A novel complement inhibitor sMAP-FH targeting both the lectin and alternative complement pathways.

Inhibition of the complement activation has emerged as an option for treatment of a range of diseases. Activation of the lectin and alternative pathways (LP and AP, respectively) contribute to the deterioration of conditions in certain diseases such as ischemia-reperfusion injuries and age-related macular degeneration (AMD). In the current study, we generated dual complement inhibitors of the pathways MAp44-FH and sMAP-FH by fusing full-length MAp44 or small mannose-binding lectin-associated protein (sMAP), LP regulators, with the N-terminal five short consensus repeat (SCR) domains of complement factor H (SCR1/5-FH), an AP regulator. The murine forms of both fusion proteins formed a complex with endogenous mannose-binding lectin (MBL) or ficolin A in the circulation when administered in mice intraperitoneally. Multiple complement activation assays revealed that sMAP-FH had significantly higher inhibitory effects on activation of the LP and AP in vivo as well as in vitro compared to MAp44-FH. Human form of sMAP-FH also showed dual inhibitory effects on LP and AP activation in human sera. Our results indicate that the novel fusion protein sMAP-FH inhibits both the LP and AP activation in mice and in human sera, and could be an effective therapeutic agent for diseases in which both the LP and AP activation are significantly involved.

Human IgG Increases Virulence of Streptococcus pyogenes through Complement Evasion.

Streptococcus pyogenes is an exclusively human pathogen that can provoke mild skin and throat infections but can also cause fatal septicemia. This gram-positive bacterium has developed several strategies to evade the human immune system, enabling S. pyogenes to survive in the host. These strategies include recruiting several human plasma proteins, such as the complement inhibitor, C4b-binding protein (C4BP), and human (hu)-IgG through its Fc region to the bacterial surface to evade immune recognition. We identified a novel virulence mechanism whereby IgG-enhanced binding of C4BP to five of 12 tested S. pyogenes strains expressed diverse M proteins that are important surface-expressed virulence factors. Importantly, all strains that bound C4BP in the absence of IgG bound more C4BP when IgG was present. Further studies with an M1 strain that additionally expressed protein H, also a member of the M protein family, revealed that binding of hu-IgG Fc to protein H increased the affinity of protein H for C4BP. Increased C4BP binding accentuated complement downregulation, resulting in diminished bacterial killing. Accordingly, mortality from S. pyogenes infection in hu-C4BP transgenic mice was increased when hu-IgG or its Fc portion alone was administered concomitantly. Electron microscopy analysis of human tissue samples with necrotizing fasciitis confirmed increased C4BP binding to S. pyogenes when IgG was present. Our findings provide evidence of a paradoxical function of hu-IgG bound through Fc to diverse S. pyogenes isolates that increases their virulence and may counteract the beneficial effects of IgG opsonization.

Comparative Analysis of Novel Complement-Targeted Inhibitors, MiniFH, and the Natural Regulators Factor H and Factor H-like Protein 1 Reveal Functional Determinants of Complement Regulation.

The serum proteins factor H (FH), consisting of 20 complement control protein modules (CCPs), and its splice product FH-like protein 1 (FHL-1; consisting of CCPs 1-7) are major regulators of the alternative pathway (AP) of complement activation. The engineered version of FH, miniFH, contains only the N- and C-terminal portions of FH linked by an optimized peptide and shows ∼ 10-fold higher ex vivo potency. We explored the hypothesis that regulatory potency is enhanced by unmasking of a ligand-binding site in the C-terminal CCPs 19-20 that is cryptic in full-length native FH. Therefore, we produced an FH variant lacking the central domains 10-15 (FHΔ10-15). To explore how avidity affects regulatory strength, we generated a duplicated version of miniFH, termed midiFH. We compared activities of FHΔ10-15 and midiFH to miniFH, FH, and FHL-1. Relative to FH, FHΔ10-15 exhibited an altered binding profile toward C3 activation products and a 5-fold-enhanced complement regulation on a paroxysmal nocturnal hemoglobinuria patient's erythrocytes. Contrary to dogma, FHL-1 and FH exhibited equal regulatory activity, suggesting that the role of FHL-1 in AP regulation has been underestimated. Unexpectedly, a substantially increased avidity for complement opsonins, as seen in midiFH, did not potentiate the inhibitory potential on host cells. In conclusion, comparisons of engineered and native FH-based regulators have identified features that determine high AP regulatory activity on host cells. Unrestricted availability of FH CCPs 19-20 and an optimal spatial orientation between the N- and C-terminal FH regions are key.

