Alternative Pathway C3 Convertase Research Articles

Abstract 2915: Targeting complement factor H renders STK11-mutant lung adenocarcinoma sensitive to anti-PD-1 therapy in mice

Abstract STK11 mutations occurs in 15-20% of lung adenocarcinoma (LUAD) and is associated with KRAS mutations, poor survival and resistance to immune checkpoint inhibitors (ICI). We observed tumor cell lines and primary human STK11-mutant LUAD upregulates of expression of C3 and complement factor H (FH). Deletion of C3 in STK11 knock-out tumor cells resulted in reduced FH expression. FH is a co-factor of Factor I, which converts C3b to proteolytically inactive iC3b thereby limiting activation of the alternative pathway (AP) C3 convertase. FH binds to proteoaminoglycans on the cell membrane and protects cells from AP complement-dependent cytotoxicity. With increased C3 generation by STK11-mutant tumor cells, we reasoned that systemic or tumor-derived FH protects tumor cells from complement attack. We hypothesized that tumor-derived C3 would promote STK11-mutant LUAD growth in immune intact mice by promoting immune evasion and would drive ICI resistance. Based on the expected tumor-protective effects of FH, we also hypothesized that anti-FH antibodies would control tumor growth and render tumor more sensitive to ICI. We evaluated growth of subcutaneously administered CMT167-STK11KO with and without C3 deletion (CMT167-STK11KO is murine KRASG12V LUAD with CRISPR/Cas9 deletion of STK11). Deletion of C3 in CMT167-STK11KO (CMT167-STK11KO/C3KO) resulted in dramatic inhibition of tumor growth and in sensitivity to anti-PD-1 therapy in vivo. In contrast, deletion of C3 in STK11-KO tumors had no effect on tumor growth in nude mice or following CD8 depletion in WT mice. These findings support a role of tumor-derived C3 in evasion of T cell anti-tumor immunity and as an obstacle to anti-PD-1 therapy. GT103 is a therapeutic antibody that binds tumor cell-attached CFH, causes complement activation, kills tumor cells, initiates a shift from a pro- to an anti-tumor immune microenvironment, and is currently being evaluated in a Phase 1b-2 clinical trial in combination with pembrolizumab (NCT05617313). We observed that single agent GT103 or anti-PD-1 has limited efficacy in CMT167-STK11KO tumor in WT mice. However, the combination of GT103 and anti-PD-1 was very effective in limiting tumor growth. Our results support a role of tumor-derived C3 in suppressing CD8 T-cell immunity and in driving ICI resistance and that the addition of GT103 renders STK11-KO tumors sensitive to anti-PD-1 in mice. These studies establish rationale for evaluating this combination in patients with STK11-mutant LUAD. Citation Format: Sora Suzuki, Anm Nazmul Khan, Thejaswini Giridharan, Catrina Ting, Ryan Bushey, Elizabeth Gottlin, Michael Campa, Edward F. Patz, Edwin H. Yau, Brahm Segal. Targeting complement factor H renders STK11-mutant lung adenocarcinoma sensitive to anti-PD-1 therapy in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2915.

Alternative Pathway Masp-3 Inhibitor OMS906 Effectively and Potently Inhibits Complement-Mediated Hemolysis in Preclinical Models Mechanistically Similar to Paroxysmal Nocturnal Hemoglobinuria

