key: cord-0954276-cjvg852h
authors: Rambaldi, Alessandro; Gritti, Giuseppe; Micò, Maria Caterina; Frigeni, Marco; Borleri, Gianmaria; Salvi, Anna; Landi, Francesco; Pavoni, Chiara; Sonzogni, Aurelio; Gianatti, Andrea; Binda, Francesca; Fagiuoli, Stefano; Di Marco, Fabiano; Lorini, Luca; Remuzzi, Giuseppe; Whitaker, Steve; Demopulos, Gregory
title: Endothelial Injury and Thrombotic Microangiopathy in COVID-19: Treatment with the Lectin-Pathway Inhibitor Narsoplimab
date: 2020-08-09
journal: Immunobiology
DOI: 10.1016/j.imbio.2020.152001
sha: 384cf0c563bd9f1e6f85b9f92f2e481b6a5b82fc
doc_id: 954276
cord_uid: cjvg852h

In COVID-19, acute respiratory distress syndrome (ARDS) and thrombotic events are frequent, life-threatening complications. Autopsies commonly show arterial thrombosis and severe endothelial damage. Endothelial damage, which can play an early and central pathogenic role in ARDS and thrombosis, activates the lectin pathway of complement. Mannan-binding lectin-associated serine protease-2 (MASP-2), the lectin pathway’s effector enzyme, binds the nucleocapsid protein of severe acute respiratory syndrome-associated coronavirus-2 (SARS-CoV-2), resulting in complement activation and lung injury. Narsoplimab, a fully human immunoglobulin gamma 4 (IgG4) monoclonal antibody against MASP-2, inhibits lectin pathway activation and has anticoagulant effects. In this study, the first time a lectin-pathway inhibitor was used to treat COVID-19, six COVID-19 patients with ARDS requiring continuous positive airway pressure (CPAP) or intubation received narsoplimab under compassionate use. At baseline and during treatment, circulating endothelial cell (CEC) counts and serum levels of interleukin-6 (IL-6), interleukin-8 (IL-8), C-reactive protein (CRP) and lactate dehydrogenase (LDH) were assessed. Narsoplimab treatment was associated with rapid and sustained reduction of CEC and concurrent reduction of serum IL-6, IL-8, CRP and LDH. Narsoplimab was well tolerated; no adverse drug reactions were reported. Two control groups were used for retrospective comparison, both showing significantly higher mortality than the narsoplimab-treated group. All narsoplimab-treated patients recovered and survived. Narsoplimab may be an effective treatment for COVID-19 by reducing COVID-19-related endothelial cell damage and the resultant inflammation and thrombotic risk.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, causing COVID-19) was identified as a clinical syndrome in Hubei province China in December 2019 and spread rapidly . By late February 2020, a fast-growing number of COVID-19 cases were diagnosed in the northern Italian region of Lombardy (Remuzzi and Remuzzi 2020) . A primary cause of death in COVID-19 is severe respiratory failure. Lung tissue in patients who died of COVID-19 shows high concentrations of SARS-CoV-2 RNA (Wichmann et al. 2020 ) and the same intense inflammatory changes seen in previously reported coronaviruses SARS-CoV (SARS) and MERS-CoV (MERS), and anti-inflammatory strategies are being evaluated for COVID-19 treatment Horby et al. 2020; Gritti et al. 2020) . Thrombosis has also been reported in SARS and COVID-19 infection (Magro et al. 2020; Ding et al. 2003; Wichmann et al. 2020 ). Similar to SARS and MERS, COVID-19 can cause life-threatening acute respiratory distress syndrome (ARDS) (Guan et al. 2020) . A central pathological component of and of the exudative phase of ARDS is endothelial injury and activation (Varga et al. 2020; Ackermann et al. 2020; Green 2020; Teuwen et al. 2020; Goshua et al. 2020; Thompson, Chambers, and Liu 2017) . Endothelial injury can also cause microvascular angiopathy and thrombosis. Endothelial activation further enhances the local inflammatory environment.

Importantly, as demonstrated in human in vitro and animal studies, endothelial injury specifically activates the lectin pathway of complement on the endothelial cell surface (Collard et al. 2000) .

