MBL-associated Serine Proteases Research Articles

A MASP-like functions as PRR to regulate the mRNA expressions of inflammatory factors in the Pacific oyster Crassostrea gigas.

Mannose-binding lectin-associated serine protease (MASP) is a type of central serine protease in the complement lectin pathway. In the present study, a MASP-like was identified from the Pacific oyster Crassostrea gigas, defined as CgMASPL-2. The cDNA sequence of CgMASPL-2 was of 3399 bp with an open reading frame of 2757 bp and encoded a polypeptide of 918 amino acids containing three CUB domains, an EGF domain, two IG domains, and a Tryp_SPC domain. In the phylogenetic tree, CgMASPL-2 was firstly clustered with Mytilus californianus McMASP-2-like, and then assigned into the invertebrate branch. CgMASPL-2 shared similar domains with M. californianus McMASP-2-like and Littorina littorea LlMReM1. CgMASPL-2 mRNA was expressed in all the tested tissues with the highest expression in haemolymph. CgMASPL-2 protein was mainly distributed in the cytoplasm of haemocytes. The mRNA expression of CgMASPL-2 increased significantly in haemocytes after Vibrio splendidus stimulation. The recombinant 3 × CUB-EGF domains of CgMASPL-2 displayed binding activities to diverse polysaccharides (lipopolysaccharide, peptidoglycan and mannose) and microbes (Staphylococcus aureus, Micrococcus luteus, Pichia pastoris, Vibrio anguillarum, V. splendidus and Escherichia coli). In anti-CgMASPL-2 treated oysters, the mRNA expressions of CgIL17-1 and CgIL17-2 in haemocytes decreased significantly after V. splendidus stimulation. The results indicated that CgMASPL-2 could directly sense microbes and regulate the mRNA expressions of inflammatory factors.

Altered profile of glycosylated proteins in serum samples obtained from patients with Hashimoto's thyroiditis following depletion of highly abundant proteins.

Hashimoto's thyroiditis (HT) is one of the most common autoimmune disorders; however, its underlying pathological mechanisms remain unclear. Although aberrant glycosylation has been implicated in the N-glycome of immunoglobulin G (IgG), changes in serum proteins have not been comprehensively characterized. This study aimed to investigate glycosylation profiles in serum samples depleted of highly abundant proteins from patients with HT and propose the potential functions of glycoproteins for further studies on the pathological mechanisms of HT. A lectin microarray containing 70 lectins was used to detect and analyze glycosylation of serum proteins using serum samples (N=27 HT; N=26 healthy control [HC]) depleted of abundant proteins. Significant differences in glycosylation status between HT patients and the HC group were verified using lectin blot analysis. A lectin-based pull-down assay combined with mass spectrometry was used to investigate potential glycoproteins combined with differentially present lectins, and an enzyme-linked immunosorbent assay (ELISA) was used to identify the expression of targeted glycoproteins in 131 patients with papillary thyroid carcinoma (PTC), 131 patients with benign thyroid nodules (BTN) patients, 130 patients with HT, and 128 HCs. Compared with the HC group, the majority of the lectin binding signals in HT group were weakened, while the Vicia villosa agglutinin (VVA) binding signal was increased. The difference in VVA binding signals verified by lectin blotting was consistent with the results of the lectin microarray. A total of 113 potential VVA-binding glycoproteins were identified by mass spectrometry and classified by gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) analyses. Using ELISA, we confirmed that lactoferrin (LTF) and mannan-binding lectin-associated serine protease 1 (MASP-1) levels were elevated in the serum of patients with HT and PTC. Following depletion of abundant proteins, remaining serum proteins in HT patients exhibited lower glycosylation levels than those observed in HCs. An increased level of potential VVA-binding glycoproteins may play an important role in HT development. LTF and MASP-1 expression was significantly higher in the serum of HT and PTC patients, providing novel insight into HT and PTC.

Association between mannose binding lectin gene polymorphisms and clinical severity of COVID-19 in children.