Understanding the Role of Anti-PEG Antibodies in the Complement Activation by Doxil in Vitro.

Infusion reactions (IRs) are common immune-mediated side effects in patients treated with a variety of drug products, including, but not limited to, nanotechnology formulations. The mechanism of IRs is not fully understood. One of the best studied mechanisms of IRs to nanomedicines is the complement activation. However, it is largely unknown why some patients develop reactions to nanomedicines while others do not, and why some nanoparticles are more reactogenic than others. One of the theories is that the pre-existing anti-polyethylene glycol (PEG) antibodies initiate the complement activation and IRs in patients. In this study, we investigated this hypothesis in the case of PEGylated liposomal doxorubicin (Doxil), which, when used in a clinical setting, is known to induce IRs; referred to as complement activation-related pseudoallergy (CARPA) in sensitive individuals. We conducted the study in vitro using plasma derived from C57BL/6 mice and twenty human donor volunteers. We used mouse plasma to test a library of well-characterized mouse monoclonal antibodies with different specificity and affinity to PEG as it relates to the complement activation by Doxil. We determined the levels of pre-existing polyclonal antibodies that bind to PEG, methoxy-PEG, and PEGylated liposomes in human plasma, and we also assessed complement activation by Doxil and concentrations of complement inhibitory factors H and I in these human plasma specimens. The affinity, specificity, and other characteristics of the human polyclonal antibodies are not known at this time. Our data demonstrate that under in vitro conditions, some anti-PEG antibodies contribute to the complement activation by Doxil. Such contribution, however, needs to be considered in the context of other factors, including, but not limited to, antibody class, type, clonality, epitope specificity, affinity, and titer. In addition, our data contribute to the knowledge base used to understand and improve nanomedicine safety.

Virulence of Group A Streptococci Is Enhanced by Human Complement Inhibitors.

Streptococcus pyogenes, also known as Group A Streptococcus (GAS), is an important human bacterial pathogen that can cause invasive infections. Once it colonizes its exclusively human host, GAS needs to surmount numerous innate immune defense mechanisms, including opsonization by complement and consequent phagocytosis. Several strains of GAS bind to human-specific complement inhibitors, C4b-binding protein (C4BP) and/or Factor H (FH), to curtail complement C3 (a critical opsonin) deposition. This results in diminished activation of phagocytes and clearance of GAS that may lead to the host being unable to limit the infection. Herein we describe the course of GAS infection in three human complement inhibitor transgenic (tg) mouse models that examined each inhibitor (human C4BP or FH) alone, or the two inhibitors together (C4BPxFH or 'double' tg). GAS infection with strains that bound C4BP and FH resulted in enhanced mortality in each of the three transgenic mouse models compared to infection in wild type mice. In addition, GAS manifested increased virulence in C4BPxFH mice: higher organism burdens and greater elevations of pro-inflammatory cytokines and they died earlier than single transgenic or wt controls. The effects of hu-C4BP and hu-FH were specific for GAS strains that bound these inhibitors because strains that did not bind the inhibitors showed reduced virulence in the 'double' tg mice compared to strains that did bind; mortality was also similar in wild-type and C4BPxFH mice infected by non-binding GAS. Our findings emphasize the importance of binding of complement inhibitors to GAS that results in impaired opsonization and phagocytic killing, which translates to enhanced virulence in a humanized whole animal model. This novel hu-C4BPxFH tg model may prove invaluable in studies of GAS pathogenesis and for developing vaccines and therapeutics that rely on human complement activation for efficacy.

CD59: A long-known complement inhibitor has advanced to a blood group system.

The blood group system number 35 is based on CD59, a 20-kDa membrane glycoprotein present on a large number of different cells, including erythrocytes. The major function of CD59 is to protect cells from complement attack. CD59 binds to complement components CS and C9 and prevents the polymerization of C9, which is required for the formation of the membrane attack complex (MAC). Other functions of CD59 in cellular immunity are less well defined. CD59 is inserted into the membrane by a glycosylphosphatidylinositol (GPI) anchor. A defect of this anchor causes lack of this protein from the cell membrane, which leads to an enhanced sensitivity towards complement attack. Patients with paroxysmal nocturnal hemoglobinuria (PNH) harbor a varying percentage of red blood cell clones with a defect in GPI-anchored proteins, including CD59. The most characteristic symptoms of this disease are episodes of hemolysis and thromboses. Although CD59 has been classified as a membrane protein for more than 25 years, an alloantibody directed against CD59 was found only recently. So far, the first and sole alloantibody described was detected in a CD59-deficient child. In 2014, CD59 received the status of a blood group system by the International Society for Blood Transfusion Red Cell Immunogenetics and Blood Group Terminology Working Party. Among a variety of almost 20 synonyms, the designation CD59 was chosen for the blood group system and CD59.l for the wild-type protein. The only three alleles published to date are null alleles. All CD59-deficient individuals recognized so far were severely ill, two of whom have died. Most of the reported cases present with a typical clinical picture within the first year of life that includes neuropathy, strokes, and mild Coombs-negative hemolysis. In one published case, the application of the complement inhibitor eculizumab caused a pronounced improvement of the clinical situation.

