Activation Of Alternative Pathway Research Articles

Association of complement pathways with COVID-19 severity and outcomes

ObjectivesComplement activation has been implicated in COVID-19 pathogenesis. This study aimed to assess the levels of complement activation products and full-length proteins in hospitalized patients with COVID-19, and evaluated whether complement pathway markers are associated with outcomes. MethodsLongitudinal measurements of complement biomarkers from 89 hospitalized adult patients, grouped by baseline disease severity, enrolled in an adaptive, phase 2/3, randomized, double-blind, placebo-controlled trial and treated with intravenous sarilumab (200 mg or 400 mg) or placebo (NCT04315298), were performed. These measurements were then correlated with clinical and laboratory parameters. ResultsAll complement pathways were activated in hospitalized patients with COVID-19. Alternative pathway activation was predominant earlier in the disease course. Complement biomarkers correlated with multiple variables of multi-organ dysfunction and inflammatory injury. High plasma sC5b-9, C3a, factor Bb levels, and low mannan-binding lectin levels were associated with increased mortality. Sarilumab treatment showed a modest inhibitory effect on complement activation. Moreover, sera from patients spontaneously deposited C5b-9 complex on the endothelial surface ex vivo, suggesting a microvascular thrombotic potential. ConclusionThese results advance our understanding of COVID-19 disease pathophysiology and demonstrate the importance of specific complement pathway components as prognostic biomarkers in COVID-19.

An Evaluation of the Complement-Regulating Activities of Human Complement Factor H (FH) Variants Associated With Age-Related Macular Degeneration.

Factor H (FH, encoded by CFH) prevents activation of the complement system's alternative pathway (AP) on host tissues. FH impedes C3 convertase (C3bBb) formation, accelerates C3bBb decay, and is a cofactor for factor I (FI)-catalyzed C3b cleavage. Numerous CFH variants are associated with age-related macular degeneration (AMD), but their functional consequences frequently remain undetermined. Here, we conduct functional comparisons between a control version of FH (not AMD linked) and 21 AMD-linked FH variants. Recombinantly produced, untagged, full-length FH versions were assayed for binding to C3b and decay acceleration of C3bBb using surface-plasmon resonance, FI-cofactor activity using a fluorescent probe of C3b integrity, suppression of C5b-9 assembly on an AP-activating surface, and inhibition of human AP-mediated lysis of sheep erythrocytes. All versions were successfully purified despite below-average yields for Arg2Thr, Arg53Cys, Arg175Pro, Arg175Gln, Ile221Val, Tyr402His, Pro503Ala, Arg567Gly, Gly1194Asp, and Arg1210Cys. Compared to control FH, Arg2Thr, Leu3Val, Ser58Ala, Asp90Gly, Asp130Asn, Gln400Lys, Tyr402His, Gly650Val, Ser890Ile, and Thr965Met showed minimal functional differences. Arg1210C, Arg53His, Arg175Gln, Gly1194Asp, Pro503Ala, Arg53Cys, Arg576Gly, and Arg175Pro (in order of decreasing efficacy) underperformed, while Ile221Val, Arg303Gln, and Arg303Trp were "marginal." We newly identified variants toward the center of the molecule, Pro503Ala and Arg567Gly, as potentially pathogenic. Our approach could be extended to other variants of uncertain significance and to assays for noncanonical FH activities, aiming to facilitate selection of cohorts most likely to benefit from therapeutic FH. This is timely as recombinant therapeutic FH is in development for intravitreal treatment of AMD in patients with reduced FH functionality.

Alternative pathway dysregulation in tissues drives sustained complement activation and predicts outcome across the disease course in COVID-19.

Complement, a critical defence against pathogens, has been implicated as a driver of pathology in COVID‐19. Complement activation products are detected in plasma and tissues and complement blockade considered for therapy. To delineate roles of complement in immunopathogenesis, we undertook the largest comprehensive study of complement in an COVID‐19 to date, a comprehensive profiling of 16 complement biomarkers, including key components, regulators and activation products, in 966 plasma samples from 682 hospitalised COVID‐19 patients collected across the hospitalisation period as part of the UK ISARIC4C study. Unsupervised clustering of complement biomarkers mapped to disease severity and supervised machine learning identified marker sets in early samples that predicted peak severity. Compared to heathy controls, complement proteins and activation products (Ba, iC3b, terminal complement complex) were significantly altered in COVID‐19 admission samples in all severity groups. Elevated alternative pathway activation markers (Ba and iC3b) and decreased alternative pathway regulator (properdin) in admission samples associated with more severe disease and risk of death. Levels of most complement biomarkers were reduced in severe disease, consistent with consumption and tissue deposition. Latent class mixed modelling and cumulative incidence analysis identified the trajectory of increase of Ba to be a strong predictor of peak COVID‐19 disease severity and death. The data demonstrate that early‐onset, uncontrolled activation of complement, driven by sustained and progressive amplification through the alternative pathway amplification loop is a ubiquitous feature of COVID‐19, further exacerbated in severe disease. These findings provide novel insights into COVID‐19 immunopathogenesis and inform strategies for therapeutic intervention.

