Treatment of Paraquat-Induced Lung Injury With an Anti-C5a Antibody: Potential Clinical Application.

OBJECTIVES: Complement activation product C5a plays a critical role in systemic inflammatory response syndrome induced by viruses, bacteria, and toxic agents including paraquat poisoning. This study is to explore the efficiency of anti-C5a-based intervention on systemic inflammatory responses induced by paraquat poisoning. DESIGN: Study of cynomolgus macaque model and plasma from paraquat-poisoning patients. SETTING: Laboratory investigation. SUBJECTS: Cynomolgus macaque (n = 12) and samples of plasma from patients (n = 16). INTERVENTIONS: The neutralizing antihuman C5a antibody (IFX-1) was administered to investigate the new treatment strategy for paraquat-induced systemic inflammatory responses in cynomolgus macaque model. In addition, C5a activation in plasma of paraquat patients was blocked by IFX-1 to investigate the blockade role of anti-C5a antibody in activation of inflammatory cells. MEASUREMENTS AND MAIN RESULTS: Dysregulated complement activation and the subsequent cytokine storm were found in patients with acute lung injury and in a primate model of paraquat poisoning. Targeted inhibition of C5a by IFX-1 led to marked alleviation of systemic inflammatory responses and multiple organ damage in the primate model. In addition, blockade of C5a activity in plasma from patients completely inhibited activation of CD11b on blood granulocytes from normal donors, suggesting that IFX-1 may alleviate the excessive activation of inflammatory responses and have clinical utility for patients with acute lung injury. CONCLUSIONS: Anti-C5a antibodies such as IFX-1 may be used as effective therapeutics for treatment of those suffering from systemic inflammatory responses induced by chemical poisoning like paraquat.

Controlling the anaphylatoxin C5a in diseases requires a specifically targeted inhibition.

The terminal complement split product C5a has been described as an important mediator in inflammatory diseases. C5a is generated upon cleavage of C5 and earlier research suggests that, besides the known C5 convertases formed upon activation of the complement pathways, various enzymes could activate C5 directly. We demonstrate that eculizumab effectively blocks C5 activation when mediated by C5-convertase formation, but fails to block C5a generation resulting from direct enzymatic cleavage by trypsin and thrombin. C5a generated by these enzymes is shown to be fully biologically functional and can be blocked by IFX-1, a specific monoclonal anti-human C5a antibody. We further report clinical cases of atypical hemolytic uremic syndrome (aHUS) and C3 Glomerulonephritis (C3GN) patients under treatment with eculizumab presenting substantially elevated C5a levels. Thus, blocking the C5 convertase mediated activation of C5 may not be efficient to control C5a-mediated effects in human disease and that a targeted approach is warranted.

A second stimulus required for enhanced antifungal activity of human neutrophils in blood is provided by anaphylatoxin C5a.

Polymorphonuclear neutrophilic granulocytes (PMN) as cellular components of innate immunity play a crucial role in the defense against systemic Candida albicans infection. To analyze stimuli that are required for PMN activity during C. albicans infection in a situation similar to in vivo, we used a human whole-blood infection model. In this model, PMN activation 10 min after C. albicans infection was largely dependent on the anaphylatoxin C5a. Most importantly, C5a enabled blood PMN to overcome filament-restricted recognition of C. albicans and allowed efficient elimination of nonfilamentous C. albicans cph1Δ/efg1Δ from blood. Major PMN effector mechanisms, including oxidative burst, release of secondary granule contents and initial fungal phagocytosis could be prevented by blocking C5a receptor signaling. Identical effects were achieved using a humanized Ab specifically targeting human C5a. Phagocytosis of C. albicans 10 min postinfection was mediated by C5a-dependent enhancement of CD11b surface expression on PMN, thus establishing the C5a-C5aR-CD11b axis as a major modulator of early anti-Candida immune responses in human blood. In contrast, phagocytosis of C. albicans by PMN 60 min postinfection occurred almost independently of C5a and mainly contributed to activation of phagocytically active PMN at later time points. Our results show that C5a is a critical mediator in human blood during C. albicans infection.

