Triple-fusion protein (TriFu): A potent, targeted, enzyme-like inhibitor of all three complement activation pathways.

The introduction of a therapeutic anti-C5 antibody into clinical practice in 2007 inspired a surge into the development of complement-targeted therapies. This has led to the recent approval of a C3 inhibitory peptide, an antibody directed against C1s and a full pipeline of several complement inhibitors in preclinical and clinical development. However, no inhibitor is available that efficiently inhibits all three complement initiation pathways and targets host cell surface markers as well as complement opsonins. To overcome this, we engineered a novel fusion protein combining selected domains of the three natural complement regulatory proteins decay accelerating factor, factor H and complement receptor 1. Such a triple fusion complement inhibitor (TriFu) was recombinantly expressed and purified alongside multiple variants and its building blocks. We analyzed these proteins for ligand binding affinity and decay acceleration activity by surface plasmon resonance. Additionally, we tested complement inhibition in several in vitro/ex vivo assays using standard classical and alternative pathway restricted hemolysis assays next to hemolysis assays with paroxysmal nocturnal hemoglobinuria erythrocytes. A novel in vitro model of the alternative pathway disease C3 glomerulopathy was established to evaluate the potential of the inhibitors to stop C3 deposition on endothelial cells. Next to the novel engineered triple fusion variants which inactivate complement convertases in an enzyme-like fashion, stoichiometric complement inhibitors targeting C3, C5, factor B, and factor D were tested as comparators. The triple fusion approach yielded a potent complement inhibitor that efficiently inhibits all three complement initiation pathways while targeting to surface markers.

SAR443809: a selective inhibitor of the complement alternative pathway, targeting complement factor Bb.

Dysregulated activation of the complement system is implicated in the onset or progression of several diseases. Most clinical-stage complement inhibitors target the inactive complement proteins present at high concentrations in plasma, which increases target-mediated drug disposition and necessitates high drug levels to sustain therapeutic inhibition. Furthermore, many efforts are aimed at inhibiting only terminal pathway activity, which leaves opsonin-mediated effector functions intact. We describe the discovery of SAR443809, a specific inhibitor of the alternative pathway C3/C5 convertase (C3bBb). SAR443809 selectively binds to the activated form of factor B (factor Bb) and inhibits alternative pathway activity by blocking the cleavage of C3, leaving the initiation of classical and lectin complement pathways unaffected. Ex vivo experiments with patient-derived paroxysmal nocturnal hemoglobinuria erythrocytes show that, although terminal pathway inhibition via C5 blockade can effectively inhibit hemolysis, proximal complement inhibition with SAR443809 inhibits both hemolysis and C3b deposition, abrogating the propensity for extravascular hemolysis. Finally, intravenous and subcutaneous administration of the antibody in nonhuman primates demonstrated sustained inhibition of complement activity for several weeks after injection. Overall, SAR443809 shows strong potential for treatment of alternative pathway-mediated disorders.

Hypoxic Processes Induce Complement Activation via Classical Pathway in Porcine Neuroretinas.

Considering the fact that many retinal diseases are yet to be cured, the pathomechanisms of these multifactorial diseases need to be investigated in more detail. Among others, oxidative stress and hypoxia are pathomechanisms that take place in retinal diseases, such as glaucoma, age-related macular degeneration, or diabetic retinopathy. In consideration of these diseases, it is also evidenced that the immune system, including the complement system and its activation, plays an important role. Suitable models to investigate neuroretinal diseases are organ cultures of porcine retina. Based on an established model, the role of the complement system was studied after the induction of oxidative stress or hypoxia. Both stressors led to a loss of retinal ganglion cells (RGCs) accompanied by apoptosis. Hypoxia activated the complement system as noted by higher C3+ and MAC+ cell numbers. In this model, activation of the complement cascade occurred via the classical pathway and the number of C1q+ microglia was increased. In oxidative stressed retinas, the complement system had no consideration, but strong inflammation took place, with elevated TNF, IL6, and IL8 mRNA expression levels. Together, this study shows that hypoxia and oxidative stress induce different mechanisms in the porcine retina inducing either the immune response or an inflammation. Our findings support the thesis that the immune system is involved in the development of retinal diseases. Furthermore, this study is evidence that both approaches seem suitable models to investigate undergoing pathomechanisms of several neuroretinal diseases.

Alternative Complement Pathway Inhibition Does Not Abrogate Meningococcal Killing by Serum of Vaccinated Individuals.