The role of alternative electron pathways in the photosynthetic chain in higher plants

Light-dependent reactions of photosynthesis takes place in the thylakoids of chloroplasts where light energy harvested from the sun drives the synthesis of ATP and NADPH. The major pathway of photosynthetic chain is the linear electron transport (LET), in which both photosystems (PSI and PSII) are involved, and ATP and NADPH are produced. However, ratio in production of those components is insufficient to cover the Calvin cycle energy requirements, depending on the metabolism of the cell. Moreover, disturbance in metabolism homeostasis, caused by environmental stress conditions, increases ATP demand, which cannot be covered by LET. Thus, in photosynthetic apparatus must exist alternative electron transport pathways, these include: cyclic electron transport (CET) mediated by NDH complex or PGR5/PGRL1 proteins, water-water cycle and PTOX enzyme. Activity of alternative pathways can optimize ratio in production of ATP/NADPH, appropriately to requirements, which allows to achieve redox balance and ATP contents.

Baseline Clinical Characteristics and Complement Biomarkers of Patients with C3 Glomerulopathy Enrolled in Two Phase 2 Studies Investigating the Factor D Inhibitor Danicopan

Introduction: C3 glomerulopathy (C3G) is a rare, progressive kidney disease resulting from dysregulation of the alternative pathway (AP) of complement. Biomarkers at baseline were investigated in patients with C3G who participated in two phase 2 studies with the factor D (FD) inhibitor, danicopan. Methods: Patients with biopsy-confirmed C3G, proteinuria ≥500 mg/day, and estimated glomerular filtration rate (eGFR) ≥30 mL/min/1.73 m2 were enrolled into two studies (NCT03369236 and NCT03459443). Biomarker analysis was performed for patients with C3G confirmed by central pathology laboratory re-evaluation. Complement and clinical biomarkers, biopsy composite score, and activity and chronicity indices were assessed at baseline and analyzed by pairwise Spearman correlation analysis. Results: Twenty-nine patients were included in the analysis (median [interquartile range] age: 24.0 [10.0] years). Systemic complement AP activation was evident by reduced median concentrations of C3 and C5, elevated sC5b-9, and normal C4, relative to reference ranges. C3 showed strong pairwise correlations with C5 and sC5b-9 (r = 0.80 and −0.73, respectively; p < 0.0001). Baseline Ba and FD concentrations were inversely correlated with eGFR (r = −0.83 and −0.87, respectively; p < 0.0001). Urinary concentrations of sC5b-9 were correlated with both plasma sC5b-9 and proteinuria (r = 0.69 and r = 0.83, respectively; p < 0.0001). Biopsy activity indices correlated strongly with biomarkers of systemic AP activation, including C3 (r = −0.76, p < 0.0001), whereas chronicity indices aligned more closely with eGFR (r = −0.57, p = 0.0021). Conclusion: Associations among complement biomarkers, kidney function, and kidney histology may add to the current understanding of C3G and assist with the characterization of patients with this heterogenous disease.

The Factor H protein family: The switchers of the complement alternative pathway.

The factor H (FH) protein family is emerging as a complex network of proteins controlling the fate of the complement alternative pathway (AP) and dictating susceptibility to a wide range of diseases including infectious, inflammatory, autoimmune, and degenerative diseases and cancer. Composed, in man, of seven highly related proteins, FH, factor H-like 1, and 5 factor H-related proteins, some of the FH family proteins are devoted to down-regulating the AP, while others exert an opposite function by promoting AP activation. Recent findings have provided insights into the molecular mechanisms defining their biological roles and their pathogenicity, illustrating the relevance that the balance between the regulators and the activators within this protein family has in defining the outcome of complement activation on cell surfaces. In this review we will discuss the emerging roles of the factor H protein family, their impact in the complement cascade, and their involvement in the pathogenesis of complement-mediated diseases associated with the AP dysregulation.