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.

Activation of Complement Components on Circulating Blood Monocytes From COVID-19 Patients.

The coronavirus disease-2019 (COVID-19) caused by the SARS-CoV-2 virus may vary from asymptomatic to severe infection with multi-organ failure and death. Increased levels of circulating complement biomarkers have been implicated in COVID-19-related hyperinflammation and coagulopathy. We characterized systemic complement activation at a cellular level in 49-patients with COVID-19. We found increases of the classical complement sentinel C1q and the downstream C3 component on circulating blood monocytes from COVID-19 patients when compared to healthy controls (HCs). Interestingly, the cell surface-bound complement inhibitor CD55 was also upregulated in COVID-19 patient monocytes in comparison with HC cells. Monocyte membrane-bound C1q, C3 and CD55 levels were associated with plasma inflammatory markers such as CRP and serum amyloid A during acute infection. Membrane-bounds C1q and C3 remained elevated even after a short recovery period. These results highlight systemic monocyte-associated complement activation over a broad range of COVID-19 disease severities, with a compensatory upregulation of CD55. Further evaluation of complement and its interaction with myeloid cells at the membrane level could improve understanding of its role in COVID-19 pathogenesis.

Ectromelia virus inhibitor of complement enzymes protects intracellular mature virus and infected cells from mouse complement.

Poxviruses produce complement regulatory proteins to subvert the host's immune response. Similar to the human pathogen variola virus, ectromelia virus has a limited host range and provides a mouse model where the virus and the host's immune response have coevolved. We previously demonstrated that multiple components (C3, C4, and factor B) of the classical and alternative pathways are required to survive ectromelia virus infection. Complement's role in the innate and adaptive immune responses likely drove the evolution of a virus-encoded virulence factor that regulates complement activation. In this study, we characterized the ectromelia virus inhibitor of complement enzymes (EMICE). Recombinant EMICE regulated complement activation on the surface of CHO cells, and it protected complement-sensitive intracellular mature virions (IMV) from neutralization in vitro. It accomplished this by serving as a cofactor for the inactivation of C3b and C4b and by dissociating the catalytic domain of the classical pathway C3 convertase. Infected murine cells initiated synthesis of EMICE within 4 to 6 h postinoculation. The levels were sufficient in the supernatant to protect the IMV, upon release, from complement-mediated neutralization. EMICE on the surface of infected murine cells also reduced complement activation by the alternative pathway. In contrast, classical pathway activation by high-titer antibody overwhelmed EMICE's regulatory capacity. These results suggest that EMICE's role is early during infection when it counteracts the innate immune response. In summary, ectromelia virus produced EMICE within a few hours of an infection, and EMICE in turn decreased complement activation on IMV and infected cells.

Complement-mediated kidney diseases.

It has long been known that the complement cascade is activated in various forms of glomerulonephritis. In many of these diseases, immune-complexes deposit in the glomeruli and activate the classical pathway. Researchers have also identified additional mechanisms by which complement is activated in the kidney, including diseases in which the alternative and lectin pathways are activated. The kidney appears to be particularly susceptible to activation of the alternative pathway, and this pathway has been implicated as a primary driver of atypical hemolytic uremic syndrome, C3 glomerulopathy, anti-neutrophil cytoplasmic antibody-associated vasculitis, as well as some forms of immune-complex glomerulonephritis. In this paper we review the shared and distinct mechanisms by which complement is activated in these different diseases. We also review the opportunities for using therapeutic complement inhibitors to treat kidney diseases.

Novel Complement Therapeutics in Development as Potential Treatment for Renal Disease.