Introduction: Mannan-binding lectin-associated serine protease-3 (MASP-3) activates pro-complement Factor D (CFD) to form mature CFD, a rate-limiting enzyme in the alternative pathway (AP) of the complement system. CFD controls cleavage of complement Factor B (CFB) to form AP C3 convertase, which drives an amplification loop of protease complexes that activates both C3b-mediated opsonization and the terminal pathway. OMS906 is a humanized IgG4 monoclonal antibody (mAb) that binds to and inhibits MASP-3, thereby blocking maturation of CFD and downstream AP activity. Data from mice and monkeys demonstrated that OMS906 is highly selective and effectively inhibits AP activity. MASP-3 inhibition could provide therapeutic benefit in a variety of AP-mediated diseases, such as paroxysmal nocturnal hemoglobinuria (PNH). In PNH, a rare and life-threatening disorder, red blood cells (RBCs) lacking CD59 and CD55 regulatory surface proteins are targeted for rapid clearance via intravascular hemolysis and extravascular hemolysis, due to AP dysregulation. Clinical efficacy of OMS906 in a study of patients with PNH has been reported. To provide mechanistic support, we conducted in vitro and in vivo studies using preclinical models of complement-mediated hemolysis that have mechanistic overlap with PNH. Methods: In vitro potency of OMS906 was assessed using RBCs deficient in CD55 and CD59 to mimic the physiological effect of PNH. Healthy human donor RBCs were treated with inhibitory CD55 and CD59 antibodies, then incubated under AP assay conditions with human serum (diluted to 50%) containing OMS906, anti-C5 IgG4 mAb, or an isotype mAb control. The potency of OMS906, expressed as the half-maximal inhibitory concentration (IC 50), was assessed based on prevention of hemolysis of the PNH-like RBCs in vitro. Lysis was quantified by measuring hemoglobin released into sample supernatants using spectrophotometric absorbance. The in vivo effect of OMS906 on survival of murine RBCs prone to rapid clearance was assessed using RBCs from Crry -/- mice, which lack a rodent-specific complement regulatory protein that blocks AP activation. C57BL/6J male mice were intravenously injected with fluorescent labelled Crry -/- RBCs and received OMS906 or isotype mAb control via subcutaneous injection, or anti-CFB mAb via intraperitoneal injection. Blood samples were taken daily until Day 14 and the remaining number of Crry -/- RBCs measured by flow cytometry. Results: In vitro, OMS906 inhibited terminal cell lysis in human PNH-like RBCs more potently than a C5 inhibitor. Comparison of representative drug concentration-response showed that the average functional potency was 58-fold greater with OMS906 (IC 50: 0.8 μg/mL; 3 experiments; 12 total RBC donors) than with anti-C5 mAb (IC 50: 46.2 μg/mL; 2 experiments; 4 total RBC donors). In addition, the maximal extent to which lysis was inhibited was consistently greater with OMS906 than with anti-C5 mAb. In vivo, the rate of decline in number of Crry -/- RBCs was significantly slowed in mice treated with OMS906, in contrast to the rapid loss observed in isotype mAb control-treated mice ( Figure). OMS906 recipients showed a rate of Crry -/- RBC survival similar to the anti-CFB mAb group. Conclusion: OMS906 inhibited RBC lysis more potently than a C5 inhibitor, providing evidence that MASP-3 inhibition blocks downstream terminal activity and thus intravascular hemolysis. In addition, OMS906 improved survival of Crry -/- RBCs comparably to a CFB inhibitor, indicating that MASP-3 inhibition prevents the AP-mediated destruction of RBCs predictive of extravascular hemolysis. OMS906 is currently in clinical development for the treatment of PNH.

Cryo-EM structures of Trypanosoma brucei gambiense ISG65 with human complement C3 and C3b and their roles in alternative pathway restriction

African Trypanosomes have developed elaborate mechanisms to escape the adaptive immune response, but little is known about complement evasion particularly at the early stage of infection. Here we show that ISG65 of the human-infective parasite Trypanosoma brucei gambiense is a receptor for human complement factor C3 and its activation fragments and that it takes over a role in selective inhibition of the alternative pathway C5 convertase and thus abrogation of the terminal pathway. No deposition of C4b, as part of the classical and lectin pathway convertases, was detected on trypanosomes. We present the cryo-electron microscopy (EM) structures of native C3 and C3b in complex with ISG65 which reveal a set of modes of complement interaction. Based on these findings, we propose a model for receptor-ligand interactions as they occur at the plasma membrane of blood-stage trypanosomes and may facilitate innate immune escape of the parasite.

Autoantibodies against complement factor B in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disorder affecting the joints. Many patients carry anti-citrullinated protein autoantibodies (ACPA). Overactivation of the complement system seems to be part of the pathogenesis of RA, and autoantibodies against the pathway initiators C1q and MBL, and the regulator of the complement alternative pathway, factor H (FH), were previously reported. Our aim was to analyze the presence and role of autoantibodies against complement proteins in a Hungarian RA cohort. To this end, serum samples of 97 ACPA-positive RA patients and 117 healthy controls were analyzed for autoantibodies against FH, factor B (FB), C3b, C3-convertase (C3bBbP), C1q, MBL and factor I. In this cohort, we did not detect any patient with FH autoantibodies but detected C1q autoantibodies in four patients, MBL autoantibodies in two patients and FB autoantibodies in five patients. Since the latter autoantibodies were previously reported in patients with kidney diseases but not in RA, we set out to further characterize such FB autoantibodies. The isotypes of the analyzed autoantibodies were IgG2, IgG3, IgGκ, IgGλ and their binding site was localized in the Bb part of FB. We detected in vivo formed FB-autoanti-FB complexes by Western blot. The effect of the autoantibodies on the formation, activity and FH-mediated decay of the C3 convertase in solid phase convertase assays was determined. In order to investigate the effect of the autoantibodies on complement functions, hemolysis assays and fluid phase complement activation assays were performed. The autoantibodies partially inhibited the complement-mediated hemolysis of rabbit red blood cells, inhibited the activity of the solid phase C3-convertase and C3 and C5b-9 deposition on complement activating surfaces. In summary, in ACPA-positive RA patients we identified FB autoantibodies. The characterized FB autoantibodies did not enhance complement activation, rather, they had inhibitory effect on complement. These results support the involvement of the complement system in the pathomechanism of RA and raise the possibility that protective autoantibodies may be generated in some patients against the alternative pathway C3 convertase. However, further analyses are needed to assess the exact role of such autoantibodies.