Pediatric Sickle Cell Disease: A Potential Role for the Complement System

Background: Sickle cell disease (SCD) is one of the most common life-threatening hereditary red blood cell (RBC) disorders, with an estimated 300,000 infants born annually with the disease. In SCD, a mutation in the gene for β-globin results in rigid sickled RBCs that have difficulty navigating the vasculature and increased risk for forming blockages in the micro-vessels within organs, leading to RBC lysis, endothelial cell (EC) damage, ischemia/reperfusion injury, and extremely painful vaso-occlusive crises (VOC). Overtime, this can lead to stroke, serious organ damage, and other complications, resulting in a reduced life expectancy. Emerging research points to the involvement in SCD of complement, an important part of the innate immune system composed of proteins that are activated to help clear invaders and damaged cells from the body. Recent work by others has demonstrated that complement activation occurs in patients with SCD in both acute and chronic phases of the disease, and may be associated with disease severity and VOC (Fig 1A). This study aims to assess the role of complement activity in children with SCD during acute crisis and disease steady state. Methods: Pediatric patients aged 0-18 with SCD (HbSS or HbS/β0) managed at The Hospital for Sick Children (Toronto, Canada) were enrolled in this REB approved study during hospital admission with diagnosed VOC or acute chest crisis (ACS) not caused by infection. Patient serum and plasma was collected during hospital admission (crisis) and during follow-up (steady state, 3 to 12 months following an acute crisis) simultaneously with routine bloodwork. Patients with >3 crisis admissions in the previous 12 months or requiring admission for >7 days were excluded. Pooled normal human serum (NHS) was collected from healthy volunteers for use as a control. Complement activity was measured using the WIESLAB Complement System Screen (Svar Life Science). Immunofluorescence (IF) imaging was used to measure the deposition of C3b and C5b-9 complement proteins on the surface of ECs exposed to patient serum or NHS. Data were analyzed using an unpaired t-test or one-way ANOVA with Tukey's multiple comparisons. Results: Thus far, 58 patients have been enrolled in the study. SCD crisis patients showed significantly decreased hemoglobin compared to steady state (Table 1). Complement activity was assessed for 6 crisis and 5 steady state patients. There was equal classical and alternative pathway activity during disease steady state and crisis, which indicates no in vivo complement protein depletion in patients, with an interesting trend showing elevated MBL pathway activity during crisis compared to steady state (Figure 1B). Complement deposition was assessed via IF in 19 crisis, 16 steady state patients, and 6 replicates of NHS. IF assay data show significantly elevated deposition of C3b on ECs when comparing patient crisis samples and healthy controls, with trends suggesting a potential difference in C5b-9 deposition between NHS and crisis patients, as well as differences in complement deposition between steady state and crisis patients (Fig 1C). Conclusion: Our preliminary data shows complement is active in SCD, resulting in elevated C3b deposition on ECs during SCD crisis. Although not statistically significant, trends suggesting a difference in C5b-9 deposition and differences between steady state and crisis patients are encouraging, as a small change in intravascular complement activity may still have significant physiological consequences. Future work is focused on further quantifying complement activity and understanding its mechanism, assessing the in vitro functional consequences of this on the surface of ECs, and investigating complement blockade as a potential treatment. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Alternative pathway activation in pregnancy, a measured amount "complements" a successful pregnancy, too much results in adverse events.

During pregnancy, the maternal host must adapt in order to enable growth of the fetus. These changes affect all organ systems and are designed both to protect the fetus and to minimize risk to the mother. One of the most prominent adaptations involves the immune system. The semi-allogenic fetoplacental unit has non-self components and must be protected against attack from the host. This requires both attenuation of adaptive immunity and protection from innate immune defense mechanisms. One of the key innate immune players is complement, and it is important that the fetoplacental unit is not identified as non-self and subjected to complement attack. Adaptation of the complement response must, however, be managed in such a way that maternal protection against infection is not compromised. As the complement system also plays a significant facilitating role in many of the stages of a normal pregnancy, it is also important that any necessary adaptation to accommodate the semi-allogenic aspects of the fetoplacental unit does not compromise this. In this review, both the physiological role of the alternative pathway of complement in facilitating a normal pregnancy, and its detrimental participation in pregnancy-specific disorders, are discussed.

A Phase 2a, Randomized, Open-Label Study to Evaluate Multiple Dosing Regimens of Subcutaneous ALXN1820 in Adult Patients with Sickle Cell Disease