Treatment with anti-C5a antibody improves the outcome of H7N9 virus infection in African green monkeys.

BACKGROUND: Patients infected with influenza A(H7N9) virus present with acute lung injury (ALI) that is due to severe pneumonia and systemic inflammation. It is often fatal because there are few effective treatment options. Complement activation has been implicated in the pathogenesis of virus-induced lung injury; therefore, we investigated the effect of targeted complement inhibition on ALI induced by H7N9 virus infection. METHODS: A novel neutralizing specific antihuman C5a antibody (IFX-1) was used. This antibody blocked the ability of C5a to induce granulocytes to express CD11b while not affecting the ability of C5b to form the membrane attack complex. African green monkeys were inoculated with H7N9 virus and treated intravenously with IFX-1. RESULTS: The virus infection led to intense ALI and systemic inflammatory response syndrome (SIRS) in association with excessive complement activation. Anti-C5a treatment in H7N9-infected monkeys substantially attenuated ALI: It markedly reduced the lung histopathological injury and decreased the lung infiltration of macrophages and neutrophils. Moreover, the treatment decreased the intensity of SIRS; the body temperature changes were minimal and the plasma levels of inflammatory mediators were markedly reduced. The treatments also significantly decreased the virus titers in the infected lungs. CONCLUSIONS: Antihuman C5a antibody treatment remarkably reduced the ALI and systemic inflammation induced by H7N9 virus infection. Complement inhibition may be a promising adjunctive therapy for severe viral pneumonia.

Inhibition of complement activation alleviates acute lung injury induced by highly pathogenic avian influenza H5N1 virus infection.

The acute lung injury (ALI) that occurs after the highly pathogenic avian influenza H5N1 virus infection is associated with an abnormal host innate immune response. Because the complement system plays a central role in innate immunity and because aberrant complement activation is associated with a variety of autoimmune and inflammatory diseases, we investigated the complement involvement in the pathogenesis of ALI induced by H5N1 virus infection. We showed that ALI in H5N1-infected mice was caused by excessive complement activation, as demonstrated by deposition of C3, C5b-9, and mannose-binding lectin (MBL)-C in lung tissue, and by up-regulation of MBL-associated serine protease-2 and the complement receptors C3aR and C5aR. Treatment of H5N1-infected mice with a C3aR antagonist led to significantly reduced inflammation in lungs, alleviating ALI. Furthermore, complement inhibition with an anti-C5a antibody or complement depletion with cobra venom factor after H5N1 challenge resulted in a similar level of protection to that seen in C3aR antagonist-treated mice. These results indicate that excessive complement activation plays an important role in mediating H5N1-induced ALI and that inhibition of complement may be an effective clinical intervention and adjunctive treatment for H5N1-induced ALI.

Protective role of tumor necrosis factor (TNF) receptors in chronic intestinal inflammation: TNFR1 ablation boosts systemic inflammatory response.