The complement system is an evolutionarily ancient arm of the innate immune system. It remains, however, one of the last major pathways in immunology for which specific pharmaceutical antagonists have been developed. In recent years, a fundamental role for complement has been described in many different renal diseases, including both pauci-immune as well as immune-complex diseases. Since the 2011 FDA approval of eculizumab, the only marketed complement antagonist, no new therapeutics have entered clinical practice. There are now multiple new agents in clinical trials, from oral molecules to small inhibitory RNA, that target the classical, lectin, and alternative pathways. Herein we summarize several potential renal diseases in which complement inhibitors may provide a therapeutic benefit, as well as specific complement inhibitors in development.

Eculizumab for the treatment of myasthenia gravis.

INTRODUCTION: Acetylcholine receptor antibody-positive generalized myasthenia gravis (gMG) is effectively treated with symptomatic and immunosuppressive drugs but a proportion of patients has a persistent disease and severe adverse events (AEs). The unmet medical needs are specific immunosuppression and AE lowering. Eculizumab blocks C5 protecting neuromuscular junction from the destructive autoantibody effects. Phase II (Study C08-001) and III (ECU-MG-301) studies, with the open-label extension (ECU-MG-302), demonstrated eculizumab efficacy and safety in refractory gMG patients. AREAS COVERED: We provide an overview of eculizumab biological features, clinical efficacy, and safety in gMG patients, highlighting our perspective on the drug positioning in the MG treatment algorithm. EXPERT OPINION: Eculizumab has the potential to significantly change the immunosuppressive approach in gMG offering the opportunity to avoid or delay corticosteroids' use due to its speed and selective mechanism of action. Eculizumab prescription will depend on: 1. ability to modify the natural disease course; 2. sustainability in the clinical practice (cost/effectiveness ratio); 3. drug-induced AE reduction. At present we are missing a controlled study on its use as a first-line treatment. We think that immunosuppression in MG will change significantly in the next years by adopting more focused 'Precision Medicine' approaches, and Eculizumab seems to satisfy such a promise.

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.

Complement in IgA Nephropathy: The Role of Complement in the Pathogenesis, Diagnosis, and Future Management of IgA Nephropathy.

Immunoglobulin A (IgA) nephropathy (IgAN) is an important cause of chronic and end-stage kidney disease. IgAN pathogenesis is incompletely understood. In particular, we cannot adequately explain the heterogeneity in clinical and histologic features and severities that characterizes IgAN. This limits patient stratification to appropriate and effective treatments and the development of disease-targeted therapies. Studies of the role of the alternative, lectin, and terminal complement pathways in IgAN have enhanced our understanding of disease pathogenesis and inform the development of novel diagnostic and therapeutic strategies. For example, recent genetic, serologic, and immunohistologic evidence suggests that imbalances between the main alternative complement pathway regulator protein (factor H) and competitor proteins that deregulate complement activity (factor H-related proteins 1 and 5, FHR1, and FHR5) associate with IgAN severity: a relative abundance of FHR1 and FHR5 amplifies complement-dependent inflammation and exacerbates kidney injury. Ongoing characterization of the mechanisms by which complement activity contributes to IgAN pathogenesis will facilitate the development of complement-based diagnostic techniques, biomarkers of disease activity and severity, and novel targeted therapies.

Complement-Based Therapy in the Management of Antibody-Mediated Rejection.

Antibody-mediated rejection (AMR) is one of the leading causes of kidney allograft failure and is usually mediated by anti-human leukocyte antigen donor-specific antibodies (DSAs). Activation of classical pathway of the complement system is responsible for downstream effects of DSA and account for significant manifestations of AMR. Currently, the treatment of AMR is based on strategies to remove preformed antibodies or to prevent their production; however, these strategies are often unsuccessful. It is theoretically possible to inhibit complement activity to prevent the effect of DSA on kidney allograft function. Complement inhibitors such as eculizumab, a complement 5 monoclonal antibody, and complement 1 esterase inhibitors (C1 INHs) have been used in prevention and treatment of AMR with variable success. Eculizumab and C1 INH seem to reduce the incidence of early AMR and allow transplantation in highly sensitized kidney transplant recipients, but data on their long-term effect on kidney allograft function are limited. Several case reports described the successful use of eculizumab in the treatment of AMR, but there are no randomized controlled studies that showed efficacy. Treatment of AMR with C1 INH, in addition to standard of care, did not change short-term outcome but long-term studies are underway.

Complement-Mediated Disorders in Pregnancy.