The lectin pathway and its effector enzyme mannan-binding lectin-associated serine protease-2 (MASP-2) have been directly linked to the lung injury in coronavirus infection. Specifically, SARS-CoV-2 nucleocapsid protein, as well as those of SARS and MERS, have been shown to activate MASP-2, and MASP-2 deposits are seen in the vasculature of lung tissue of patients Magro et al. 2020) . Complement activation has been demonstrated to contribute to pulmonary injury in SARS and MERS infections in murine models, with increased complement factors seen in pulmonary tissue and complement blockade mitigating lung injury (Gralinski et al. 2018; Jiang et al. 2018 ). The rapid time-course to protection of respiratory function in these models suggests that the lectin pathway drives complement activation in both SARS and MERS (Gralinski et al. 2018; Jiang et al. 2018) . Additionally, lung tissue from deceased COVID-19 patients shows components of the lectin and terminal complement pathways, specifically MASP-2, complement factor 4d (C4d) and C5b-9 (i.e., the membrane attack complex) Magro et al. 2020) .

Narsoplimab (Omeros Corporation) is a high-affinity fully human immunoglobulin gamma 4 (IgG4) monoclonal antibody that binds MASP-2 and blocks lectin pathway activation ( Figure 1 ). Narsoplimab is the subject of a rolling Biologics License Application with the United States Food and Drug Administration (FDA) for the treatment of hematopoietic stem cell transplantassociated thrombotic microangiopathy (HSCT-TMA) and has been granted FDA's Breakthrough Therapy designation for this indication. As with COVID-19, endothelial injury is a central component of the pathophysiology of HSCT-TMA (Jodele et al. 2014 ) and activates the lectin pathway (Collard et al. 2000) . In its pivotal, single-arm clinical trial for HSCT-TMA, narsoplimab demonstrated marked clinical and laboratory improvement. In a high-risk population and using a rigorous response-based composite measure consisting of organ function, transfusion J o u r n a l P r e -p r o o f 5 burden, and laboratory values (i.e., platelet and lactate dehydrogenase (LDH)), 54% of all narsoplimab-treated patients and 65% of those receiving at least 4 weeks of protocol-specified treatment achieved a complete response compared to the FDA-agreed efficacy threshold of 15% . Narsoplimab also is in Phase 3 clinical trials for immunoglobulin A (IgA) nephropathy and atypical hemolytic uremic syndrome for which the drug has received FDA's Breakthrough Therapy and Fast Track designations, respectively. The complement activation pathways and narsoplimab mechanism of action. Narsoplimab, by blocking MASP-2, inhibits activation of the lectin pathway (LP). Part of the innate immune system, the LP is activated by microorganisms or injured cells. Microorganisms display carbohydrate-based pathogenassociated molecular patterns (PAMPs) and injured host cells display damage-associated molecular patterns (DAMPs) on their surfaces. DAMPs are not displayed on healthy cells but become exposed with cell injury. Lectins, carrying MASP-2, bind to the PAMPs or DAMPs, localizing lectin pathway activation to the vicinity of the cell surface. Activated MASP-2 cleaves complement factor 2 (C2) and C4, initiating a series of enzymatic steps that result in the production of the anaphylatoxins C3a and C5a and formation J o u r n a l P r e -p r o o f of C5b-9 (the membrane attack complex), and can also directly cleave C3 through the C4-bypass mechanism. The alternative pathway acts as an amplification loop, further enhancing lectin pathwaymediated complement activation. Unlike C3 or C5 inhibition, MASP-2 inhibition does not interfere with the classical pathway, preserving the adaptive immune response and the antigen-antibody complexmediated lytic response needed to fight infection.

MASP-2 also acts directly on the coagulation cascade and the contact system, cleaving prothrombin to thrombin and forming fibrin clots. Narsoplimab not only inhibits lectin pathway activation but also blocks microvascular injury-associated thrombus formation as well as MASP-2-mediated activation of kallikrein and factor XII.

Complement inhibition has been proposed as a treatment for severe COVID-19, but clinical data supporting this therapeutic approach are scant (Campbell and Kahwash 2020; Mastaglio et al. 2020; Diurno et al. 2020) . Given the heavy disease burden in Italy, evidence linking lectin pathway activation to coronavirus-related pathophysiology, and the efficacy and safety of narsoplimab in the endothelial-injury syndrome HSCT-TMA, we treated patients with severe

Giovanni XXIII Hospital in Bergamo. This represents the first time that a lectin pathway inhibitor has been used to treat patients with COVID-19, and here we report our initial clinical experience.