Tumor necrosis factor-α (TNF-α) acts as a key factor for the development of inflammatory bowel diseases (IBDs), whose function is known to be mediated by TNF receptor 1 (TNFR1) or TNFR2. However, the precise role of the two receptors in IBD remains poorly understood. Herein, chronic colitis was established by oral administration of dextran sulfate sodium (DSS) in TNFR1 or TNFR2-/- mice. Unexpectedly, TNFR1 or TNFR2 deficiency led to exacerbation of signs of colitis compared with wild-type (WT) counterparts. Of note, TNFR1 ablation rendered significantly increased mortality compared with TNFR2 and WT mice after DSS. Aggravated pathology of colitis in TNFR1-/- or TNFR2-/- mice correlated with elevated colonic expression of proinflammatory cytokines and chemokines. Importantly, ablation of TNFR1 or TNFR2 increased apoptosis of colonic epithelial cells, which might be due to the heightened ratio of Bax/Bcl-2 and increased expression of caspase-8. Intriguingly, despite comparable intensity of intestinal inflammation in TNFR-deficient mice after DSS, systemic inflammatory response (including splenomegaly and myeloid expansion) was augmented dramatically in TNFR1-/- mice, instead of TNFR2-/- mice. Granulocyte-macrophage colony-stimulating factor (GMCSF) was identified as a key mediator in this process, as neutralization of GMCSF dampened peripheral inflammatory reaction and reduced mortality in TNFR1-/- mice. These data suggest that signaling via TNFR1 or TNFR2 has a protective role in chronic intestinal inflammation, and that lacking TNFR1 augments systemic inflammatory response in GMCSF-dependent manner.

Highly accurate and large-scale collision cross sections prediction with graph neural networks.

The collision cross section (CCS) values derived from ion mobility spectrometry can be used to improve the accuracy of compound identification. Here, we have developed the Structure included graph merging with adduct method for CCS prediction (SigmaCCS) based on graph neural networks using 3D conformers as inputs. A model was trained, evaluated, and tested with >5,000 experimental CCS values. It achieved a coefficient of determination of 0.9945 and a median relative error of 1.1751% on the test set. The model-agnostic interpretation method and the visualization of the learned representations were used to investigate the chemical rationality of SigmaCCS. An in-silico database with 282 million CCS values was generated for three different adduct types of 94 million compounds. Its source code is publicly available at https://github.com/zmzhang/SigmaCCS . Altogether, SigmaCCS is an accurate, rational, and off-the-shelf method to directly predict CCS values from molecular structures.

Pharmacokinetic analysis of vilobelimab, anaphylatoxin C5a and antidrug antibodies in PANAMO: a phase 3 study in critically ill, invasively mechanically ventilated COVID-19 patients.

BACKGROUND: Vilobelimab, a complement 5a (C5a)-specific monoclonal antibody, reduced mortality in critically ill COVID-19 patients in a phase 3 multicentre, randomized, double-blind, placebo-controlled study. As part of the study, vilobelimab concentrations and C5a levels as well as antidrug antibodies (ADAs) to vilobelimab were analysed. RESULTS: From Oct 1, 2020 to Oct 4, 2021, 368 invasively mechanically ventilated COVID-19 patients were randomized: 177 patients were randomly assigned to receive vilobelimab while 191 patients received placebo. Pharmacokinetic sampling was only performed at sites in Western Europe. Blood samples for vilobelimab measurements were available for 93 of 177 (53%) patients in the vilobelimab group and 99 of 191 (52%) patients in the placebo group. On day 8, after three infusions, mean vilobelimab (trough) concentrations ranged from 21,799.3 to 302,972.1 ng/mL (geometric mean 137,881.3 ng/mL). Blood samples for C5a measurements were available for 94 of 177 (53%) patients in the vilobelimab group and 99 of 191 (52%) patients in the placebo group. At screening, C5a levels were highly elevated and comparable between groups. In the vilobelimab group, median C5a levels were 118.3 ng/mL [IQR 71.2-168.2 ng/mL] and in the placebo group, median C5a levels were 104.6 ng/mL [IQR 77.5-156.6 ng/mL]. By day 8, median C5a levels were reduced by 87% in the vilobelimab group (median 14.5 ng/mL [IQR 9.5-21.0 ng/mL], p < 0.001) versus an 11% increase in the placebo group (median 119.2 ng/mL [IQR 85.9-152.1 ng/mL]). Beyond day 8, though plasma sampling was sparse, C5a levels did not reach screening levels in the vilobelimab group while C5a levels remained elevated in the placebo group. Treatment-emergent ADAs were observed in one patient in the vilobelimab group at hospital discharge on day 40 and in one patient in the placebo group at hospital discharge on day 25. CONCLUSIONS: This analysis shows that vilobelimab efficiently inhibits C5a in critically ill COVID-19 patients. There was no evidence of immunogenicity associated with vilobelimab treatment. Trial registration ClinicalTrials.gov, NCT04333420. Registered 3 April 2020, https://clinicaltrials.gov/ct2/show/NCT04333420.

