Inhibition of mannan-binding lectin associated serine protease (MASP)-2 reduces the cognitive deficits in a mouse model of severe traumatic brain injury.

The lectin pathway (LP) of complement mediates inflammatory processes linked to tissue damage and loss of function following traumatic brain injury (TBI). LP activation triggers a cascade of proteolytic events initiated by LP specific enzymes called MASPs (for Mannan-binding lectin Associated Serine Proteases). Elevated serum and brain levels of MASP-2, the effector enzyme of the LP, were previously reported to be associated with the severity of tissue injury and poor outcomes in patients with TBI. To evaluate the therapeutic potential of LP inhibition in TBI, we first conducted a pilot study testing the effect of an inhibitory MASP-2 antibody (α-MASP-2), administered systemically at 4 and 24 h post-TBI in a mouse model of controlled cortical impact (CCI). Treatment with α-MASP-2 reduced sensorimotor and cognitive deficits for up to 5 weeks post-TBI. As previous studies by others postulated a critical role of MASP-1 in LP activation, we conducted an additional study that also assessed treatment with an inhibitory MASP-1 antibody (α-MASP-1). A total of 78 mice were treated intraperitoneally with either α-MASP-2, or α-MASP-1, or an isotype control antibody 4 h and 24 h after TBI or sham injury. An amelioration of the cognitive deficits assessed by Barnes Maze, prespecified as the primary study endpoint, was exclusively observed in the α-MASP-2-treated group. The behavioral data were paralleled by a reduction of the lesion size when evaluated histologically and by reduced systemic LP activity. Our data suggest that inhibition of the LP effector enzyme MASP-2 is a promising treatment strategy to limit neurological deficits and tissue loss following TBI. Our work has translational value because a MASP-2 antibody has already completed multiple late-stage clinical trials in other indications and we used a clinically relevant treatment protocol testing the therapeutic mechanism of MASP-2 inhibition in TBI.

Development and characterization of narsoplimab, a selective MASP-2 inhibitor, for the treatment of lectin-pathway-mediated disorders.

Introduction: Overactivation of the lectin pathway of complement plays a pathogenic role in a broad range of immune-mediated and inflammatory disorders; mannan-binding lectin-associated serine protease-2 (MASP-2) is the key effector enzyme of the lectin pathway. We developed a fully human monoclonal antibody, narsoplimab, to bind to MASP-2 and specifically inhibit lectin pathway activation. Herein, we describe the preclinical characterization of narsoplimab that supports its evaluation in clinical trials. Methods and results: ELISA binding studies demonstrated that narsoplimab interacted with both zymogen and enzymatically active forms of human MASP-2 with high affinity (KD 0.062 and 0.089 nM, respectively) and a selectivity ratio of >5,000-fold relative to closely related serine proteases C1r, C1s, MASP-1, and MASP-3. Interaction studies using surface plasmon resonance and ELISA demonstrated approximately 100-fold greater binding affinity for intact narsoplimab compared to a monovalent antigen binding fragment, suggesting an important contribution of functional bivalency to high-affinity binding. In functional assays conducted in dilute serum under pathway-specific assay conditions, narsoplimab selectively inhibited lectin pathway-dependent activation of C5b-9 with high potency (IC50 ~ 1 nM) but had no observable effect on classical pathway or alternative pathway activity at concentrations up to 500 nM. In functional assays conducted in 90% serum, narsoplimab inhibited lectin pathway activation in human serum with high potency (IC50 ~ 3.4 nM) whereas its potency in cynomolgus monkey serum was approximately 10-fold lower (IC50 ~ 33 nM). Following single dose intravenous administration to cynomolgus monkeys, narsoplimab exposure increased in an approximately dose-proportional manner. Clear dose-dependent pharmacodynamic responses were observed at doses >1.5 mg/kg, as evidenced by a reduction in lectin pathway activity assessed ex vivo that increased in magnitude and duration with increasing dose. Analysis of pharmacokinetic and pharmacodynamic data revealed a well-defined concentration-effect relationship with an ex vivo EC50 value of approximately 6.1 µg/mL, which was comparable to the in vitro functional potency (IC50 33 nM; ~ 5 µg/mL). Discussion: Based on these results, narsoplimab has been evaluated in clinical trials for the treatment of conditions associated with inappropriate lectin pathway activation, such as hematopoietic stem cell transplantation-associated thrombotic microangiopathy.