Background:Sickle cell disease (SCD) is a group of inherited disorders that result in chronic hemolysis/anemia and vaso-occlusive crises (VOC). Current management options for SCD do not adequately address both anemia and VOC. Hematopoietic stem cell transplant is the only curative treatment but is associated with life-threatening complications. Complement activation may play an important role in the pathophysiology of SCD since intravascular hemolysis of sickle red blood cells (RBCs) leads to release of free hemoglobin triggering activation of the complement alternative pathway (CAP). Intravascular hemolysis and CAP activation promotes anemia and VOC in patients with SCD. Properdin is an endogenous activator of the CAP (Roumenina LT, et al. Am J Hematol. 2020;95(5):456-464; Vercellotti GM, et al. Am J Hematol. 2019;94(3):327-33). ALXN1820 is a novel, humanized, bispecific, VHH antibody that simultaneously binds human albumin and properdin. The antibody effectively and selectively inhibits CAP activation while binding to albumin, which confers an extended circulatory half-life to the molecule. Pretreatment with a mouse anti-properdin antibody significantly ameliorated signs of hemolysis and VOC in a mouse model of SCD. The safety, pharmacokinetics and pharmacodynamics of ALXN1820 are being evaluated in an ongoing study in healthy volunteers (NCT04631562). Study design, methods, and study population: This open-label Phase 2a study will evaluate the safety, tolerability, efficacy, pharmacokinetics (PK), pharmacodynamics (PD), and immunogenicity of multiple doses and dosing regimens of ALXN1820 administered subcutaneously to patients with SCD (see Figure for study schema). Key inclusion criteria are confirmed diagnosis of SCD (HbSS and HbSß0-thalassemia), hemoglobin levels 5.5-10g/dL at screening, 1-10 VOC episodes in the past 12 months, and vaccination requirements (meningococcal, Haemophilus influenzae type B (Hib) and Streptococcus pneumoniae). Concomitant treatment with stable dose of hydroxyurea is permitted during the study as applicable. Key exclusion criteria are receiving voxelotor or crizanlizumab within 60 days of providing informed consent and, prior treatment with complement inhibitors within 6 months of first study dose. Key endpoints of the study are shown in the Table. Safety analyses will be performed on the Safety Population and will be reported by each cohort and treatment arm. Efficacy analyses will include absolute and percentage change from baseline to the end of the Treatment Period (12 weeks) in complement biomarkers, hemoglobin, hemolysis markers, and hemopexin for Cohort 1 and Cohort 2. Conclusion: Complement activation may play an important role in the pathophysiology of SCD. Treatment with an anti-properdin antibody significantly ameliorated signs of hemolysis and VOC in a mouse model of SCD supporting the potential of ALXN1820 to treat SCD. Data from this Phase 2 study are anticipated to confirm the potential of ALXN1820 to treat SCD and guide the design of future studies in patients with SCD and other complement-mediated diseases. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Complement Activation Is Associated with Endothelial Damage in Hematopoietic Stem Cell Transplant Associated-Thrombotic Microangiopathy

Introduction: Hematopoietic stem cell transplant-associated thrombotic microangiopathy (HSCT-TMA) is a life-threatening complication of HSCT. Identification of at-risk patients is challenging, leading to delays in diagnosis and poor outcomes. Degradation of the glycocalyx and dysregulated complement activation are integral to the pathophysiology. The glycocalyx components thrombomodulin (TM) and syndecan-1 (SYND1) are shed during cell damage, and detection in patient plasma may serve as biomarkers of endothelial cell (EC) dysfunction. Therapeutic intervention to preserve glycocalyx integrity may protect the endothelium and also improve the clinical outcome during HSCT-TMA. Additionally, plasma Ba, an alternative complement pathway (AP) activation product, has been identified as a prognostic biomarker of HSCT-TMA. Our objective was to explore the link between excessive AP activation and endothelial damage in the context of HSCT-TMA and to potentially identify biomarkers to aid in its diagnosis. Methods: Biomarker levels of soluble TM, SYND1, and Ba were measured in plasma of pediatric HSCT patients with and without (controls) TMA obtained from Texas Children's Hospital. Sample acquisition was approved by Baylor College of Medicine Institutional Review Board. Biomarkers were also measured in a pre-clinical in vivo model of inflammation-mediated complement activation similar to HSCT-TMA, in which C57BL/6J mice were injected with lipopolysaccharide (LPS) to induce complement activation, followed by treatment with AP (anti-fB IgG) and terminal complement pathway (anti-C5 IgG) inhibitors or an IgG isotype control. Finally, as the immunosuppressant cyclosporin A (CsA) is a known trigger of HSCT-TMA, the role of complement in CsA-mediated damage in human ECs (HMEC-1 cells and HUVECs) was assessed in vitro. ECs were incubated with CsA followed by normal human serum (NHS) in the presence or absence of the C5 complement inhibitor eculizumab. TM was then measured by fluorescent staining on the cell surface and quantified using mean fluorescence intensity (MFI). Results: HSCT-TMA patients (n=11), compared to HSCT control patients (n=7), had significantly elevated levels of biomarkers of glycocalyx damage (TM: 18.3 ± 10.9 ng/mL and 6.6 ± 2.9 ng/mL, respectively; P<0.01; SYND1: 175.3 ± 177.0 ng/mL and 35.8 ± 18.9, respectively; P<0.05). A strong positive correlation between TM and SYND1 was observed (Pearson r=0.89). Plasma Ba was also significantly elevated in patients with TMA compared to those without (1790.0 ± 1262.0 ng/mL and 652.1 ± 233.5 ng/mL, respectively; P<0.05), and correlated positively with both TM and SYND1 levels (Pearson r=0.72 and 0.50, respectively). We have previously shown in an in vivo model of inflammation-mediated complement activation that LPS-injected mice, compared to saline-injected control mice, had significantly increased plasma Ba levels. Here we demonstrated LPS-injected mice (which also received IgG isotype control) had higher circulating levels of TM than untreated mice (35.1 ± 5.8 ng/mL [n=6] and 10.9 ± 1.6 ng/mL [n=6], respectively; P<0.0001). Inhibition of alternative (anti-fB IgG) and terminal (anti-C5 IgG) pathways of complement significantly attenuated TM levels (compared to isotype control [35.1 ± 5.8 ng/mL]; anti-FB: 25.2 ± 5.3 ng/mL, P<0.01; anti-C5: 26.2 ± 4.9 ng/mL, P<0.05, Fig. 1). TM was positively correlated with Ba (Pearson r=0.68). In vitro treatment of HMEC-1 cells with CsA led to deposition of iC3b and C5b-9, and C5 inhibition with eculizumab significantly reduced C5b-9 deposition (P< 0.0001) but had no effect on iC3b deposition. Treatment of HUVECs with CsA also reduced surface expression of TM (P<0.0001), and TM loss was partially restored by eculizumab (P<0.05, Fig 2). Conclusion: Plasma biomarkers of endothelial damage were elevated in patients with HSCT-TMA and in mice with inflammatory-mediated complement activation. Furthermore, AP activation positively correlated with biomarkers of endothelial damage. Blockade of complement activation significantly reduced markers of endothelial damage in both in vivo and in vitro models. These results provide further evidence linking complement activation and endothelial damage in HSCT-TMA and suggest that the use of these biomarkers may aid in the diagnosis of HSCT-TMA. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Dose-Exposure-Response Relationships of Biomarkers and Efficacy Measures with Iptacopan, a Complement Factor B Inhibitor, in Patients (pts) with Paroxysmal Nocturnal Hemoglobinuria (PNH) with or without Concomitant Anti-C5 Therapy