The anti-C5a antibody vilobelimab efficiently inhibits C5a in patients with severe COVID-19.

Recently, we reported the phase II portion of the adaptive phase II/III PANAMO trial exploring potential benefit and safety of selectively blocking C5a with the monoclonal antibody vilobelimab (IFX-1) in patients with severe coronavirus disease 2019 (COVID-19). The potent anaphylatoxin C5a attracts neutrophils and monocytes to the infection site, causes tissue damage by oxidative radical formation and enzyme releases, and leads to activation of the coagulation system. Results demonstrated that C5a inhibition with vilobelimab was safe and secondary outcomes appeared in favor of vilobelimab. We now report the pharmacokinetic/pharmacodynamic (PK/PD) analysis of the phase II study. Between March 31 and April 24, 2020, 30 patients with severe COVID-19 pneumonia confirmed by real-time polymerase chain reaction were randomly assigned 1:1 to receive vilobelimab plus best supportive care or best supportive care only. Samples for measurement of vilobelimab, C3a and C5a blood concentrations were taken. Vilobelimab predose (trough) drug concentrations in plasma ranged from 84,846 to 248,592 ng/ml (571 to 1674 nM) with a geometric mean of 151,702 ng/ml (1022 nM) on day 2 and from 80,060 to 200,746 ng/ml (539 to 1352 nM) with a geometric mean of 139,503 ng/ml (939 nM) on day 8. After the first vilobelimab infusion, C5a concentrations were suppressed in the vilobelimab group (median 39.70 ng/ml 4.8 nM, IQR 33.20-45.55) as compared to the control group (median 158.53 ng/ml 19.1 nM, IQR 60.03-200.89, p = 0.0006). The suppression was maintained on day 8 (p = 0.001). The current PK/PD analysis shows that vilobelimab efficiently inhibits C5a in patients with severe COVID-19.

Anti-C5a antibody (vilobelimab) therapy for critically ill, invasively mechanically ventilated patients with COVID-19 (PANAMO): a multicentre, double-blind, randomised, placebo-controlled, phase 3 trial.

