Inhibition Of C5a Research Articles

Avacopan's potential to decrease MPO-ANCA titres concurrent with ameliorated activity in ANCA-associated vasculitis.

Avacopan, an orally administered C5a receptor antagonist, is effective in microscopic polyangiitis via the inhibition of neutrophil priming induced by C5a. However, the exact effect of avacopan on the production of myeloperoxidase antineutrophil cytoplasmic antibody (MPO-ANCA) is yet to be clearly established. This report presents a microscopic polyangiitis patient without major organ damage where high levels of MPO-ANCA persisted with high-dose steroid therapy and azathioprine, but the addition of avacopan led to a reduction in MPO-ANCA titres. The present case implies that avacopan-mediated inhibition of C5a may lead to a reduction in MPO-ANCA levels, thereby potentially ameliorating the pathophysiology of ANCA-associated vasculitis. Nevertheless, the impact of avacopan on MPO-ANCA production cannot be asserted solely based on this report; therefore, further examination is necessary through subgroup analysis using data from larger-scale studies.

TMIC-06. GLIOBLASTOMA PROGRESSION IS PROMOTED BY C5AR1 SIGNALING THROUGH EMT INDUCTION, AND ITS BLOCKADE BY W54011 ATTENUATES MALIGNANT PHENOTYPES

Abstract BACKGROUND Glioblastoma multiforme (GBM) is an aggressive brain tumor with poor prognosis. Tumor mesenchymal stem-like cells (tMSLCs) in GBM tissues secrete complement component 5a (C5a), which promotes tumor malignancy. This study aimed to investigate the impact of C5a on GBM tumorspheres (GBM-TS) and to evaluate the therapeutic potential of C5a inhibition using the C5a antagonist, W54011. METHODS RNA sequencing (RNAseq) was performed on tumor tissues to identify differentiaaly expressing genes (DEG) and perform gene set enrichment analysia (GSEA) to correlate with patient survival based on C5aR1 expression. GBM-TS and tMSLCs were cultured to create a conditioned medium (CM) containing C5a, which was used to stimulate GBM-TS. The effect of C5a on GBM-TS proliferation, invasion, and stemness was determined using WST/ATP assay, 3D invasion assay, and neurosphere formation assay. Western blot and RNAseq analysis were performed to validate the results. In vivo studies were conducted using orthotopic xenograft mouse models injected with GBM-TS alone or with CM and treated with W54011. RESULTS Patients with high C5aR1 expression had a poor prognosis. Treatment with CM increased proliferation, invasion, and stemness of GBM-TS, but W54011 reversed these effects. Treatment with CM induced epithelial-messenchymal transition (EMT) in GBM TSs, whereas W54011 restored the spherical morphology and induced apoptosis. Our results were supported by transcriptome analysis and marker expression in Western blots. In the orthotopic xenograft mouse model, co-injection of GBM-TS and CM resulted in larger tumors and poorer survival, while treatment with W54011 reduced tumor size. CONCLUSIONS C5a promotes the growth, invasion, and stemness of GBM-TS, and inhibition of C5a using W54011 may have therapeutic potential for patients with high C5a expression. These results provide insight into the underlying mechanisms of GBM progression and establish a knowledge base for the clinical efficacy of W54011 in GBM treatment.

A prospective phase 2 clinical trial of a C5a complement inhibitor for acute GVHD with lower GI tract involvement.

