculitides (AAVs) are small vessel

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vasculitides involving predominantly ear-nose-throat, kidneys, lungs and nerves. AAVs are life-

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threatening diseases, especially in their most severe forms such as necrotizing crescentic glomerulonephritis (GN) and/or intra-alveolar hemorrhage.

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Unlike immune complex GN or anti-glomerular basement membrane GN, AAVs are classified as pauciimmune GN. However, based on recent insights from animal models, the view of AAVs as a

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complement-unrelated disease has been challenged. Indeed, complement activation, and especially complement alternative pathway (cAP) activation, has been shown to be determinant in AAV pathogenesis through C5a generation, a potent chemoattractant for neutrophils with priming capacities. Here, we review in vitro and in vivo data supporting the role of cAP in murine models and in human AAVs. These findings, together with the need to eradicate glucocorticoid toxicity, led to the development of an anti-C5aR molecule, CCX168, also known as avacopan. Its development and future opportunities are also discussed.

Keywords ANCA-associated vasculitis – Complement alternative pathway – C5aR – Neutrophils – Avacopan.

Journal Pre-proof Abbreviations AAV: ANCA-associated vasculitis ANCA: anti-neutrophil cytoplasmic autoantibodies BVAS: Birmingham vasculitis activity score cAP: complement alternative pathway CFH: complement factor H eGFR: estimated glomerular filtration rate

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EGPA: eosinophilic granulomatosis with polyangiitis ESRD: end stage renal disease GN: glomerulonephritis

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GPA: granulomatosis with polyangiitis

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MAC: membrane attack complex MPA: micropolyangiitis

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MPO: myeloperoxidase

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NCGN: necrotizing and crescentic glomerulonephritis NET: neutrophil extracellular trap

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PR3: proteinase 3

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ROS: reactive oxygen species

Journal Pre-proof I.

Introduction 1. Clinical picture

Granulomatosis with polyangiitis (GPA), micropolyangiitis (MPA), eosinophilic granulomatosis with polyangiitis (EGPA), necrotizing and crescentic glomerulonephritis (NCGN) are manifestations of antineutrophil cytoplasmic autoantibodies (ANCA)-associated vasculitides (AAVs) and glomerulonephritis [1,2]. The main autoantigens in AAVs are myeloperoxidase (MPO-ANCA) and proteinase 3 (PR3ANCA). Indeed, 80 to 90 percent of patients with GPA or MPA are tested positive for ANCA [3,4].

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PR3-ANCAs are found in 70 to 80 percent of GPA patients, while MPO-ANCAs are detected in 60 percent of MPA patients [5]. Other neutrophil antigens, the so-called minor ANCA antigens, are also described (e.g. cathepsin G, lactoferrin, bactericidal permeability-increasing protein, elastase) but they

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have no specific clinical or prognostic value when detected in AAV patients [6].

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AAVs are characterized by pauci-immune necrotizing inflammation of the small blood vessels, with kidneys and lungs being the most affected organs. Rapidly progressive renal failure and pulmonary

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hemorrhage constitute the two main conditions that may be fatal for these patients unless rapidly

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managed. Current therapies used for remission induction rely mainly on immunosuppressive drugs, with high-dose steroids in addition to cyclophosphamide or rituximab [5]. Azathioprine and rituximab are

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the two main drugs used to maintain remission and to prevent relapse when remission is achieved. However, considerable treatment-related morbidity and failure to achieve sustained remission for all

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AAV patients underline the importance of developing less toxic and more effective therapies.

Until recently, the lack of indirect signs of complement consumption in plasma of AAV patients by routine tests (e.g., C3 and C4) led most clinicians to conclude that the complement system is of negligible significance to AAV pathophysiology [7]. Supporting this view, AAV pathological lesions are characterized by a paucity of immunoglobulin and complement deposition within tissues, especially when compared with typical immune complex or anti–glomerular basement membrane–mediated GN. However, low-intensity deposition of complement fractions is quite common, especially at focal sites of inflammation and necrosis [8–10].

Journal Pre-proof 2. AAV pathophysiology The pathogenesis of AAVs is not yet fully understood. The classical pathophysiological view involves a trigger that may be an ear-nose-throat infection or another infectious or inflammatory stimulus resulting in neutrophil activation through the secretion of pro-inflammatory cytokines [11,12]. This induces adhesion molecule expression and endothelial cell activation [13]. These triggers favor the expression of PR3 and MPO, the ANCA auto-antigens, that translocate to the neutrophil cytoplasmic membrane [14,15], where they become accessible to circulating ANCAs. Endothelium injury by

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activated neutrophils is the next step, responsible for the release of reactive oxygen species (ROS) and proteases [16,17]. NETosis, a particular from of neutrophil death [18], is also involved in endothelial cell injury. Indeed, if NETs (neutrophil extracellular traps) classically contribute to fight extracellular

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infectious agents, they can also be cytotoxic for endothelial cells and tissues in sterile conditions. Firstly,

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NETosis is involved in loss of tolerance and autoimmunity promotion as it can induce ANCA through dendritic cells cross-presentation to T and B cells [19,20]. Secondly, it has been shown that ANCAs can

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induce NETosis, which can, in turn, activate the complement alternative pathway (cAP) [21,22].

Although AAVs are commonly considered as pauci-immune diseases, i.e. without immune-complex

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deposition in tissues, it is possible that antigen-antibody complexes are formed locally with ANCAs enhancing the inflammatory response and neutrophil recruitment. It has been suggested that neutrophils

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from AAV patients have a defective apoptosis regulation, thus hindering their clearance by macrophages [23]. The combination of these two mechanisms may result in the persistence of apoptotic and/or necrotic neutrophils at the inflammatory sites disrupting the resolution of inflammation and promoting autoimmunity [23]. Neutrophil activation also results in cAP activation with C5aR as the main receptor involved, as we will detail further [24]. The anaphylatoxin C5a not only activates neutrophils, but also vascular endothelial cells, promoting their retraction and increasing vascular permeability [25,26].

Activated neutrophils synthesize many pro-inflammatory and/or immunomodulatory cytokines, including BAFF (B-cell activating factor) which promotes the differentiation and the activation of Blymphocytes [27,28]. Inflammation is also amplified by an imbalance in the different subpopulations of

Journal Pre-proof T cells, in particular a decrease of regulatory T cells [29]. Activated Th1 T cells produce IFNγ and TNFα, inducing macrophage migration and maturation. The activity of Th17 T cells has been shown to be increased in AAVs, also contributing to neutrophil recruitment [29]. In this inflammatory context, activated B and T cells, macrophages and dendritic cells may form a granuloma, a condition that occurs in GPA.

3. Overview of the complement system

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The complement system has a central and critical role in innate and humoral immunity. It is composed of more than 30 plasma and membrane-bound proteins. Activation of the complement system mainly follows three individualized pathways: the classical, the lectin, and the alternative pathways. These

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pathways are distinguished according to the factor that triggers their activation (Figure 1) [30,31].

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The classic pathway is initiated by immune complexes (antibody bound with antigen) creating a link between adaptive and innate immune responses. The lectin pathway is activated by pathogen surface

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components such as mannose. These two pathways share a common feature: they are unidirectional, i.e.

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activation is broken if the stimulus is eliminated. In contrast, the alternative pathway remains activated until it is down-regulated by specific regulatory factors [32]. In their absence, this self-sustaining

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pathway acts as an amplification loop [33]. Activation of the alternative pathway begins with the “tickover”: a physiological and constitutive low-level C3 auto-activation. It is contained by regulatory

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proteins, such as factor H and factor I, unless the amplification loop is fully activated by exogenous pathogens, especially encapsulated bacteria. Activation of either one of these pathways leads to C3 convertase formation (C4 and C2 by-products form C4bC2a in the classical and lectin pathway, C3 and factor B by-products form C3bBbP in the alternative pathway). This enzyme allows the conversion of C3 into the anaphylatoxin C3a and C3b. C3b is an opsonin, but it also allows the formation of another enzyme, C5 convertase (C3bC4bC2a in the classical and lectin pathway, C3b2BbP in the alternative pathway). This C5 convertase cleaves C5 into C5a, another anaphylatoxin with a powerful neutrophil chemoattractant action and an important neutrophil activating potential [34], and C5b, initiating the assembly of the membrane attack complex (MAC), also known as C5b-9 [35].

Journal Pre-proof 4. Purpose of the review cAP appears to be pivotal in the pathogenesis of anti-MPO NCGN in mice as we will review in detail. The relevance of cAP activation in human AAVs is supported by immunohistochemical demonstration of cAP components at sites of AAV injury and by association between plasma cAP activation fragments and AAV disease activity. Thus, the purpose of this in-depth review is to summarize the latest evidence of cAP involvement in human AAVs. These recent discoveries represent the scientific background that led to the development of the C5aR antagonist (avacopan) that is currently being evaluated in AAV

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patients.

Murine models of AAVs and complement alternative pathway involvement

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1. Murine models of AAVs

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Most studies that investigated the role of cAP in AAVs used a murine model developed more that fifteen years ago [36]. Xiao et al. immunized MPO-deficient mice with purified murine MPO and passively

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transferred the subsequently produced auto-antibodies or splenocytes to wild-type or recombinase

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activating gene 2 (RAG2)-deficient recipients (lacking B and T cells). After approximately 1 week, mice receiving anti-MPO IgG developed focal necrotizing crescentic glomerulonephritis in about 5-15%

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glomeruli in wild-type mice and 80% in RAG2-deficient mice. This study was an important

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breakthrough in the field, demonstrating the direct pathogenicity of ANCAs in the murine model.