Inhibition of the lectin pathway of complement activation reduces LPS-induced acute respiratory distress syndrome in mice.

Acute respiratory distress syndrome (ARDS) is a life-threatening disorder with a high rate of mortality. Complement activation in ARDS initiates a robust inflammatory reaction that can cause progressive endothelial injury in the lung. Here, we tested whether inhibition of the lectin pathway of complement could reduce the pathology and improve the outcomes in a murine model of LPS-induced lung injury that closely mimics ARDS in human. In vitro, LPS binds to murine and human collectin 11, human MBL and murine MBL-A, but not to C1q, the recognition subcomponent of the classical pathway. This binding initiates deposition of the complement activation products C3b, C4b and C5b-9 on LPS via the lectin pathway. HG-4, a monoclonal antibody that targets MASP-2, a key enzyme in the lectin pathway, inhibited lectin pathway functional activity in vitro, with an IC50 of circa 10nM. Administration of HG4 (5mg/kg) in mice led to almost complete inhibition of the lectin pathway activation for 48hrs, and 50% inhibition at 60hrs post administration. Inhibition of the lectin pathway in mice prior to LPS-induced lung injury improved all pathological markers tested. HG4 reduces the protein concentration in bronchoalveolar lavage fluid (p<0.0001) and levels of myeloid peroxide (p<0.0001), LDH (p<0.0001), TNFα and IL6 (both p<0.0001). Lung injury was significantly reduced (p<0.001) and the survival time of the mice increased (p<0.01). From the previous findings we concluded that inhibition of the lectin pathway has the potential to prevent ARDS pathology.

MASP-2 and MASP-3 inhibitors block complement activation, inflammation, and microvascular stasis in a murine model of vaso-occlusion in sickle cell disease.

Patients with sickle cell disease (SCD) have ongoing hemolysis that promotes endothelial injury, complement activation, inflammation, vaso-occlusion, ischemia-reperfusion pathophysiology, and pain. Complement activation markers are increased in SCD in steady-state and further increased during vaso-occlusive crisis (VOC). However, the mechanisms driving complement activation in SCD have not been completely elucidated. Ischemia-reperfusion and heme released from hemoglobin during hemolysis, events that characterize SCD pathophysiology, can activate the lectin pathway (LP) and alternative pathway (AP), respectively. Here we evaluated the role of LP and AP in Townes sickle (SS) mice using inhibitory monoclonal antibodies (mAb) to mannose binding lectin (MBL)-associated serine protease (MASP)-2 or MASP-3, respectively. Townes SS mice were pretreated with MASP-2 mAb, MASP-3 mAb, isotype control mAb, or PBS before they were challenged with hypoxia-reoxygenation or hemoglobin. Pretreatment of SS mice with MASP-2 or MASP-3 mAb, markedly reduced Bb fragments, C4d and C5a in plasma and complement deposition in the liver, kidneys, and lungs collected 4 hours after challenge compared to control mAb-treated mice. Consistent with complement inhibition, hepatic inflammation markers NF-ĸB phospho-p65, VCAM-1, ICAM-1, and E-selectin were significantly reduced in SS mice pretreated with MASP-2 or MASP-3 mAb. Importantly, MASP-2 or MASP-3 mAb pretreatment significantly inhibited microvascular stasis (vaso-occlusion) induced by hypoxia-reoxygenation or hemoglobin. These studies suggest that the LP and the AP are both playing a role in promoting inflammation and vaso-occlusion in SCD. Inhibiting complement activation via the LP or the AP might inhibit inflammation and prevent VOC in SCD patients.

Role of the lectin pathway of complement in hematopoietic stem cell transplantation-associated endothelial injury and thrombotic microangiopathy.