BACKGROUND: Vilobelimab, an anti-C5a monoclonal antibody, was shown to be safe in a phase 2 trial of invasively mechanically ventilated patients with COVID-19. Here, we aimed to determine whether vilobelimab in addition to standard of care improves survival outcomes in this patient population. METHODS: This randomised, double-blind, placebo-controlled, multicentre phase 3 trial was performed at 46 hospitals in the Netherlands, Germany, France, Belgium, Russia, Brazil, Peru, Mexico, and South Africa. Participants aged 18 years or older who were receiving invasive mechanical ventilation, but not more than 48 h after intubation at time of first infusion, had a PaO2/FiO2 ratio of 60-200 mm Hg, and a confirmed SARS-CoV-2 infection with any variant in the past 14 days were eligible for this study. Eligible patients were randomly assigned (1:1) to receive standard of care and vilobelimab at a dose of 800 mg intravenously for a maximum of six doses (days 1, 2, 4, 8, 15, and 22) or standard of care and a matching placebo using permuted block randomisation. Treatment was not continued after hospital discharge. Participants, caregivers, and assessors were masked to group assignment. The primary outcome was defined as all-cause mortality at 28 days in the full analysis set (defined as all randomly assigned participants regardless of whether a patient started treatment, excluding patients randomly assigned in error) and measured using Kaplan-Meier analysis. Safety analyses included all patients who had received at least one infusion of either vilobelimab or placebo. This study is registered with ClinicalTrials.gov, NCT04333420. FINDINGS: From Oct 1, 2020, to Oct 4, 2021, we included 368 patients in the ITT analysis (full analysis set; 177 in the vilobelimab group and 191 in the placebo group). One patient in the vilobelimab group was excluded from the primary analysis due to random assignment in error without treatment. At least one dose of study treatment was given to 364 (99%) patients (safety analysis set). 54 patients (31%) of 177 in the vilobelimab group and 77 patients (40%) of 191 in the placebo group died in the first 28 days. The all-cause mortality rate at 28 days was 32% (95% CI 25-39) in the vilobelimab group and 42% (35-49) in the placebo group (hazard ratio 0·73, 95% CI 0·50-1·06; p=0·094). In the predefined analysis without site-stratification, vilobelimab significantly reduced all-cause mortality at 28 days (HR 0·67, 95% CI 0·48-0·96; p=0·027). The most common TEAEs were acute kidney injury (35 [20%] of 175 in the vilobelimab group vs 40 [21%] of 189 in the placebo), pneumonia (38 [22%] vs 26 [14%]), and septic shock (24 [14%] vs 31 [16%]). Serious treatment-emergent adverse events were reported in 103 (59%) of 175 patients in the vilobelimab group versus 120 (63%) of 189 in the placebo group. INTERPRETATION: In addition to standard of care, vilobelimab improves survival of invasive mechanically ventilated patients with COVID-19 and leads to a significant decrease in mortality. Vilobelimab could be considered as an additional therapy for patients in this setting and further research is needed on the role of vilobelimab and C5a in other acute respiratory distress syndrome-causing viral infections. FUNDING: InflaRx and the German Federal Government.

Blockade of complement activation product C5a activity using specific antibody attenuates intestinal damage in trinitrobenzene sulfonic acid induced model of colitis.

Complement represents a chief component of innate immunity in host defense. However, excessive complement activation has been involved in the pathogenesis of inflammatory diseases. In this study, we investigated the contribution of complement to intestinal pathology of patients and rodents with inflammatory bowel disease. The expression of complement effectors (C3a and C3) was increased remarkably in inflamed colons of IBD patients compared with those of normal counterparts. In accordance with this, the sustained activation of complement in serum and colon (including elevated C3a and C5a levels, enhanced hemolytic activity, downregulated expression of C5a receptors) was observed, following the establishment of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis, which peaked at 24 h. Mice pretreated with neutralizing anti-C5a antibodies (-2, 0, and 2 days after TNBS instillation) had significantly reduced weight loss and improved macroscopic/microscopic scores, comparable to the efficacy of prednisolone treatment. Strikingly, treatment with anti-C5a at 24 h after TNBS instillation showed remarkable therapeutic effects, whereas prednisolone did not. The efficacy of anti-C5a administration was associated with decreased release of proinflammatory chemokines and cytokines, inhibition of infiltration of neutrophils into colons, and enhanced Th2 response. These findings suggest a disease-promoting role of complement, particular C5a, in the pathology of TNBS-induced colitis in mice, indicating possible therapeutic potentials for C5a-specific antibody in IBD.

Complement C5a regulates IL-17 by affecting the crosstalk between DC and gammadelta T cells in CLP-induced sepsis.

Complement 5a (C5a) and Interleukin-17 (IL-17) are two important inflammatory mediators in sepsis. Here we studied the mechanisms underlying regulation of IL-17 by anaphylatoxin C5a. We found that C5a blockade increased the survival rate of mice following cecal ligation and puncture (CLP)-induced sepsis and decreased IL-17 expression in vivo. IL-17 was secreted mainly by gammadelta T cells in this model. Importantly, our data suggest that C5a participates in the regulation of IL-17 secretion by gammadelta T cells. Dendritic cells (DC) were found to act as a "bridge" between C5a and gammadelta T cells in a mechanism involving IL-6 and transforming growth factor beta (TGF-beta). These results imply that C5a affects the crosstalk between DC and gammadelta T cells during sepsis development, and this may result in a large production of inflammatory mediators such as IL-17.