Because PR3 structure and expression are different between rodents and humans, no relevant murine model of PR3-ANCA AAV is currently available [37–39]. It is to be noted that there is also a rat model of , in which immunization of WKY rats with human MPO led to the generation of anti-human MPO antibodies that cross-reacted with rat MPO and caused a mild form of NCGN [40]. Although this model shows that MPO-ANCAs contribute to inducing or aggravating renal vasculitis lesions, the use of human MPO has the disadvantage of promoting the formation of immune complexes. These models are therefore not considered as optimal autoimmune models of AAV and complement activation has not been studied in this context.

Journal Pre-proof 2. Complement alternative pathway in murine models of AAVs Xiao et al. were the first to demonstrate the involvement of the cAP [41] in their murine model of antiMPO induced AAV [36]. The depletion of C3 (using cobra venom factor) and the invalidation of C5 or factor B completely prevented NCGN development. Conversely, C4-deficient mice developed glomerulonephritis to the same extent as wild-type mice. This indicates the central role of cAP in glomerulonephritis pathogenesis, as factor B is essential for cAP, whereas C4 is required only for activation of the classic pathway and the lectin-binding pathway. This study [41] did not identify which

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factor was responsible for the complement activation, but a number of candidates were suggested earlier, including reactive oxygen radicals [42], MPO [43] and properdin [44]. However, neither properdin nor MASP-2 (Mannan-binding lectin serine protease 2) involved in lectin pathway, were found to be

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necessary for AAV development in the model [45]. Xiao et al. then transferred anti-MPO IgG into C6-

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deficient mice, which did not result in any protection against NCGN. This demonstrated that the MAC had no critical role in the disease and that its targeting does not represent a therapeutic option [46].

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The pathophysiologic importance of the cAP was further evidenced when the anaphylatoxin C5a and its

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receptor (C5aR/CD88) were proven to be essential for the mediation of NCGN [24,34,46].

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3. C5a in NCGN pathogenesis

To further decipher the involvement of the cAP in AAVs, BB5.1, a C5-inhibiting monoclonal antibody

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and MAC formation inhibitor, was studied in the anti-MPO passive transfer model. It was shown that pre-treatment with BB5.1 before anti-MPO transfer blocked NCGN development and decreased glomerular cell influx. When treatment was given after MPO transfer, the phenotype and histological features of NCGN were less severe (80% reduction in glomerular crescent formation and more than 85% reduction in albuminuria on day 7) [34].

C5a is involved in various inflammatory processes. Indeed, it is one of the most pro-inflammatory peptides, acting on many cell types, especially on myeloid cells. First, C5a is a powerful chemoattractant for neutrophils, monocytes and macrophages [47]. C5a also enhances neutrophil survival by delaying apoptosis [48], favors adhesion molecule expression on neutrophils [49], and activates respiratory burst

Journal Pre-proof and phagocytosis [50]. Finally, C5a participates in neutrophil degranulation, a process that is essential for their effector functions [51]. C5a is also a strong inducer of properdin release by neutrophils [44] and thus might be part of a positive feedback loop stabilizing the alternative pathway convertase, thereby increasing inflammation. It is to be highlighted that Freeley et al. recently showed that the key mediator of inflammation in antiMPO vasculitis is C5 derived from sources outside of the bone marrow, and especially circulating liverderived C5 [45]. These data supported the development of a molecule preventing C5 secretion from the

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liver. A clinical trial (NCT02352493) is ongoing in patients suffering from paroxysmal nocturnal hemoglobinuria with inadequate complement activation caused by dysfunctional GPI impeding CD55

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or CD59-mediated complement activation regulation.

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4. C5aR (CD88) in NCGN pathogenesis

The anaphylatoxin C5a binds to the C5a receptor 1 (C5aR or CD88) or to the C5a receptor 2 (C5aR2,

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also known as C5L2 or GPR77) [52,53]. Both receptors are seven-transmembrane receptors and interact

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with C5a with the same high affinity. C5aR mediates most of the C5a actions and is expressed on myeloid cells, especially neutrophils, mast cells/basophils, monocyte/macrophages, and dendritic cells

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[54,55]. It has also been found on parenchymal cells, such as epithelial cells, keratinocytes and smooth muscle cells [56]. C5aR is involved in various pro-inflammatory pathways: T cell activation, antigen

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presenting cell maturation, phagocytosis impairment, pro-inflammatory cytokine secretion and inhibition of Treg generation [56]. Only a few biological responses are reported for the more modestlyexpressed C5L2 [53,57]. The C5L2 receptor might serve as a decoy receptor for C5a without any biological effect as suggested by the fact it lacks G protein coupling, as opposed to C5aR [58]. However, C5L2 may be more than a simple decoy receptor given that an anti-inflammatory function [58–60] and recently a proinflammatory action have been suggested for this receptor [61,62].

To underline the pivotal role of C5aR in NCGN, a modified model of AAV was used [63] : myeloperoxidase-deficient mice were immunized with myeloperoxidase, then irradiated and transplanted with a bone marrow from wild-type mice or C5aR-deficient mice. Schreiber et al. then

Journal Pre-proof demonstrated that these MPO-/- mice, producing MPO-ANCAs, irradiated and then transplanted with a bone marrow from C5aR-/- mice, developed a milder disease as compared with animals reconstituted with wild-type bone marrow. Thus, the C5aR expressed on circulating neutrophils plays a major role in glomerular neutrophil accumulation and in NCGN development [24].

Using this latter model [36], Xiao et al. demonstrated that C5aR/CD88 deficiency improves NCGN while NCGN is exacerbated when C5L2 is lacking [46]. Indeed, mice deficient in this latter receptor

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more than doubled the percentage of glomerular crescents after anti-MPO–antibody transfer. This suggests that C5L2 has indeed an inhibitory function in ANCA vasculitis, as also suggested earlier [58– 60]. These data suggest that inhibiting C5 itself would eradicate a potential protective effect of C5a

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mediated by C5L2 receptor [46]. This led the authors to test an oral antagonist of C5aR, the CCX168,

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in humanized mice with knocked-in human C5aR/CD88. This compound improved ANCA-induced

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NCGN and gave credit for its evaluation in humans [46].

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Taken together, these data highlight the pivotal role of C5 and of its receptor, C5aR, in AAVs. This also

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led numerous authors to revisit the role of cAP in AAV patients (Figure 2).

Human data supporting complement alternative pathway involvement in AAVs

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1. In vitro studies

The production of complement factors by ANCA-activated neutrophils was first demonstrated by Xiao et al. [41]. Using TNFα-primed heathy human neutrophils incubated with IgG isolated from AAV patients, they demonstrated that ANCAs induced neutrophils to release undetermined factors that caused complement activation with generation of C3a [41]. Eventually, this anaphylatoxin amplifies in turn the recruitment and activation of more neutrophils, responsible for an amplification loop [64] (Figure 3).

To further investigate the role of complement activation, the same group focused on C3a and C5a anaphylatoxins [24]. Supernatants from ANCA-activated neutrophils initiated the complement cascade in normal serum, resulting in the generation of C3a and C5a. This supernatant subsequently primed

Journal Pre-proof neutrophils for ANCA-induced activation (respiratory burst). In the meantime, they showed that only neutrophil C5aR blockade abrogated this priming (C3aR blockade did not). Furthermore, recombinant C5a, but not C3a, dose-dependently primed neutrophils for ANCA-induced respiratory burst. These data indicate that C5a, but not C3a, upregulates the membrane expression of ANCA antigens and primes neutrophils for a subsequent ANCA-induced respiratory burst [24]. A latterly published study found consistent results demonstrating that neutrophils, primed by cytokines or coagulation factors, were able to activate the cAP and released C5a, thus amplifying the inflammatory response [64]. In this process,

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activation of p38 mitogen-activated protein kinase (p38MAPK), extracellular signal-regulated kinase (ERK) and phosphoinositol 3-kinase (PI3K) is involved in signal transduction [65]. Altogether, these data demonstrate that activated neutrophils produce C5a amplifying the cAP activation

2. In vivo: blood analysis

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and in turn the C5a generation, in the manner of an auto-amplifying loop.

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a. C3, C4 and MAC in blood

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Gou et al. were the first to analyze plasma level of various complement components in both patients with active (66 patients) and remittent (54 patients) AAV, and to correlate levels with clinical and

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pathological parameters [66].

First, active AAVs were associated with increased levels of circulating C3a, C5a, soluble C5b-9 (sC5b-

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9) and Bb. This was consistent with previous data focusing on C5a [67]. In patients with remittent AAVs, C5b-9 and C5a levels were comparable to those of control patients, whereas C3a levels remained elevated [66]. They suggested that the C3a remains high because the opsonin C3b, involved in the clearance of apoptotic bodies, continues to be produced. The other components (C5a and C5b-9) would be inactivated by complement regulatory factors. Secondly, C4d, a C4 by-product necessary in the classical and lectin pathways, was not found to differ between active and remittent AAVs but was always at a higher level as compared to healthy subjects. This suggests that the classic and/or lectin pathways were also activated in patients with active disease, but they were probably not pathogenic: inflammatory syndrome could increase C4d levels in a non-specific way. They then showed that plasma levels of C2

Journal Pre-proof and C4 stayed higher in patients with AAVs (in active as well as in inactive form), arguing against consumption of C2 or C4 during activation of the classical and/or lectin pathways [66].