Hematopoietic stem cell transplantation-associated thrombotic microangiopathy (HSCT-TMA) is a life-threatening syndrome that occurs in adult and pediatric patients after hematopoietic stem cell transplantation. Nonspecific symptoms, heterogeneity within study populations, and variability among current diagnostic criteria contribute to misdiagnosis and underdiagnosis of this syndrome. Hematopoietic stem cell transplantation and associated risk factors precipitate endothelial injury, leading to HSCT-TMA and other endothelial injury syndromes such as hepatic veno-occlusive disease/sinusoidal obstruction syndrome, idiopathic pneumonia syndrome, diffuse alveolar hemorrhage, capillary leak syndrome, and graft-versus-host disease. Endothelial injury can trigger activation of the complement system, promoting inflammation and the development of endothelial injury syndromes, ultimately leading to organ damage and failure. In particular, the lectin pathway of complement is activated by damage-associated molecular patterns (DAMPs) on the surface of injured endothelial cells. Pattern-recognition molecules such as mannose-binding lectin (MBL), collectins, and ficolins-collectively termed lectins-bind to DAMPs on injured host cells, forming activation complexes with MBL-associated serine proteases 1, 2, and 3 (MASP-1, MASP-2, and MASP-3). Activation of the lectin pathway may also trigger the coagulation cascade via MASP-2 cleavage of prothrombin to thrombin. Together, activation of complement and the coagulation cascade lead to a procoagulant state that may result in development of HSCT-TMA. Several complement inhibitors targeting various complement pathways are in clinical trials for the treatment of HSCT-TMA. In this article, we review the role of the complement system in HSCT-TMA pathogenesis, with a focus on the lectin pathway.

Lectin Pathway Mediates Complement Activation by SARS-CoV-2 Proteins.

Early and persistent activation of complement is considered to play a key role in the pathogenesis of COVID-19. Complement activation products orchestrate a proinflammatory environment that might be critical for the induction and maintenance of a severe inflammatory response to SARS-CoV-2 by recruiting cells of the cellular immune system to the sites of infection and shifting their state of activation towards an inflammatory phenotype. It precedes pathophysiological milestone events like the cytokine storm, progressive endothelial injury triggering microangiopathy, and further complement activation, and causes an acute respiratory distress syndrome (ARDS). To date, the application of antiviral drugs and corticosteroids have shown efficacy in the early stages of SARS-CoV-2 infection, but failed to ameliorate disease severity in patients who progressed to severe COVID-19 pathology. This report demonstrates that lectin pathway (LP) recognition molecules of the complement system, such as MBL, FCN-2 and CL-11, bind to SARS-CoV-2 S- and N-proteins, with subsequent activation of LP-mediated C3b and C4b deposition. In addition, our results confirm and underline that the N-protein of SARS-CoV-2 binds directly to the LP- effector enzyme MASP-2 and activates complement. Inhibition of the LP using an inhibitory monoclonal antibody against MASP-2 effectively blocks LP-mediated complement activation. FACS analyses using transfected HEK-293 cells expressing SARS-CoV-2 S protein confirm a robust LP-dependent C3b deposition on the cell surface which is inhibited by the MASP-2 inhibitory antibody. In light of our present results, and the encouraging performance of our clinical candidate MASP-2 inhibitor Narsoplimab in recently published clinical trials, we suggest that the targeting of MASP-2 provides an unsurpassed window of therapeutic efficacy for the treatment of severe COVID-19.

Absence of the Lectin Activation Pathway of Complement Ameliorates Proteinuria-Induced Renal Injury.

Proteinuria is an adverse prognostic feature in renal diseases. In proteinuric nephropathies, filtered proteins exert an injurious effect on the renal tubulointerstitium, resulting in inflammation and fibrosis. In the present study, we assessed to what extent complement activation via the lectin pathway may contribute to renal injury in response to proteinuria-related stress in proximal tubular cells. We used the well-established mouse model of protein overload proteinuria (POP) to assess the effect of lectin pathway inhibition on renal injury and fibrotic changes characteristic of proteinuric nephropathy. To this end, we compared experimental outcomes in wild type mice with MASP-2-deficient mice or wild type mice treated with MASP-2 inhibitor to block lectin pathway functional activity. Multiple markers of renal injury were assessed including renal function, proteinuria, macrophage infiltration, and cytokine release profiles. Both MASP-2-deficient and MASP-2 inhibitor-treated wild type mice exhibited renoprotection from proteinuria with significantly less tubulointerstitial injury when compared to isotype control antibody treated mice. This indicates that therapeutic targeting of MASP-2 in proteinuric nephropathies may offer a useful strategy in the clinical management of proteinuria associated pathologies in a variety of different underlying renal diseases.