A Monoclonal Antibody That Provides a Model for C3 Nephritic Factors.

Complement is a major innate defense system that protects the intravascular space from microbial invasion. Complement activation results in the assembly of C3 convertases, serine proteases that cleave complement protein C3, generating bioactive fragments C3a and C3b. The complement response is rapid and robust, largely due to a positive feedback regulatory loop mediated by alternative pathway (AP) C3 convertase. C3 nephritic factors (C3NEFs) are autoantibodies that stabilize AP convertase, resulting in uncontrolled C3 cleavage, which, in principle, can promote critical tissue injury similar to that seen in certain renal conditions. Investigations of C3NEFs are hampered by a challenging issue: each C3NEF is derived from a different donor source, and there is no method to compare one C3NEF to another. We have identified a widely available mouse anti-C3 mAb that, similar to many C3NEFs, can stabilize functional AP convertase in a form resistant to decay acceleration by multiple complement regulators. The antibody requires the presence of properdin to confer convertase stability, and hampers the activity of Salp20, a tic salivary protein that accelerates convertase dissociation by displacing properdin from the convertase complex. This mAb can serve as an urgently needed standard for the investigation of C3NEFs. This study also provides novel insights into the dynamics of AP convertase.

Modeling C3 glomerulopathies: C3 convertase regulation on an extracellular matrix surface.

C3 glomerulopathies (C3G) are ultra-rare complement-mediated diseases that lead to end-stage renal disease (ESRD) within 10 years of diagnosis in ~50% of patients. Overactivation of the alternative pathway (AP) of complement in the fluid phase and on the surface of the glomerular endothelial glycomatrix is the underlying cause of C3G. Although there are animal models for C3G that focus on genetic drivers of disease, in vivo studies of the impact of acquired drivers are not yet possible. Here we present an in vitro model of AP activation and regulation on a glycomatrix surface. We use an extracellular matrix substitute (MaxGel) as a base upon which we reconstitute AP C3 convertase. We validated this method using properdin and Factor H (FH) and then assessed the effects of genetic and acquired drivers of C3G on C3 convertase. We show that C3 convertase readily forms on MaxGel and that this formation was positively regulated by properdin and negatively regulated by FH. Additionally, Factor B (FB) and FH mutants impaired complement regulation when compared to wild type counterparts. We also show the effects of C3 nephritic factors (C3Nefs) on convertase stability over time and provide evidence for a novel mechanism of C3Nef-mediated C3G pathogenesis. We conclude that this ECM-based model of C3G offers a replicable method by which to evaluate the variable activity of the complement system in C3G, thereby offering an improved understanding of the different factors driving this disease process.

Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of the Alternative Pathway Masp-3 Inhibitor OMS906 in a Phase 1 Study of Healthy Subjects

