key: cord-0930119-7lfeg4kk
authors: Mastellos, Dimitrios C.; Pires da Silva, Bruno G.P.; Fonseca, Benedito A.L.; Fonseca, Natasha P.; Martins, Maria A.; Mastaglio, Sara; Ruggeri, Annalisa; Sironi, Marina; Radermacher, Peter; Chrysanthopoulou, Akrivi; Skendros, Panagiotis; Ritis, Konstantinos; Manfra, Ilenia; Iacobelli, Simona; Huber-Lang, Markus; Nilsson, Bo; Yancopoulou, Despina; Connolly, E. Sander; Garlanda, Cecilia; Ciceri, Fabio; Risitano, Antonio M.; Calado, Rodrigo T.; Lambris, John D.
title: Complement C3 vs C5 inhibition in severe COVID-19: Early clinical findings reveal differential biological efficacy
date: 2020-09-19
journal: Clin Immunol
DOI: 10.1016/j.clim.2020.108598
sha: f00a4d9c8a359ffc8de27f800abecffed1609629
doc_id: 930119
cord_uid: 7lfeg4kk

Growing clinical evidence has implicated complement as a pivotal driver of COVID-19 immunopathology. Deregulated complement activation may fuel cytokine-driven hyper-inflammation, thrombotic microangiopathy and NET-driven immunothrombosis, thereby leading to multi-organ failure. Complement therapeutics have gained traction as candidate drugs for countering the detrimental consequences of SARS-CoV-2 infection. Whether blockade of terminal complement effectors (C5, C5a, or C5aR1) may elicit similar outcomes to upstream intervention at the level of C3 remains debated. Here we compare the efficacy of the C5-targeting monoclonal antibody eculizumab with that of the compstatin-based C3-targeted drug candidate AMY-101 in small independent cohorts of severe COVID-19 patients. Our exploratory study indicates that therapeutic complement inhibition abrogates COVID-19 hyper-inflammation. Both C3 and C5 inhibitors elicit a robust anti-inflammatory response, reflected by a steep decline in C-reactive protein and IL-6 levels, marked lung function improvement, and resolution of SARS-CoV-2-associated acute respiratory distress syndrome (ARDS). C3 inhibition afforded broader therapeutic control in COVID-19 patients by attenuating both C3a and sC5b-9 generation and preventing FB consumption. This broader inhibitory profile was associated with a more robust decline of neutrophil counts, attenuated neutrophil extracellular trap (NET) release, faster serum LDH decline, and more prominent lymphocyte recovery. These early clinical results offer important insights into the differential mechanistic basis and underlying biology of C3 and C5 inhibition in COVID-19 and point to a broader pathogenic involvement of C3-mediated pathways in thromboinflammation. They also support the evaluation of these complement-targeting agents as COVID-19 therapeutics in large prospective trials.

As the COVID-19 pandemic sweeps through the globe with an increasing death toll, pressing questions about its intricate immunobiology arise, pointing to the urgent need for effective therapeutic measures against its systemic consequences 1, 2, 3 .

Initially perceived as exclusively targeting the respiratory system, COVID-19 has been revealed as a complex, multiorgan disorder with a plethora of thomboinflammatory manifestations in key vital organs, including the lungs, heart, liver, kidney, and brain 2 . The common denominator driving pathology in these organs appears to be an extensive and deregulated activation of innate immune pathways causing massive monocyte and neutrophil infiltration into infected tissues and a disseminated thromboinflammatory response of the microvascular endothelium (thrombotic microangiopathy) 4, 5, 6 . This derailed inflammatory response, marked by a systemic increase in proinflammatory cytokines (known as cytokine storm) reflects a maladaptive host immune response to SARS-CoV-2 that is instigated by pathogen recognition systems, such as the complement cascade, which become overwhelmingly active in their attempt to thwart the infectious agent 5, 7 . The lack of effective vaccines and the growing appreciation that a multitude of host immune factors contribute to infection risk, disease severity and therapeutic outcomes have galvanized efforts to develop tailored and stage-specific COVID-19 therapies exploiting several antiviral, anti-inflammatory and immune modulating treatments 8, 9, 10, 11 . neutrophil activation, and a NET-dependent, Tissue Factor (TF)-driven hypercoagulable phenotype that disseminates through the vascular bed of multiple organs 12 . Mounting clinical data have implicated deregulated complement and coagulation pathways as risk factors for adverse outcomes in COVID-19 13 .

Increased C5a and sC5b-9 levels and prominent activation of the C5a-C5aR1 axis have been reported both in the infected lungs and systemically 14, 15, 16 . While these studies have propelled the clinical evaluation of terminal pathway therapeutics (anti-C5, C5a/C5aR1 blockade), key C3-mediated processes that fuel monocyte/neutrophil-driven inflammatory damage, cytokine responses and TF-driven thrombosis in COVID-19 remain operative 12 . These include upstream C3 convertase activity, leading to C3b-opsonization and alternative pathway (AP) amplification via any of the triggering routes that SARS-CoV-2 infection engages.

Earlier studies in animal models of SARS-CoV infection underscored the pivotal role of C3 activation in the pathogenesis of SARS-CoV associated ARDS 17 . Given that C3 activation is the convergence point of all complement pathways, we hypothesized that C3 targeting may afford broader and more comprehensive therapeutic coverage in COVID-19-associated ARDS 5 .