Several teams, including ours, then demonstrated that a low C3 level at the initial diagnosis of AAV was associated with poorer renal outcomes and an increased risk of death [68–71]. Interestingly, renal thrombotic microangiopathy lesions were found in half of the patients with low C3 levels (5 patients/10), versus 15% in patients with normal C3 levels (3 patients/20). Almost all of those patients developed

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fulminant end stage renal disease (ESRD) unresponsive to conventional treatment [69]. Our group found that C3 level was associated with the intensity of extracapillary glomerular proliferation, leading to the identification of a subset of patients with worse renal prognosis [70]. Interestingly, in these studies,

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serum complement C4 was not associated with AAV prognosis, once again supporting complement

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activation via cAP in humans [68–70]. Patients with undifferentiated hypocomplementemia (low C3 level, low C4 level or low CH50, total complement activity) at disease onset presented with significantly

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higher rates of diffuse alveolar hemorrhage, skin lesions, blood evidence of thrombotic microangiopathy

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and intraglomerular immune complexes deposition [71], and, in another study, with a significantly lower

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overall and renal survival rates [72].

b. Complement alternative pathway in blood

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In the light of recent findings brought by the murine model of AAVs [73–75], Gou et al. then focused on cAP and especially on two characteristic markers: properdin and factor Bb. On the one hand, plasma Bb levels were higher only in patients with active AAVs: this correlated with biological data (erythrocyte sedimentation rate), clinical scores (Birmingham Vasculitis Activity Score, BVAS) and with anatomopathological findings. The more elevated the Bb, the more cellular crescents were found in glomeruli. It was also correlated with plasma levels of C3a, C5a, and sC5b-9, reflecting the systemic activation of the complement system. On the other hand, they found decreased levels of circulating properdin in patients with AAV compared with healthy controls and an inverse correlation with the proportion of crescents in the renal specimen. Taken together, these results suggest a properdin consumption by an overactivation of the cAP [66].

Journal Pre-proof The same team showed that urinary levels of C3a, C5a and sC5b-9 were higher during active disease than during remission [76]. This is also consistent with previous data focusing on C5a [67]. Increased urinary C5a might come from (i) the circulation because of glomerular basement membrane damage and/or (ii) the local activation of complement in kidneys, thus releasing C5a into urine (51). Interestingly, a correlation between urinary levels of Bb and serum creatinine was found [76], reinforcing the idea that cAP is involved in glomerulonephritis genesis.

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c. Complement regulatory proteins Among the regulators of the cAP, complement factor H (CFH) is essential as it competes with factor B for binding to C3b, thus preventing the formation of C3bBb, the alternative-pathway C3 convertases. It

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also accelerates its decay and acts as a cofactor for the factor I-mediated proteolytic inactivation of C3b

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[77]. CFH is found in blood and on cell surfaces and contains specific binding sites for C-reactive protein (CRP) [78]. The dysfunction of this regulatory factor is involved in complement-dependent auto-

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immunes diseases, such as atypical Hemolytic and Uremic Syndrome [79] or C3 glomerulonephritis

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[80].

Chen et al. studied the role of CFH in 82 patients with MPO-ANCA AAV [81]. They found that plasma

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CFH levels were significantly lower in active AAV patients when compared to AAV patients in remission and normal controls, and longitudinally that plasma levels of CFH were significantly higher

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in remission than those in the active stage. Also, CFH inversely correlated with initial serum creatinine, BVAS, proportion of total crescents and cellular crescents in renal specimens. Consequently, CFH levels were lower in patients with renal treatment failure compared with those achieving recovery of renal function. Above all, multivariate survival analysis revealed that plasma CFH levels were independently associated with composite outcome of death or ESRD in AAV patients, after adjustment. However, it is not known if decreased plasma CFH levels are related to excessive consumption because of complement overactivation, or if a low base level of CFH is a predisposing factor that favors cAP activation [81]. Nevertheless, the first hypothesis is more probable. Indeed, in vitro, MPO inhibits the interaction between CFH and C3b, and the decay-accelerating activity of CFH. Thus, MPO-CFH interaction may participate in the pathogenesis of AAVs by contributing to activation of the cAP [82].

Journal Pre-proof 3. In vivo: pathological analysis Aside from the Chapel Hill classification [2], glomerulonephritis (GN) are classified in 5 subgroups by pathologists [83] : immune-complex GN (e.g. lupus nephritis, IgA nephropathy, infection-related GN), anti-glomerular basement membrane GN, monoclonal immunoglobulin (Ig) GN (e.g. Monoclonal Ig deposition disease, proliferative GN with monoclonal Ig deposits), C3 GN and pauci-immune GN [83]. Pauci–immune necrotizing and crescentic GN, as found in AAVs, are characterized by negative or few

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Ig deposits when assessed by immunofluorescence microscopy or immunohistochemistry [84].

In 1977, Hu et al. reported the case of a patient with GPA and renal C3 deposition [85]. Later, evidence accumulated to support the idea that AAVs are not so “pauci-immune”. Indeed, immunoglobulins and/or

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complement components have been detected in a large proportion of kidney biopsies from AAV

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patients, using electron microscopy or immunofluorescence [10,86].

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Haas and Eustache were the first to study 126 renal biopsies of patients with necrotizing crescentic

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glomerulonephritis with positive ANCA serology with electron microscopy [87]. Glomerular immune deposits were found in 68 biopsies (54%). Almost all (87%) biopsies were also positives for

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immunoglobulin or complement component (IgG, IgA, IgM, C3 or C1q), however at a relatively weak level, in immunofluorescence analysis [87]. Although those results were contested (Hilhorst et al. later

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found less than 5% of electron dense deposits in their cohort [88]), this emphasized once again the role of complement in ANCA pathogenesis. In light of evidence found in blood studies, many authors then focused on cAP involvement in kidney biopsies of AAV patients.

a. C3, C4 and MAC deposition in kidneys C3c and C3d are by-products of C3b cleavage by complement regulator Factor I. C3c localization in glomerulus indicates ongoing immune deposit formation and complement activation [89] and C3d seems to be found at active lesions at the time of ongoing complement activation and after complement activation [9].

Journal Pre-proof In a cohort of 112 renal biopsies from AAV patients, one third showed C3c deposition. These patients had more proteinuria and worse renal function at presentation [90]. C3c deposition correlated with a poorer renal outcome in the smaller cohort of Gigante et al. [10]. C3d and C5b-9 deposition were found in glomeruli and small blood vessels of patients with active AAV [9,66]. It correlated with a higher amount of cellular crescents and more proteinuria at onset [88]. If these results point toward an activation of the cAP, C4d was found in the majority of renal biopsies [88,91] as well as mannose-binding lectin [91] suggesting that both the classical pathway and the

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mannose-binding lectin pathway may be involved. However, these results are still debatable [9,92].

b. Complement alternative pathway in kidneys

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Xing et al. found that deposition of factor B and properdin could be detected in glomeruli and small

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vessels of 7 patients with active MPO-ANCA associated glomerulonephritis. Colocalization with C3d and the membrane attack complex suggest that activation of the cAP leads to renal damage [9]. Further

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studies consistent with this finding found (i) that deposition of Bb in glomeruli correlated with the

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proportion of total crescents in the renal biopsy [66], (ii) that properdin deposition was associated with cellular crescents and proteinuria [88]. These findings are compatible with the hypothesis that

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complement activation in AAVs occurs predominantly via cAP activation.

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Performing laser microdissection and proteomic analysis of glomeruli from 13 patients with AAVs and ANCA-negative pauci-immune GN, Sethi et al. confirmed the previous immunofluorescence findings [93]. Evidence for cAP activation was found for both MPO- and PR3-positive patients. There was more C3 deposition compared to small amounts of C4 detected, and variable amounts of C5 and C9 in the kidneys of these patients. Even if no factor B or properdin was detected, suggesting that the cAP activation occurs in blood and not locally in the tissues, these results confirm predominant cAP activation in kidneys of AAV patients [93]. It should be noted that C3 deposition was found to be more pronounced in double negative ANCA patients with NCGN, suggesting the co-existence of another pathological entity or even of innate cAP dysregulation in these patients [94].

Journal Pre-proof c. C5aR in kidneys In a cohort of 24 patients with active AAVs and 19 patients with inactive AAVs, Yuan et al. demonstrated that the expression of C5aR/CD88 was significantly lower in kidneys of patients with AAVs than in normal controls, whereas neutrophil infiltration was, as expected, elevated [67]. Moreover, the expression of C5aR/CD88 was closely correlated with renal function at presentation and to the extent of interstitial infiltration [67]. It is known that CD88 is rapidly internalized after treatment with C5a [95]. Thus, the low expression of CD88 in AAV patients might be attributed to C5a-mediated

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internalization, reflecting the significant amount of C5a produced by activated neutrophil in this context. Interestingly, expression of C5L2 in glomeruli was higher in AAV patients than that in normal controls [67]. The significance of this finding remains unexplained as the pro- or anti-inflammatory role of C5L2

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is still a matter of discussion [58–62], but it is known that C5L2 is not internalized after C5a binding

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[96]. Even if the exact mechanism of CD88 and C5L2 regulation in AAV patients remains to be

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explored, this could reflect a self-protection mechanism, to dampen C5a-mediated inflammation.

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4. Complement regulatory proteins

Recently, Cheng et al. demonstrated that the expression of complement regulatory proteins CD46, CD55

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and CD59 in kidneys of 51 AAV patients was associated with the severity of renal injury: CD46 (also known as MCP, Membrane Cofactor Protein) expression level in glomeruli inversely correlated with

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the proportion of normal glomeruli while CD55 (also known as DAF, Decay Accelerating Factor) and CD59 (also known as MAC-IP, MAC-Inhibitory Protein) expression level in glomeruli correlated with the proportion of total crescents [97].