Novel strategies for the treatment of sepsis.

The history of therapeutic interventions in clinical trials for sepsis has been referred to as the "graveyard for pharmaceutical companies." That is now set to change, as research provides hope for new approaches that will be therapeutically effective in humans with sepsis.

Efficacy and Safety of Vilobelimab (IFX-1), a Novel Monoclonal Anti-C5a Antibody, in Patients With Early Severe Sepsis or Septic Shock-A Randomized, Placebo-Controlled, Double-Blind, Multicenter, Phase IIa Trial (SCIENS Study).

Anaphylatoxin C5a, a proinflammatory complement split product, plays a central role in mediating organ dysfunction. OBJECTIVES: This phase II clinical trial was conducted to study safety, tolerability, pharmacokinetics, and pharmacodynamics of vilobelimab, a recombinant monoclonal antibody against C5a, in patients with severe sepsis or septic shock. DESIGN: Multicenter, randomized, and placebo-controlled study. SETTING AND PARTICIPANTS: Eleven multidisciplinary ICUs across Germany. Adult patients with severe sepsis or septic shock and with early onset of infection-associated organ dysfunction. MAIN OUTCOMES AND MEASURES: Patients were randomly assigned in a ratio of 2:1 to three subsequent dosing cohorts for IV vilobelimab or placebo receiving either 2 × 2 mg/kg (0 and 12 hr), 2 × 4 mg/kg (0 and 24 hr), and 3 × 4 mg/kg (0, 24, and 72 hr). Co-primary endpoints were pharmacodynamics (assessed by C5a concentrations), pharmacokinetics (assessed by vilobelimab concentrations), and safety of vilobelimab. Preliminary efficacy was evaluated by secondary objectives. RESULTS: Seventy-two patients were randomized (16 patients for each vilobelimab dosing cohort and eight patients for each placebo dosing cohort). Vilobelimab application was associated with dosing dependent decrease in C5a compared with baseline (p < 0.001). Duration of C5a decrease increased with more frequent dosing. Membrane attack complex lysis capacity measured by 50% hemolytic complement was not affected. Vilobelimab was well tolerated with similar safety findings in all dose cohorts. No vilobelimab-specific adverse events emerged. For vilobelimab-treated patients, investigators attributed less treatment-emergent adverse events as related compared with placebo. Dosing cohorts 2 and 3 had the highest ICU-free and ventilator-free days. There was no difference in mortality, vasopressor-free days, or renal replacement therapy-free days between the groups. CONCLUSIONS AND RELEVANCE: Administration of vilobelimab in patients with severe sepsis and septic shock selectively neutralizes C5a in a dose-dependent manner without blocking formation of the membrane attack complex and without resulting in detected safety issues. The data warrant further investigation of C5a inhibition in sepsis.

Interleukin-17-producing gammadelta+ T cells protect NOD mice from type 1 diabetes through a mechanism involving transforming growth factor-beta.

Whether interleukin (IL)-17 promotes a diabetogenic response remains unclear. Here we examined the effects of neutralization of IL-17 on the progress of adoptively transferred diabetes. IL-17-producing cells in non-obese diabetic (NOD) mice were identified and their role in the pathogenesis of diabetes examined using transfer and co-transfer assays. Unexpectedly, we found that in vivo neutralization of IL-17 did not protect NOD-severe combined immunodeficiency (SCID) mice against diabetes transferred by diabetic splenocytes. In NOD mice, gammadelta(+) T cells were dominated by IL-17-producing cells and were found to be the major source of IL-17. Interestingly, these IL-17-producing gammadelta T cells did not exacerbate diabetes in an adoptive transfer model, but had a regulatory effect, protecting NOD mice from diabetes by up-regulating transforming growth factor (TGF)-beta production. Our data suggest that the presence of IL-17 did not increase the chance of the development of diabetes; gammadelta T cells protected NOD mice from diabetes in a TGF-beta-dependent manner, irrespective of their role as major IL-17 producers.