This investigation and all assessments in all patients, including those in the control groups, were conducted at Azienda Socio-Sanitaria Territoriale Papa Giovanni XXIII in Bergamo, Italy and approved by the Institutional Ethics Committee and the Agenzia Italiana del Farmaco. All study patients provided informed consent. All data were collected and analyzed by the authors.

Standard hematoxylin and eosin staining (H&E) and immunohistochemistry were performed on formalin-fixed, paraffin-embedded samples obtained from pathological autopsies of COVID-19 J o u r n a l P r e -p r o o f patients. H&E-stained sections were reviewed by two pathologists (A.G. and A.S.).

Bond Ready-to-Use Antibody CD34 (Clone QBEnd/10, Leica Biosystems, Germany), an antibody optimized for use with Bond Polymer Refine Detection. The assay was performed on an automated stainer platform (Leica Bond-3, Leica Biosystems, Germany) using a heat-based antigen retrieval technique (Bond Epitope Retrieval) as recommended by the manufacturer.

Cytoplasmatic staining of endothelium in the capillaries of pulmonary alveoli indicated positive results.

Circulating Endothelial Cells (CEC) identification and count CEC were measured by flow cytometry using peripheral blood collected with EDTA. After an erythrocyte bulky-lysis step, samples were labeled for 20 minutes at room temperature with the following: anti-CD45 V500-C (clone 2D1), anti-CD34 PerCP-CY5.5 (clone 8G12) (BD Biosciences, USA), and anti-CD146 PE (clone P1H12) (BD Biosciences-Pharmingen, USA). At least 1×10 6 events/sample with total leukocyte morphology were acquired by flow cytometry (FACSLyric, BD Biosciences, USA). To reduce operator-induced variability, all samples were analyzed by the same laboratory technician. CEC values were calculated by a dual-platform counting method using the lymphocyte subset as reference population as previously reported (Almici et al. 2017) .

Levels of interleukin-1β, interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10, tumor necrosis factor, and interleukin-12p70 were analyzed in a single serum sample by flow cytometry (BD CBA Human Inflammatory Cytokines Kit, BD Biosciences, USA). Narsoplimab treatment, supportive therapy and outcome assessment Narsoplimab 4 mg/kg was administered intravenously twice weekly for 2 to 4 weeks. At study initiation, dosing duration was set at 2 weeks but was increased empirically when the first patient J o u r n a l P r e -p r o o f treated with narsoplimab experienced a clinical and laboratory-marker recurrence after cessation of treatment at 2 weeks, subsequently resolving with an additional week of dosing. All patients received routine supportive care per our hospital's guidelines at the time of the study, including prophylactic enoxaparin (Clexane, Sanofi Aventis) 4,000 IU/0.4 mL, azithromycin (Zitromax,Pfizer SpA, Italy) 500 mg once daily, hydroxychloroquine (Plaquenil, Sanofi Aventis) 200 mg twice daily, darunavir and cobicistat (Rezolsta, Janssen-Cilag S.p.A., Italy) 800/150 mg once daily. Beginning March 27, 2020 per updated institutional guidelines, all COVID-19 patients in our hospital received methylprednisolone 1 mg/kg, which was administered to five of the six narsoplimab-treated patients. All respiratory support was provided according to institutional treatment algorithms.

In addition to CEC counts and cytokine levels, clinical and laboratory measures, including blood counts, LDH and C-reactive protein (CRP) levels, were collected on all narsoplimab-treated patients per standard clinical practice. Routine blood examinations were collected prior to each narsoplimab dose and then twice weekly. Respiratory function was evaluated daily. All patients received chest computed tomography (CT) at hospital admission to document interstitial pneumonia and, if clinically indicated, during hospitalization to document pulmonary embolism.