Background: Standard of care treatment for pts with PNH targets C5 in the complement pathway; however, residual intravascular hemolysis (IVH) and emerging C3-mediated extravascular hemolysis (EVH) can still occur. In two Phase II trials (X2201 [NCT03439839] and X2204 [NCT03896152]), the oral, selective, reversible complement factor B inhibitor iptacopan provided hematologic benefit in pts with PNH who had active hemolysis, both as add-on therapy in anti-C5-treated pts and monotherapy in anti-C5-naïve pts (Risitano et al. Lancet Haematol 2021; Jang et al. Blood Adv 2022). Using data from these two studies, this integrated analysis aimed to evaluate dose-exposure-response relationships of biomarkers and efficacy measures to support dose selection for iptacopan. Methods: X2201 and X2204 were open-label studies that enrolled pts with PNH who had active hemolysis, despite treatment with anti-C5 therapy, or in the absence of prior anti-C5 therapy, respectively. In X2201, pts with lactate dehydrogenase (LDH) ≥1.5 x upper limit of normal (ULN) received iptacopan 200 mg twice daily (bid; Cohort 1) and pts with LDH ≥1.25 x ULN and hemoglobin (Hb) <10.5 g/dL received iptacopan 50 mg bid (Cohort 2). Pts in Cohort 2 could have their dose increased to 200 mg bid if hemolysis persisted. Both cohorts received iptacopan as add-on therapy to eculizumab; eculizumab treatment could be adjusted or withdrawn after 26 weeks (wks) at the investigator's discretion. In X2204, pts with LDH ≥1.5 x ULN and Hb <10.5 g/dL who were naïve to anti-C5 treatment were randomized to receive iptacopan 25 mg bid for 4 wks followed by iptacopan 100 mg bid for 8 wks (Cohort 1), or iptacopan 50 mg bid for 4 wks followed by iptacopan 200 mg bid for 8 wks (Cohort 2). To quantify the exposure-response relationship, trough concentrations of iptacopan (Ctrough) were correlated with LDH, Hb, factor Bb fragments, Wieslab® alternative complement pathway activity assay data, C3 fragment deposition on red blood cells (RBCs) and PNH RBC clone size using sigmoid maximum effect (Emax) models. Analyses were performed after pts had received iptacopan at the same dose for 4 wks. Results: A total of 29 pts with PNH were enrolled (X2201, N=16; X2204, N=13). The pharmacokinetic parameters of iptacopan were in line with previous reports in these two studies (Risitano et al. Lancet Haematol 2021; Jang et al.Blood Adv 2022) and were similar between the two study populations. In the dose range 25-200 mg bid, increases in dose resulted in a less than proportional increase in exposure, probably because of on-target binding to the highly abundant plasma factor B. Age and sex did not significantly impact exposure; the impact of ethnicity was not assessed because it was confounded by the study populations (15/16 pts in X2201 were Caucasian; all pts in X2204 were Asian). When assessing LDH, Hb, factor Bb fragments and Wieslab® assay data, 95% of the maximum response was reached with Ctrough >700 ng/mL, >650 ng/mL, >500 ng/mL and >750 ng/mL (EC95), respectively. C3 deposition was only informative in X2201; in X2204, C3 deposition was negligible at baseline and throughout the study because the pts were anti-C5-naïve. Complete abrogation of C3 deposition was reached at all dose levels in X2201 (Ctrough >600 ng/mL). PNH RBC cell clone size increased in both studies, but no clear dose-dependent relationship was observed. Accounting for interpatient variability in exposure, iptacopan 200 mg bid was identified as the optimum regimen to achieve Ctrough >750 ng/mL in most pts. Conclusions: Results from this pooled dose-exposure-response analysis of the X2201 and X2204 studies indicate that iptacopan 200 mg bid is the optimum dose to achieve Ctrough >750 ng/mL and consequently to provide maximal disease control via alternative complement pathway inhibition. The dose-exposure-response relationship was similar between pts regardless of whether they had a partial response to C5 inhibition (X2201) or were anti-C5-naïve (X2204), supporting the use of iptacopan 200 mg bid in both populations. Based on clinical efficacy and safety data from these two studies (Risitano et al. Lancet Haematol 2021; Jang et al. Blood Adv 2022) and supported by data from this integrated dose-exposure-response analysis, two ongoing Phase III trials are investigating iptacopan monotherapy at a dose of 200 mg bid in anti-C5 treated (APPLY-PNH; NCT04558918) and anti-C5-naïve (APPOINT-PNH; NCT04820530) pts with PNH.

