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Activation of classical and lectin complement pathways contributes to several human diseases. Empasiprubart is a humanized recycling monoclonal antibody that inhibits both pathways by binding to the CCP2 domain of complement factor 2 (C2), an interaction that is dependent on both Ca 2+ and pH. Here, we resolve the crystal structure of empasiprubart complexed with C2, providing the molecular basis of its Ca 2+ dependency, and report a randomized, double-blind, placebo-controlled trial to assess the safety and tolerability (primary objectives) in addition to pharmacokinetics, pharmacodynamics, and immunogenicity (secondary objectives) of empasiprubart in 78 healthy participants (NCT04532125). A single intravenous (IV) dose of empasiprubart reduces circulating C2 levels by up to 99% and dose-dependently inhibits the classical and lectin pathways. Multiple IV empasiprubart doses reinforce reductions in free C2 levels, which persist until the endpoint of the study at 41 weeks. This prolonged reduction is in line with the empasiprubart elimination half-life (70–88 days). Single and multiple ascending doses of empasiprubart are generally safe and well tolerated. Overall, our results reveal in atomic detail the mechanism of empasiprubart and demonstrate that it is a first-in-class anti-C2 therapeutic antibody for use in complement-mediated diseases. Though the complement system is pivotal in the defence against infections, pathologic activation of the system contributes to disease. Here, authors show that their recently developed monoclonal antibody against complement factor 2, empasiprubart, inhibits the classical and lectin pathways in a clinical trial, and its crystal structure provides basis for its inhibitory properties, such as Ca 2+ binding.

The complement system, composed of the three activation pathways, has both protective and pathogenic roles in the development of systemic lupus erythematosus (or lupus), a prototypic autoimmune disease. The classical pathway contributes to the clearance of immune complexes (ICs) and apoptotic cells, whereas the alternative pathway (AP) exacerbates renal inflammation. The role of the lectin pathway (LP) in lupus has remained largely unknown. Mannose-binding lectin (MBL)-associated serine proteases (MASPs), which are associated with humoral pattern recognition molecules (MBL or ficolins), are the enzymatic constituents of the LP and AP. MASP-1 encoded by the gene significantly contributes to the activation of the LP. After the binding of MBL/ficolins to pathogens or self-altered cells, MASP-1 autoactivates first, then activates MASP-2, and both participate in the formation of the LP C3 convertase C4b2a, whereas, MASP-3, the splice variant of the gene, is required for the activation of the zymogen of factor D (FD), and finally participates in the formation of the AP C3 convertase C3bBb. To investigate the roles of MASP-1 and MASP-3 in lupus, we generated gene knockout lupus-prone MRL/ mice ( MRL/ mice), lacking both MASP-1 and MASP-3, and analyzed their renal disease. As expected, sera from MRL/ mice had no or markedly reduced activation of the LP and AP with zymogen forms of complement FD. Compared to their wild-type littermates, the MRL/ mice had maintained serum C3 levels, little-to-no albuminuria, as well as significantly reduced glomerular C3 deposition levels and glomerular pathological score. On the other hand, there were no significant differences in the levels of serum anti-dsDNA antibody, circulating ICs, glomerular IgG and MBL/ficolins deposition, renal interstitial pathological score, urea nitrogen, and mortality between the wild-type and MRL/ mice. Our data indicate that MASP-1/3 plays essential roles in the development of lupus-like glomerulonephritis in MRL/ mice, most likely activation of the LP and/or AP.

Physiochemical stress induces tissue injury as a result of the detection of abnormal molecular patterns by sensory molecules of the innate immune system. Here, we have described how the recently discovered C-type lectin collectin-11 (CL-11, also known as CL-K1 and encoded by COLEC11) recognizes an abnormal pattern of L-fucose on postischemic renal tubule cells and activates a destructive inflammatory response. We found that intrarenal expression of CL-11 rapidly increases in the postischemic period and colocalizes with complement deposited along the basolateral surface of the proximal renal tubule in association with L-fucose, the potential binding ligand for CL-11. Mice with either generalized or kidney-specific deficiency of CL-11 were strongly protected against loss of renal function and tubule injury due to reduced complement deposition. Ex vivo renal tubule cells showed a marked capacity for CL-11 binding that was induced by cell stress under hypoxic or hypothermic conditions and prevented by specific removal of L-fucose. Further analysis revealed that cell-bound CL-11 required the lectin complement pathway-associated protease MASP-2 to trigger complement deposition. Given these results, we conclude that lectin complement pathway activation triggered by ligand-CL-11 interaction in postischemic tissue is a potent source of acute kidney injury and is amenable to sugar-specific blockade.