Involvement of lower gastrointestinal tract (LGI) occurs in 60% of patients with graft-versus-host-disease (GVHD). Complement components C3 and C5 are involved in GVHD pathogenesis. In this phase 2a study, we evaluated the safety and efficacy of ALXN1007, a monoclonal antibody against C5a, in patients with newly diagnosed LGI acute GVHD receiving concomitant corticosteroid. Twenty-five patients were enrolled; one was excluded from the efficacy analysis based upon negative biopsy. Most patients (16/25, 64%) had acute leukemia; 52% (13/25) had an HLA-matched unrelated donor; and 68% (17/25) received myeloablative conditioning. Half the patients (12/24) had a high biomarker profile,Ann Arbor score 3; 42% (10/24) had high-risk GVHD per Minnesota classification. Day-28 overall response was 58% (13/24 complete response, 1/24 partial response), and 63% by Day-56 (all complete responses). Day-28 overall response was 50% (5/10) in Minnesota high-risk and 42% (5/12) in high-risk Ann Arbor patients, increasing to 58% (7/12) by Day-56. Non-relapse mortality at 6-months was 24% (95% CI 11-53). The most common treatment-related adverse event was infection (6/25, 24%). Neither baseline complement levels (except for C5), activity, nor inhibition of C5a with ALXN1007 correlated with GVHD severity or responses. Further studies are needed to evaluate the role of complement inhibition in GVHD treatment.

Long non-coding RNA reprogramming (LncRNA-ROR) attenuate inflammation induced by LPS through the regulation of miRNA-124-3p and inhibition of C5a and TLR4/Myd88/NF-κB pathwaysin C28/12 cells

Purpose: Osteoarthritis (OA), inflammation of joint and articular cartilage, is a serious joint disorder and a major cause of pain and disability. LncRNAs exert their role in OA progression. The purpose of this present research is to investigate the impact and molecular mechanism of lncRNA regulator of reprogramming (LncRNA-ROR) on inflammatory damage induced by lipopolysaccharide (LPS) in C28/12 cells. 
 Methods: The expressions of lncRNA-ROR and miR-124-3p and the mRNA expressions of COX-2, iNOS, IL-8, C5a, and TNF-α were determined in C28/12 cells stimulated with LPS using RT-qPCR. Viability of C28/12 cells was evaluated using CCK-8 assay and C28/12 cell apoptosis was assessed by flow cytometry in LPS-induced C28/12 cells. Moreover, the concentration of IL-8, C5a, and TNF-α and the protein expressions of TLR4/Myd88/NF-κB pathways were examined using Elisa assay. 
 Results: Results revealed that LPS inhibited the proliferation, enhanced rate of cell apoptosis, and promoted mRNA expressions of iNOS, IL-8, C5a, and TNF-α in C28/12 cells. Besides, the expression of lncRNA-ROR was expressively reduced in C28/12 cells stimulated with LPS and the lncRNA-ROR overexpression markedly reduced the inflammatory injury stimulated by LPS in C28/12 cells. Besides, lncRNA-ROR significantly down-regulated the miR-124-3p expression and miR-124-3p exposure inhibited the protecting impact of lncRNA-ROR on LPS-induced C28/12 cells. Furthermore, lncRNA-ROR suppressed the TLR4/Myd88/NF-κB pathways via suppressing the expression of miR-124-3p in LPS-induced C28/12 cells. 
 Conclusion: The finding revealed that lncRNA-ROR protected against LPS-stimulated inflammatory injury in C28/12 cells by suppressing TLR4/Myd88/NF-κB pathways via inhibiting miR-124-3p suggested an efficient therapeutic strategy for OA treatment.

Effects of heat shock protein 90 on complement activation in myocardial ischemia/reperfusion injury after pioglitazone preconditioning.