Mannose-binding lectin (MBL) is a member of innate immunity and acts with MASP (MBL-associated serine protease) to activate the lectin pathway of the complement system. MBL gene polymorphisms are associated with susceptibility to infectious diseases. This study investigated whether MBL2 genotype, serum MBL levels, and serum MASP-2 levels affect the course of SARS-CoV-2 infection. Pediatric patients diagnosed with COVID-19 by positive real-time polymerase chain reaction (PCR) were included in the study. Single nucleotide polymorphisms in the promoter and exon 1 in the MBL2 gene (rs11003125, rs7096206, rs1800450, rs1800451, rs5030737) were identified by a PCR and restriction fragment length polymorphisms analysis. Serum MBL and MASP-2 levels were measured by ELISA. COVID-19 patients were divided into asymptomatic and symptomatic. Variables were compared between these two groups. A total of 100 children were included in the study. The mean age of the patients was 130 ± 67.2 months. Of the patients, 68 (68%) were symptomatic, and 32 (32%) were asymptomatic. The polymorphisms in the - 221nt and - 550nt promoter regions did not differ between groups (p > 0.05). All codon 52 and codon 57 genotypes were determined as wild-type AA. AB genotypes were found 45.6% in symptomatic patients while 23.5% in asymptomatics. Moreover, BB genotype was detected 9.4% in symptomatic and 6.3% in asymptomatic patients (p < 0.001). B allele was more frequent in symptomatic patients (46.3%) compared to asymptomatic patients (10.9%). (p < 0.001). Serum MBL and MASP-2 levels did not differ statistically between the groups (p = 0.295, p = 0.073). These findings suggest that codon 54 polymorphism in the MBL2 gene exon-1 region can be associated with the symptomatic course of COVID-19.

Cooperation of Complement MASP-1 with Other Proinflammatory Factors to Enhance the Activation of Endothelial Cells.

Endothelial cells play an important role in sensing danger signals and regulating inflammation. Several factors are capable of inducing a proinflammatory response (e.g., LPS, histamine, IFNγ, and bradykinin), and these factors act simultaneously during the natural course of the inflammatory reaction. We have previously shown that the complement protein mannan-binding lectin-associated serine protease-1 (MASP-1) also induces a proinflammatory activation of the endothelial cells. Our aim was to investigate the possible cooperation between MASP-1 and other proinflammatory mediators when they are present in low doses. We used HUVECs and measured Ca2+ mobilization, IL-8, E-selectin, VCAM-1 expression, endothelial permeability, and mRNA levels of specific receptors. LPS pretreatment increased the expression of PAR2, a MASP-1 receptor, and furthermore, MASP-1 and LPS enhanced each other's effects in regulating IL-8, E-selectin, Ca2+ mobilization, and changes in permeability in a variety of ways. Cotreatment of MASP-1 and IFNγ increased the IL-8 expression of HUVECs. MASP-1 induced bradykinin and histamine receptor expression, and consequently, increased Ca2+ mobilization was found. Pretreatment with IFNγ enhanced MASP-1-induced Ca2+ mobilization. Our findings highlight that well-known proinflammatory mediators and MASP-1, even at low effective doses, can strongly synergize to enhance the inflammatory response of endothelial cells.

Mannose-binding lectin gene 2 variant DD (rs 5030737) is associated with susceptibility to COVID-19 infection in the urban population of Patna City (India).

The study aimed to measure plasma levels of Mannose-Binding Lectin (MBL) and MBL-associated serine protease-2 (MASP-2) and theirpolymorphisms in COVID-19 patients and controls to detect association. As MBL is a protein of immunological importance, it may contribute to the first-line host defence against SARS-CoV-2. MBL initiates the lectin pathway of complement activation with help of MASP-1 and MASP-2. Hence, appropriate serum levels of MBL and MASPs are crucial in getting protection from the disease. The polymorphisms of MBL and MASP genes affect their plasma levels, impacting their protective function and thus may manifest susceptibility, extreme variability in the clinical symptoms and progression of COVID-19 disease. The present study was conducted to find plasma levels and genetic variations in MBL and MASP-2 in COVID-19 patients and controls using PCR-RFLP and ELISA, respectively.The present study was conducted to find plasma levels and genetic variations in MBL and MASP-2 in COVID-19 patients and controls using PCR-RFLP and ELISA, respectively. Our results indicate that median serum levels of MBL and MASP-2 were significantly low in diseased cases but attained normal levels on recovery. Only genotypeDD was found to be associated with COVID-19 cases in the urban population of Patna city.