Introduction: The alternative pathway of the complement system is implicated in a wide variety of diseases. Complement Factor D (CFD) drives formation of alternative pathway C3 convertase, generating C3b and creating a positive feedback loop of protease complexes. Left unchecked, this amplification cycle leads to inflammation, tissue destruction, and the intravascular and extravascular hemolysis observed in paroxysmal nocturnal hemoglobinuria (PNH). Mannan-binding lectin-associated serine protease-3 (MASP-3) is the activator of CFD, responsible for conversion of its zymogen form (pro-CFD) to mature CFD. OMS906 is a humanized IgG4 monoclonal antibody (mAb) that binds to and inhibits MASP-3. Preclinical studies of OMS906 in mice and cynomolgus monkeys have demonstrated inhibition of alternative pathway activity via MASP-3 inhibition, suggesting therapeutic potential of OMS906 in the treatment of diseases associated with alternative pathway dysregulation. Here we report the safety, tolerability, pharmacokinetics (PK) and pharmacodynamics (PD) of OMS906 in healthy subjects. Methods: This was a Phase 1, first-in-human, randomized, double-blind, single-center, placebo-controlled study of OMS906 in healthy subjects. Males or females aged 18-64 years with a body mass index (BMI) of 20-32 kg/m2 were enrolled. Subjects were grouped into 9 single-ascending dose cohorts from 0.1-5 mg/kg intravenous (IV) OMS906 and 3-8 mg/kg subcutaneous (SC) OMS906. Cohorts comprised 8 subjects each (6:2 active:placebo). Study drug was delivered by 30-min infusion (50 mL volume for IV and no dilution for SC). The primary objective was to assess safety and tolerability of OMS906 in healthy subjects. The secondary objectives were to characterize PK and PD of single-ascending IV and SC doses of OMS906, and to assess the presence of anti-drug antibodies (ADAs) against OMS906 in healthy subjects. Results: Seventy-two healthy subjects were studied; 54 received OMS906 (30 IV and 24 SC) and 18 received placebo. Mean age was 43 years (range, 20-63), mean BMI 26.7 kg/m2 (range, 21.0-31.4) and mean weight 77.7 kg (range, 50.8-105.7). Males (n=35, 48.6%) and females (n=37, 51.4%) were represented in nearly equal numbers. Subjects were White (n=40, 55.6%), Black or African American (n=22, 30.6%), Asian (n=3, 4.2%), American Indian (n=2, 2.8%) or designated as Multiple Ethnicity (n=5, 6.9%). There were no serious adverse events (AEs) or discontinuations due to AEs. Most treatment-emergent AEs (TEAEs) were mild in severity; there were no severe TEAEs. Most TEAEs were determined unlikely due to OMS906 with exception of injection site reactions (ISRs), which were the most frequently reported AEs. Reported ISRs comprised: 27 ISRs in 17 of 24 subjects receiving SC OMS906 (70.8%); 1 ISR in a subject receiving IV OMS906 (3.3%); 3 ISRs in 2 of 8 subjects receiving SC placebo (25.0%); and none in subjects receiving IV placebo. There were no hypersensitivity reactions. ADAs were detected in subjects receiving OMS906 and the degree of reactivity increased to Day 85. The overall confirmed positive rate of ADAs was 20.4% (n=11/54 receiving OMS906), consistent with the reported frequency of other mAbs in development (Vaisman-Mentesh A, et al. Front. Immunol. 2020;11:1951). Among subjects with ADAs, there was no obvious dose response or difference in ADA frequencies between IV and SC OMS906 administration and no evidence of neutralizing activity. OMS906 displayed consistent PK properties with dose proportionality and non-linearity for both IV and SC administration (Table). Tmax for SC cohorts was 96-239 hours (median). A long half-life (103-423 hours) was observed, with measurable drug concentrations detected at Day 85 for the 5 mg/kg IV and 8 mg/kg SC OMS906 cohorts. The key pharmacodynamic marker, mature CFD, showed a dose-proportional response with rapid suppression and substantial degree of suppression of long duration. Conclusion: In this Phase 1 study of healthy subjects, MASP-3 inhibitor OMS906 was well tolerated with no safety concerns. There were no serious AEs. The most common AEs were ISRs, predominantly observed in the SC OMS906 cohorts. The observed PK and PD profiles were consistent with predictions from preclinical assessments, showing predictable systemic exposure, evidence of alternative pathway inhibition, and a long duration of action. Further clinical study of OMS906 is warranted, including in patients with PNH. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Scaffold hopping via ring opening enables identification of acyclic compounds as new complement Factor D inhibitors