Demographic and clinical patient data are presented as frequency with percentage for categorical variables and median with range for continuous ones. Difference in CEC value between normal and COVID-19 patients was assessed with Mann-Whitney U-test. Repeated measures analysis was performed to test differences in CEC and cytokine levels during narsoplimab treatment at appropriate timepoints; non-parametric Friedman test was used, and pairwise-comparisons were performed using paired Wilcoxon signed-rank test. Decreasing trend of LDH and CRP levels J o u r n a l P r e -p r o o f during treatment were evaluated with non-parametric Spearman test between the observations and time. Significance at 5% was fixed. Analysis was performed using R software (version 3.6.2). Table 1 summarizes the clinical characteristics of the six narsoplimab-treated patients. Median age was 56.5 years, and 83% were male. All patients were overweight or obese based on a body mass index (BMI) ≥ 25 and ≥ 30, respectively. At enrollment, all had pneumonia/ARDS requiring CPAP with two patients rapidly deteriorating and requiring intubation soon after enrollment.

Narsoplimab treatment was started within 48 hours of CPAP initiation.

J o u r n a l P r e -p r o o f Figure 2 summarizes the clinical outcomes observed in these narsoplimab-treated patients. In four patients, enoxaparin was given at therapeutic doses (100 IU/kg twice daily) due to CT scan-documented pulmonary thromboses (patients #4 and #6), medical decision (patient #3) or rapidly deteriorating respiratory function requiring intubation (patient #5). Median follow-up was 27 days (range 16-90), and patients were administered narsoplimab twice weekly with a median of 8 total narsoplimab doses (range 5-8). Following treatment, all patients improved clinically.

Four patients (67%) reduced ventilatory support from CPAP to non-rebreather or Venturi oxygen mask after a median of 3 narsoplimab doses (range 2-3). In three of these patients, oxygen support was weaned and then discontinued, and discharge followed a median of 6 (range 5-8) total narsoplimab doses. In patient #4, a contrast-enhanced CT scan documented massive bilateral pulmonary thromboses 4 days following enrollment. Enoxaparin was added to ongoing narsoplimab dosing, and rapid clinical and radiographic (repeat CT scan) improvement was documented 11 days later (Figure 3 ), subsequently allowing discharge from the hospital. In the two remaining patients (#5 and #6), rapidly worsening severe ARDS was documented soon after enrollment. In patient #5, severe ARDS (PaO2/FiO2 of 55) required intubation at day 4.

Nonetheless, subsequent clinical outcome was rapidly favorable, and the patient was discharged from the intensive care unit after 3 days. Based on these initial observations and published findings in acute graft-versus-host disease (GVHD) in which immune-mediated attack of vascular endothelial cells leads to their detachment from the vessel wall and release into circulation (Almici et al. 2017) , prior to initiation of the study with narsoplimab we began measuring CEC counts in a non-study cohort of molecularly confirmed COVID-19 patients randomly selected in our hospital. In this non-study cohort of 33 COVID-19 patients, we found that CEC/mL of peripheral blood (median 110, range 38-877) were significantly increased compared to healthy controls (median 7, range 0-37) (P = 0·0004), ( Figure   5 ). 

The findings in this study further indicate that endothelial injury is central to the pathophysiology of COVID-19-related lung injury (Varga et al. 2020; Green 2020; Ackermann et al. 2020; Teuwen et al. 2020; Goshua et al. 2020) GVHD and infections (Ackermann et al. 2020; Varga et al. 2020) . In COVID-19, endothelial injury appears to be caused by direct viral infection. Given the known relationship between endothelial injury and multi-organ thrombotic microangiopathy in HSCT-TMA and the mounting evidence for a similar relationship in COVID-19, it is reasonable that the pathophysiologic events that follow endothelial injuryand lead to diffuse TMAare also similar. Endothelial injury, regardless of cause, activates the lectin pathway of complement on the endothelial cell surface (Collard et al. 2000) . In its pivotal HSCT-TMA trial, the MASP-2 inhibitor narsoplimab demonstrated marked improvement in laboratory and clinical endpoints, including survival ). In the current study, inhibition of MASP-2 and the lectin pathway by narsoplimab was associated with clinical improvement and survival in all COVID-19 patients treated with the drug.