MASP2 inhibition by narsoplimab suppresses endotheliopathies characteristic of transplant-associated thrombotic microangiopathy: in vitro and ex vivo evidence.

Transplant-associated thrombotic microangiopathy (TA-TMA) is an endotheliopathy complicating up to 30% of allogeneic hematopoietic stem cell transplants (alloHSCT). Positive feedback loops among complement, pro-inflammatory, pro-apoptotic, and coagulation cascade likely assume dominant roles at different disease stages. We hypothesized that mannose-binding lectin-associated serine protease 2 (MASP2), principal activator of the lectin complement system, is involved in the microvascular endothelial cell (MVEC) injury characteristic of TA-TMA through pathways that are susceptible to suppression by anti-MASP2 monoclonal antibody narsoplimab. Pre-treatment plasmas from 8 of 9 TA-TMA patients achieving a complete TMA response in a narsoplimab clinical trial activated caspase 8, the initial step in apoptotic injury, in human MVEC. This was reduced to control levels following narsoplimab treatment in 7 of the 8 subjects. Plasmas from 8 individuals in an observational TA-TMA study, but not 8 alloHSCT subjects without TMA, similarly activated caspase 8, which was blocked in vitro by narsoplimab. mRNA sequencing of MVEC exposed to TA-TMA or control plasmas with and without narsoplimab suggested potential mechanisms of action. The top 40 narsoplimab-affected transcripts included upregulation of SerpinB2, which blocks apoptosis by inactivating procaspase 3; CHAC1, which inhibits apoptosis in association with mitigation of oxidative stress responses; and pro-angiogenesis proteins TM4SF18, ASPM, and ESM1. Narsoplimab also suppressed transcripts encoding pro-apoptotic and pro-inflammatory proteins ZNF521, IL1R1, Fibulin-5, aggrecan, SLC14A1, and LOX1, and TMEM204, which disrupts vascular integrity. Our data suggest benefits to narsoplimab use in high-risk TA-TMA and provide a potential mechanistic basis for the clinical efficacy of narsoplimab in this disorder.

Urinary complement proteins in IgA nephropathy progression from a relative quantitative proteomic analysis.

IgA nephropathy (IgAN) is one of the leading causes of end-stage renal disease (ESRD). Urine testing is a non-invasive way to track the biomarkers used for measuring renal injury. This study aimed to analyse urinary complement proteins during IgAN progression using quantitative proteomics. In the discovery phase, we analysed 22 IgAN patients who were divided into three groups (IgAN 1-3) according to their estimated glomerular filtration rate (eGFR). Eight patients with primary membranous nephropathy (pMN) were used as controls. Isobaric tags for relative and absolute quantitation (iTRAQ) labelling, coupled with liquid chromatography-tandem mass spectrometry, was used to analyse global urinary protein expression. In the validation phase, western blotting and parallel reaction monitoring (PRM) were used to verify the iTRAQ results in an independent cohort (N = 64). In the discovery phase, 747 proteins were identified in the urine of IgAN and pMN patients. There were different urine protein profiles in IgAN and pMN patients, and the bioinformatics analysis revealed that the complement and coagulation pathways were most activated. We identified a total of 27 urinary complement proteins related to IgAN. The relative abundance of C3, the membrane attack complex (MAC), the complement regulatory proteins of the alternative pathway (AP), and MBL (mannose-binding lectin) and MASP1 (MBL associated serine protease 2) in the lectin pathway (LP) increased during IgAN progression. This was especially true for MAC, which was found to be involved prominently in disease progression. Alpha-N-acetylglucosaminidase (NAGLU) and α-galactosidase A (GLA) were validated by western blot and the results were consistent with the iTRAQ results. Ten proteins were validated in a PRM analysis, and these results were also consistent with the iTRAQ results. Complement factor B (CFB) and complement component C8 alpha chain (C8A) both increased with the progression of IgAN. The combination of CFB and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) also showed potential as a urinary biomarker for monitoring IgAN development. There were abundant complement components in the urine of IgAN patients, indicating that the activation of AP and LP is involved in IgAN progression. Urinary complement proteins may be used as biomarkers for evaluating IgAN progression in the future.

MASP3 Deficiency in Mice Reduces but Does Not Abrogate Alternative Pathway Complement Activity Due to Intrinsic Profactor D Activity.

