Complement activation promoted by the lectin pathway mediates C3aR-dependent sarcoma progression and immunosuppression.

Complement has emerged as a component of tumor promoting inflammation. We conducted a systematic assessment of the role of complement activation and effector pathways in sarcomas. C3-/-, MBL1/2-/- and C4-/- mice showed reduced susceptibility to 3-methylcholanthrene sarcomagenesis and transplanted sarcomas, whereas C1q and factor B deficiency had marginal effects. Complement 3a receptor (C3aR), but not C5aR1 and C5aR2, deficiency mirrored the phenotype of C3-/- mice. C3 and C3aR deficiency were associated with reduced accumulation and functional skewing of tumor-associated macrophages, increased T cell activation and response to anti-PD-1 therapy. Transcriptional profiling of sarcoma infiltrating macrophages and monocytes revealed the enrichment of MHC II-dependent antigen presentation pathway in C3-deficient cells. In patients, C3aR expression correlated with a macrophage population signature and C3 deficiency-associated signatures predicted better clinical outcome. These results suggest that the lectin pathway and C3a/C3aR axis are key components of complement and macrophage-mediated sarcoma promotion and immunosuppression.

C3 complement inhibition prevents antibody-mediated rejection and prolongs renal allograft survival in sensitized non-human primates.

Sensitized kidney transplant recipients experience high rates of antibody-mediated rejection due to the presence of donor-specific antibodies and immunologic memory. Here we show that transient peri-transplant treatment with the central complement component C3 inhibitor Cp40 significantly prolongs median allograft survival in a sensitized nonhuman primate model. Despite donor-specific antibody levels remaining high, fifty percent of Cp40-treated primates maintain normal kidney function beyond the last day of treatment. Interestingly, presence of antibodies of the IgM class associates with reduced median graft survival (8 vs. 40 days; p = 0.02). Cp40 does not alter lymphocyte depletion by rhesus-specific anti-thymocyte globulin, but inhibits lymphocyte activation and proliferation, resulting in reduced antibody-mediated injury and complement deposition. In summary, Cp40 prevents acute antibody-mediated rejection and prolongs graft survival in primates, and inhibits T and B cell activation and proliferation, suggesting an immunomodulatory effect beyond its direct impact on antibody-mediated injury.

C5a-C5aR1 Axis Activation Drives Envenomation Immunopathology by the Snake Naja annulifera.

Systemic complement activation drives a plethora of pathological conditions, but its role in snake envenoming remains obscure. Here, we explored complement's contribution to the physiopathogenesis of Naja annulifera envenomation. We found that N. annulifera venom promoted the generation of C3a, C4a, C5a, and the soluble Terminal Complement Complex (sTCC) mediated by the action of snake venom metalloproteinases. N. annulifera venom also induced the release of lipid mediators and chemokines in a human whole-blood model. This release was complement-mediated, since C3/C3b and C5a Receptor 1 (C5aR1) inhibition mitigated the effects. In an experimental BALB/c mouse model of envenomation, N. annulifera venom promoted lipid mediator and chemokine production, neutrophil influx, and swelling at the injection site in a C5a-C5aR1 axis-dependent manner. N. annulifera venom induced systemic complementopathy and increased interleukin and chemokine production, leukocytosis, and acute lung injury (ALI). Inhibition of C5aR1 with the cyclic peptide antagonist PMX205 rescued mice from these systemic reactions and abrogated ALI development. These data reveal hitherto unrecognized roles for complement in envenomation physiopathogenesis, making complement an interesting therapeutic target in envenomation by N. annulifera and possibly by other snake venoms.

C5a-C5aR1 axis activation drives envenomation immunopathology by the snake naja annulifera

Systemic complement activation drives a plethora of pathological conditions, but its role in snake envenoming remains obscure. Here, we explored complement’s contribution to the physiopathogenesis of Naja annulifera envenomation. We found that N. annulifera venom promoted the generation of C3a, C4a, C5a, and the soluble Terminal Complement Complex (sTCC) mediated by the action of snake venom metalloproteinases. N. annulifera venom also induced the release of lipid mediators and chemokines in a human whole-blood model. This release was complement-mediated, since C3/C3b and C5a Receptor 1 (C5aR1) inhibition mitigated the effects. In an experimental BALB/c mouse model of envenomation, N. annulifera venom promoted lipid mediator and chemokine production, neutrophil influx, and swelling at the injection site in a C5a-C5aR1 axis-dependent manner. N. annulifera venom induced systemic complementopathy and increased interleukin and chemokine production, leukocytosis, and acute lung injury (ALI). Inhibition of C5aR1 with the cyclic peptide antagonist PMX205 rescued mice from these systemic reactions and abrogated ALI development. These data reveal hitherto unrecognized roles for complement in envenomation physiopathogenesis, making complement an interesting therapeutic target in envenomation by N. annulifera and possibly by other snake venoms.