Heat shock protein 90 (HSP90) appears to have a pivotal function in ischemic preconditioning. Pioglitazone preconditioning (PioC) attenuates myocardial ischemia/reperfusion (I/R) injuries. The current study aims to investigate the role of HSP90, complement C3 and C5a, and nuclear factor kappa-B (NF-κB) in PioC-induced cardioprotection. A total of 80 rats were randomly categorized into 4 groups, as follows: sham, I/R, PioC, and PioC+HSP90 inhibitor geldanamycin (PioC+GA). The sham group rats had a thoracotomy, in which the ligature was passed by the heart with no ligation (150 min). The other 3 groups were exposed to ischemia (30 min) followed by reperfusion (2 h). In the PioC group, pioglitazone (3 mg/kg) was administered intravenously 24 h before ischemia. In the PioC+GA group, after being pretreated with pioglitazone, GA was administered (intraperitoneally, 1 mg/kg) 30 min before ischemia. Myocardial infarct sizes (ISs), apoptosis rates, creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH), and cardiac troponin I (cTnI) serum levels were determined. The HSP90, C3, NF-κB, C5a, B-cell lymphoma-2 (Bcl-2), and Bax expression levels, as well as interleukin (IL)-1β, IL-6, intercellular cell adhesion molecule-1 (ICAM-1), and tumor necrosis factor alpha (TNF-α) mRNA levels were measured. The myocardial ISs, serum CK-MB, cTnI and LDH levels, apoptosis rates, IL-1β, IL-6, TNF-α, ICAM-1 release, as well as Bax, C5a, C3, and NF-κB protein expression were considerably lower in the PioC group than in the I/R group (p < 0.05). The Bcl-2 and HSP90 expression was higher in the PioC group than in the I/R group (p < 0.05). Geldanamycin inhibited the effects of PioC. These data strongly demonstrate that the PioC-induced is dependent upon HSP90 activity. The HSP90 is indispensable for PioC-mediated cardioprotection. The HSP90 attenuates I/R-induced ISs, apoptosis of cardiomyocytes and myocardial inflammation through C3, C5a and NF-κB activation inhibition.

Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of Anti-C5a Antibody BDB-001 for Severe COVID-19: A Randomized, Double-Blind, Placebo-Controlled Phase 1 Clinical Trial in Healthy Chinese Adults.

Severe Coronavirus Disease 2019 (COVID-19) progresses with inflammation and coagulation, due to an overactive complement system. Complement component 5a (C5a) plays a key role in the complement system to trigger a powerful "cytokine and chemokine storm" in viral infection. BDB-001, a recombinant human immunoglobulin G4 (IgG4) that specially binds to C5a, has the potential to inhibit the C5a-triggered cytokine storm in treating COVID-19 patients and other inflammation diseases. Here, we have explored its safety, tolerability, pharmacokinetics, and pharmacodynamics in healthy adults. This trial is registered with http://www.chinadrugtrials.org.cn/(CTR20200429 ). Thirty-two enrolled participants were randomized into three single-dose cohorts (2, 4, and 8mg/kg) and 1 multi-dose cohort (4mg/kg), and received either BDB-001 or placebo (3:1) double-blindly. The safety and tolerability after administration were evaluated for 21days for single-dose cohorts and 28days for the multi-dose cohort. The pharmacokinetics of BDB-001 in plasma and pharmacodynamics as free C5a in plasma were analyzed. The incidence of drug-related adverse events (AEs) was low, and all AEs were mild or moderate: neither AEs≥3 (NCI-Common Terminology Criteria For Adverse Events, CTCAE 5.0) nor serious adverse events (SAEs) were found. The area under the concentration-time curve from time zero to 480h (AUC0-480h), that from time zero to infinity (AUCinf), and peak plasma concentration ©max) increased dose-dependently from 2 to 8mg/kg in the single-dose cohorts and were characterized by a nonlinear pharmacokinetics of target-mediated drug disposal (TMDD). The accumulation index by AUC0-tau after five administrations (4mg/kg) from the multi-dose cohort was 6.42, suggesting an accumulation effect. Furthermore, inhibition of C5a at the plasma level was observed. The results of this phase I study supported that BDB-001 is a potent anti-C5a inhibitor with safety, tolerability, and no immunogenicity. TRIALREGISTRATION NUMBER: CTR20200429.

Complement C5a inhibition: a new form of COVID-19 treatment for mechanically ventilated patients?

Complement C5a inhibition: a new form of COVID-19 treatment for mechanically ventilated patients?