Cardioprotection by an anti-MASP-2 antibody in a murine model of myocardial infarction.

Background: Myocardial ischaemia-reperfusion injury is a major cause of mortality and morbidity in the developed world. Many approaches have been investigated to counteract the pathological consequences associated with acute myocardial infarction (AMI) and cardiac remodelling. It is accepted that inflammation, and therefore activation of the complement pathway, is a crucial step in the pathogenesis of this injury, and many attempts have been made to ameliorate the infarction and consequent dysfunction using anticomplement therapy, with mixed success. Recently, the lectin complement activation pathway involving the mannose-binding lectin-associated serine protease 2 (MASP-2) has been shown to be an important mediator of the inflammatory response in ischaemia/reperfusion injury in the heart. In this study, therefore, we aimed to investigate the feasibility of using monoclonal antibodies raised against MASP-2 in a murine model of AMI. Methods: Mice were injected with anti-MASP-2 antibody or control 18 hours prior to experimental infarction by ligation of the left anterior descending coronary artery for 30 min followed by 120 min reperfusion. The developed infarct was measured, and blood was collected for analysis of lectin pathway functional activity. Results and conclusions: We found that mice treated with anti-MASP-2 antibody had smaller infarcts than those treated with control antibody. We believe this may represent a valuable step forward in the protection of the myocardium against ischaemia-reperfusion injury.

Lectin pathway effector enzyme mannan-binding lectin-associated serine protease-2 can activate native complement C3 in absence of C4 and/or C2.

All 3 activation pathways of complement-the classic pathway (CP), the alternative pathway, and the lectin pathway (LP)- converge into a common central event: the cleavage and activation of the abundant third complement component, C3, via formation of C3-activating enzymes (C3 convertases). The fourth complement component, C4, and the second component, C2, are indispensable constituents of the C3 convertase complex, C4bC2a, which is formed by both the CP and the LP. Whereas in the absence of C4, CP can no longer activate C3, LP retains a residual but physiologically critical capacity to convert native C3 into its activation fragments, C3a and C3b. This residual C4 and/or C2 bypass route is dependent on LP-specific mannan-binding lectin-associated serine protease-2. By using various serum sources with defined complement deficiencies, we demonstrate that, under physiologic conditions LP-specific C4 and/or C2 bypass activation of C3 is mediated by direct cleavage of native C3 by mannan-binding lectin-associated serine protease-2 bound to LP-activation complexes captured on ligand-coated surfaces.-Yaseen, S., Demopulos, G., Dudler, T., Yabuki, M., Wood, C. L., Cummings, W. J., Tjoelker, L. W., Fujita, T., Sacks, S., Garred, P., Andrew, P., Sim, R. B., Lachmann, P. J., Wallis, R., Lynch, N., Schwaeble, W. J. Lectin pathway effector enzyme mannan-binding lectin-associated serine protease-2 can activate native complement C3 in absence of C4 and/or C2.

Mannan binding lectin-associated serine protease-2 (MASP-2) critically contributes to post-ischemic brain injury independent of MASP-1.