Dysregulation of complement activation causes a number of diseases, including paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome. These conditions can be treated with monoclonal antibodies (mAbs) that bind to the complement component C5 and prevent formation of the membrane attack complex (MAC). While MAC is involved in uncontrolled lysis of erythrocytes in these patients, it is also required for serum bactericidal activity (SBA), i.e. clearance of encapsulated bacteria. Therefore, terminal complement blockage in these patients increases the risk of invasive disease by Neisseria meningitidis more than 1000-fold compared to the general population, despite obligatory vaccination. It is assumed that alternative instead of terminal pathway inhibition reduces the risk of meningococcal disease in vaccinated individuals. To address this, we investigated the SBA with alternative pathway inhibitors. Serum was collected from adults before and after vaccination with a meningococcal serogroup A, C, W, Y capsule conjugate vaccine and tested for meningococcal killing in the presence of factor B and D, C3, C5 and MASP-2 inhibitors. B meningococci were not included in this study since the immune response against protein-based vaccines is more complex. Unsurprisingly, inhibition of C5 abrogated killing of meningococci by all sera. In contrast, both factor B and D inhibitors affected meningococcal killing in sera from individuals with low, but not with high bactericidal anti-capsular titers. While the anti-MASP-2 mAb did not impair SBA, inhibition of C3 impeded meningococcal killing in most, but not in all sera. These data provide evidence that vaccination can provide protection against invasive meningococcal disease in patients treated with alternative pathway inhibitors.

Complement Factor D as a Strategic Target for Regulating the Alternative Complement Pathway.

The complement system is central to first-line defense against invading pathogens. However, excessive complement activation and/or the loss of complement regulation contributes to the development of autoimmune diseases, systemic inflammation, and thrombosis. One of the three pathways of the complement system, the alternative complement pathway, plays a vital role in amplifying complement activation and pathway signaling. Complement factor D, a serine protease of this pathway that is required for the formation of C3 convertase, is the rate-limiting enzyme. In this review, we discuss the function of factor D within the alternative pathway and its implication in both healthy physiology and disease. Because the alternative pathway has a role in many diseases that are characterized by excessive or poorly mediated complement activation, this pathway is an enticing target for effective therapeutic intervention. Nonetheless, although the underlying disease mechanisms of many of these complement-driven diseases are quite well understood, some of the diseases have limited treatment options or no approved treatments at all. Therefore, in this review we explore factor D as a strategic target for advancing therapeutic control of pathological complement activation.

The Sez6 Family Inhibits Complement by Facilitating Factor I Cleavage of C3b and Accelerating the Decay of C3 Convertases.

The Sez6 family consists of Sez6, Sez6L, and Sez6L2. Its members are expressed throughout the brain and have been shown to influence synapse numbers and dendritic morphology. They are also linked to various neurological and psychiatric disorders. All Sez6 family members contain 2-3 CUB domains and 5 complement control protein (CCP) domains, suggesting that they may be involved in complement regulation. We show that Sez6 family members inhibit C3b/iC3b opsonization by the classical and alternative pathways with varying degrees of efficacy. For the classical pathway, Sez6 is a strong inhibitor, Sez6L2 is a moderate inhibitor, and Sez6L is a weak inhibitor. For the alternative pathway, the complement inhibitory activity of Sez6, Sez6L, and Sez6L2 all equaled or exceeded the activity of the known complement regulator MCP. Using Sez6L2 as the representative family member, we show that it specifically accelerates the dissociation of C3 convertases. Sez6L2 also functions as a cofactor for Factor I to facilitate the cleavage of C3b; however, Sez6L2 has no cofactor activity toward C4b. In summary, the Sez6 family are novel complement regulators that inhibit C3 convertases and promote C3b degradation.

Natural immunoglobulin M-based delivery of a complement alternative pathway inhibitor in mouse models of retinal degeneration.