Success in Navigating Hurdles to Oral Delivery of a Bioactive Peptide Complement Antagonist through Use of Nanoparticles to Increase Bioavailability and In Vivo Efficacy

AbstractSubstantial preclinical data have validated cyclic hexapeptide complement C5a receptor 1 antagonists (C5aRAs) that target immune cells, as novel therapies for a range of inflammatory diseases that currently have limited effective treatment options. However, like most small‐molecule peptides, their poor oral bioavailability and short circulation half‐life are major hurdles that have limited their clinical translation. Here, a single emulsion technique is employed to produce poly(lactic‐co‐glycolic) acid nanoparticles (NPs) with exceptionally high peptide C5aRA (PMX205) loading efficiency (over 50%). Strikingly, the PMX205‐NPs not only facilitate prolonged release of the encapsulated PMX205 but also dramatically increase its oral bioavailability (from ≈25% to ≈50%), and therapeutic potential (≈95% inhibition of C5a induces neutrophilia in mice and maintenance of neuroprotective barrier integrity). The enhanced in vivo pharmacological activity of PMX205 in the form of NPs opens an exciting opportunity for the clinical application of peptide C5aRAs and possibly other therapeutic peptides.

Complement C5a induces the formation of neutrophil extracellular traps by myeloid-derived suppressor cells to promote metastasis.

Myeloid-derived suppressor cells (MDSCs) play a major role in cancer progression. In this study, we investigated the mechanisms by which complement C5a increases the capacity of polymorphonuclear MDSCs (PMN-MDSCs) to promote tumor growth and metastatic spread. Stimulation of PMN-MDSCs with C5a favored the invasion of cancer cells via a process dependent on the formation of neutrophil extracellular traps (NETs). NETosis was dependent on the production of high mobility group box 1 (HMGB1) by cancer cells. Moreover, C5a induced the surface expression of the HMGB1 receptors TLR4 and RAGE in PMN-MDSCs. In a mouse lung metastasis model, inhibition of C5a, C5a receptor-1 (C5aR1) or NETosis reduced the number of circulating-tumor cells (CTCs) and the metastatic burden. In support of the translational relevance of these findings, C5a was able to stimulate migration and NETosis in PMN-MDSCs obtained from lung cancer patients. Furthermore, myeloperoxidase (MPO)-DNA complexes, as markers of NETosis, were elevated in lung cancer patients and significantly correlated with C5a levels. In conclusion, C5a induces the formation of NETs from PMN-MDSCs in the presence of cancer cells, which may facilitate cancer cell dissemination and metastasis.

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.

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).

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. Multicenter, randomized, and placebo-controlled study. Eleven multidisciplinary ICUs across Germany. Adult patients with severe sepsis or septic shock and with early onset of infection-associated organ dysfunction. 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. 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. 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.

Efficacy matters: broadening complement inhibition in COVID-19 - Authors' reply.

Efficacy matters: broadening complement inhibition in COVID-19 - Authors' reply.

Efficacy matters: broadening complement inhibition in COVID-19.

Efficacy matters: broadening complement inhibition in COVID-19.

Essential role of the C5a receptor in E coli–induced oxidative burst and phagocytosis revealed by a novel lepirudin-based human whole blood model of inflammation