Complement factor D (FD) is a rate-limiting enzyme of the alternative pathway (AP). Recent studies have suggested that it is synthesized as an inactive precursor and that its conversion to enzymatically active FD is catalyzed by mannan-binding lectin-associated serine protease 3 (MASP3). However, whether MASP3 is essential for AP complement activity remains uncertain. It has been shown that Masp1/3 gene knockout did not prevent AP complement overactivation in a factor H-knockout mouse, and a human patient lacking MASP3 still retained AP complement activity. In this study, we have assessed AP complement activity in a Masp3-knockout mouse generated by CRISPR/Cas9 editing of the Masp1/3 gene. We confirmed specific Masp3 gene inactivation by showing intact MASP1 protein expression and absence of mature FD in the mutant mice. Using several assays, including LPS- and zymosan-induced C3b deposition and rabbit RBC lysis tests, we detected plasma concentration-dependent AP complement activity in Masp3 gene-inactivated mice. Thus, although not measurable in 5% plasma, significant AP complement activity was detected in 20-50% plasma of Masp3 gene-inactivated mice. Furthermore, whereas FD gene deletion provided more than 90% protection of CD55/Crry-deficient RBCs from AP complement-mediated extravascular hemolysis, Masp3 gene deletion only provided 30% protection in the same study. We also found pro-FD to possess intrinsic catalytic activity, albeit at a much lower level than mature FD. Our data suggest that MASP3 deficiency reduces but does not abrogate AP complement activity and that this is explained by intrinsic pro-FD activity, which can be physiologically relevant invivo.

Proteomics analysis of serum from thymoma patients

Thymoma is the most common malignant tumor in thymic epithelial tumors (TETS). This study aimed to identify the changes in serum proteomics in patients with thymoma. Proteins were extracted from twenty patients with thymoma serum and nine healthy controls and prepared for mass spectrometry (MS) analysis. Data independent acquisition (DIA) quantitative proteomics technique was used to examine the serum proteome. Differential proteins of abundance changes in the serum were identified. Bioinformatics was used to examine the differential proteins. Functional tagging and enrichment analysis were conducted using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. The string database was used to assess the interaction of different proteins. In all, 486 proteins were found in all samples. There were differences in 58 serum proteins between patients and healthy blood donors, 35 up-regulated and 23 down-regulated. These proteins are primarily exocrine and serum membrane proteins involved in controlling immunological responses and antigen binding, according to GO functional annotation. KEGG functional annotation showed that these proteins play a significant role in the complement and coagulation cascade and the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signal pathway. Notably, the KEGG pathway (complement and coagulation cascade) is enriched, and three key activators were up-regulated: von willebrand factor (VWF), coagulation factor v (F5) and vitamin k-dependent protein c (PC). Protein–protein interaction (PPI) analysis showed that six proteins ((VWF, F5, thrombin reactive protein 1 (THBS1), mannose-binding lectin-associated serine protease 2 (MASP2), apolipoprotein B (APOB), and apolipoprotein (a) (LPA)) were up-regulated and two proteins (Metalloproteinase inhibitor 1(TIMP1), ferritin light chain (FTL)) were down-regulated. The results of this study showed that several proteins involved in complement and coagulation cascades were up-regulated in the serum of patients.