The three complement pathways comprising the early phase of the complement system (the classical, lectin, and alternative pathways) act together with the innate and adaptive immune systems to protect against foreign entities and maintain tissue homeostasis. While these systems are normally under tight regulatory control, several diseases have been reported to correlate with uncontrolled activation and amplification of the alternative pathway, including paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, C3 glomerulopathy, and age-related macular degeneration. Complement FactorD (CFD), a serine protease, is the rate-limiting enzyme for the activity of alternative pathway. CFD activates the alternative pathway by cleaving Complement Factor B complexed to C3b (C3bB) to generate alternative pathway C3 convertase (C3bBb). In our search for novel CFD inhibitors with therapeutic potential, we employed a hot-spot analysis of an ensemble of apo and holo CFD structures. This analysis identified potential pharmacophore features that aided in the design of a series of compounds based on an l-proline core. While these compounds inhibited CFD in an esterolytic assay (for example, a proline-based compound, IC50 = 161 nM), the pharmacokinetic (PK) properties were poor. A strategy of scaffold hopping via ring opening led to a novel series of acyclic compounds, with subsequent structure-based ligand design and lead optimization producing several novel CFD inhibitors. One of these inhibitors, 1-(2-((2-(3-chloro-2-fluorobenzylamino)-2-oxoethyl)(cyclopropyl)amino)-2-oxoethyl)-5-(3-methyl-3-(1-methylpiperidin-4-yl)ureido)-1H-indazole-3-carboxamide, showed good potency with IC50s of 37 nM in the esterolytic assay and 30 nM in a hemolytic assay and PK assessments following oral administration to rats revealed a Cmax of 113 ng/mL and an AUC0-24h of 257 hr.ng/mL.

Structure-Guided Engineering of a Complement Component C3-Binding Nanobody Improves Specificity and Adds Cofactor Activity.

The complement system is a part of the innate immune system, where it labels intruding pathogens as well as dying host cells for clearance. If complement regulation is compromised, the system may contribute to pathogenesis. The proteolytic fragment C3b of complement component C3, is the pivot point of the complement system and provides a scaffold for the assembly of the alternative pathway C3 convertase that greatly amplifies the initial complement activation. This makes C3b an attractive therapeutic target. We previously described a nanobody, hC3Nb1 binding to C3 and its degradation products. Here we show, that extending the N-terminus of hC3Nb1 by a Glu-Trp-Glu motif renders the resulting EWE-hC3Nb1 (EWE) nanobody specific for C3 degradation products. By fusing EWE to N-terminal CCP domains from complement Factor H (FH), we generated the fusion proteins EWEnH and EWEµH. In contrast to EWE, these fusion proteins supported Factor I (FI)-mediated cleavage of human and rat C3b. The EWE, EWEµH, and EWEnH proteins bound C3b and iC3b with low nanomolar dissociation constants and exerted strong inhibition of alternative pathway-mediated deposition of complement. Interestingly, EWEnH remained soluble above 20 mg/mL. Combined with the observed reactivity with both human and rat C3b as well as the ability to support FI-mediated cleavage of C3b, this features EWEnH as a promising candidate for in vivo studies in rodent models of complement driven pathogenesis.

POS-041 BCX9930, AN ORAL FACTOR D INHIBITOR, SUPPRESSES COMPLEMENT ALTERNATIVE PATHWAY ACTIVITY IN PATIENTS WITH COMPLEMENT 3 GLOMERULOPATHY

POS-041 BCX9930, AN ORAL FACTOR D INHIBITOR, SUPPRESSES COMPLEMENT ALTERNATIVE PATHWAY ACTIVITY IN PATIENTS WITH COMPLEMENT 3 GLOMERULOPATHY

POS-017 A POPULATION PERSPECTIVE OF NEPHRITIC FACTORS AS SYSTEMIC DRIVERS OF COMPLEMENT DYSREGULATION IN C3 GLOMERULOPATHY

POS-017 A POPULATION PERSPECTIVE OF NEPHRITIC FACTORS AS SYSTEMIC DRIVERS OF COMPLEMENT DYSREGULATION IN C3 GLOMERULOPATHY

Factor H-Related Protein 1 Drives Disease Susceptibility and Prognosis in C3 Glomerulopathy.

C3 glomerulopathy (C3G) is a heterogeneous group of chronic renal diseases characterized predominantly by glomerular C3 deposition and complement dysregulation. Mutations in factor H-related (FHR) proteins resulting in duplicated dimerization domains are prototypical of C3G, although the underlying pathogenic mechanism is unclear. Using in vitro and in vivo assays, we performed extensive characterization of an FHR-1 mutant with a duplicated dimerization domain. To assess the FHR-1 mutant's association with disease susceptibility and renal prognosis, we also analyzed CFHR1 copy number variations and FHR-1 plasma levels in two Spanish C3G cohorts and in a control population. Duplication of the dimerization domain conferred FHR-1 with an increased capacity to interact with C3-opsonized surfaces, which resulted in an excessive activation of the alternative pathway. This activation does not involve C3b binding competition with factor H. These findings support a scenario in which mutant FHR-1 binds to C3-activated fragments and recruits native C3 and C3b; this leads to formation of alternative pathway C3 convertases, which increases deposition of C3b molecules, overcoming FH regulation. This suggests that a balanced FHR-1/FH ratio is crucial to control complement amplification on opsonized surfaces. Consistent with this conceptual framework, we show that the genetic deficiency of FHR-1 or decreased FHR-1 in plasma confers protection against developing C3G and associates with better renal outcome. Our findings explain how FHR-1 mutants with duplicated dimerization domains result in predisposition to C3G. They also provide a pathogenic mechanism that may be shared by other diseases, such as IgA nephropathy or age-related macular degeneration, and identify FHR-1 as a potential novel therapeutic target in C3G.