Complement plays an essential role in inflammation and tissue damage. However, it is largely unknown to what extent the system acts as a primary inducer of secondary mediator systems in the inflammatory network of human whole blood. Here we describe a novel in vitro model using the thrombin-specific hirudin analog lepirudin as anticoagulant, which, in contrast to heparin, did not interfere with complement activation. The model was used to study the role of complement in Escherichia coli-induced inflammatory responses. Granulocyte and monocyte oxidative burst was complement dependent as it was reduced by 85% and 70%, respectively, by the C3 [corrected] binding peptide compstatin. A similar reduction was found by inhibition of C5, C5a, and C5a receptor (C5aR). Furthermore, anti-CR3 antibodies were as efficient as the C5aR antagonist in reducing granulocyte oxidative burst, whereas blocking CD14 or C3aR had no effect. Up-regulation of granulocyte CR3 was virtually abolished by a C5aR antagonist. Opsonization and phagocytosis was completely inhibited by blocking of C5aR or CR3, whereas blocking of the FcgammaRs (CD16, CD32, CD64) had no effect. In contrast to oxidative burst and phagocytosis, cytokine secretion was largely complement independent. Thus, anti-CD14 abolished tumor necrosis factor-alpha, interleukin-6 (IL-6), and IL-10 secretion, whereas IL-8 was equally inhibited by anti-CD14 and compstatin. In conclusion, the present model is particularly useful for studying complement as part of the inflammatory network. The results emphasize a crucial role for C5a-C5aR interaction in E coli-induced up-regulation of CR3 and the subsequent oxidative burst and phagocytosis. Complement inhibition may have therapeutic implications in oxidative burst-induced tissue damage.

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.

BackgroundSevere 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.MethodsWe 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).FindingsBetween 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.InterpretationIn 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.FundingInflaRx.

Anti-C5a Antibody (IFX-1) Treatment of Severe COVID-19: An Exploratory Phase 2 Randomized Controlled Trial

Background: Severe coronavirus disease 2019 (Covid-19) is characterized by inflammation and coagulation in the presence of complement activation. Methods: We conducted an explorative phase 2 randomized, open label first part of an adaptive phase 2/3 trial of intravenous IFX-1, a monoclonal antibody selectively blocking the anaphylatoxin C5a, in adults with severe Covid-19. Patients were randomized between IFX-1 plus best supportive care (BSC) or BSC only. Results: 30 patients underwent randomization: 15 assigned to IFX-1 and 15 to BSC. PaO2/FiO2 ratio improvement on day 5, chosen as primary outcome parameter, did not show significant differences between groups. However, IFX-1 treatment was associated with consistent trends of improvement as evidenced by lower mortality rate, reduction in renal impairment, normalization of lymphocyte counts, and lowering of plasma lactate dehydrogenase concentrations. Kaplan-Meier estimates of mortality by 28 days were 13% for IFX-1 and 27% for BSC (HR for death, 0.56; 95%CI 0.09-3.74). Serious adverse events rates were comparable between groups but the rate of pulmonary embolisms was three-fold lower in the IFX-1 group (13%) compared to BSC group (40%). IFX-1 treatment was associated with significant increase of D-dimer levels suggesting a potential pro-fibrinolytic activity of anti-C5a treatment. Conclusion: In this exploratory part of the study, C5a inhibition with IFX-1 was shown to be safe in severe Covid-19. PaO2/FiO2 ratio at day five was comparable between groups, but consistent signals of benefit including a lower 28-day all-cause mortality rate, lower rate in impaired kidney function and a lower rate of pulmonary embolism warrant investigating C5a-inhibition with IFX-1 within a phase 3 trial.Trial Registration: This trial has been registered with the NIH, U.S. National Library of Medicine at ClinicalTrials.gov (NCT04333420). Funding Statement: The trial is funded by InflaRx GmbH.Declaration of Interests: NR and RG are founders, active officers and executive directors of InflaRx (InflaRx GmbH, InflaRx Pharmaceuticals Inc. and InflaRx N.V.) and hold shares and stock options in InflaRx. KP is Global Head of Clinical Development of InflaRx and holds stock options in InflaRx. SR is employee at Metronomia, a contracted statistical service provider for InflaRx. MW is supported by grants from the German Research Foundation, SFB-TR84 C6 and C9 and by the German Ministry of Education and Research in the framework of the CAPSyS (01ZX1304B) and the PROVID project (FKZ 01KI20160A). DvdB reports receiving departmental honoraria for serving on a scientific advisory board for InflaRx in 2017, paid to Amsterdam UMC. All other authors have no Conflict of Interest. Ethics Approval Statement: The study protocol was approved by the institutional review board of the Academic Medical Center, part of Amsterdam UMC, Amsterdam, the Netherlands (IRB: 2020_067#B2020179). If direct informed consent of patients was not feasible, patients could be included with a deferred consent procedure. All patients or their legally authorized representatives gave written informed consent for the study.