PURPOSE: Age-related macular degeneration is a slowly progressing disease. Studies have tied disease risk to an overactive complement system. We have previously demonstrated that pathology in two mouse models, the choroidal neovascularization (CNV) model and the smoke-induced ocular pathology (SIOP) model, can be reduced by specifically inhibiting the alternative complement pathway (AP). Here we report on the development of a novel injury-site targeted inhibitor of the alternative pathway, and its characterization in models of retinal degeneration. METHODS: Expression of the danger associated molecular pattern, a modified annexin IV, in injured ARPE-19 cells was confirmed by immunohistochemistry and complementation assays using B4 IgM mAb. Subsequently, a construct was prepared consisting of B4 single chain antibody (scFv) linked to a fragment of the alternative pathway inhibitor, fH (B4-scFv-fH). ARPE-19 cells stably expressing B4-scFv-fH were microencapsulated and administered intravitreally or subcutaneously into C57BL/6 J mice, followed by CNV induction or smoke exposure. Progression of CNV was analyzed using optical coherence tomography, and SIOP using structure-function analyses. B4-scFv-fH targeting and AP specificity was assessed by Western blot and binding experiments. RESULTS: B4-scFv-fH was secreted from encapsulated RPE and inhibited complement in RPE monolayers. B4-scFv-fH capsules reduced CNV and SIOP, and western blotting for breakdown products of C3α, IgM and IgG confirmed a reduction in complement activation and antibody binding in RPE/choroid. CONCLUSIONS: Data supports a role for natural antibodies and neoepitope expression in ocular disease, and describes a novel strategy to target AP-specific complement inhibition to diseased tissue in the eye. PRECIS: AMD risk is tied to an overactive complement system, and ocular injury is reduced by alternative pathway (AP) inhibition in experimental models. We developed a novel inhibitor of the AP that targets an injury-specific danger associated molecular pattern, and characterized it in disease models.

IL-1/IL-1R signaling induced by all-trans-retinal contributes to complement alternative pathway activation in retinal pigment epithelium.

The underlying mechanisms of complement activation in Stargardt disease type 1 (STGD1) and age-related macular degeneration (AMD) are not fully understood. Overaccumulation of all-trans-retinal (atRAL) has been proposed as the pathogenic factor in both diseases. By incubating retinal pigment epithelium (RPE) cells with atRAL, we showed that C5b-9 membrane attack complexes (MACs) were generated mainly through complement alternative pathway. An increase in complement factor B (CFB) expression as well as downregulation of complement regulatory proteins CD46, CD55, CD59, and CFH were observed in RPE cells after atRAL treatment. Furthermore, interleukin-1ß production was provoked in both atRAL-treated RPE cells and microglia/macrophages. Coincubation of RPE cells with interleukin-1 receptor antagonist (IL1Ra) and atRAL ameliorated complement activation and downregulated CFB expression by attenuating both p38 and c-Jun N-terminal kinase (JNK) signaling pathways. Our findings demonstrate that atRAL induces an autocrine/paracrine IL-1/IL-1R signaling to promote complement alternative pathway activation in RPE cells and provide a novel perspective on the pathomechanism of macular degeneration.

Effect of red seaweed sulfated galactans on initial steps of complement activation in vitro.

The research described here presents data on the effect of galactans of red algae, carrageenans (λ/µ/ν-, κ-, κ/ß-, and ι/κ-types), and agar on complement system activation in normal human serum. The experiments were based on well surfaces coated with triggering agents for binding initiating complement components -C3 and C4. The sulfated galactans inhibited C3 binding to lipopolysaccharide with direct dependence on the sulfation degree of polysaccharides. Sulfation degree was also important in carrageenans' capacity to reduce C4 binding to mannan. However, C4 binding to antibodies was considerably activated by carrageenans, especially with 3,6-anhydrogalactose. The gelling carrageenans were able to block antigen binding centers of total serum IgM and with more intensity than non-gelling. No structural characteristics mattered in ameliorating C5 cleavage by plasmin in extrinsic protease complement activation, but λ/µ/ν- and κ/ß-carrageenans almost completely inhibited C5 cleavage. Thus, galactans participated in cell surface biology by imitating surface glycans in inhibition of C3 binding and mannose binding lectin, but as to the tthe heclassical pathway these substances stimulated complement, probably due to their structure based on carrabiose.

Modeling the activation of the alternative complement pathway and its effects on hemolysis in health and disease.

The complement system is a powerful mechanism of innate immunity poised to eliminate foreign cells and pathogens. It is an intricate network of >35 proteins, which, once activated, leads to the tagging of the surface to be eliminated, produces potent chemoattractants to recruit immune cells, and inserts cytotoxic pores into nearby lipid surfaces. Although it can be triggered via different pathways, its net output is largely based on the direct or indirect activation of the alternative pathway. Complement dysregulation or deficiencies may cause severe pathologies, such as paroxysmal nocturnal hemoglobinuria (PNH), where a lack of complement control proteins leads to hemolysis and life-threatening anemia. The complexity of the system poses a challenge for the interpretation of experimental data and the design of effective pharmacological therapies. To address this issue, we developed a mathematical model of the alternative complement pathway building on previous modelling efforts. The model links complement activation to the hemolytic activity of the terminal alternative pathway, providing an accurate description of pathway activity as observed in vitro and in vivo, in health and disease. Through adjustment of the parameters describing experimental conditions, the model was capable of reproducing the results of an array of standard assays used in complement research. To demonstrate its clinical applicability, we compared model predictions with clinical observations of the recovery of hematological biomarkers in PNH patients treated with the complement inhibiting anti-C5 antibody eculizumab. In conclusion, the model can enhance the understanding of complement biology and its role in disease pathogenesis, help identifying promising targets for pharmacological intervention, and predict the outcome of complement-targeting pharmacological interventions.