Regulation of IL-8 production by complement-activated product, C5a, in vitro and in vivo during sepsis.

Excessive complement-activated product complement 5a (C5a) has been implicated in the pathogenesis of sepsis development. Herein, we employed in vitro and in vivo models of sepsis to investigate the functional relationship between overtly produced C5a and IL-8. Our data revealed that C5a could strongly amplify IL-8 expression from human whole blood cells induced by LPS and other types of TLR agonists. ERK1/2 and p38, but not JNK, were mainly participated in signaling pathways for IL-8 production. In the whole blood stimulated by Escherichiacoli, C5a levels were quickly elevated and blockage of C5a significantly decreased E. coli-elicited IL-8 production. In the mouse model of sepsis induced by cecal ligation and puncture (CLP), the markedly increased keratinocyte-derived cytokine (KC) could be strongly suppressed by blockage of C5a. These data suggest that excessive C5a functions as a critical inflammatory mediator to enhance IL-8 production mainly through MAPK signaling pathways.

Attenuation of IgG immune complex-induced acute lung injury by silencing C5aR in lung epithelial cells.

Acute lung injury (ALI) in mouse lung occurs after distal airway deposition of IgG immune complexes (IgGICs), resulting in a breakdown of the vascular-airway barrier, causing intrapulmonary edema, hemorrhage, and accumulation of neutrophils [polymorphonuclear leukocytes (PMNs)] in the alveolar compartment, these changes being complement (C5a) and C5a receptor (C5aR) dependent. In this ALI model, C5aR expression (protein) was found to occur on upper (bronchial) and lower (alveolar) airway epithelial cells. An adenovirus construct (siRNA) was used to silence mRNA for C5aR in the lung. Under such conditions, C5aR protein was markedly reduced on lung epithelial cells, resulting in much reduced leakage of albumin into the lung, diminished buildup of PMNs, and lower levels of proinflammatory mediators in bronchoalveolar lavage fluids. These studies indicate that bronchial and alveolar epithelial cell C5aR is up-regulated and greatly contributes to inflammation and injury in the lung. The use of siRNA administered into the airways avoids systemic suppression of C5aR, which might compromise innate immunity. It is possible that such an intervention might be employed in humans with ALI or acute respiratory distress syndrome as well as in upper-airway inflammatory diseases, such as chronic obstructive pulmonary disease and asthma, where there is evidence for complement activation and buildup of PMNs.

Anti-C5a antibody IFX-1 (vilobelimab) treatment versus best supportive care for patients with severe COVID-19 (PANAMO): an exploratory, open-label, phase 2 randomised controlled trial.