L-ficolin-MASP arm of the complement system in schizophrenia

The abnormal neurodevelopment secondary to in utero adversities, such as hypoxia, malnutrition and maternal infections, underlies schizophrenia (SZ) etiology. As the genes of MBL-associated serine proteases (MASP) of the complement lectin pathway, MASP1 and MASP2, are expressed in the developing cortex and are functionally important for neuronal migration, we hypothesize that the malfunction ofl-ficolin-MASP arm may also be involved in schizophrenia pathophysiology as it was shown for MBL-MASP complexes. We investigated serum l-ficolin and plasma MASP-2 levels, the activity of l-ficolin-bound MASP-2, as well as an array of the complement-related variables in chronic schizophrenic patients in the acute phase of the disease and controls without physical or mental diagnoses.The median concentration of l-ficolin in Armenian controls was 3.66 μg/ml and similar to those reported for other Caucasian populations. SZ-cases had ∼40 % increase in serum l-ficolin (median 5.08 μg/ml; P < 0.0024). In the pooled sample, l-ficolin level was higher in males than in females (P < 0.0031), but this gender dichotomy was not affecting the variable association with schizophrenia (P < 0.016). Remarkably, MASP-2 plasma concentration showed gender-dependent significant variability in the group of patients but not in controls. When adjusted for gender and gender*diagnosis interaction, a significantly high MASP-2 level in female patients versus female controls was observed (median: 362 ng/ml versus 260 ng/ml, respectively; P < 0.0020). A significant increase in l-ficolin-bound MASP-2 activity was also observed in schizophrenia (on the median, cases vs controls: 7.60 vs 6.50 RU; P < 0.021). Correlation analyses of the levels of l-ficolin and MASP-2, l-ficolin-(MASP-2) activity and the demographic data did not show any significant association with the age of individuals, family history, age at onset and duration of the illness, and smoking. Noteworthy, the levels of l-ficolin and MASP-2 in circulation were significantly associated with the type of schizophrenia (paranoid SZ-cases had much higher l-ficolin (P < 0.0035) and lower MASP-2 levels than the other types combined (P < 0.049)). Correlations were also found between: (i) the classical pathway functional activity and l-ficolin level (rs = 0.19, P < 0.010); (ii) the alternative pathway functional activity and MASP-2 level (rs = 0.26, P < 0.00035); (iii) the activity of l-ficolin-bound MASP2 and the downstream C2 component haemolytic activity (rs = −0.19, P < 0.017); and (iv) l-ficolin and the upstream C-reactive protein (CRP) serum concentrations (r = 0.28, P < 0.018).Overall, the results showed l-ficolin-related lectin pathway alterations in schizophrenia pathophysiology. It is likely that in addition to the MBL-MASP component over-activity reported previously, the alterations of the lectin pathway in schizophrenia also involve variations of l-ficolin-(MASP-2) on protein concentration and activity levels.

Analysis of Mannose-Binding Lectin Protein Levels and their MIC Profile in Patients with Dermatophytosis.

Analysis of Mannose-Binding Lectin Protein Levels and their MIC Profile in Patients with Dermatophytosis.

FCN3 inhibits the progression of hepatocellular carcinoma by suppressing SBDS-mediated blockade of the p53 pathway.

Hepatocellular carcinoma (HCC) is the third-leading cause of cancer deaths globally. Although considerable progress has been made in the treatment, clinical outcomes of HCC patients are still poor. Therefore, it is necessary to find novel prognostic factors upon which prevention and treatment strategies can be formulated. Ficolin-3 (FCN3) protein is a member of the human ficolin family. It activates complement through pathways associated with mannose-binding lectin-associated serine proteases. Herein, we identified that FCN3 was downregulated in HCC tissues and decreased FCN3 expression was closely related to poor prognosis. Overexpression of FCN3 induced apoptosis and inhibited cell proliferation via the p53 signaling pathway. Mechanistically, FCN3 modulated the nuclear translocation of eukaryotic initiation factor 6 (EIF6) by binding ribosome maturation factor (SBDS), which induced ribosomal stress and activation of the p53 pathway. In addition, Y-Box Binding Protein 1 (YBX1) involved in the transcription and translation level regulation of FCN3 to SBDS. Besides, a negative feedback loop in the downstream of FCN3 involving p53, YBX1 and SBDS was identified.

Thrombotic microangiopathy mediates poor prognosis among lupus nephritis via complement lectin and alternative pathway activation.