Taken together, these results are compatible with the hypothesis that complement activation in AAVs occurs predominantly by the activation of the cAP. In line with these findings, the following model has been proposed: C5a, produced following cAP activation, causes priming of neutrophils [24], induces ANCA antigens translocation to the neutrophil cell surface allowing interaction with ANCAs [98], resulting in C5a production in an amplifying loop manner (Figure 3). This proposed pathophysiological model led to the development of therapies targeting C5a and especially targeting its receptor, C5aR.

Journal Pre-proof

IV.

Evidence-based data of cAP targeting through C5aR blockade: from mice to humans 1. Pre-clinical development

In the murine model of MPO-ANCA-associated glomerulonephritis, blocking the common complement pathway through C5 or C5a receptor resulted in less glomerular damage [34,46]. First, Huugen et al. showed that administration of a C5-inhibiting monoclonal antibody (BB5.1) resulted in NCGN avoidance and a lesser glomerular cell influx when given before AAV induction, and a less severe

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phenotype and histologic NCGN when given after [34]. Targeting the C5a-receptor but not C5 itself has the advantage that the final common pathway of complement activation (MAC) remains fully functional, thus conserving the innate immune response

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toward microbial agents, especially encapsulated bacteria. Thus, Xiao et al. evaluated the effect of

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CCX168, an antagonist of C5aR in mice with the murine C5a receptor knocked out and replaced with the human C5a receptor (hC5aR mice) with MPO-induced NCGN [46]. CCX168 reduced glomerular

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crescents from 30.4% to 3.3%. CCX168 intake resulted in a reduction of neutrophil infiltration in glomeruli and a reduction of affected glomeruli [46]. These results gave credit for CCX168 development

ur

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in humans, under the name “avacopan”.

2. C5aR blockade in humans: Phase I trial data

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Forty-eight healthy volunteers (24 men and 24 women, 47 Caucasians, 1 Asian, mean age 38, range 2145 years) were enrolled in a Phase I clinical trial. Given at sequential doses from 1 to 100 mg, avacopan was well tolerated and its pharmacokinetics was dose-dependent. Seven days’ exposure to multiple doses of avacopan (up to 50 mg twice daily) was not associated with any serious adverse event and no apparent dose-dependent adverse events were noted. Reported adverse events (headache, diarrhea, dizziness and nausea) were rare and not significantly different between avacopan- and placebo-treated subjects [99].

3. C5aR blockade in humans: Phase II studies (CLEAR and CLASSIC trials)

Journal Pre-proof After the phase I trial, two Phase II trials evaluating avacopan were initiated: CLEAR and CLASSIC trials. The European Phase II CLEAR trial was designed to evaluate the safety and efficacy of avacopan for steroid minimization or elimination from regimens of AAV patients treated with standard of care. The North American Phase II CLASSIC trial was mainly a safety study.

The CLEAR trial (NCT01363388) was a double-blind, placebo-controlled trial, that enrolled 67 patients with biopsy-proven, newly-diagnosed or relapsing ANCA-associated vasculitis (anti-PR3 or anti-MPO

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ANCA) [100]. The aim of this trial was to test the efficacy and safety of avacopan in patients with active AAVs. Induction treatment relied on cyclophosphamide followed by azathioprine as maintenance regimen, or rituximab as remission induction treatment.

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Patients were sequentially randomized to receive either (i) placebo + 60 mg prednisolone (20 patients),

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(ii) avacopan 30 mg twice daily + prednisolone 20 mg, to assess if avacopan could safely allow a glucocorticoid dose reduction (22 patients), (iii) avacopan 30 mg twice daily + prednisolone 60 mg, to

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assess if avacopan could fully replace glucocorticoid (21 patients). After 12 weeks of avacopan (or

na

placebo), prednisolone was tapered to be stopped at the latest after 14 (group ii) or 20 weeks (group i). At baseline, all three groups were similar. Most patients presented with newly-diagnosed AAVs and

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renal involvement (> 95%). It is to be noted that renal function was only moderately impaired (estimated glomerular filtration rate (eGFR) around 50 ml/min/1.73m², urinary albumin/creatinine ratio around 300

Jo

mg/g) and BVAS was around 14 in each group. The primary endpoint was a 50% or more decrease in Birmingham Vasculitis Activity Score (BVAS). In a non-inferiority, intention-to-treat analysis, it was achieved in 70%, 86% (p=0.002 vs placebo) and 81% (p=0.01 vs placebo) of patients in group i, ii and iii [100]. Secondary endpoints focused on renal responses. First, the decrease of albuminuria/creatininuria ratio was higher in group ii and iii (-56%, p<0.01 and -43% respectively) than in placebo group i (-21%) at week 12. Moreover, a significant decrease in proteinuria was noted as soon as 2 weeks after avacopan initiation in both groups. Secondly, the decrease of MCP-1 (monocyte chemotactic proteine-1) to creatinine ratio was higher in group ii (-70%, p<0.001) than in placebo group i (-43%), suggesting a faster resolution of renal inflammatory lesions under avacopan. Moreover, an improvement of physical,

Journal Pre-proof mental and emotional quality of life was also noted in avacopan groups. Finally, adverse events were noted at similar frequency in all groups with 17% (4/23) patients in control group i and 25% (11/44) patients in avacopan groups ii and iii having serious adverse events. Side effects (mostly psychiatric disorders or new-onset diabetes) related to prednisolone were less common in avacopan groups (34% vs 65%). Very few patients had worsening vasculitis and no death was noted [100]. In conclusion, the CLEAR trial showed that avacopan could effectively and safely replace high-dose glucocorticoids in patients with AAVs. In addition to a “steroid-sparing” effect, urinary findings suggest

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that avacopan may also add some benefits to the standard induction regimen in terms of efficacy [100].

The CLASSIC trial (NCT02222155) was a Phase II dose assessment study of CCX168, randomized,

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double-blinded and placebo-controlled. The aim of this trial was to test the safety and efficacy of two

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dose regimens of avacopan (10 or 30 mg, twice daily) in addition to standard induction treatment (rituximab or cyclophosphamide plus full-dose corticoid) [101].

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Forty-two patients with new or relapsing AAV with anti-PR3 or anti-MPO ANCAs were randomized

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into three groups: standard of care + placebo (n=13), standard of care + 10 mg avacopan twice daily (n=13) or standard of care + 30 mg avacopan twice daily (n=16). All patients received either rituximab

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or cyclophosphamide as remission induction regimen [100], in addition to 60 mg of prednisone tapered to 10 mg daily by week 12. The primary endpoint was safety (incidence of adverse events). Despite the

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lack of power to show any difference in efficacy, secondary endpoints were a decrease of BVAS from baseline to week 12 of at least 50% and no worsening in any organ. Once again, patients had mild renal impairment (eGFR 59 ml/min) and mean BVAS was 15.3. There was no difference between groups in terms of adverse events or serious adverse events [101]. In conclusion, the CLASSIC trial demonstrated that avacopan was safe in patients with AAV if added to high-dose corticosteroids and standard of care.

Some limitations of these studies must be underlined. First, follow-up of patients was short (3 months). Secondly, change from baseline was used as primary endpoint and a proportion of the secondary endpoint with its known limitations [102–104]. Despite these limitations, the results of the CLEAR and

Journal Pre-proof CLASSIC trials, summarized in Table 1, suggested the safety of avacopan (30 mg twice daily) and its potential steroid sparing potential.

4. C5aR blockade in humans: Phase III study (ADVOCATE trial) To further evaluate the efficacy of avacopan, a large multicenter, double-blinded, double-placebo, randomized study has been set up: the ADVOCATE trial (NCT02994927) [105]. Patients from 19 countries, with severe GPA or MPA and anti-MPO or anti-PR3 ANCA and an eGFR > 15

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ml/min/1.73m² were eligible. In combination with rituximab or cyclophosphamide followed by azathioprine, two strategies are being evaluated: (i) avacopan placebo + usual dose of prednisone, (ii) avacopan 30 mg twice daily + prednisone placebo. Primary endpoints include the proportion of patients

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reaching remission (BVAS=0) at week 26 and those with sustained remission at week 52. Secondary

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endpoints are glucocorticoid toxicity (assessed by a score, the glucocorticoid toxicity index), BVAS at week 4, quality of life improvement, proportion and delay to relapse, eGFR evolution over 52 weeks.

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Inclusions were achieved in summer 2018 with more than 300 patients included, and results are eagerly

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awaited for the end of 2019.

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Of note, the use of avacopan has been authorized by the FDA and the EMA and is currently under investigation for two other complement mediated diseases: C3 glomerulonephritis (NCT03301467, ECT

Jo

2017-001821-42) and atypical Hemolytic and Uremic Syndrome (ACCESS trial, NCT02464891).

V.