Prolonged intraocular residence and retinal tissue distribution of a fourth-generation compstatin-based C3 inhibitor in non-human primates.

Age-related macular degeneration (AMD) is a leading cause of irreversible vision loss among the elderly population. Genetic studies in susceptible individuals have linked this ocular disease to deregulated complement activity that culminates in increased C3 turnover, retinal inflammation and photoreceptor loss. Therapeutic targeting of C3 has therefore emerged as a promising strategy for broadly intercepting the detrimental proinflammatory consequences of complement activation in the retinal tissue. In this regard, a PEGylated second-generation derivative of the compstatin family of C3-targeted inhibitors is currently in late-stage clinical development as a treatment option for geographic atrophy, an advanced form of AMD which lacks approved therapy. While efficacy has been strongly suggested in phase 2 clinical trials, crucial aspects still remain to be defined with regard to the ocular bioavailability, tissue distribution and residence, and dosing frequency of such inhibitors in AMD patients. Here we report the intraocular distribution and pharmacokinetic profile of the fourth-generation compstatin analog, Cp40-KKK in cynomolgus monkeys following a single intravitreal injection. Using a sensitive surface plasmon resonance (SPR)-based competition assay and ELISA, we have quantified both the amount of inhibitor and the concentration of C3 retained in the vitreous of Cp40-KKK-injected animals. Cp40-KKK displays prolonged intraocular residence, being detected at C3-saturating levels for over 3 months after a single intravitreal injection. Moreover, we have probed the distribution of Cp40-KKK within the ocular tissue by means of immunohistochemistry and highly specific anti-Cp40-KKK antibodies. Both C3 and Cp40-KKK were detected in the retinal tissue of inhibitor-injected animals, with prominent co-localization in the choroid one-month post intravitreal injection. These results attest to the high retinal tissue penetrance and target-driven distribution of Cp40-KKK. Given its subnanomolar binding affinity and prolonged ocular residence, Cp40-KKK constitutes a promising drug candidate for ocular pathologies underpinned by deregulated C3 activation.

New insights into the immune functions of complement.

The recognition of microbial or danger-associated molecular patterns by complement proteins initiates a cascade of events that culminates in the activation of surface complement receptors on immune cells. Such signalling pathways converge with those activated downstream of pattern recognition receptors to determine the type and magnitude of the immune response. Intensive investigation in the field has uncovered novel pathways that link complement-mediated signalling with homeostatic and pathological T cell responses. More recently, the observation that complement proteins also act in the intracellular space to shape T cell fates has added a new layer of complexity. Here, we consider fundamental mechanisms and novel concepts at the interface of complement biology and immunity and discuss how these affect the maintenance of homeostasis and the development of human pathology.

Complement-Dependent Mechanisms and Interventions in Periodontal Disease.

Periodontitis is a prevalent inflammatory disease that leads to the destruction of the tooth-supporting tissues. Current therapies are not effective for all patients and this oral disease continues to be a significant public health and economic burden. Central to periodontal disease pathogenesis is a reciprocally reinforced interplay between microbial dysbiosis and destructive inflammation, suggesting the potential relevance of host-modulation therapies. This review summarizes and discusses clinical observations and pre-clinical intervention studies that collectively suggest that complement is hyperactivated in periodontitis and that its inhibition provides a therapeutic benefit. Specifically, interception of the complement cascade at its central component, C3, using a locally administered small peptidic compound (Cp40/AMY-101) protected non-human primates from induced or naturally occurring periodontitis. These studies indicate that C3-targeted intervention merits investigation as an adjunctive treatment of periodontal disease in humans.

Protective Effects of the Complement Inhibitor Compstatin CP40 in Hemorrhagic Shock.

Trauma-induced hemorrhagic shock (HS) plays a decisive role in the development of immune, coagulation, and organ dysfunction often resulting in a poor clinical outcome. Imbalanced complement activation is intricately associated with the molecular danger response and organ damage after HS. Thus, inhibition of the central complement component C3 as turnstile of both inflammation and coagulation is hypothesized as a rational strategy to improve the clinical course after HS.Applying intensive care conditions, anaesthetized, monitored, and protectively ventilated nonhuman primates (NHP; cynomolgus monkeys) received a pressure-controlled severe HS (60 min at mean arterial pressure 30 mmHg) with subsequent volume resuscitation. Thirty minutes after HS, animals were randomly treated with either an analog of the C3 inhibitor compstatin (i.e., Cp40) in saline (n = 4) or with saline alone (n = 4). The observation period lasted 300 min after induction of HS.We observed improved kidney function in compstatin Cp40-treated animals after HS as determined by improved urine output, reduced damage markers and a tendency of less histopathological signs of acute kidney injury. Sham-treated animals revealed classical signs of mucosal edema, especially in the ileum and colon reflected by worsened microscopic intestinal injury scores. In contrast, Cp40-treated HS animals exhibited only minor signs of organ edema and significantly less intestinal damage. Furthermore, early systemic inflammation and coagulation dysfunction were both ameliorated by Cp40.The data suggest that therapeutic inhibition of C3 is capable to significantly improve immune, coagulation, and organ function and to preserve organ-barrier integrity early after traumatic HS. C3-targeted complement inhibition may therefore reflect a promising therapeutic strategy in fighting fatal consequences of HS.