Complement-mediated disorders in pregnancy span a large spectrum and have been implicated in all three complement pathways: classical, lectin, and alternative. Our understanding of these disorders in recent years has advanced due to a better understanding of complement regulatory proteins, such as complement factor H, complement factor I, membrane cofactor protein, and thrombomodulin that particularly affect the alternative complement pathway. Enthusiasm in genotyping for mutations that encode these proteins has allowed us to study the presence of genetic variants which may predispose women to develop conditions such as pregnancy-associated hemolytic uremic syndrome (P-aHUS), thrombotic thrombocytopenic purpura, preeclampsia/hemolysis, elevated liver enzymes, low platelets (HELLP), systemic lupus erythematosus/antiphospholipid syndrome, and peripartum cardiomyopathy. The advent of the anti-C5-antibody eculizumab to quench the complement cascade has already proven in small case series to improve maternal kidney outcomes in complement-mediated obstetric catastrophes such as P-aHUS and HELLP. In this review, we will detail the pathogenesis behind these complement-mediated pregnancy disorders, the role of complement variants in disease phenotype, and the most up-to-date experience with eculizumab in this population.

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.

Complement: an efficient sword of innate immunity.

Complement is vital for protecting individuals against pathogens and any disturbance of homeostasis associated with appearance of foreign antigens. Four antenna molecules seek for putative danger and subsequently start three activation pathways to eliminate the hostile triggering signal. To achieve this mission the complement arsenal contains soluble plasma factors as well as membrane-bound receptor molecules. Fulfilling a broad spectrum of biological functions, complement participates to construct and orchestrate an immunological network with extensive links to other elements of innate immunity, but also to its younger brother, the adaptive immune system. The body generously supports the complement activity with a high level of complement production; not only the liver as 'the capital of complement expression' but also decentralized synthesis sites guarantee its all-over presence. On the other hand, it is of fundamental interest for the organism to limit this powerful immunological regiment by establishing a tight surveillance composed of redundantly acting regulator molecules. To find the appropriate dimension of complement activity is critical, as shown by the spectrum of diseases associated with an excess or a lack. Numerous therapeutic approaches aim to correct such an imbalance and to re-establish the antimicrobial capacity of complement without induction of chronic inflammation and autoimmunity.

Complement inhibition keeps mothers calm and avoids fetal rejection.

The paternal antigens presented by the fetus could be considered foreign by the mother's immune system and elicit an immune response. Here we show that the complement system functions as an effector in fetal rejection in two different mouse models of pregnancy loss. In a mouse model of fetal loss and growth restriction (IUGR) induced by antiphospholipid antibodies (aPL), we found that complement activation is a crucial and early mediator of pregnancy loss. We demonstrated that C5a-C5aR interaction and neutrophils are key mediators of fetal injury. We identified tissue factor (TF) as a critical intermediate that, acting downstream of C5 activation, enhances neutrophil activity and trophoblast injury. In an antibody-independent mouse model of spontaneous miscarriage and IUGR (CBAxDBA) we also identified C5a as an essential mediator. Complement activation caused dysregulation of the angiogenic factors (deficiency of free vascular endothelial growth factor (VEGF) and elevated levels of soluble VEGF receptor 1) required for normal placental development. Inhibition of complement activation prevented angiogenesis failure and rescued pregnancies. Our studies in antibody-dependent and antibody-independent models of pregnancy complications identified complement activation as the crucial mediator of damage and will allow development of new interventions to prevent pregnancy loss and IUGR.

A Gyrolab Assay for the Quantitation of Free Complement Protein C5a in Human Plasma.

Complement protein C5a is recognized as an important component of the alternative complement pathway. Its role is prominent enough to garner interest not only as a biomarker, but also as a potential therapeutic target. Bioanalytical challenges have been posed in proper quantitation of free C5a due to interference from its precursor, C5. Additionally, free therapeutic target quantitation can be difficult due to effects of sample dilution and prolonged sample incubation when therapeutic is used as capture reagent. Gyrolab technology enables quantitation of free target analyte with minimal sample dilution and rapid sample incubations, thus enabling in vitro results that are more representative of in vivo pharmacodynamics. When coupled with strategic sample pretreatment, Gyrolab offers an opportunity to quantitate free C5a in human plasma with an assay that vastly diminishes C5 interference. A Gyrolab assay for the quantitation of free C5a in human plasma was developed and validated. Validation results confirmed that proper sample pretreatment and use of the Gyrolab platform yield accurate and reliable results. Due to the advantages that it provides, Gyrolab has become our default technology of choice for quantitation of free target.