Direct activation of the alternative complement pathway by SARS-CoV-2 spike proteins is blocked by factor D inhibition.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly contagious respiratory virus that can lead to venous/arterial thrombosis, stroke, renal failure, myocardial infarction, thrombocytopenia, and other end-organ damage. Animal models demonstrating end-organ protection in C3-deficient mice and evidence of complement activation in humans have led to the hypothesis that SARS-CoV-2 triggers complement-mediated endothelial damage, but the mechanism is unclear. Here, we demonstrate that the SARS-CoV-2 spike protein (subunit 1 and 2), but not the N protein, directly activates the alternative pathway of complement (APC). Complement-dependent killing using the modified Ham test is blocked by either C5 or factor D inhibition. C3 fragments and C5b-9 are deposited on TF1PIGAnull target cells, and complement factor Bb is increased in the supernatant from spike protein-treated cells. C5 inhibition prevents the accumulation of C5b-9 on cells, but not C3c; however, factor D inhibition prevents both C3c and C5b-9 accumulation. Addition of factor H mitigates the complement attack. In conclusion, SARS-CoV-2 spike proteins convert nonactivator surfaces to activator surfaces by preventing the inactivation of the cell-surface APC convertase. APC activation may explain many of the clinical manifestations (microangiopathy, thrombocytopenia, renal injury, and thrombophilia) of COVID-19 that are also observed in other complement-driven diseases such as atypical hemolytic uremic syndrome and catastrophic antiphospholipid antibody syndrome. C5 inhibition prevents accumulation of C5b-9 in vitro but does not prevent upstream complement activation in response to SARS-CoV-2 spike proteins.

C3 Glomerulopathy: Pathogenesis and Treatment.

C3 glomerulopathy (C3G) is a rare set of kidney diseases with 2 patterns: C3 glomerulonephritis (C3GN) and dense deposit disease. Pathogenesis of both diseases is due to complement dysregulation in the alternative pathway. Acquired or genetic alterations of the regulatory proteins of the complement pathway result in C3G. Although the disease is characterized by low C3 levels in serum and C3-dominant staining by immunofluorescence on biopsy, other disease entities such as infection-related glomerulonephritis and masked monoclonal deposits can present similarly. Both the C3GN and dense deposit disease variants of C3G are progressive and recur in transplanted kidneys. Although no direct treatment is available, complement blockers are either available or in the clinical trial phase. This review will survey the pathogenesis of C3GN and current treatment options.

Peri- and Post-operative Evaluation and Management of Atypical Hemolytic Uremic Syndrome (aHUS) in Kidney Transplantation.

Atypical hemolytic uremic syndrome (aHUS) is a severe thrombotic microangiopathy characterized by over-activation of the alternative complement pathway. The etiology of the dysregulated complement system is commonly a genetic variant in one or more complement proteins as identified in ∼ 60%-70% patients. The risk of recurrence after a kidney transplantation is high and depends on the underlying complement abnormality. For a long time, kidney transplantation was contraindicated in these patients because of the high rate of recurrence and subsequent allograft loss. Over the past decade, advancements in the understanding of etiopathogenesis of aHUS and approval of the anti-complement drug, eculizumab, have allowed for successful kidney transplantation in these patients. All patients with ESRD due to aHUS should undergo screening for complement genetic variants. Patients in whom a genetic variant is not identified or in whom a genetic variant of uncertain significance is identified should undergo further testing to determine etiology of disease. This review aims to shed light on the diagnostic and therapeutic considerations in patients with aHUS preceding and following kidney transplantation.

Effects of Lactobacillus rhamnosus ATCC 7469 on Different Parameters Related to Health Status of Rainbow Trout (Oncorhynchus mykiss) and the Protection Against Yersinia ruckeri.