Here we performed a comparative assessment of key clinical and biochemical correlates in two small COVID- 19 Table S1 ). The Ecu-cohort also received concomitant treatment with corticosteroids according to physician's assessment. All ecu-patients received penicillin for prophylaxis.

Complement activity assay (APH50): Complement hemolytic activity via the alternative pathway (APH50) was monitored in all patient samples as previously described 21 .

Complement protein levels and C3/C5 activation fragments: C3, C4, and FB levels were determined by nephelometry in plasma using an IMMAGE 800 protein chemistry analyzer (Beckman Coulter). C3dg levels were measured by nephelometry following PEG precipitation of plasma (11% w/v) and subsequent incubation with an anti-C3d antibody (Dako). C3a and sC5b-9 levels were measured in EDTA-plasma collected from the patients at predetermined time points by human C3a-and C5b-9 -specific ELISAs according to the manufacturer's instructions (Quidel). sC5b-9 levels were also determined using a modified ELISA method described in 22 . To enable comparison of C5b-9 measurements derived from different laboratories and to normalize values generated from C5b-9 ELISA-based assays with different dynamic ranges of detection, we plotted the fold change of C5b-9 levels over baseline values for each patient/cohort.

Assay for IL-6: IL-6 levels were quantified in patient EDTA-plasma using an ELISA automated immunoassay (R&D Systems) following manufacturer's instructions. Marburg, Germany) as previously described 22 . Neutrophil extracellular traps (NETs)

were quantified in patient EDTA-plasma by an MPO/DNA complex ELISA, as previously described 24 .

Statistical analysis: In view of the small sample size no formal comparisons for statistical significance were performed between the two patient cohorts. However, statistical analysis between two data sets (i.e., treatments, days) within the same group was performed using the unpaired (two-tailed) student's t-test (Prism,

GraphPad v 8.0). The level of statistical significance was set to 0.05. Data are presented as mean values +/-standard deviation (SD).

In the AMY-101 CUP, three patients were recruited from April 10 th to May 21 st , 2020 after providing written informed consent. Treatment was initiated in patients fulfilling the following criteria: COVID-19, diagnosed with qRT-PCR and chest X-ray and/or CT scan; ARDS, defined as acute-onset respiratory failure with bilateral infiltrates on chest radiograph or CT scan, hypoxemia as defined by a A total of 12 patients met criteria, but two refused to participate. A total of 10 patients gave written informed consent and were enrolled during this period of time. Two patients who were on mechanical ventilation before enrollment died of mechanical ventilation-associated pneumonia on days +19 (Ecu-1) and +18 (Ecu-9). The other eight patients showed clinical improvement, were discharged, and are alive until last follow-up on August 12 th , 2020. One patient (Ecu-5) was intubated within 12 hours after the initial dose, required mechanical ventilation until day +9, and was discharged on day +17. All other patients did not require mechanical ventilation, but oxygen supplementation (nasal catheter, high flow oxygen mask), and were discharged on days +2 to +27 (median, 10 days). Three patients received one dose, three patients received two doses, and four patients received three doses of eculizumab. Eculizumab administration was halted when patients showed significant clinical improvement not requiring oxygen supplementation. Eight out of ten patients were alive and discharged at day +28. No severe adverse event (CTCAE grade III or IV) attributable to the drug was observed. Other severe adverse events are described in Table S2 (supplemental data).

Both C3 and C5 inhibition elicited a robust anti-inflammatory response in COVID-19

patients marked by a rapid decline of C-reactive protein (CRP) levels that led to normalization with 6-8 days after treatment initiation. (Fig 1, panel A) . This rapid antiinflammatory response also was reflected in a reciprocal decrease of IL-6 levels in AMY-101-and Ecu-treated patients that led to an almost 50% reduction of baseline values within 48 h from the start of treatment ( ranging between 9-18 days) (Fig. 4) .

We 29 . The reasons why sC5b-9 increases during eculizumab treatment, apparently diverging from clinical course, remain to be fully elucidated, but argue against using sC5b-9 as a reliable biomarker of ongoing disease activity in COVID-19.

Both inhibitors led to sustained inhibition of ex vivo AP-mediated complement hemolytic activity (AP50) in COVID-19 patient sera (Fig. 5, C) . While AMY-101 treatment resulted in complete abrogation of AP activity throughout the treatment, a residual hemolytic activity (ranging between 7-11.5%) was detected in patient sera dosed with eculizumab on days 2 and 7 respectively (Fig 5, C) . This 'leakage' in activity likely correlates with the rebound of terminal pathway activation products (sC5b-9) on day 7.

Complement intervention has emerged as a promising strategy for ameliorating 

Alexion Pharmaceuticals, Novartis, Alnylam and Ra Pharma and lecture fees from

Pfizer and Apellis, and served as member of advisory-investigator boards for Alexion, Roche, Achillion, Novartis, Apellis and Samsung, and as a consultant for Amyndas

holds a patent on compositions of matter and methods for the diagnosis and treatment of sepsis by C5a inhibitory strategies licensed to InflaRx

acted as a speaker for Alexion Pharma Brazil. The other authors declare no References

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competing interests.

Supplementary material