BACKGROUND: Complement activation via the lectin activation pathway (LP) has been identified as the key mechanism behind post-ischemic tissue inflammation causing ischemia-reperfusion injury (IRI) which can significantly impact the clinical outcome of ischemic disease. This work defines the contributions of each of the three LP-associated enzymes-mannan-binding lectin-associated serine protease (MASP)-1, MASP-2, and MASP-3-to ischemic brain injury in experimental mouse models of stroke. METHODS: Focal cerebral ischemia was induced in wild-type (WT) mice or mice deficient for defined complement components by transient middle cerebral artery occlusion (tMCAO) or three-vessel occlusion (3VO). The inhibitory MASP-2 antibody was administered systemically 7 and 3.5 days before and at reperfusion in WT mice in order to assure an effective MASP-2 inhibition throughout the study. Forty-eight hours after ischemia, neurological deficits and infarct volumes were assessed. C3 deposition and microglia/macrophage morphology were detected by immunohistochemical, immunofluorescence, and confocal analyses. RESULTS: MASP-2-deficient mice (MASP-2(-/-)) and WT mice treated with an antibody that blocks MASP-2 activity had significantly reduced neurological deficits and histopathological damage after transient ischemia and reperfusion compared to WT or control-treated mice. Surprisingly, MASP-1/3(-/-) mice were not protected, while mice deficient in factor B (fB(-/-)) showed reduced neurological deficits compared to WT mice. Consistent with behavioral and histological data, MASP-2(-/-) had attenuated C3 deposition and presented with a significantly higher proportion of ramified, surveying microglia in contrast to the hypertrophic pro-inflammatory microglia/macrophage phenotype seen in the ischemic brain tissue of WT mice. CONCLUSIONS: This work demonstrates the essential role of the low-abundant MASP-2 in the mediation of cerebral ischemia-reperfusion injury and demonstrates that targeting MASP-2 by an inhibitory therapeutic antibody markedly improved the neurological and histopathological outcome after focal cerebral ischemia. These results contribute to identifying the key lectin pathway component driving brain tissue injury following cerebral ischemia and call for a revision of the presently widely accepted view that MASP-1 is an essential activator of the lectin pathway effector component MASP-2.

The lectin pathway of complement activation is a critical component of the innate immune response to pneumococcal infection.

The complement system plays a key role in host defense against pneumococcal infection. Three different pathways, the classical, alternative and lectin pathways, mediate complement activation. While there is limited information available on the roles of the classical and the alternative activation pathways of complement in fighting streptococcal infection, little is known about the role of the lectin pathway, mainly due to the lack of appropriate experimental models of lectin pathway deficiency. We have recently established a mouse strain deficient of the lectin pathway effector enzyme mannan-binding lectin associated serine protease-2 (MASP-2) and shown that this mouse strain is unable to form the lectin pathway specific C3 and C5 convertases. Here we report that MASP-2 deficient mice (which can still activate complement via the classical pathway and the alternative pathway) are highly susceptible to pneumococcal infection and fail to opsonize Streptococcus pneumoniae in the none-immune host. This defect in complement opsonisation severely compromises pathogen clearance in the lectin pathway deficient host. Using sera from mice and humans with defined complement deficiencies, we demonstrate that mouse ficolin A, human L-ficolin, and collectin 11 in both species, but not mannan-binding lectin (MBL), are the pattern recognition molecules that drive lectin pathway activation on the surface of S. pneumoniae. We further show that pneumococcal opsonisation via the lectin pathway can proceed in the absence of C4. This study corroborates the essential function of MASP-2 in the lectin pathway and highlights the importance of MBL-independent lectin pathway activation in the host defense against pneumococci.

Targeting of mannan-binding lectin-associated serine protease-2 confers protection from myocardial and gastrointestinal ischemia/reperfusion injury.

Complement research experienced a renaissance with the discovery of a third activation route, the lectin pathway. We developed a unique model of total lectin pathway deficiency, a mouse strain lacking mannan-binding lectin-associated serine protease-2 (MASP-2), and analyzed the role of MASP-2 in two models of postischemic reperfusion injury (IRI). In a model of transient myocardial IRI, MASP-2-deficient mice had significantly smaller infarct volumes than their wild-type littermates. Mice deficient in the downstream complement component C4 were not protected, suggesting the existence of a previously undescribed lectin pathway-dependent C4-bypass. Lectin pathway-mediated activation of C3 in the absence of C4 was demonstrated in vitro and shown to require MASP-2, C2, and MASP-1/3. MASP-2 deficiency also protects mice from gastrointestinal IRI, as do mAb-based inhibitors of MASP-2. The therapeutic effects of MASP-2 inhibition in this experimental model suggest the utility of anti-MASP-2 antibody therapy in reperfusion injury and other lectin pathway-mediated disorders.