Ecotin is a serine protease inhibitor produced by hundreds of microbial species, including pathogens. Here we show, that ecotin orthologs from Escherichia coli, Yersinia pestis, Pseudomonas aeruginosa and Leishmania major are potent inhibitors of MASP-1 and MASP-2, the two key activator proteases of the complement lectin pathway. Factor D is the key activator protease of another complement activation route, the alternative pathway. We show that ecotin inhibits MASP-3, which is the sole factor D activator in resting human blood. In pathway-specific ELISA tests, we found that all ecotin orthologs are potent lectin pathway inhibitors, and at high concentration, they block the alternative pathway as well. In flow cytometry experiments, we compared the extent of complement-mediated opsonization and lysis of wild-type and ecotin-knockout variants of two E. coli strains carrying different surface lipopolysaccharides. We show, that endogenous ecotin provides significant protections against these microbicidal activities for both bacteria. By using pathway specific complement inhibitors, we detected classical-, lectin- and alternative pathway-driven complement attack from normal serum, with the relative contributions of the activation routes depending on the lipopolysaccharide type. Moreover, in cell proliferation experiments we observed an additional, complement-unrelated antimicrobial activity exerted by heat-inactivated serum. While ecotin-knockout cells are highly vulnerable to these activities, endogenous ecotin of wild-type bacteria provides complete protection against the lectin pathway-related and the complement-unrelated attack, and partial protection against the alternative pathway-related damage. In all, ecotin emerges as a potent, versatile self-defense tool that blocks multiple antimicrobial activities of the serum. These findings suggest that ecotin might be a relevant antimicrobial drug target.

Ficolins are a group of proteins containing both a collagen-like domain and a fibrinogen-like domain and are found in varying tissues. Ficolins present in sera have a lectin activity toward N-acetylglucosamine through their fibrinogen-like domains. The domain organizations between ficolins and mannose-binding lectin (MBL) are very similar in that both consist of a collagen-like domain and a carbohydrate-binding domain, although their carbohydrate-binding moieties are different. MBL acts as an opsonin and activates complement in association with MBL-associated serine proteases (MASPs) and sMAP, a truncated protein of MASP-2, via the lectin pathway. Like MBL, two types of human serum ficolins, L-ficolin/P35 and H-ficolin, are associated with MASPs and sMAP, and activate the lectin pathway. In addition, L-ficolin/P35 acts as an opsonin. These findings indicate that serum ficolins play an important a role in innate immunity in a similar manner to MBL.

BackgroundIgA nephropathy (IgAN) and Henoch-Schönlein purpura are common glomerular disorders in children sharing the same histopathologic pattern of IgA deposits within the mesangium, even if their physiopathology may be different. Repeated exposure to pathogens induces the production of abnormal IgA1. The immune complex deposition in the renal mesangium in IgAN or potentially in small vessels in Henoch-Schönlein purpura induces complement activation via the alternative and lectin pathways. Recent studies suggest that levels of membrane attack complex (MAC) in the urine might be a useful indicator of renal injury. Because of the emerging availability of therapies that selectively block complement activation, the aim of the present study is to investigate whether MAC immunostaining might be a useful marker of IgA-mediated renal injury.MethodsWe conducted immunohistochemistry analysis of the MAC on renal biopsies from 67 pediatric patients with IgAN and Henoch-Schönlein purpura. We classified their renal biopsies according to the Oxford classification, retrieved symptoms, biological parameters, treatment, and follow-up.ResultsWe found MAC expression was significantly related to impaired renal function and patients whose clinical course required therapy. MAC deposits tend to be more abundant in patients with decreased glomerular filtration rate (p = 0.02), patients with proteinuria > 0.750 g/day/1.73 m2, and with nephrotic syndrome. No correlation with histological alterations was observed.ConclusionsWe conclude that MAC deposition could be a useful additional indicator of renal injury in patients with IgAN and Henoch-Schönlein purpura, independent of other indicators.

ObjectiveSLE is a systemic autoimmune disease in which the complement system plays a key pathogenic role, yet the contribution of the lectin pathway remains unclear. Lectin pathway-dependent complement activation is initiated by pattern-recognition molecules complexed with mannose-binding lectin (MBL)-associated serine proteases (MASPs) and MBL-associated proteins (MAPs). Here, we combined biochemical and genetic analyses to explore associations between MASP/MAP proteins, SLE manifestations and autoantibody specificities.MethodsSerum concentrations of MASP-3, MAP-1 and MASP-2 were measured using ELISA in Swedish patients with SLE (n=522) and population-based matched controls (n=322). Serum type I interferon activity was measured by a cell-based reporter assay. Associations with SLE manifestations and autoantibodies were explored using logistic regression models. Single-nucleotide genetic variants spanning the MASP1 and MASP2 genes were analysed for associations with MASP/MAP levels and SLE manifestations.ResultsPatients with MAP-1 serum concentrations in the highest quartile had significantly higher rates of discoid rash (OR 2.8 (95% CI 1.4 to 5.7)), haematological manifestations (OR 2.1 (95% CI 1.1 to 3.7)) and autoantibodies against Sm, RNP, SSA and SSB (ORs 2.4 (95% CI 1.3 to 4.6) to 3.6 (95% CI 1.7 to 7.7)). Patients in the highest quartiles of MAP-1 and MASP-2 had lower rates of anti-β2GP1 and anti-cardiolipin IgG and IgA anti-phospholipid antibodies (ORs 0.29 (95% CI 0.12 to 0.68) to 0.56 (95% CI 0.31 to 1.0)). Serum MAP-1 levels correlated with type I interferon activity (Spearman’s rho 0.34, p<0.0001), which mediated the associations of MAP-1 with haematological manifestations and Sm/RNP autoantibodies. Significant protein quantitative trait loci for MAP-1 and MASP-2 were identified; however, these did not show consistent associations with SLE or specific SLE manifestations.ConclusionsThese results demonstrate a distinct clinical and serological SLE profile associated with components of the lectin pathway. The lectin pathway-regulatory protein MAP-1 displayed the strongest associations and may serve as a marker of SLE manifestations with a type I interferon signature.