POS-040 BCX9930, AN ORAL FACTOR D INHIBITOR IN DEVELOPMENT FOR THE TREATMENT OF COMPLEMENT-MEDIATED KIDNEY DISEASES, INHIBITS COMPLEMENT ALTERNATIVE PATHWAY ACTIVITY IN HEALTHY SUBJECTS

Complement Factor D, a serine protease, is the rate-limiting enzyme of the alternative pathway (AP) and has the lowest concentration in plasma among all the complement proteins. Factor D is essential for the formation of AP C3 convertase, as well as for the amplification of complement activity initiated by all 3 complement pathways, including amplification of up to 80% of classical and lectin pathways’ complement responses (Harboe et al, Clin Exp Immunol 2004). BCX9930 is an orally administered, potent and selective inhibitor of Factor D under development for the treatment of complement-mediated kidney diseases, including C3 glomerulopathy, IgA nephropathy, and primary membranous nephropathy.

Structure and function of a family of tick-derived complement inhibitors targeting properdin

Activation of the serum-resident complement system begins a cascade that leads to activation of membrane-resident complement receptors on immune cells, thus coordinating serum and cellular immune responses. Whilst many molecules act to control inappropriate activation, Properdin is the only known positive regulator of the human complement system. By stabilising the alternative pathway C3 convertase it promotes complement self-amplification and persistent activation boosting the magnitude of the serum complement response by all triggers. In this work, we identify a family of tick-derived alternative pathway complement inhibitors, hereafter termed CirpA. Functional and structural characterisation reveals that members of the CirpA family directly bind to properdin, inhibiting its ability to promote complement activation, and leading to potent inhibition of the complement response in a species specific manner. We provide a full functional and structural characterisation of a properdin inhibitor, opening avenues for future therapeutic approaches.

Human Properdin Released By Infiltrating Neutrophils Can Modulate Influenza A Virus Infection.

The complement system is designed to recognise and eliminate invading pathogens via activation of classical, alternative and lectin pathways. Human properdin stabilises the alternative pathway C3 convertase, resulting in an amplification loop that leads to the formation of C5 convertase, thereby acting as a positive regulator of the alternative pathway. It has been noted that human properdin on its own can operate as a pattern recognition receptor and exert immune functions outside its involvement in complement activation. Properdin can bind directly to microbial targets via DNA, sulfatides and glycosaminoglycans, apoptotic cells, nanoparticles, and well-known viral virulence factors. This study was aimed at investigating the complement-independent role of properdin against Influenza A virus infection. As one of the first immune cells to arrive at the site of IAV infection, we show here that IAV challenged neutrophils released properdin in a time-dependent manner. Properdin was found to directly interact with haemagglutinin, neuraminidase and matrix 1 protein Influenza A virus proteins in ELISA and western blot. Furthermore, modelling studies revealed that properdin could bind HA and NA of the H1N1 subtype with higher affinity compared to that of H3N2 due to the presence of an HA cleavage site in H1N1. In an infection assay using A549 cells, properdin suppressed viral replication in pH1N1 subtype while promoting replication of H3N2 subtype, as revealed by qPCR analysis of M1 transcripts. Properdin treatment triggered an anti-inflammatory response in H1N1-challenged A549 cells and a pro-inflammatory response in H3N2-infected cells, as evident from differential mRNA expression of TNF-α, NF-κB, IFN-α, IFN-β, IL-6, IL-12 and RANTES. Properdin treatment also reduced luciferase reporter activity in MDCK cells transduced with H1N1 pseudotyped lentiviral particles; however, it was increased in the case of pseudotyped H3N2 particles. Collectively, we conclude that infiltrating neutrophils at the site of IAV infection can release properdin, which then acts as an entry inhibitor for pandemic H1N1 subtype while suppressing viral replication and inducing an anti-inflammatory response. H3N2 subtype can escape this immune restriction due to altered haemagglutinin and neuraminindase, leading to enhanced viral entry, replication and pro-inflammatory response. Thus, depending on the subtype, properdin can either limit or aggravate IAV infection in the host.