COVID-19 Associated Atypical Hemolytic Uremic Syndrome

Introduction: Atypical hemolytic uremic syndrome (atypical HUS) is a complement-mediated thrombotic microangiopathy (TMA) that affects multiple organs including the kidneys. We present a case of 37-year-old male with recent COVID-19 infection who presented with epistaxis. His blood work was consistent with TMA and acute kidney injury (AKI). Hemodialysis and plasmapheresis (PLEX) were initiated for presumed thrombotic thrombocytopenic purpura (TTP) but after excluding ADAMTS-13 deficiency, eculizumab was started for possible atypical HUS. Renal biopsy and genetic testing ultimately confirmed diagnosis of atypical HUS. Case presentation: A 37-year-old male with past medical history of recent COVID infection presented to the emergency room with complaint of epistaxis. Physical exam showed mild epistaxis from right naris and bilateral leg swelling. Initial laboratory findings were hemoglobin (Hb) 5 g/dL, platelets 70 B/L, Lactate dehydrogenase (LDH) 581 IU/L, haptoglobin < 10 mg/dL, creatinine (Cr) 36.80 mg/dL, blood urea nitrogen (BUN) 232 mg/dL, potassium (K) 6.6 mmol/L, calcium (Ca) 6 mg/dL and normal prothrombin time (PT) and partial thromboplastin time (PTT). Complement C3 levels were low but normal C4 levels. Peripheral blood smear showed schistocytes confirming TMA. He was started on both PLEX and hemodialysis (HD) along with prednisone 80 mg daily. PLEX was stopped on day 4 after normal ADAMTS-13 levels resulted, helping rule out TTP. Based on history and laboratory findings, diagnosis of atypical HUS was favored and eculizumab was started. He received meningococcal vaccine and prophylactic penicillin while on eculizumab. Hemodialysis was continued with eventual improvement of his labs to Hb 7, platelets 119, LDH 339, haptoglobin < 10, Cr 9.07, BUN 85, K 5.8, and Ca 8.0. He was discharged on bimonthly dose of eculizumab and hemodialysis three times a week. Two months later, his labs were Hb 10.4, platelets 184, LDH 490, haptoglobin < 10, Cr 11.60, BUN 82, K 5.2, and Ca 9.2. PT and PTT remained normal. Given persistence of laboratory findings, renal biopsy and genetic testing for complement proteins was done. Kidney biopsy showed diffuse global renal microangiopathy with negative C3. Genetic testing showed a heterozygous C3 variant, homozygous deletion of CFHR1-CFHR4, negative factor H autoantibodies (FHAA), and single nucleotide variant in C5 gene. Discussion: Atypical HUS is a disease characterized by the triad of microangiopathic hemolytic anemia, thrombocytopenia, and AKI. The complement system is a part of the immune system that enhances the ability of phagocytic cells to clear microbes and damaged cells. Atypical HUS is caused by dysregulation of the alternative complement pathway that leads to microvascular thrombosis. Various mutations in complement and its regulatory proteins have been discovered in atypical HUS patients. Heterozygous C3 variants result in persistently low C3 levels and cause severe disease. Most individuals with a homozygous deletion of CFHR1 and/or CFHR3, CFHR4 genes have a pathogenic role in the development of the anti-factor H autoantibodies. These autoantibodies interfere with the binding of CFH to the C3 convertase and are associated with defective CFH-dependent cell protection. Some case reports of COVID-19 patients showed that complement activation was significantly stronger in lung alveolar septa, hyaline membranes, and in alveolar lumina via the lectin and alternative complement pathways. Treatment of atypical HUS is symptomatic but in severe disease eculizumab or ravulizumab have roles by binding to the complement protein C5, inhibiting its cleavage to C5a and C5b, and preventing formation of terminal complement complex C5b-9. Patients on these therapies are at risk of meningococcal disease, thus it is recommended to get meningococcal vaccines and prophylactic penicillin for the duration of therapy. Our patient had homozygous deletion of CFHR1-CFHR4 genes with no factor H autoantibodies. However, he did have C3 variant with gain of function mutation which caused overt activation of alternative pathway as indicated by his low C3 levels with normal C4 levels. He responded partially to eculizumab and discussion on possible kidney transplant has been started.