The lectin pathway of the complement system has been implicated in secondary ischemic/inflammatory injury after traumatic brain injury (TBI). However, previous experimental studies have yielded conflicting results, and human studies are scarce. In this exploratory study, we investigated associations of several lectin pathway proteins early after injury and single-nucleotide polymorphisms (SNP) with outcomes after severe TBI (mortality at 14 days [primary outcome] and consciousness assessed with the Glasgow Coma Scale [GCS] at 14 days, disability assessed with the Glasgow Outcome Scale Extended [GOSE] at 90 days). Forty-four patients with severe TBI were included. Plasma levels of lectin pathway proteins were sampled at 6, 12, 24, and 48 h after injury and eight mannose-binding lectin (MBL) and ficolin (FCN)2 SNPs were analyzed by enzyme-linked immunosorbent assay (ELISA) and genotyping, respectively. Plasma protein levels were stable with only a slight increase in mannose-binding protein-associated serine protease (MASP)-2 and FCN2 levels after 48 h (p < 0.05), respectively. Neither lectin protein plasma levels (6 h or mean levels) nor MBL2 genotypes or FCN2 variant alleles were associated with 14 day mortality or 14 day consciousness. However, FCN2, FCN3, and MASP-2 levels were higher in patients with an unfavorable outcome (GOSE 1–4) at 90 days (p < 0.05), whereas there was no difference in MBL2 genotypes or FCN2 variant alleles. In particular, higher mean MASP-2 levels over 48 h were independently associated with a GOSE score < 4 at 90 days after adjustment (odds ratio 3.46 [95% confidence interval 1.12–10.68] per 100 ng/mL increase, p = 0.03). No association was observed between the lectin pathway of the complement system and 14 day mortality or 14 day consciousness. However, higher plasma FCN2, FCN3, and, in particular, MASP-2 levels early after injury were associated with an unfavorable outcome at 90 days (death, vegetative state, and severe disability) which may be related to an increased activation of the lectin pathway.

The objective of the present study was to elucidate the association between glomerular complement depositions belonging to the alternative (AP) and lectin (LP) pathways, and clinical findings of lupus nephritis (LN). Immunofluorescence (IF) was performed on 17 LN patients using antibodies against factor B, factor H, properdin, mannose-binding lectin (MBL) and L-ficolin. Compared with factor B/factor H negative patients (n = 9), positive patients (n = 8) showed longer duration of LN (p < 0.05) and more severe interstitial fibrosis (p < 0.05). Eleven patients had properdin deposition in glomeruli, and in three of them, with a duration of LN of less than 1 month, factor B was undetectable. Compared with properdin negative patients (n = 6), positive patients (n = 11) showed significantly higher urinary protein excretion (p < 0.01). MBL/L-ficolin positive patients (n = 11) also had significantly higher urinary protein excretion (p < 0.05) compared with negative patients (n = 6). An independent association was found between glomerular deposition of properdin and that of MBL/L-ficolin (p < 0.01) in addition to factor B/factor H. Traces of glomerular activation of AP and LP reflected the clinical status of LN. It appears that glomerular deposition of each complement component, especially properdin, may be an index of the histological activity of LN.

Ficolins are a group of oligomeric lectins with subunits consisting of both collagen-like and fibrinogen-like domains. The majority of ficolins identified in vertebrates and invertebrates to date recognize N-acetylglucosamine (GlcNAc). X-ray crystallographic analysis of human ficolins has shown that the fibrinogen-like domain binds to the N-acetylated moiety. Ficolins in serum are associated with MBL-associated serine protease (MASP). The ficolin-MASP complex binds directly to carbohydrates present on the surface of a variety of Gram-positive and Gram-negative bacteria through ficolin. Binding of the complex initiates complement activation via the lectin pathway, leading to generation of opsonic fragments of complement components, such as C3b, and to lysis of the bacteria by the membrane attack complex. Thus, serum ficolins play an important role in innate immunity.