Conclusion

Currently available options for treating AAVs allow more than 80% of patients to achieve remission, with a 5-year overall mortality rate of 10 to 15% [106]. As in other auto-immune diseases, the use of high dose corticosteroids has been the cornerstone of AAV treatment for more than five decades. Their use allows for fast control of disease-related inflammation. However, it has been well demonstrated, in AAVs and in other conditions, that large cumulative doses of corticosteroids are associated with many short- and long-term complications and comorbidities [107]. Side effects may even exceed the risks related to the disease or the likelihood of relapse [108,109]. Based on these observations, several

Journal Pre-proof strategies have been tested to reduce the cumulated administered doses [110,111] and many others are still ongoing. Accumulating evidence from murine models and in vitro studies has highlighted the central role of cAP in neutrophil activation and ANCA-associated vasculitis pathogenesis. This was further evidenced by human studies, emphasizing the point that cAP components, and especially Bb fragments, could be useful markers for monitoring the disease activity and severity. In view of these results, targeting the C5a-mediated neutrophil activation, resulting from cAP activation, appears to be an effective option. In

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this context, avacopan, a C5a receptor blocking molecule, is currently under evaluation in a Phase III controlled trial and constitutes the most promising therapy for AAV patients to achieve sustained

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na

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remission and to decrease glucocorticoid-related morbidity.

Journal Pre-proof VI. [1]

References

Jennette JC, Falk RJ. Small-Vessel Vasculitis. N Engl J Med 1997;337:1512–23. doi:10.1056/NEJM199711203372106.

[2]

Jennette JC, Falk RJ, Bacon PA, Basu N, Cid MC, Ferrario F, et al. 2012 revised International Chapel Hill Consensus Conference Nomenclature of Vasculitides. Arthritis Rheum 2013;65:1– 11. doi:10.1002/art.37715.

[3]

Guillevin L, Durand-Gasselin B, Cevallos R, Gayraud M, Lhote F, Callard P, et al. Microscopic

ro of

polyangiitis: clinical and laboratory findings in eighty-five patients. Arthritis Rheum 1999;42:421–30. doi:10.1002/1529-0131(199904)42:3<421::AID-ANR5>3.0.CO;2-6. [4]

Finkielman JD, Lee AS, Hummel AM, Viss MA, Jacob GL, Homburger HA, et al. ANCA are

-p

detectable in nearly all patients with active severe Wegener’s granulomatosis. Am J Med

[5]

re

2007;120:643.e9-14. doi:10.1016/j.amjmed.2006.08.016. Jennette JC, Nachman PH. ANCA Glomerulonephritis and Vasculitis. Clin J Am Soc Nephrol

Talor MV, Stone JH, Stebbing J, Barin J, Rose NR, Burek CL. Antibodies to selected minor

na

[6]

lP

CJASN 2017;12:1680–91. doi:10.2215/CJN.02500317.

target antigens in patients with anti-neutrophil cytoplasmic antibodies (ANCA): Minor target

ur

antigens in ANCA-associated vasculitis. Clin Exp Immunol 2007;150:42–8. doi:10.1111/j.13652249.2007.03453.x.

Kettritz R. With Complements from ANCA Mice. J Am Soc Nephrol 2014;25:207–9.

Jo

[7]

doi:10.1681/ASN.2013101043. [8]

Jennette JC, Wilkman AS, Falk RJ. Anti-neutrophil cytoplasmic autoantibody-associated glomerulonephritis and vasculitis. Am J Pathol 1989.

[9]

Xing G, Chen M, Liu G, Heeringa P, Zhang J, Zheng X, et al. Complement Activation Is Involved in Renal Damage in Human Antineutrophil Cytoplasmic Autoantibody Associated PauciImmune Vasculitis. J Clin Immunol 2009;29:282–91. doi:10.1007/s10875-008-9268-2.

[10]

Gigante A, Salviani C, Giannakakis K, Rosato E, Barbano B, Moroso A, et al. Clinical and histological outcome predictors in renal limited pauci-immune crescentic glomerulonephritis: a

Journal Pre-proof single

centre

experience.

Int

J

Immunopathol

Pharmacol

2012;25:287–92.

doi:10.1177/039463201202500133. [11]

Falk RJ, Terrell RS, Charles LA, Jennette JC. Anti-neutrophil cytoplasmic autoantibodies induce neutrophils to degranulate and produce oxygen radicals in vitro. Proc Natl Acad Sci 1990;87:4115–9. doi:10.1073/pnas.87.11.4115.

[12]

Huugen D, Xiao H, van Esch A, Falk RJ, Peutz-Kootstra CJ, Buurman WA, et al. Aggravation of anti-myeloperoxidase antibody-induced glomerulonephritis by bacterial lipopolysaccharide:

[13]

ro of

role of tumor necrosis factor-alpha. Am J Pathol 2005;167:47–58. Reumaux D, Vossebeld PJ, Roos D, Verhoeven AJ. Effect of tumor necrosis factor-induced integrin activation on Fc gamma receptor II-mediated signal transduction: relevance for

-p

activation of neutrophils by anti-proteinase 3 or anti-myeloperoxidase antibodies. Blood

[14]

re

1995;86:3189–95.

Witko-Sarsat V, Lesavre P, Lopez S, Bessou G, Hieblot C, Prum B, et al. A large subset of

lP

neutrophils expressing membrane proteinase 3 is a risk factor for vasculitis and rheumatoid

[15]

na

arthritis. J Am Soc Nephrol JASN 1999;10:1224–33. Brachemi S, Mambole A, Fakhouri F, Mouthon L, Guillevin L, Lesavre P, et al. Increased

ur

Membrane Expression of Proteinase 3 during Neutrophil Adhesion in the Presence of Anti Proteinase 3 Antibodies. J Am Soc Nephrol 2007;18:2330–9. doi:10.1681/ASN.2006121309. Porges AJ, Redecha PB, Kimberly WT, Csernok E, Gross WL, Kimberly RP. Anti-neutrophil

Jo

[16]

cytoplasmic antibodies engage and activate human neutrophils via Fc gamma RIIa. J Immunol Baltim Md 1950 1994;153:1271–80. [17]

Schreiber A, Luft FC, Kettritz R. Phagocyte NADPH Oxidase Restrains the Inflammasome in ANCA-Induced GN. J Am Soc Nephrol 2015;26:411–24. doi:10.1681/ASN.2013111177.

[18]

Berthelot J-M, Le Goff B, Neel A, Maugars Y, Hamidou M. NETosis: At the crossroads of rheumatoid

arthritis,

lupus,

doi:10.1016/j.jbspin.2016.05.013.

and

vasculitis.

Joint

Bone

Spine

2017;84:255–62.

Journal Pre-proof [19]

Kessenbrock K, Krumbholz M, Schönermarck U, Back W, Gross WL, Werb Z, et al. Netting neutrophils

in

autoimmune

small-vessel

vasculitis.

Nat

Med

2009;15:623–5.

doi:10.1038/nm.1959. [20]

Sangaletti S, Tripodo C, Chiodoni C, Guarnotta C, Cappetti B, Casalini P, et al. Neutrophil extracellular traps mediate transfer of cytoplasmic neutrophil antigens to myeloid dendritic cells toward

ANCA

induction

and

associated

autoimmunity.

Blood

2012;120:3007–18.

doi:10.1182/blood-2012-03-416156. Söderberg D, Segelmark M. Neutrophil Extracellular Traps in ANCA-Associated Vasculitis.

ro of

[21]

Front Immunol 2016;7. doi:10.3389/fimmu.2016.00256. [22]

Lange C, Csernok E, Moosig F, Holle JU. Immune stimulatory effects of neutrophil extracellular

Witko-Sarsat V, Reuter N, Mouthon L. Interaction of proteinase 3 with its associated partners:

re

[23]

-p

traps in granulomatosis with polyangiitis. Clin Exp Rheumatol 2017;35 Suppl 103:33–9.

implications in the pathogenesis of Wegener’s granulomatosis. Curr Opin Rheumatol 2010;22:1–

Schreiber A, Xiao H, Jennette JC, Schneider W, Luft FC, Kettritz R. C5a Receptor Mediates

na

[24]

lP

7. doi:10.1097/BOR.0b013e3283331594.

Neutrophil Activation and ANCA-Induced Glomerulonephritis. J Am Soc Nephrol

[25]

ur

2009;20:289–98. doi:10.1681/ASN.2008050497. Foreman KE, Vaporciyan AA, Bonish BK, Jones ML, Johnson KJ, Glovsky MM, et al. C5a-

Jo

induced expression of P-selectin in endothelial cells. J Clin Invest 1994;94:1147–55. doi:10.1172/JCI117430. [26]

Schraufstatter IU, Trieu K, Sikora L, Sriramarao P, DiScipio R. Complement c3a and c5a induce different signal transduction cascades in endothelial cells. J Immunol Baltim Md 1950 2002;169:2102–10.

[27]

Lenert P, Lenert A. Current and emerging treatment options fo ANCA-associated vasculitis: potential role of belimumab and other BAFF/APRIL targeting agents. Drug Des Devel Ther 2015:333. doi:10.2147/DDDT.S67264.

Journal Pre-proof [28]

McClure M, Gopaluni S, Jayne D, Jones R. B cell therapy in ANCA-associated vasculitis: current and emerging treatment options. Nat Rev Rheumatol 2018;14:580–91. doi:10.1038/s41584-0180065-x.

[29]

Abdulahad WH, Lamprecht P, Kallenberg CGM. T-helper cells as new players in ANCAassociated vasculitides. Arthritis Res Ther 2011;13:236. doi:10.1186/ar3362.

[30]

Morgan BP, Harris CL. Complement, a target for therapy in inflammatory and degenerative diseases. Nat Rev Drug Discov 2015;14:857–77. doi:10.1038/nrd4657. Trouw LA, Pickering MC, Blom AM. The complement system as a potential therapeutic target

ro of

[31]

in rheumatic disease. Nat Rev Rheumatol 2017;13:538–47. doi:10.1038/nrrheum.2017.125. [32]

Thurman JM, Holers VM. The Central Role of the Alternative Complement Pathway in Human

Chen M, Daha MR, Kallenberg CGM. The complement system in systemic autoimmune disease.

re

[33]

-p

Disease. J Immunol 2006;176:1305–10. doi:10.4049/jimmunol.176.3.1305.