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

The susceptibility of the kidney to alternative pathway activation-A hypothesis.

The glomerulus is often the prime target of dysregulated alternative pathway (AP) activation. In particular, AP activation is the key driver of two severe kidney diseases: atypical hemolytic uremic syndrome and C3 glomerulopathy. Both conditions are associated with a variety of predisposing molecular defects in AP regulation, such as genetic variants in complement regulators, autoantibodies targeting AP proteins, or autoantibodies that stabilize the AP convertases (C3- and C5-activating enzymes). It is noteworthy that these are systemic AP defects, yet in both diseases pathologic complement activation primarily affects the kidneys. In particular, AP activation is often limited to the glomerular capillaries. This tropism of AP-mediated inflammation for the glomerulus points to a unique interaction between AP proteins in plasma and this particular anatomic structure. In this review, we discuss the pre-clinical and clinical data linking the molecular causes of aberrant control of the AP with activation in the glomerulus, and the possible causes of this tropism. Based on these data, we propose a model for why the kidney is so uniquely and frequently targeted in patients with AP defects. Finally, we discuss possible strategies for preventing pathologic AP activation in the kidney.

Bispecific Antibodies: A Novel Approach for the Treatment of Solid Tumors.

Advancement in sequencing technologies allows for the identification of molecular pathways involved in tumor progression and treatment resistance. Implementation of novel agents targeting these pathways, defined as targeted therapy, significantly improves the prognosis of cancer patients. Targeted therapy also includes the use of monoclonal antibodies (mAbs). These drugs recognize specific oncogenic proteins expressed in cancer cells. However, as with many other types of targeting agents, mAb-based therapy usually fails in the long-term control of cancer progression due to the development of resistance. In many cases, resistance is caused by the activation of alternative pathways involved in cancer progression and the development of immune evasion mechanisms. To overcome this off-target resistance, bispecific antibodies (bsAbs) were developed to simultaneously target differential oncogenic pathway components, tumor-associated antigens (TAA) and immune regulatory molecules. As a result, in the last few years, several bsAbs have been tested or are being tested in cancer patients. A few of them are currently approved for the treatment of some hematologic malignancies but no bsAbs are approved in solid tumors. In this review, we will provide an overview of the state-of-the-art of bsAbs for the treatment of solid malignancies outlining their classification, design, main technologies utilized for production, mechanisms of action, updated clinical evidence and potential limitations.

Alternative pathway amplification and infections.

The alternative pathway (AP) is the phylogenetically oldest arm of the complement system and may have evolved to mark pathogens for elimination by phagocytes. Studies using purified AP proteins or AP-specific serum showed that C3b amplification on bacteria commenced following a lag phase of about 5 min and was highly dependent on the concentration of complement. Most pathogens have evolved several elegant mechanisms to evade complement, including expressing proteases that degrade AP proteins and secreting proteins that block function of C3 convertases. In an example of convergent evolution, many microbes recruit the AP inhibitor factor H (FH) using molecular mechanisms that mimic FH interactions with host cells. In most instances, the AP serves to amplify C3b deposited on microbes by the classical pathway (CP). The role of properdin on microbes appears to be restricted to stabilization of C3 convertases; scant evidence exists for its role as an initiator of the AP on pathogens in the context of serum. Therapeutic complement inhibition carries with it an increased risk of infection. Antibody (Ab)-dependent AP activation may be critical for complement activation by vaccine-elicited Ab when the CP is blocked, and its molecular mechanism is discussed.

The echinoid complement system inferred from genome sequence searches

The vertebrate complement cascade is an essential host protection system that functions at the intersection of adaptive and innate immunity. However, it was originally assumed that complement was present only in vertebrates because it was activated by antibodies and functioned with adaptive immunity. Subsequently, the identification of the key component, SpC3, in sea urchins plus a wide range of other invertebrates significantly expanded the concepts of how complement functions. Because there are few reports on the echinoid complement system, an alternative approach to identify complement components in echinoderms is to search the deduced proteins encoded in the genomes. This approach identified known and putative members of the lectin and alternative activation pathways, but members of the terminal pathway are absent. Several types of complement receptors are encoded in the genomes. Complement regulatory proteins composed of complement control protein (CCP) modules are identified that may control the activation pathways and the convertases. Other regulatory proteins without CCP modules are also identified, however regulators of the terminal pathway are absent. The expansion of genes encoding proteins with Macpf domains is noteworthy because this domain is a signature of perforin and proteins in the terminal pathway. The results suggest that the major functions of the echinoid complement system are detection of foreign targets by the proteins that initiate the activation pathways resulting in opsonization by SpC3b fragments to augment phagocytosis and destruction of the foreign targets by the immune cells.