The pathogenesis of thrombotic microangiopathy (TMA) in lupus nephritis (LN) remains complicated. This study aimed to detect the deposition of complement lectin pathway (LP) and alternative pathway (AP) components in renal tissues, then evaluate the clinicopathological characteristics and risk factors for renal survival between patients with or without TMA in LN cohorts. We included 79 patients with biopsy-proven LN-associated TMA and matched the same number of LN patients without TMA as the control group. The deposition of mannose binding lectin (MBL), MBL-associated serine proteases 1/3 (MASP1/3), complement factor B (CFB), complement factor D (CFD), C4d, and von Willebrand factor (VWF) in renal tissue was assessed by immunohistochemistry and immunofluorescence. Besides, co-localization of C5b-9 and CD34 was detected by confocal microscopy. In our retrospective cohort, the incidence of acute kidney injury (30% vs. 14%, p = 0.013), acute hemodialysis (35% vs. 5%, p < 0.001), and interstitial fibrosis (43% vs. 13%, p < 0.001) is higher in the TMA, compared with the control group. Despite aggressive steroids pulse, plasma exchange, and immunosuppressive therapy among TMA group, they still had significantly inferior 3-year renal survival rates (68% vs. 89%, p = 0.002) than those in the non-TMA group. COX regression analysis identified that TMA (HR 4.807, 95% CI [2.052, 11.263], p < 0.001) is a risk factor in LN. MBL, MASP1/3, CFB, CFD, C4d, and VWF deposited along the glomerulus among LN, while TMA had stronger staining intensity and deposition. The co-localized expression of CD34 and C5b-9 in the endothelial cells was also observed in the renal tissues. TMA is an independent risk factor for renal survival in LN patients. Moreover, LP and AP activation are involved in the pathogenesis of LN-associated TMA.

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

Trial in Progress: An Open-Label, Multi-Center Phase 2 Study Evaluating Efficacy and Safety of the Masp-2 Inhibitor Narsoplimab in Pediatric Patients with High-Risk Hematopoietic Stem Cell Transplant-Associated Thrombotic Microangiopathy (HSCT-TMA)

Background: Hematopoietic stem cell transplant-associated thrombotic microangiopathy (HSCT-TMA; also known as TA-TMA) is a potentially life-threatening complication associated with multi-organ injury and significant morbidity and mortality. HSCT-TMA incidence rates of up to 39% have been reported in both pediatric and adult allogeneic HSCT (alloHSCT) recipients; high-risk pediatric patients with proteinuria and evidence of terminal complement activation (elevated sC5b-9) had a 1-year post-HSCT survival of <20% versus 100% in those with normal sC5b-9 levels and no proteinuria (Jodele S, et al. Blood. 2014;124:645-653). In the HSCT setting, toxic conditioning regimens, infection, and graft-versus-host disease (GVHD) can cause endothelial injury, which triggers activation of the lectin pathway of complement and in turn the coagulation cascade, together leading to TMA. Narsoplimab (OMS721) is a fully human IgG4 monoclonal antibody that inhibits mannan-binding lectin-associated serine protease-2 (MASP-2), the effector enzyme of the lectin pathway and an activator of the coagulation cascade. Narsoplimab was previously evaluated for safety and efficacy in adults with high-risk HSCT-TMA in an open-label pivotal trial (NCT02222545); narsoplimab treatment was well tolerated and resulted in clinical response and favorable overall survival (Khaled SK, et al. J Clin Oncol. 2022; JCO2102389). Currently, HSCT-TMA has no approved therapy, and therefore represents a significant unmet medical need. Here, we describe the design of the first clinical trial evaluating the efficacy and safety of narsoplimab in pediatric patients with HSCT-TMA. Study Design and Methods: This is an open-label, multi-center Phase 2 trial of narsoplimab in pediatric patients (Figure 1). Eligible patients are aged ≥28 days to <18 years, have received alloHSCT for the treatment of non-malignant or malignant disease, and have been diagnosed with high-risk HSCT-TMA. Exclusion criteria include prior treatment with eculizumab, ravulizumab, or defibrotide within 3 months prior to screening, having Shiga toxin-producing Escherichia coli hemolytic uremic syndrome (STEC-HUS), or ADAMTS13 activity <10%. Patients will receive narsoplimab 4 mg/kg via intravenous (IV) infusion twice weekly during the 8-week treatment period (Days 1-56). If a patient meets all clinical response criteria prior to 8 weeks of treatment, dosing may be decreased to 4 mg/kg IV once weekly until the end of the treatment period. Narsoplimab can be used in conjunction with standard-of-care treatments. At the end of the treatment period, patients will enter a 44-week follow-up period (Days 60-365). The planned recruitment is at least 18 pediatric patients, distributed across the age range. The primary endpoint is 100-day survival from date of HSCT-TMA diagnosis. Secondary endpoints include: safety, survival at 52 weeks, overall survival from date of HSCT-TMA diagnosis, anti-drug antibody response, clinical response rate, narsoplimab pharmacokinetics and measurement of lectin pathway activity. Conclusion: Following the favorable results obtained in a pivotal Phase 2 study in adults with HSCT-TMA, further evaluation of narsoplimab efficacy and safety in pediatric patients with high-risk HSCT-TMA is warranted. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Mannan-binding lectin serine protease-2 (MASP-2) in human kidney and its relevance for proteolytic activation of the epithelial sodium channel