J Autoimmun 2010;34:J276–86. doi:10.1016/j.jaut.2009.11.014. Huugen D, van Esch A, Xiao H, Peutz-Kootstra C-J, Buurman W-A, Tervaert JWC, et al.

lP

[34]

na

Inhibition of complement factor C5 protects against anti-myeloperoxidase antibody-mediated glomerulonephritis in mice. Kidney Int 2007;71:646–54. doi:10.1038/sj.ki.5002103. Mathern DR, Heeger PS. Molecules Great and Small: The Complement System. Clin J Am Soc

ur

[35]

Nephrol 2015;10:1636–50. doi:10.2215/CJN.06230614. Xiao H, Heeringa P, Hu P, Liu Z, Zhao M, Aratani Y, et al. Antineutrophil cytoplasmic

Jo

[36]

autoantibodies specific for myeloperoxidase cause glomerulonephritis and vasculitis in mice. J Clin Invest 2002;110:955–63. doi:10.1172/JCI200215918. [37]

Pfister H, Ollert M, Fröhlich LF, Quintanilla-Martinez L, Colby TV, Specks U, et al. Antineutrophil cytoplasmic autoantibodies against the murine homolog of proteinase 3 (Wegener autoantigen) are pathogenic in vivo. Blood 2004;104:1411–8. doi:10.1182/blood-2004-01-0267.

[38]

van der Geld YM, Hellmark T, Selga D, Heeringa P, Huitema MG, Limburg PC, et al. Rats and mice immunised with chimeric human/mouse proteinase 3 produce autoantibodies to mouse Pr3 and rat granulocytes. Ann Rheum Dis 2007;66:1679–82. doi:10.1136/ard.2006.064626.

Journal Pre-proof [39]

Salama AD, Little MA. Animal models of antineutrophil cytoplasm antibody-associated vasculitis. Curr Opin Rheumatol 2012;24:1–7. doi:10.1097/BOR.0b013e32834d2d52.

[40]

Little MA. Antineutrophil cytoplasm antibodies directed against myeloperoxidase augment leukocyte-microvascular interactions in vivo. Blood 2005;106:2050–8. doi:10.1182/blood-200503-0921.

[41]

Xiao H, Schreiber A, Heeringa P, Falk RJ, Jennette JC. Alternative Complement Pathway in the Pathogenesis of Disease Mediated by Anti-Neutrophil Cytoplasmic Autoantibodies. Am J Pathol

[42]

ro of

2007;170:52–64. doi:10.2353/ajpath.2007.060573. Shingu M, Nonaka S, Nishimukai H, Nobunaga M, Kitamura H, Tomo-Oka K. Activation of complement in normal serum by hydrogen peroxide and hydrogen peroxide-related oxygen

Vogt W. Complement activation by myeloperoxidase products released from stimulated human polymorphonuclear

leukocytes.

1996;195:334–46.

doi:10.1016/S0171-

Wirthmueller U, Dewald B, Thelen M, Schäfer MK, Stover C, Whaley K, et al. Properdin, a

na

[44]

Immunobiology

lP

2985(96)80050-7.

re

[43]

-p

radicals produced by activated neutrophils. Clin Exp Immunol 1992;90:72–8.

positive regulator of complement activation, is released from secondary granules of stimulated

[45]

ur

peripheral blood neutrophils. J Immunol Baltim Md 1950 1997;158:4444–51. Freeley SJ, Popat RJ, Parmar K, Kolev M, Hunt BJ, Stover CM, et al. Experimentally-induced

Jo

anti-myeloperoxidase vasculitis does not require properdin, MASP-2 or bone marrow-derived C5: Complement and vasculitis. J Pathol 2016;240:61–71. doi:10.1002/path.4754. [46]

Xiao H, Dairaghi DJ, Powers JP, Ertl LS, Baumgart T, Wang Y, et al. C5a Receptor (CD88) Blockade Protects against MPO-ANCA GN. J Am Soc Nephrol 2014;25:225–31. doi:10.1681/ASN.2013020143.

[47]

Marder SR, Chenoweth DE, Goldstein IM, Perez HD. Chemotactic responses of human peripheral blood monocytes to the complement-derived peptides C5a and C5a des Arg. J Immunol Baltim Md 1950 1985;134:3325–31.

Journal Pre-proof [48]

Perianayagam MC, Balakrishnan VS, King AJ, Pereira BJG, Jaber BL. C5a delays apoptosis of human neutrophils by a phosphatidylinositol 3-kinase-signaling pathway. Kidney Int 2002;61:456–63. doi:10.1046/j.1523-1755.2002.00139.x.

[49]

Laudes IJ, Chu JC, Sikranth S, Huber-Lang M, Guo R-F, Riedemann N, et al. Anti-c5a ameliorates coagulation/fibrinolytic protein changes in a rat model of sepsis. Am J Pathol 2002;160:1867–75. doi:10.1016/S0002-9440(10)61133-9.

[50]

Mollnes TE, Brekke O-L, Fung M, Fure H, Christiansen D, Bergseth G, et al. Essential role of

ro of

the C5a receptor in E coli-induced oxidative burst and phagocytosis revealed by a novel lepirudin-based human whole blood model of inflammation. Blood 2002;100:1869–77. [51]

Becker EL, Kermode JC, Naccache PH, Yassin R, Marsh ML, Munoz JJ, et al. The inhibition of

-p

neutrophil granule enzyme secretion and chemotaxis by pertussis toxin. J Cell Biol

[52]

re

1985;100:1641–6.

Gerard NP, Gerard C. The chemotactic receptor for human C5a anaphylatoxin. Nature

Ohno M, Hirata T, Enomoto M, Araki T, Ishimaru H, Takahashi TA. A putative chemoattractant

na

[53]

lP

1991;349:614–7. doi:10.1038/349614a0.

receptor, C5L2, is expressed in granulocyte and immature dendritic cells, but not in mature

[54]

ur

dendritic cells. Mol Immunol 2000;37:407–12. Dunkelberger J, Zhou L, Miwa T, Song W-C. C5aR expression in a novel GFP reporter gene

Jo

knockin mouse: implications for the mechanism of action of C5aR signaling in T cell immunity. J Immunol Baltim Md 1950 2012;188:4032–42. doi:10.4049/jimmunol.1103141. [55]

Karsten CM, Laumonnier Y, Eurich B, Ender F, Bröker K, Roy S, et al. Monitoring and cellspecific deletion of C5aR1 using a novel floxed GFP-C5aR1 reporter knock-in mouse. J Immunol Baltim Md 1950 2015;194:1841–55. doi:10.4049/jimmunol.1401401.

[56]

Collectif. Encyclopedia of Immunobiology. 2016.

[57]

Okinaga S, Slattery D, Humbles A, Zsengeller Z, Morteau O, Kinrade MB, et al. C5L2, a nonsignaling C5A binding protein. Biochemistry 2003;42:9406–15. doi:10.1021/bi034489v.

Journal Pre-proof [58]

Gerard NP, Lu B, Liu P, Craig S, Fujiwara Y, Okinaga S, et al. An anti-inflammatory function for the complement anaphylatoxin C5a-binding protein, C5L2. J Biol Chem 2005;280:39677– 80. doi:10.1074/jbc.C500287200.

[59]

Scola A-M, Johswich K-O, Morgan BP, Klos A, Monk PN. The human complement fragment receptor, C5L2, is a recycling decoy receptor.

Mol Immunol 2009;46:1149–62.

doi:10.1016/j.molimm.2008.11.001. [60]

Bamberg CE, Mackay CR, Lee H, Zahra D, Jackson J, Lim YS, et al. The C5a receptor (C5aR)

ro of

C5L2 is a modulator of C5aR-mediated signal transduction. J Biol Chem 2010;285:7633–44. doi:10.1074/jbc.M109.092106. [61]

Chen N-J, Mirtsos C, Suh D, Lu Y-C, Lin W-J, McKerlie C, et al. C5L2 is critical for the

-p

biological activities of the anaphylatoxins C5a and C3a. Nature 2007;446:203–7.

[62]

re

doi:10.1038/nature05559.

Rittirsch D, Flierl MA, Nadeau BA, Day DE, Huber-Lang M, Mackay CR, et al. Functional roles

Schreiber A, Xiao H, Falk RJ, Jennette JC. Bone marrow-derived cells are sufficient and

na

[63]

lP

for C5a receptors in sepsis. Nat Med 2008;14:551–7. doi:10.1038/nm1753.

necessary targets to mediate glomerulonephritis and vasculitis induced by anti-myeloperoxidase

[64]

ur

antibodies. J Am Soc Nephrol JASN 2006;17:3355–64. doi:10.1681/ASN.2006070718. Camous L, Roumenina L, Bigot S, Brachemi S, Frémeaux-Bacchi V, Lesavre P, et al.

Jo

Complement alternative pathway acts as a positive feedback amplification of neutrophil activation. Blood 2011;117:1340–9. doi:10.1182/blood-2010-05-283564. [65]

Hao J, Meng L-Q, Xu P-C, Chen M, Zhao M-H. p38MAPK, ERK and PI3K Signaling Pathways Are Involved in C5a-Primed Neutrophils for ANCA-Mediated Activation. PLoS ONE 2012;7:e38317. doi:10.1371/journal.pone.0038317.