BACKGROUND: Severe COVID-19 is characterised by inflammation and coagulation in the presence of complement system activation. We aimed to explore the potential benefit and safety of selectively blocking the anaphylatoxin and complement protein C5a with the monoclonal antibody IFX-1 (vilobelimab), in patients with severe COVID-19. METHODS: We did an exploratory, open-label, randomised phase 2 trial (part of the adaptive phase 2/3 PANAMO trial) of intravenous IFX-1 in adults with severe COVID-19 at three academic hospitals in the Netherlands. Eligibility criteria were age 18 years or older; severe pneumonia with pulmonary infiltrates consistent with pneumonia, a clinical history of severe shortness of breath within the past 14 days, or a need for non-invasive or invasive ventilation; severe disease defined as a ratio of partial pressure of arterial oxygen to fractional concentration of oxygen in inspired air (PaO2/FiO2) between 100 mm Hg and 250 mm Hg in the supine position; and severe acute respiratory syndrome coronavirus 2 infection confirmed by RT-PCR. Patients were randomly assigned 1:1 to receive IFX-1 (up to seven doses of 800 mg intravenously) plus best supportive care (IFX-1 group) or best supportive care only (control group). The primary outcome was the percentage change in PaO2/FiO2 in the supine position between baseline and day 5. Mortality at 28 days and treatment-emergent and serious adverse events were key secondary outcomes. The primary analysis was done in the intention-to-treat population and safety analyses were done in all patients according to treatment received. This trial is registered at ClinicalTrials.gov (NCT04333420). FINDINGS: Between March 31 and April 24, 2020, 30 patients were enrolled and randomly assigned to the IFX-1 group (n=15) or the control group (n=15). During the study it became clear that several patients could not be assessed regularly in the supine position because of severe hypoxaemia. It was therefore decided to focus on all PaO2/FiO2 assessments (irrespective of position). At day 5 after randomisation, the mean PaO2/FiO2 (irrespective of position) was 158 mm Hg (SD 63; range 84-265) in the IFX-1 group and 189 mm Hg (89; 71-329) in the control group. Analyses of the least squares mean relative change in PaO2/FiO2 at day 5 showed no differences between treatment groups (17% change in the IFX-1 group vs 41% in the control group; difference -24% [95% CI -58 to 9], p=0·15. Kaplan-Meier estimates of mortality by 28 days were 13% (95% CI 0-31) for the IFX-1 group and 27% (4-49) for the control group (adjusted hazard ratio for death 0·65 [95% CI 0·10-4·14]). The frequency of serious adverse events were similar between groups (nine [60%] in the IFX-1 group vs seven [47%] in the control group) and no deaths were considered related to treatment assignment. However, a smaller proportion of patients had pulmonary embolisms classed as serious in the IFX-1 group (two [13%]) than in the control group (six [40%]). Infections classed as serious were reported in three (20%) patients in the IFX-1 group versus five (33%) patients in the control group. INTERPRETATION: In this small exploratory phase 2 part of the PANAMO trial, C5a inhibition with IFX-1 appears to be safe in patients with severe COVID-19. The secondary outcome results in favour of IFX-1 are preliminary because the study was not powered on these endpoints, but they support the investigation of C5a inhibition with IFX-1 in a phase 3 trial using 28-day mortality as the primary endpoint. FUNDING: InflaRx.

Effect of IL-10 on neutrophil recruitment and survival after Pseudomonas aeruginosa challenge.

IL-10 is a potent, endogenous anti-inflammatory cytokine known to decrease cytokine and keratinocyte-derived chemokine (KC) expression. Traditionally, in vivo effects of IL-10 were extrapolated from studies employing systemic antibody neutralization. As a result, divergent data regarding the protective and/or harmful roles of IL-10 have been reported. In this study, we used a lung-specific, tetracycline-inducible IL-10 overexpression-transgenic (IL-10 OE) mouse to study the effects of IL-10 overexpression on Pseudomonas aeruginosa-induced lung inflammation and corresponding survival in mice. Overexpression of IL-10 in the lung significantly increased mortality. During the early phase after infection (6-hours after infection), neutrophil recruitment as well as cytokine (TNF-alpha) and chemokine (KC) expression were significantly decreased in the IL-10 OE mice, which resulted in attenuated bacterial clearance. In contrast, overzealous production of KC and TNF-alpha intensified neutrophil infiltration and increased vascular leakage in IL-10 OE mice at the later stage of infection (24 hours after infection). Neutrophil depletion showed impaired bacterial clearance in both control and IL-10 OE mice, and further enhanced mouse mortality, whereas exogenous administration of KC reversed this finding. Our data indicate that early neutrophil recruitment is important for combating bacterial infection, and that the inhibition of neutrophil recruitment by IL-10 results in insufficient bacteria clearance in the lung, leading to excessive development of inflammation and increased mortality.