Different Aspects of Classical Pathway Overactivation in Patients With C3 Glomerulopathy and Immune Complex-Mediated Membranoproliferative Glomerulonephritis.

The rare and heterogeneous kidney disorder C3 glomerulopathy (C3G) is characterized by dysregulation of the alternative pathway (AP) of the complement system. C3G is often associated with autoantibodies stabilizing the AP C3 convertase named C3 nephritic factors (C3NeF). The role of classical pathway (CP) convertase stabilization in C3G and related diseases such as immune complex-mediated membranoproliferative glomerulonephritis (IC-MPGN) remains largely unknown. Here, we investigated the CP convertase activity in patients with C3G and IC-MPGN. Using a refined two-step hemolytic assay, we measured the stability of CP convertases directly in the serum of 52 patients and 17 healthy controls. In four patients, CP convertase activity was prolonged compared to healthy controls, i.e. the enzymatic complex was stabilized. In three patients (2 C3G, 1 IC-MPGN) the convertase stabilization was caused by immunoglobulins, indicating the presence of autoantibodies named C4 nephritic factors (C4NeFs). Importantly, the assay also enabled detection of non-immunoglobulin-mediated stabilization of the CP convertase in one patient with C3G. Prolonged CP convertase activity coincided with C3NeF activity in all patients and for up to 70 months of observation. Crucially, experiments with C3-depleted serum showed that C4NeFs stabilized the CP C3 convertase (C4bC2a), that does not contain C3NeF epitopes. All patients with prolonged CP convertase activity showed clear signs of complement activation, i.e. lowered C3 and C5 levels and elevated levels of C3d, C3bc, C3bBbP, and C5b-9. In conclusion, this work provides new insights into the diverse aspects and (non-)immunoglobulin nature of factors causing CP convertase overactivity in C3G/IC-MPGN.

CFH and CFHR Copy Number Variations in C3 Glomerulopathy and Immune Complex-Mediated Membranoproliferative Glomerulonephritis.

C3 Glomerulopathy (C3G) and Immune Complex-Mediated Membranoproliferative glomerulonephritis (IC-MPGN) are rare diseases characterized by glomerular deposition of C3 caused by dysregulation of the alternative pathway (AP) of complement. In approximately 20% of affected patients, dysregulation is driven by pathogenic variants in the two components of the AP C3 convertase, complement C3 (C3) and Factor B (CFB), or in complement Factor H (CFH) and Factor I (CFI), two genes that encode complement regulators. Copy number variations (CNVs) involving the CFH-related genes (CFHRs) that give rise to hybrid FHR proteins also have been described in a few C3G patients but not in IC-MPGN patients. In this study, we used multiplex ligation-dependent probe amplification (MLPA) to study the genomic architecture of the CFH-CFHR region and characterize CNVs in a large cohort of patients with C3G (n = 103) and IC-MPGN (n = 96) compared to healthy controls (n = 100). We identified new/rare CNVs resulting in structural variants (SVs) in 5 C3G and 2 IC-MPGN patients. Using long-read single molecule real-time sequencing (SMRT), we detected the breakpoints of three SVs. The identified SVs included: 1) a deletion of the entire CFH in one patient with IC-MPGN; 2) an increased number of CFHR4 copies in one IC-MPGN and three C3G patients; 3) a deletion from CFHR3-intron 3 to CFHR3-3′UTR (CFHR34–6Δ) that results in a FHR3-FHR1 hybrid protein in a C3G patient; and 4) a CFHR31–5-CFHR410 hybrid gene in a C3G patient. This work highlights the contribution of CFH-CFHR CNVs to the pathogenesis of both C3G and IC-MPGN.

Autoantibodies Against the Complement Regulator Factor H in the Serum of Patients With Neuromyelitis Optica Spectrum Disorder.