[66]

Gou S-J, Yuan J, Chen M, Yu F, Zhao M-H. Circulating complement activation in patients with anti-neutrophil cytoplasmic antibody–associated vasculitis. Kidney Int 2013;83:129–37. doi:10.1038/ki.2012.313.

Journal Pre-proof [67]

Yuan J, Gou S-J, Huang J, Hao J, Chen M, Zhao M-H. C5a and its receptors in human antineutrophil

cytoplasmic

antibody

(ANCA)-associated

vasculitis.

Arthritis

Res

Ther

2012;14:R140. doi:10.1186/ar3873. [68]

Molad Y, Tovar A, Ofer-Shiber S. Association of low serum complement C3 with reduced patient and renal survival in antimyeloperoxidase-associated small-vessel vasculitis. Nephron Clin Pract 2014;126:67–74. doi:10.1159/000357154.

[69]

Manenti L, Vaglio A, Gnappi E, Maggiore U, Allegri L, Allinovi M, et al. Association of Serum

ro of

C3 Concentration and Histologic Signs of Thrombotic Microangiopathy with Outcomes among Patients with ANCA-Associated Renal Vasculitis. Clin J Am Soc Nephrol CJASN 2015;10:2143–51. doi:10.2215/CJN.00120115.

Augusto J-F, Langs V, Demiselle J, Lavigne C, Brilland B, Duveau A, et al. Low Serum

-p

[70]

re

Complement C3 Levels at Diagnosis of Renal ANCA-Associated Vasculitis Is Associated with Poor Prognosis. PLOS ONE 2016;11:e0158871. doi:10.1371/journal.pone.0158871. Fukui S, Iwamoto N, Umeda M, Nishino A, Nakashima Y, Koga T, et al. Antineutrophilic

lP

[71]

na

cytoplasmic antibody-associated vasculitis with hypocomplementemia has a higher incidence of serious organ damage and a poor prognosis. Medicine (Baltimore) 2016;95:e4871.

[72]

ur

doi:10.1097/MD.0000000000004871.

Deshayes S, Aouba A, Khoy K, Mariotte D, Lobbedez T, Martin Silva N. Hypocomplementemia

Jo

is associated with worse renal survival in ANCA-positive granulomatosis with polyangiitis and microscopic polyangiitis. PLOS ONE 2018;13:e0195680. doi:10.1371/journal.pone.0195680. [73]

Watanabe H, Noguchi E, Shio K, Iwadate H, Kobayashi H, Ohira H. Usefulness of complement split product, Bb, as a clinical marker for disease activity of lupus nephritis. Fukushima J Med Sci 2006;52:103–9.

[74]

Ganter MT, Brohi K, Cohen MJ, Shaffer LA, Walsh MC, Stahl GL, et al. Role of the alternative pathway in the early complement activation following major trauma. Shock Augusta Ga 2007;28:29–34. doi:10.1097/shk.0b013e3180342439.

[75]

Pavlov IY, De Forest N, Delgado JC. Specificity of EIA immunoassay for complement factor Bb testing. Clin Lab 2011;57:225–8.

Journal Pre-proof [76]

Gou S-J, Yuan J, Wang C, Zhao M-H, Chen M. Alternative complement pathway activation products in urine and kidneys of patients with ANCA-associated GN. Clin J Am Soc Nephrol CJASN 2013;8:1884–91. doi:10.2215/CJN.02790313.

[77]

Pangburn MK, Schreiber RD, Müller-Eberhard HJ. Human complement C3b inactivator: isolation, characterization, and demonstration of an absolute requirement for the serum protein beta1H for cleavage of C3b and C4b in solution. J Exp Med 1977;146:257–70.

[78]

Jarva H, Jokiranta TS, Hellwage J, Zipfel PF, Meri S. Regulation of complement activation by

ro of

C-reactive protein: targeting the complement inhibitory activity of factor H by an interaction with short consensus repeat domains 7 and 8-11. J Immunol Baltim Md 1950 1999;163:3957–62. [79]

Noris M, Remuzzi G. Atypical hemolytic-uremic syndrome. N Engl J Med 2009;361:1676–87.

Servais A, Noël L-H, Roumenina LT, Le Quintrec M, Ngo S, Dragon-Durey M-A, et al. Acquired

re

[80]

-p

doi:10.1056/NEJMra0902814.

and genetic complement abnormalities play a critical role in dense deposit disease and other C3

Chen S-F, Wang F-M, Li Z-Y, Yu F, Zhao M-H, Chen M. Plasma complement factor H is

na

[81]

lP

glomerulopathies. Kidney Int 2012;82:454–64. doi:10.1038/ki.2012.63.

associated with disease activity of patients with ANCA-associated vasculitis. Arthritis Res Ther

[82]

ur

2015;17. doi:10.1186/s13075-015-0656-8. Chen S-F, Wang F-M, Li Z-Y, Yu F, Chen M, Zhao M-H. Myeloperoxidase influences the

Jo

complement regulatory activity of complement factor H. Rheumatol Oxf Engl 2018. doi:10.1093/rheumatology/kex529. [83]

Sethi S, Haas M, Markowitz GS, D’Agati VD, Rennke HG, Jennette JC, et al. Mayo Clinic/Renal Pathology Society Consensus Report on Pathologic Classification, Diagnosis, and Reporting of GN. J Am Soc Nephrol JASN 2016;27:1278–87. doi:10.1681/ASN.2015060612.

[84]

Jennette JC, Falk RJ. Pathogenesis of antineutrophil cytoplasmic autoantibody-mediated disease. Nat Rev Rheumatol 2014;10:463–73. doi:10.1038/nrrheum.2014.103.

[85]

Hu C-H. Cutaneous Manifestations of Wegener Granulomatosis. Arch Dermatol 1977;113:175. doi:10.1001/archderm.1977.01640020047008.

Journal Pre-proof [86]

Van Timmeren MM, Chen M, Heeringa P. Review article: Pathogenic role of complement activation in anti-neutrophil cytoplasmic auto-antibody-associated vasculitis. Nephrology 2009;14:16–25. doi:10.1111/j.1440-1797.2009.01086.x.

[87]

Haas M, Eustace JA. Immune complex deposits in ANCA-associated crescentic glomerulonephritis: A study of 126 cases. Kidney Int 2004;65:2145–52. doi:10.1111/j.15231755.2004.00632.x.

[88]

Hilhorst M, van Paassen P, van Rie H, Bijnens N, Heerings-Rewinkel P, van Breda Vriesman P,

ro of

et al. Complement in ANCA-associated glomerulonephritis. Nephrol Dial Transplant 2017;32:1302–13. doi:10.1093/ndt/gfv288. [89]

Schulze M, Pruchno CJ, Burns M, Baker PJ, Johnson RJ, Couser WG. Glomerular C3c

-p

localization indicates ongoing immune deposit formation and complement activation in

[90]

re

experimental glomerulonephritis. Am J Pathol 1993;142:179–87. Chen M, Xing G-Q, Yu F, Liu G, Zhao M-H. Complement deposition in renal histopathology of

lP

patients with ANCA-associated pauci-immune glomerulonephritis. Nephrol Dial Transplant

[91]

na

2008;24:1247–52. doi:10.1093/ndt/gfn586.

Xing G, Chen M, Liu G, Zheng X, E J, Zhao M. Differential Deposition of C4d and MBL in

ur

Glomeruli of Patients with ANCA-Negative Pauci-Immune Crescentic Glomerulonephritis. J Clin Immunol 2010;30:144–56. doi:10.1007/s10875-009-9344-2. Sethi S, Nasr SH, De Vriese AS, Fervenza FC. C4d as a Diagnostic Tool in Proliferative GN. J

Jo

[92]

Am Soc Nephrol JASN 2015;26:2852–9. doi:10.1681/ASN.2014040406. [93]

Sethi S, Zand L, De Vriese AS, Specks U, Vrana JA, Kanwar S, et al. Complement activation in pauci-immune necrotizing and crescentic glomerulonephritis: results of a proteomic analysis. Nephrol Dial Transplant 2017;32:i139–45. doi:10.1093/ndt/gfw299.

[94]

Fervenza FC, Smith RJH, Sethi S. Association of a novel complement factor H mutation with severe crescentic and necrotizing glomerulonephritis. Am J Kidney Dis Off J Natl Kidney Found 2012;60:126–32. doi:10.1053/j.ajkd.2012.03.007.

[95]

Huey R, Hugli TE. Characterization of a C5a receptor on human polymorphonuclear leukocytes (PMN). J Immunol Baltim Md 1950 1985;135:2063–8.