Proteolytic activation of the renal epithelial sodium channel (ENaC) is increased by aldosterone. The aldosterone-sensitive protease remains unidentified. In humans, elevated circulating aldosterone is associated with increased urinary extracellular vesicle (uEVs) excretion of mannan-binding lectin associated serine protease-2 (MASP-2). We hypothesized that MASP-2 is a physiologically relevant ENaC-activating protease. It was confirmed that MASP2 mRNA is abundantly present in liver but not in human and mouse kidneys. Aldosterone-stimulation of murine cortical colleting duct (mCCD) cells did not induce MASP-2 mRNA. In human kidney collecting duct, MASP-2 protein was detected in AQP2-negative/ATP6VB1-positive intercalated cells suggestive of MASP2 protein uptake. Plasma concentration of full-length MASP-2 and the short splice variant MAp19 were not changed in a cross-over intervention study in healthy humans with low (70 mmol/day) versus high (250 mmol/day) Na+ intake despite changes in aldosterone. The ratio of MAp19/MASP-2 in plasma was significantly increased with a high Na+ diet and the ratio correlated with changes in aldosterone and fractional Na+ excretion. MASP-2 was not detected in crude urine or in uEVs. MASP2 activated an amiloride-sensitive current when co-expressed with ENaC in Xenopus oocytes, but not when added to the bath solution. In monolayers of collecting duct M1 cells, MASP2 expression did not increase amiloride-sensitive current and in HEK293 cells, MASP-2 did not affect γENaC cleavage. MASP-2 is neither expressed nor co-localized and co-regulated with ENaC in the human kidney or in urine after low Na+ intake. MASP-2 does not mediate physiological ENaC cleavage in low salt/high aldosterone settings.

MASP1 Gene Polymorphism and MASP-3 Serum Levels in Patients with Chronic Chagas Disease

ABSTRACT Introduction Chagas disease (CD), caused by Trypanosoma cruzi, is a major public health issue worldwide affecting 6–7 million people, mainly in Latin America. The complement system plays a crucial role in host immune defense against T. cruzi infection and during the chronic phase of CD; however, the role of the MBL-associated serine protease 1 (MASP1) gene encoding MASP-1, MASP-3, and MAp44 complement proteins has not yet been reported in CD. This study investigated the possible association between MASP1 gene polymorphisms and MASP-3 protein serum levels in chronic CD and its clinical forms. Methods Five polymorphisms of MASP1 gene regulatory regions were genotyped in 214 patients with CD and 197 healthy controls (rs7609662 G>A, rs13064994 C>T, rs72549262 C>G, rs1109452 C>T and rs850314 G>A). MASP-3 serum levels were assessed in 70 patients and 66 healthy controls. Clinical data, serum levels of complement proteins (ficolin-2, ficolin-3 and MBL) and inflammatory markers (pentraxin-3 and hsCRP) were also included in the analyses. Results A significant association of the MASP1 GC_CCA haplotype with CD (padj= 0.002; OR 3.17 [1.19–8.39]) and chronic chagasic cardiomyopathy (CCC) (padj= 0.013; OR 4.57 [1.37–15.16] was observed. MASP-3 and pentraxin-3 levels were positively correlated in the patients (rho = 0.62; p = 0.0001). MASP-3 levels were not associated with MASP1 polymorphisms or CD and its clinical forms. Furthermore, no correlation was observed between MASP-3 levels and that of ficolin-2, ficolin-3, MBL and hsCRP. Conclusion Our findings suggest a possible role for the MASP1 GC_CCA haplotype in susceptibility to chronic CD and CCC clinical forms.

Significance of Mannan Binding Lectin-Associated Serine Protease 2 in Urinary Extracellular Vesicles in IgA Nephropathy.