Effect of complement C5a on the expression of MCP-1 and NGAL in immune kidney injury of trichloroethylene sensitized mice

Objective: To explore the possible role of C5a in the pathogenesis of renal injury in TCE- sensitized mice, to analyze the impact of expression of neutrophil gelatinase-associated lipocalin (NGAL) and monocyte chemotactic protein-1 (MCP-1) in the presence or absence of C5a receptor antagonist (C5aRA) pretreatment. Methods: A total of 50 female specific pathogens free(SPF) BALB/c mice were randomly divided into blank control group (n=5) , solvent control group (n=5) , TCE group (n=20) , and TCE+C5aRA group (n= 20) . After one week for adaptive feeding, a mouse model of TCE-induced skin sensitization was established by treating with 50% TCE and 30% TCE in turn. The mice in solvent control group accept same reagents without TCE and the mice in blank control group underwent nothing. In TCE +C5aRA group, except for the TCE solution treatment, mice were intraperitoneally injected with 0.5 mg/kg C5aRA solution at the time of challenge. And the skin erythema and edema reaction were scored 24 h after the last challenge. The mice were divided into sensitization positive group and sensitization negative group according to the scoring result. The mice were aseptically sacrificed 72 h after the last challenge to obtain the kidneys. The structural damage of kidney was observed after histopathological staining. The levels of NGAL and MCP-1 mRNA and proteins were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC) , respectively. Results: The sensitization rate of mice in TCE group and TCE+C5aRA group was 45.0% (9/20) and 40.0% (8/20) , respectively. No skin lesions was found in the mice of blank control group and solvent control group. The results of histopathological staining showed that the TCE sensitization positive mice showed renal tubular dilatation, vacuolar degeneration of renal tubular epithelial cells, and infiltration of interstitial cells. The pathological damage of the kidney in TCE sensitization positive group was mild, and no inflammatory cell infiltration was seen. The data of qRT-PCR showed that the expression levels of NGAL and MCP-1 mRNA in the TCE sensitization positive group were significantly increased than in solvent control group and TCE sensitization negative group (P<0.05) , while the levels of NGAL and MCP-1 mRNA in TCE+C5aRA sensitization positive group were decreased than TCE sensitization positive group (P <0.05) . The results of IHC showed that the expression levels of NGAL and MCP-1 in TCE protein sensitization positive group were significantly higher than those in solvent control group and TCE sensitization negative group (P<0.05) . After C5aRA pretreatment, the expression levels of NGAL and MCP-1 protein were decreased than the mice in TCE sensitization positive group (P<0.05) . Conclusion: The regulation of C5a on the expression of MCP-1 and NGAL may participate in TCE- induced mice kidney damage, and pharmacological inhibition of C5a seems to be an effective way to protect the kidney injury in TCE-sensitized mice.

Production of a bispecific antibody targeting TNF-α and C5a in Pichia pastoris and its therapeutic potential in rheumatoid arthritis.

To provide an alternative therapeutic modality for rheumatoid arthritis (RA), a novel bispecific antibody (BsAb) targeting human tumor necrosis factor α (TNF-α) and human complement component C5a was constructed. BsAb was expressed in Pichia pastoris and secreted into the culture medium as a functional protein. In vitro functional study demonstrated that BsAb could simultaneously bind to TNF-α and C5a and neutralize their biological actions. Furthermore, BsAb showed significant improvements in both the antigen-binding affinity and the neutralizing ability as compared to its original antibodies produced in E. coli. It was also found that TNF-α and C5a had an additive/synergistic effect on promoting the production of inflammatory cytokines and chemokines and C5a receptor (C5aR) expression in human macrophages. Compared to single inhibition of TNF-α or C5a with respective antibody, BsAb showed a superior efficacy in blocking inflammatory cytokines, chemokines, and C5aR response, as well as in lowering the C5a-mediated chemotaxis of macrophages via C5aR in vitro. With improved production processing and the ability to simultaneously block TNF-α and C5a action, BsAb has a great potential to be developed into a therapeutic agent and may offer a better therapeutic index for RA.