Journal Pre-proof [96]

Cain SA, Monk PN. The Orphan Receptor C5L2 Has High Affinity Binding Sites for Complement Fragments C5a and C5a des-Arg

74

. J Biol Chem 2002;277:7165–9.

doi:10.1074/jbc.C100714200. [97]

Cheng L, Gou S-J, Qiu H-Y, Ma L, Fu P. Complement regulatory proteins in kidneys of patients with anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis: Complement regulatory proteins in AAV. Clin Exp Immunol 2018;191:116–24. doi:10.1111/cei.13051. Wilde B, van Paassen P, Witzke O, Tervaert JWC. New pathophysiological insights and treatment

of

ANCA-associated

vasculitis.

doi:10.1038/ki.2010.472. [99]

Kidney

Int

2011;79:599–612.

ro of

[98]

Bekker P, Dairaghi D, Seitz L, Leleti M, Wang Y, Ertl L, et al. Characterization of Pharmacologic

-p

and Pharmacokinetic Properties of CCX168, a Potent and Selective Orally Administered

re

Complement 5a Receptor Inhibitor, Based on Preclinical Evaluation and Randomized Phase 1 Clinical Study. PloS One 2016;11:e0164646. doi:10.1371/journal.pone.0164646.

lP

[100] Jayne DRW, Bruchfeld AN, Harper L, Schaier M, Venning MC, Hamilton P, et al. Randomized

na

Trial of C5a Receptor Inhibitor Avacopan in ANCA-Associated Vasculitis. J Am Soc Nephrol 2017;28:2756–67. doi:10.1681/ASN.2016111179.

ur

[101] Merkel PA, Niles J, Jimenez R, Spiera RF, Rovin BH, Bomback A, et al. A Randomized Clinical Trial of CCX168, an Orally Administered C5aR Inhibitor for Treatment of Patients with ANCA-

Jo

Associated Vasculitis (abstract). Arthritis Rheumatol 2016 2016:68 (suppl 10). [102] Vickers AJ. The use of percentage change from baseline as an outcome in a controlled trial is statistically inefficient: a simulation study. BMC Med Res Methodol 2001;1. doi:10.1186/14712288-1-6. [103] Harrell FE, Slaughter JC. Biostatistics for Biomedical Research. 2016. [104] Bland JM, Altman DG. Comparisons against baseline within randomised groups are often used and can be highly misleading. Trials 2011;12:264. doi:10.1186/1745-6215-12-264. [105] ChemoCentryx. A Phase 3 Clinical Trial of CCX168 (Avacopan) in Patients With ANCAAssociated Vasculitis (ADVOCATE) (NCT02994927), 2016.

Journal Pre-proof [106] Guillevin L. Maintenance treatment of ANCA-associated vasculitides. Clin Exp Rheumatol 2017;35 Suppl 103:199–201. [107] King C, Harper L. Avoidance of Harm From Treatment for ANCA-Associated Vasculitis. Curr Treat Options Rheumatol 2017;3:230–43. doi:10.1007/s40674-017-0082-y. [108] Little MA, Nightingale P, Verburgh CA, Hauser T, De Groot K, Savage C, et al. Early mortality in systemic vasculitis: relative contribution of adverse events and active vasculitis. Ann Rheum Dis 2010;69:1036–43. doi:10.1136/ard.2009.109389.

ro of

[109] McGregor JG, Hogan SL, Hu Y, Jennette CE, Falk RJ, Nachman PH. Glucocorticoids and relapse and infection rates in anti-neutrophil cytoplasmic antibody disease. Clin J Am Soc Nephrol CJASN 2012;7:240–7. doi:10.2215/CJN.05610611.

-p

[110] Pepper RJ, McAdoo SP, Moran SM, Kelly D, Scott J, Hamour S, et al. A novel glucocorticoid-

re

free maintenance regimen for anti-neutrophil cytoplasm antibody–associated vasculitis. Rheumatology 2018. doi:10.1093/rheumatology/key288.

lP

[111] McAdoo SP, Medjeral-Thomas N, Gopaluni S, Tanna A, Mansfield N, Galliford J, et al. Long-

na

term follow-up of a combined rituximab and cyclophosphamide regimen in renal anti-neutrophil cytoplasm antibody-associated vasculitis. Nephrol Dial Transplant Off Publ Eur Dial Transpl

Jo

ur

Assoc - Eur Ren Assoc 2018. doi:10.1093/ndt/gfx378.

Journal Pre-proof VII.

Tables and figures legends

Figure 1 - The complement system Three pathways lead to complement activation: the classical, the lectin and the alternative pathways. The classical pathway is activated by immune complexes (antigen-antibody complexes) binding to C1q. Subsequent activation of C1r and C1s leads to C2 and C4 activation, thus forming the classical C3 convertase (C4bC2a). The lectin pathway is activated by microbial surfaces, through MBL and MASP, and leads to the same classical C3 convertase. The alternative pathway has a special feature: it is

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constitutively activated at a low level (C3 tick-over, resulting from the C3 hydrolysis). Activated C3, helped by properdin, associates with activated factor B to form the alternative C3 convertase (C3bBb). Both types of C3 convertase further amplify C3b generation, essential for the classical (C4bC2aC3b)

-p

and alternative (C3bBbC3b) C5 convertase. This key enzyme orchestrates the proteolysis of C5 to C5a,

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a potent anaphylatoxin involved in inflammation, chemotaxis and neutrophil activation, and C5b,

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essential for MAC assembly (C5b-9). Complement regulatory factors are shown in red boxes.

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Abbreviation: Ab, antibody; Ag, antigen; C1-inh, C1-inhibitor; CD, cluster of differentiation; MAC, membrane attack complex; MASP, mannan-binding lectin serine protease; MBL, mannose binding

ur

lectin.

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Figure 2 - Complement alternative pathway in AAVs: two decades from bench to bedside References are in top-right corner. Abbreviation: AAV, ANCA-associated vasculitis; CCX168, avacopan; Ig, immunoglobulin; NCGN, Necrotizing and Crescentic Glomerulonephritis; ↓, low level; ↑, high level; ↔, is correlated to.

Journal Pre-proof Figure 3 - Complement alternative pathway activation in ANCA-associated vasculitis pathogenesis (1) An inflammatory stimulus, such as an ear-nose-throat infection leads to neutrophil priming through pro-inflammatory cytokine secretion such as TNFα. (2) This induces the translocation of PR3 and MPO to the neutrophil cytoplasmic membrane, which become accessible to circulating ANCAs. (3) ANCAs induce neutrophil activation leading to (4) endothelial adhesion, favored by the increased expression of adhesion molecule secondary to pro-inflammatory stimuli, followed by vascular destruction by the

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release of ROS and lytic enzymes creating the vasculitis; (5) NETs production, perpetuating the presentation of auto-antigens to the innate immune system and complement activating factor production, especially from the alternative pathway such as C3, factor B or properdin. (6) Eventually, the

re

by favoring neutrophil chemotaxis and priming.

-p

subsequently generated C5a, secondary to alternative pathway activation, will further amplify this loop

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Abbreviations: B, factor B; NETs, neutrophil extracellular traps; Prop, properdin; ROS, reactive oxygen

Jo

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species.

Journal Pre-proof

Table 1 – Phase II and III trials testing avacopan in AAV patients

Trial (patients)

Inclusion criteria

CLEAR (Phase II) N=67

proven new or relapsing AAVs and PR3 or MPO ANCA

CYC+AZA or RTX and:

(ii) ava 30mg bid + pred 20mg (n=22) (iii) ava 30mg bid + pred 60mg

(Phase II)

relapsing AAVs and PR3

CYC (i) SoC + placebo (n=13)

or MPO ANCA

(ii) SoC + ava 10 mg bid (n=13)

N>300

Anti-MPO or -Pr3 ANCA eGFR>15 ml/min/1.73m²

19/22*

17/21*

i

ii

p e

r P

f o

% ↓ uACR -21%

-56%*

u o

J

(ii) ava 30mg bid + pred placebo

2/13

l a

-43%

-70%*

-50%

[100]

AEs-SAEs 21 (91%)

19 (86%)

21 (96%)

4 (17%)

3 (14%)

8 (36%)

↓ in BVAS >50%: 3/16

10/13

New/relapsing AAV: 64%/36%

11/13

12/16

Δ eGFR from BL:

PR3/MPO-ANCA: 50%/50%

+0.8

-0.8

[101] +3.1

BVAS: 15.3±6.6 eGFR: 59±27 mL/min/1.73 m²

+ 8 weeks follow-up:

(i) ava placebo + usual dose of pred

-43%

Safety (SAEs): 2/13

iii

% ↓ uMCP-1

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BVAS ≈ 13.8±6.1

eGFR ≈ 52±20 mL/min/1.73 m²

rn

(iii) SoC + ava 30 mg bid (n=16)

(Phase III)

PR3/MPO-ANCA: 45%/55%

Female/male: 55%/45%.

Patients with new or

Need of CYC or RTX

14/20

Age: 58±13 years.

52 weeks intervention

iii

↓ in BVAS >50%:

New/relapsing AAV: 73%/27%

(i) placebo + pred 60mg (n=20)

CLASSIC

ADVOCATE

ii

Female/male: 30%/70%.

12 weeks:

Severe GPA or MPA

i

Age ≈ 58±14 years.

(n=21)

N=42

Secondary endpoints Ref.

inclusion1

12 weeks: Patients with biopsy-

Primary endpoint

Patient characteristics at

Intervention

Remission (BVAS=0)

GTI

at week 26

BVAS=0 at week 4

NA

QoL improvement Remission (BVAS=0)

Number and % relapse

at week 52

eGFR over 52 weeks

Abbreviations: ava, avacopan; AZA, azathioprine; bid, twice daily; BVAS, Birmingham Vasculitis Activity Score; CYC, cyclophosphamide; eGFR, estimated Glomerular Filtration Rate; GTI, glucocorticoid toxicity index; NA, not available; pred, prednisone; QoL, quality of life; ref, reference; RTX, rituximab; SAE, serious adverse events; SoC, standard of care: rituximab or cyclophosphamide plus full-dose corticoid; uACR, urinary albuminuria/creatininuria ratio; uMCP: urinary MCP (monocyte chemotactic protein); Δ, change from baseline (BL). *: p<0.05 vs group i. 1

for CLEAR trial, data from the three groups have been averaged.