Neuromyelitis optica spectrum disorder (NMOSD) is an autoimmune inflammatory disease of the central nervous system (CNS), characterized by pathogenic, complement-activating autoantibodies against the main water channel in the CNS, aquaporin 4 (AQP4). NMOSD is frequently associated with additional autoantibodies and antibody-mediated diseases. Because the alternative pathway amplifies complement activation, our aim was to evaluate the presence of autoantibodies against the alternative pathway C3 convertase, its components C3b and factor B, and the complement regulator factor H (FH) in NMOSD. Four out of 45 AQP4-seropositive NMOSD patients (~9%) had FH autoantibodies in serum and none had antibodies to C3b, factor B and C3bBb. The FH autoantibody titers were low in three and high in one of the patients, and the avidity indexes were low. FH-IgG complexes were detected in the purified IgG fractions by Western blot. The autoantibodies bound to FH domains 19-20, and also recognized the homologous FH-related protein 1 (FHR-1), similar to FH autoantibodies associated with atypical hemolytic uremic syndrome (aHUS). However, in contrast to the majority of autoantibody-positive aHUS patients, these four NMOSD patients did not lack FHR-1. Analysis of autoantibody binding to FH19-20 mutants and linear synthetic peptides of the C-terminal FH and FHR-1 domains, as well as reduced FH, revealed differences in the exact binding sites of the autoantibodies. Importantly, all four autoantibodies inhibited C3b binding to FH. In conclusion, our results demonstrate that FH autoantibodies are not uncommon in NMOSD and suggest that generation of antibodies against complement regulating factors among other autoantibodies may contribute to the complement-mediated damage in NMOSD.

Duck Complement Factor H Binds to Outer Membrane Protein Omp24 of Riemerella anatipestifer.

The resistance to serum complement-mediated killing is a vital virulence property of microbial pathogens. Complement factor H (FH) is a key negative regulator of the complement alternative pathway (AP) that prevents formation and accelerates the decay of AP C3 convertase and acts as a cofactor in the inactivation of C3b. Pathogens can recruit host FH through their surface proteins to escape the clearance of the complement system. Riemerella anatipestifer could also evade the complement system attack to achieve host infection, but the mechanism is still unclear. In this study, the R. anatipestifer proteins that could interact with FH in host serum were screened and analyzed, and the functions were determined. Affinity chromatography with a Ni-nitrilotriacetic acid Sefinose column and mass spectrometry identified three outer membrane proteins (Omp) of R. anatipestifer, Omp54, Omp53, and Omp24, as potential FH-binding proteins. We then successfully conducted the prokaryotic expression and polyclonal antibody preparation of three candidate proteins. Indirect immunofluorescence assay showed that three candidate proteins were all present in R. anatipestifer. The affinity blotting assay, anti-serum-inhibiting assay, and serum bactericidal assay presented evidence that Omp24 could bind FH. Moreover, FH bound to Omp24 was associated with resistance to the alternative pathway and functional for R. anatipestifer survival in the normal duck serum. These results suggested that R. anatipestifer Omp24 was a FH-binding protein and the interaction with FH blocked the alternative pathway. Recruitment of complement regulatory proteins may facilitate better R. anatipestifer resistance to this vital line of host defense.

Inhibition of the Complement Alternative Pathway by Chemically Modified DNA Aptamers That Bind with Picomolar Affinity to Factor B.

The complement system is a conserved component of innate immunity that fulfills diverse roles in defense and homeostasis. Inappropriate activation of complement contributes to many inflammatory diseases, however, which has led to a renewed emphasis on development of therapeutic complement inhibitors. Activation of complement component C3 is required for amplification of complement and is achieved through two multisubunit proteases called C3 convertases. Of these, the alternative pathway (AP) C3 convertase is responsible for a majority of the C3 activation products in vivo, which renders it an attractive target for inhibitor discovery. In this study, we report the identification and characterization of two related slow off-rate modified DNA aptamers (SOMAmer) reagents that inhibit formation of the AP C3 convertase by binding to the proprotease, factor B (FB). These aptamers, known as SL1102 (31 bases) and SL1103 (29 bases), contain uniform substitutions of 5-(N-2-naphthylethylcarboxyamide)-2'-deoxyuridine for deoxythymidine. SL1102 and SL1103 bind FB with K d values of 49 and 88 pM, respectively, and inhibit activation of C3 and lysis of rabbit erythrocytes under AP-specific conditions. Cocrystal structures of SL1102 (3.4 Å) and SL1103 (3.1 Å) bound to human FB revealed that SL1102 and SL1103 recognize a site at the juncture of the CCP1, CCP3, and vWF domains of FB. Consistent with these structures and previously published information, these aptamers inhibited FB binding to C3b and blocked formation of the AP C3 convertase. Together, these results demonstrate potent AP inhibition by modified DNA aptamers and expand the pipeline of FB-binding molecules with favorable pharmacologic properties.