The lectin pathway of complement is part of the innate immune response. Activation of the lectin pathway is initiated by members of the MASP enzyme family (MASP-1, MASP-2 and MASP-3) (Schwaeble et al. 2002) , which complex in blood with lectins, specifically mannan-binding lectin, the ficolins and collectins. These lectins recognize and bind to carbohydrate patterns found on surfaces of pathogenic microorganisms or injured host cells, including damaged endothelial cells (Collard et al. 2000) , targeting MASPs to their site(s) of action and leading to their activation.

MASP-2, the key enzyme responsible for lectin pathway activation, binds and undergoes activation by the COVID-19 N protein (Gao T 2020) and has been found in the microvasculature of lung tissue in patients with severe COVID-19 (Magro et al. 2020) . Activated MASP-2 initiates a series of enzymatic steps that results in production of the anaphylatoxins C3a and C5a and in formation of the membrane attack complex C5b-9 (Dobo, Kocsis, and Gal 2018) , which can induce proinflammatory responses and cause cell lysis and death. MASP-2 can also cleave C3 directly through the C4 bypass (Yaseen et al. 2017) . Importantly, MASP-2 is located upstream in the lectin pathway, so inhibition of MASP-2 does not interfere with the lytic arm of the classical pathway (i.e., C1r/C1s-driven formation of the C3 and C5 convertases), preserving the adaptive immune response needed to fight infection (Schwaeble et al. 2011) (Figure 1 ).

In addition to its role in complement, MASP-2 acts directly on the coagulation cascade and the contact system, cleaving prothrombin to thrombin and forming fibrin clots (Gulla et al. 2010; Krarup et al. 2007) . Narsoplimab not only inhibits lectin pathway activation but also blocks microvascular injury-associated thrombus formation as well as MASP-2-mediated activation of kallikrein and factor XII (Demopulos, Dudler, and Nilsson 2020; Omeros Corporation 2013 . License Application to the U.S. Food and Drug Administration), and no bleeding was observed in the patients we treated. It appears that narsoplimab may block coagulation resulting from endothelial damage (associated with factor XII activation) but not extracellular matrix-related (factor VII-driven) coagulation. Additional studies are underway to determine in more detail the mechanism(s) by which narsoplimab affects coagulation.

Lectin pathway inhibition has not previously been investigated as a treatment for COVID-19. All patients in this study had COVID-19-related respiratory failure. Following treatment with the MASP-2 inhibitor narsoplimab, all patients recovered and were able to be discharged from the hospital, further supporting the importance of the lectin pathway in COVID-19 pathophysiology.

In each case, COVID-19 lung injury had progressed to ARDS requiring CPAP prior to narsoplimab treatment. Two patients continued to deteriorate following the first dose and required invasive mechanical ventilation. Both patients were subsequently able to discontinue invasive mechanical ventilation with continued narsoplimab treatment. Two patients (one intubated and the other on CPAP) experienced massive bilateral pulmonary thromboses, and both patients completely recovered with narsoplimab, possibly benefitting from the drug's anticoagulant effects. The temporal patterns of laboratory markers (CEC, IL-6, IL-8, CRP and LDH) were consistent with the observed clinical improvement and with the hypothesized mechanism of action. In particular, CEC counts appear to be a reliable tool to evaluate endothelial damage and J o u r n a l P r e -p r o o f treatment response in this disease. The temporal improvement of IL-6 and IL-8 with narsoplimab treatment suggests that lectin pathway activation may precede cytokine elevation in and that lectin pathway inhibition has a beneficial effect on the cytokine storm described in patients with COVID-19 infection (Xiong et al. 2020) . Two weeks of narsoplimab dosing was planned initially but was increased to 3 to 4 weeks following the rise in CEC in patient #1 when dosing was first discontinued. With the third week of dosing, the patient's CEC counts again improved. Rebound pulmonary signs and symptoms have not been observed following 4 weeks of narsoplimab treatment. No narsoplimab-related adverse events were observed.

Use of other complement inhibitors in COVID-19 have been reported. AMY-101, a compstatinbased C3 inhibitor (Mastaglio et al. 2020) , was used in one patient and eculizumab was administered together with antiviral and anticoagulant therapy to four patients (Diurno et al. 2020 ). These five patients were on CPAP and survived. Two COVID-19 patients on high-flow nasal oxygen received a C5a antibody in conjunction with supportive therapy, including antiviral therapy, following steroid treatment. These two patients also survived .