Critical role of C5a in sickle cell disease.

Innate immune complement activation may contribute to sickle cell disease (SCD) pathogenesis. Ischemia-reperfusion physiology is a key component of the inflammatory and vaso-occlusive milieu in SCD and is associated with complement activation. C5a is an anaphylatoxin, a potent pro-inflammatory mediator that can activate leukocytes, platelets, and endothelial cells, all of which play a role in vaso-occlusion. We hypothesize that hypoxia-reoxygenation (H/R) in SCD mice activates complement, promoting inflammation and vaso-occlusion. At baseline and after H/R, sickle Townes-SS mice had increased C3 activation fragments and C5b-9 deposition in kidneys, livers and lungs and alternative pathway Bb fragments in plasma compared to control AA-mice. Activated complement promoted vaso-occlusion (microvascular stasis) in SS-mice; infusion of zymosan-activated, but not heat-inactivated serum, induced substantial vaso-occlusion in the skin venules of SS-mice. Infusion of recombinant C5a induced stasis in SS, but not AA-mice that was blocked by anti-C5a receptor (C5aR) IgG. C5a-mediated stasis was accompanied by inflammatory responses in SS-mice including NF-κB activation and increased expression of TLR4 and adhesion molecules VCAM-1, ICAM-1, and E-selectin in the liver. Anti-C5aR IgG blocked these inflammatory responses. Also, C5a rapidly up-regulated Weibel-Palade body P-selectin and von Willebrand factor on the surface of human umbilical vein endothelial cells in vitro and on vascular endothelium in vivo. In SS-mice, a blocking antibody to P-selectin inhibited C5a-induced stasis. Similarly, an antibody to C5 that blocks murine C5 cleavage or an antibody that blocks C5aR inhibited H/R-induced stasis in SS-mice. These results suggest that inhibition of C5a may be beneficial in SCD.

Inhibition of C5a prevents IL-1β-induced alternations in rat synoviocytes in vitro

C5a is an important pro-inflammatory peptide involved in complement activation, membrane attack complex formation, immune cell chemotaxis, and allergic responses. Osteoarthritis is a disease characterized by degenerative changes in articular cartilage. It has recently been found that inflammatory responses play an important role in the pathogenesis of osteoarthritis and also in rheumatoid arthritis, where dysfunctional synoviocytes are involved. We performed a series of studies to verify our hypothesis that inhibition of C5a would prevent IL-1β-induced alternations in rat synoviocytes. In vitro studies were performed with RSC-364 cells to examine the role of C5a in the function of synoviocytes. RSC-364 cells (a rat derived synovial cell line) were treated with IL-1β, IL-1β+siC5a, IL-1β+PMX205 that is antagonist of C5aR, or left untreated. Cell cycle, proliferation, apoptosis, invasion, as well as levels of C5a, IL-17A and TNF-α expression were evaluated. We found that IL-1β could significantly increase the proliferation and invasion capabilities of RSC-364 cells, as well as of C5a IL-17A and TNF-α expression. In contrast, inhibition of C5a by siRNA or application of antagonist of C5aR PMX205 reversed the IL-1β-induced changes in C5a expression, cell cycle, proliferation, apoptosis, invasion, and cytokines releases. Taken together, our study results suggest that IL-1β can increase C5a expression in RSC-364 cells, and that C5a exerts a proinflammatory effect in RSC-364 cells. Inhibition of C5a might represent a new strategy for treating rheumatoid arthritis.