Immunoglobulin A (IgA) nephropathy (IgAN) is a common chronic glomerulonephritis and the main cause of end-stage renal diseases. Recent evidence suggests that mannan binding lectin associated serine proteases 2 (MASP2) is related to IgAN; therefore, we investigated the expression and significance of MASP2 in serum and urinary extracellular vesicles (UEVs) in patients with IgAN. Thirty-eight patients with IgAN and 17 healthy controls were enrolled in this study. UEVs were extracted by ultracentrifugation. The separation by ultra-high-speed centrifuge was verified by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Candidate internal references (TSG101, CD9, flotillin, β-actin and GAPDH) were identified by western blotting in the control group, and the expression of MASP2 in the UEVs was compared. The levels of MASP2 in the serum and UEVs in the IgAN and control groups were determined by enzyme-linked immunosorbent assay (ELISA). TEM and NTA results demonstrated that UEVs were successfully extracted. Western blotting results confirmed that TSG101 was suitable as an internal reference for this study. Compared with the control group, the IgAN group showed positive expression of MASP2. MASP2 levels in the UEVs, determined by ELISA, showed significant differences between IgAN and control groups, which were significantly positively correlated with the level of urinary microalbumin. The level of MASP2 in UEVs was related to IgAN and shows promise as a biomarker for evaluating the severity of renal injury and prognosis of IgAN, thereby helping to elucidate the role of MASP2 in the mannan-binding lectin pathway.

Identification of substrates of MBL Associated Serine Protease-1 (MASP-1) from human plasma using N-terminomics strategy

MBL Associated Serine Protease-1 (MASP-1) is an abundant enzyme of the lectin complement pathway. MASP-1 cleaves numerous substrates like MASP-2, MASP-3, C2, C3i, fibrinogen, FXIII and prothrombin. It has thrombin-like specificity and can cleave thrombin substrates. Owing to its high concentration and relaxed substrate specificity, MASP-1 has substrates outside the complement system and can influence other proteolytic cascades and physiological processes. The unidentified substrates may assist us to ascertain the role(s) of MASP-1. In this study, we used a high-throughput N-terminomics method to identify substrates of MASP-1 from human plasma. We have identified 35 putative substrates of MASP-1. Among the identified proteins, alpha 2-antiplasmin, alpha-1-acid glycoprotein, antithrombin III, and siglec-6 were demonstrated to be cleaved by MASP-1. We have discussed the physiological relevance of cleavage of these substrates by MASP-1. The expression of Siglec-6 and MASP-1 has been reported in the B cells. Alpha-1-acid glycoprotein cleavage by MASP-1 may occur in the acute phase as it is known to be an inhibitor of platelet aggregation, whereas MASP-1 triggers platelet aggregation. The cleavage alpha2 antiplasmin by MASP-1 implies that MASP-1 may be promoting plasmin-mediated fibrinolysis. Our study supports that MASP-1 may be implicated in thrombosis as well as thrombolysis.

Mannose binding lectin-associated serine protease 2 (MASP2) gene polymorphism and susceptibility to human T-lymphotropic virus type 1 (HTLV-1) infection in blood donors from Mashhad, Iran.

Mannose binding lectin-associated serine protease 2 (MASP2) is the effector part of mannose binding lectin (MBL) that activates the complement system in an antibody-independent manner. We aimed to investigate the role of genetic polymorphisms in the MASP2 gene and susceptibility to HTLV-1 infection. A total of 172 HTLV-1 infected individuals and 170 healthy blood donors were analyzed in this case-control study. Nine single nucleotide polymorphisms (SNPs) encompassing different regions of the MASP2 gene were genotyped with a polymerase chain reaction-sequence-specific primer (PCR-SSP) assay. The relation between the SNPs genotype and the susceptibility to HTLV-1 infection was investigated with a χ2 test considering P < 0.05 as statistically significant. Two of nine tested SNPs were associated with the risk of HTLV-1 infection. The genotype TT at rs17409276 decreased the risk of HTLV-1 (P = 0.005, OR = 0.301, 95% CI = 0.124-0.728). The genotypes CC and CT at rs2273346 were also associated with a higher risk of HTLV-1 acquisition (P = 0.004, OR = 2.225, 95% CI = 1.277-3.877). These findings highlight the importance of MASP2 genetic polymorphisms in the lectin pathway of complement activation and susceptibility to HTLV-1 infection.