In the current study, we investigated the effect of a probiotic bacterium (Lactobacillus rhamnosus ATCC 7469) microencapsulated with alginate and hi-maize starch and coated with chitosan on improving growth factors, body composition, blood chemistry, and the immune response of rainbow trout (initial weight: 18.41 ± 0.32 g). Four experimental diets were formulated to feed fish for 60 days. They were control diet without any additive (C), diet added with beads without probiotic (E), a probiotic sprayed to the diet (L.r), and encapsulated probiotic supplemented diet (E-L.r). The results indicated that feeding with E-Lr significantly improved weight gain (84.98 g) and feed conversion ratio (0.95) compared to the other groups (P < 0.05). Also, fish fed E-Lr diet had a significantly higher value of whole-body protein (17.51%), total protein in the blood (4.98 g/dL), lysozyme (30.66 U/mL), alternative complement pathway hemolytic activity (134 U/mL), superoxide dismutase (203 U/mg protein), and catalase (528.33 U/mg protein) (P < 0.05) as compared to those fed the control diet. Similarly, a higher relative expression of immune-related genes such as interleukin-1 (Il-1) and tumor necrosis factor-alpha (TNF-1α) were reported in those fed E-L.r and L.r diets respectively. Interestingly, the fish fed dietary E-L.r had a significantly lower value of lipid in the whole body (4.82%) and cholesterol in the blood (160.67%) in comparison with those fed the control diet (P < 0.05). At the end of the experiment, all groups were challenged by Yersinia ruckeri where the survival rate of rainbow trout fed dietary E-L.r (70.36%) was statistically higher than that of the others (P < 0.05). Overall, the results suggested that encapsulated probiotic Lact. rhamnosus ATCC 7469 acted better than unencapsulated probiotic and has a potential to improve growth performance, flesh quality, and the immune response of rainbow trout.

Paroxysmal nocturnal hemoglobinuria: Test to monitor the action of eculizumab treatment.

INTRODUCTION: Paroxysmal nocturnal hemoglobinuria (PNH) is caused by a somatic mutation in the PIG-A gene, which encodes for glycosylphosphatidylinositol, a phospholipid membrane that anchors proteins like CD55 and CD59. These proteins are inhibitors of the complement-mediated lysis. PNH is diagnosed by flow cytometry, and treatment with eculizumab improves the life quality of patients with severe clinical compromise. The aim of this work was to evaluate a hemolytic test that allows monitoring the blockade of the alternative complement pathway caused by eculizumab (herein MET test). METHODS: There were analyzed a total of 163 serum samples from nine patients with PNH under treatment with eculizumab and ten healthy volunteers like controls. The patients were evaluated for 6 months. The MET test consisted in incubating red blood cells from patients (RBCPNH ) with either acidified serum from healthy volunteers and from patients with PNH. The results can be (a) Positive, (b) Blockade profile, or (c) Negative. RESULTS: Seven patients responded favorably to the eculizumab, and the test evidenced the blockade profile. The two remaining patients were nonresponders to the treatment, with a positive MET test. In these patients, the dose was increased. One responded favorably with a blockade profile, and the other continued to be nonresponder. CONCLUSIONS: The MET test proved to be a useful tool to monitor the blockade of complement by eculizumab.

Recurrence and Outcomes of Complement-Related Renal Disease After Pediatric Renal Transplantation.

Complement dysregulation is related to different glomerular pathologies. Patients with complement dysregulation have high recurrence risk after transplant; however, with trough-effective therapeutics, renal transplant can be an option for these patients. Here, we present 2 boys with renal disease related to complement dysregulation and their outcomes after renal transplant. Patient 1 had atypical hemolytic uremic syndrome, which was treated with eculizumab before renal transplant; eculizumab therapy was also continued after transplant as preventive therapy. Eculizumab therapy was stopped at year 2 post-transplant. At year 4 post-transplant, his serum creatinine level was 0.87 mg/dL. Patient 2, who had chronic renal disease related to C3 glomerulopathy, was not responsive to eculizumab before renal transplant. At month 4 posttransplant, C3 glomerulopathy recurrence was demonstrated with biopsy, and serum creatinine level was 1.96 mg/dL at this time. Eculizumab was started as a rescue therapy. At year 4 posttransplant, his serum creatinine level was 2.07 mg/dL. In our 2 patients with complement dysregulation, eculizumab was an effective and preventive therapy after renal transplant. However, more studies are needed to understand the long-term efficacy and safety of eculizumab after renal transplant.

Clinical relevance of membrane attack complex deposition in children with IgA nephropathy and Henoch-Schönlein purpura.

BACKGROUND: IgA 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. METHODS: We 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. RESULTS: We 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. CONCLUSIONS: We 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.