Collectively, these reports support our findings with narsoplimab. Notably, unlike C3 and C5

inhibitors, the MASP-2 antibody narsoplimab fully maintains classical complement pathway function and does not interfere with the adaptive immune response or the antigen-antibody complex-mediated lytic response (Schwaeble et al. 2011) . No evidence of narsoplimab-related infection risk has been observed in narsoplimab clinical trials.

While this was a compassionate use, single-arm study, two different control groups provide a retrospective comparison. The first was described in a recently published article by Gritti et al. (Gritti et al. 2020 ) evaluating the use of siltuximab, an IL-6 inhibitor, in COVID-19 patients. The siltuximab study and our narsoplimab study share the same lead investigators (G.G. and A.R.), J o u r n a l P r e -p r o o f entry criteria and patient characteristics (i.e., demographics, symptoms, comorbidities, ARDS severity, laboratory values and respiratory support at enrollment). In that study, mortality rates in the siltuximab-treated and the control groups were 33% and 53%, respectively. The second retrospective comparator is represented by the 33 patients who were randomly selected within our hospital to assess the viability of CEC measurements in COVID-19 patients. Of these 33 patients, 22 met the same entry criteria and had similar baseline characteristics as the narsoplimab-treated patients. Median baseline CEC count, however, in the control group compared to that in the narsoplimab-treated group was 101/mL versus 334/mL, respectively. Interestingly, 20 of these 22 patients (91%) were treated with IL-6 inhibitors (tocilizumab or siltuximab) and/or steroids, and the group had an overall 30-day mortality of 32%. The mortality rate was still 31% when the outcome analysis was restricted to 16 patients matched for age to narsoplimab-treated patients (median 58 years, range 51-65 years). In this latter group, 94% received IL-6 and/or steroid therapy and the median baseline CEC count at 55/mL was six-fold lower than in the narsoplimabtreated patients.

The use of steroids in COVID-19 has resulted in reports of mixed outcomes (Veronese et al. 2020 ). Most recently, the Randomised Evaluation of COVID-19 therapy (RECOVERY) trial, demonstrated that dexamethasone reduced 28-day mortality in patients on invasive mechanical ventilation by 28.7% (29.0% versus 40.7% with usual care), by 14% (21.5% versus 25.0% with usual care) in those receiving oxygen support without invasive mechanical ventilation and had no effect on mortality in patients not receiving respiratory support at randomization (17.0% versus 13.2% with usual care) (Horby et al. 2020) . Based on these data and the experience at our hospital, we believe that steroids have a role to play in treating COVID-19 patients with respiratory dysfunction, acting to tamp down the inflammatory response. In the narsoplimab-J o u r n a l P r e -p r o o f treated group, one (patient #1) of the six patients did not receive steroids. Subsequently, in late

March 2020, institutional guidelines were updated, requiring that all patients in our hospital receive steroids. Of the five narsoplimab-treated patients who received steroids, two (patients #2 and #3) initiated them after already improving such that CPAP was no longer required or was discontinued the following day ( Figure 2) . As described previously, we evaluated CEC counts in a separate group of four patients receiving only steroids for a short duration, and the counts were found to be unaffected by steroid administration. This suggests that any beneficial effect of steroids on COVID-19-associated endothelial damage may be delayed and had little effect on the recovery course of patients #2 and #3.

Our findings have several limitations. First, this is a small, uncontrolled case series and patients were heterogeneous in clinical presentation. Second, although COVID-19 treatment was standardized at our institution, data collection in this compassionate-use program was not prospectively defined. Third, the treatment regimen was empirical, and it is not known if a longer or more frequent treatment regimen would affect our findings. Finally, the narsoplimab-treated patients received other therapies as part of supportive care.

Despite these limitations, our findings strongly suggest that endothelial injury-induced activation of MASP-2 and the lectin pathway play a central role in the pathophysiology of COVID-19related lung injury. The improvements in clinical status and laboratory findings following narsoplimab treatment are notable. While not definitive, these findings strongly suggest meaningful clinical efficacy and provide supportive evidence related to the drug's mechanism of action and the pathophysiology of the disease. Lectin pathway inhibition by narsoplimab appears to be a promising treatment of COVID-19-related lung injury and endothelial damage-associated thromboses, and further investigation is warranted.

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