Distinction of early complement classical and lectin pathway activation via quantification of C1s/C1-INH and MASP-1/C1-INH complexes using novel ELISAs.

The most commonly used markers to assess complement activation are split products that are produced through activation of all three pathways and are located downstream of C3. In contrast, C4d derives from the cleavage of C4 and indicates either classical (CP) or lectin pathway (LP) activation. Although C4d is perfectly able to distinguish between CP/LP and alternative pathway (AP) activation, no well-established markers are available to differentiate between early CP and LP activation. Active enzymes of both pathways (C1s/C1r for the CP, MASP-1/MASP-2 for the LP) are regulated by C1 esterase inhibitor (C1-INH) through the formation of covalent complexes. Aim of this study was to develop validated immunoassays detecting C1s/C1-INH and MASP-1/C1-INH complex levels. Measurement of the complexes reveals information about the involvement of the respective pathways in complement-mediated diseases. Two sandwich ELISAs detecting C1s/C1-INH and MASP-1/C1-INH complex were developed and tested thoroughly, and it was investigated whether C1s/C1-INH and MASP-1/C1-INH complexes could serve as markers for either early CP or LP activation. In addition, a reference range for these complexes in healthy adults was defined, and the assays were clinically validated utilizing samples of 414 COVID-19 patients and 96 healthy controls. The immunoassays can reliably measure C1s/C1-INH and MASP-1/C1-INH complex concentrations in EDTA plasma from healthy and diseased individuals. Both complex levels are increased in serum when activated with zymosan, making them suitable markers for early classical and early lectin pathway activation. Furthermore, measurements of C1-INH complexes in 96 healthy adults showed normally distributed C1s/C1-INH complex levels with a physiological concentration of 1846 ± 1060 ng/mL (mean ± 2SD) and right-skewed distribution of MASP-1/C1-INH complex levels with a median concentration of 36.9 (13.18 - 87.89) ng/mL (2.5-97.5 percentile range), while levels of both complexes were increased in COVID-19 patients (p<0.0001). The newly developed assays measure C1-INH complex levels in an accurate way. C1s/C1-INH and MASP-1/C1-INH complexes are suitable markers to assess early classical and lectin pathway activation. An initial reference range was set and first studies showed that these markers have added value for investigating and unraveling complement activation in human disease.

Factor D.

Complement factor D (FD) is a serine protease that plays an essential role in the activation of the alternative pathway (AP) by cleaving complement factor B (FB) and generating the C3 convertases C3(H2 O)Bb and C3bBb. FD is produced mainly from adipose tissue and circulates in an activated form. On the contrary, the other serine proteases of the complement system are mainly synthesized in the liver. The activation mechanism of FD has long been unknown. Recently, a serendipitous discovery in the mechanism of FD activation has been provided by a generation of Masp1 gene knockout mice lacking both the serine protease MASP-1 and its alternative splicing variant MASP-3, designated MASP-1/3-deficient mice. Sera from the MASP-1/3-deficient mice had little-to-no lectin pathway (LP) and AP activity with circulating zymogen or proenzyme FD (pro-FD). Sera from patients with 3MC syndrome carrying mutations in the MASP1 gene also had circulating pro-FD, suggesting that MASP-1 and/or MASP-3 are involved in activation of FD. Here, we summarize the current knowledge of the mechanism of FD activation that was finally elucidated using the sera of mice monospecifically deficient for MASP-1 or MASP-3. Sera of the MASP-1-deficient mice lacked LP activity, but those of the MASP-3-deficient mice lacked AP activity with pro-FD. This review illustrates the pivotal role of MASP-3 in the physiological activation of the AP via activation of FD.

Alternative pathway diagnostics.

Uncontrolled alternative pathway activation is the primary driver of several diseases, and it contributes to the pathogenesis of many others. Consequently, diagnostic tests to monitor this arm of the complement system are increasingly important. Defects in alternative pathway regulation are strong risk factors for disease, and drugs that specifically block the alternative pathway are entering clinical use. A range of diagnostic tests have been developed to evaluate and monitor the alternative pathway, including assays to measure its function, expression of alternative pathway constituents, and activation fragments. Genetic studies have also revealed many disease-associated variants in alternative pathway genes that predict the risk of disease and prognosis. Newer imaging modalities offer the promise of non-invasively detecting and localizing pathologic complement activation. Together, these various tests help in the diagnosis of disease, provide important prognostic information, and can help guide therapy with complement inhibitory drugs.