Taming hemodialysis-induced inflammation: Are complement C3 inhibitors a viable option?

Owing to an increasing shortage of donor organs, the majority of patients with end-stage kidney disease remains reliant on extracorporeal hemodialysis (HD) in order to counter the lifelong complications of a failing kidney. While HD remains a life-saving option for these patients, mounting evidence suggests that it also fuels a vicious cycle of thromboinflammation that can increase the risk of cardiovascular disease. During HD, blood-borne innate immune systems become inappropriately activated on the biomaterial surface, instigating proinflammatory reactions that can alter endothelial and vascular homeostasis. Complement activation, early during the HD process, has been shown to fuel a multitude of detrimental thromboinflammatory reactions that collectively contribute to patient morbidity. Here we discuss emerging aspects of complement's involvement in HD-induced inflammation and put forth the concept that targeted intervention at the level of C3 might constitute a promising therapeutic approach in HD patients.

Complement C5a-Mediated TAM-ing of Antitumor Immunity Drives Squamous Carcinogenesis.

In this issue of Cancer Cell, Medler et al. demonstrate that fibrinolytic enzyme-mediated generation of complement C5a reprograms tumor-infiltrating C5aR1+ macrophages into an immunosuppressive phenotype that dampens CD8+ T cell responses during squamous carcinogenesis. C5aR1 blockade combined with chemotherapy offers a promising immunomodulatory strategy for treating squamous cell carcinoma.

Safety profile after prolonged C3 inhibition.

The central component of the complement cascade, C3, is involved in various biological functions, including opsonization of foreign bodies, clearance of waste material, activation of immune cells, and triggering of pathways controlling development. Given its broad role in immune responses, particularly in phagocytosis and the clearance of microbes, a deficiency in complement C3 in humans is often associated with multiple bacterial infections. Interestingly, an increased susceptibility to infections appears to occur mainly in the first two years of life and then wanes throughout adulthood. In view of the well-established connection between C3 deficiency and infections, therapeutic inhibition of complement at the level of C3 is often considered with caution or disregarded. We therefore set out to investigate the immune and biochemical profile of non-human primates under prolonged treatment with the C3 inhibitor compstatin (Cp40 analog). Cynomolgus monkeys were dosed subcutaneously with Cp40, resulting in systemic inhibition of C3, for 1 week, 2 weeks, or 3 months. Plasma concentrations of both C3 and Cp40 were measured periodically and complete saturation of plasma C3 was confirmed. No differences in hematological, biochemical, or immunological parameters were identified in the blood or tissues of animals treated with Cp40 when compared to those injected with vehicle alone. Further, skin wounds showed no signs of infection in those treated with Cp40. In fact, Cp40 treatment was associated with a trend toward accelerated wound healing when compared with the control group. In addition, a biodistribution study in a rhesus monkey indicated that the distribution of Cp40 in the body is associated with the presence of C3, concentrating in organs that accumulate blood and produce C3. Overall, our data suggest that systemic C3 inhibition in healthy adult non-human primates is not associated with a weakened immune system or susceptibility to infections.

Gingival Exudatome Dynamics Implicate Inhibition of the Alternative Complement Pathway in the Protective Action of the C3 Inhibitor Cp40 in Nonhuman Primate Periodontitis.

Periodontitis is a prevalent chronic inflammatory disease associated with dysbiosis. Although complement inhibition has been successfully used to treat periodontitis in animal models, studies globally analyzing inflamed tissue proteins to glean insight into possible mechanisms of action are missing. Using quantitative shotgun proteomics, we aimed to investigate differences in composition of inflammatory gingival tissue exudate ("gingival crevicular fluid"; GCF), before and after local administration of an inhibitor of the central complement component, C3, in nonhuman primates. The C3 inhibitor, Cp40 (also known as AMY-101) was administered locally in the maxillary gingival tissue of cynomolgus monkeys with established periodontitis, either once a week (1×-treatment; n = 5 animals) or three times per week (3×-treatment; n = 10 animals), for 6 weeks followed by another 6 weeks of observation in the absence of treatment. 45 GCF samples were processed for FASP digestion and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Data were processed using the ProgenesisQI software. The statistical significance of differences between the groups was determined by RM-ANOVA, and a protein expression change was considered as a true regulation at >2-fold and p < 0.05. The human orthologues were subjected to Gene Ontology analyses using PANTHER. Data are available via ProteomeXchange with identifier PXD009502. 573 proteins with >2 peptides were longitudinally quantified. Both 3× and 1× administration of Cp40 resulted in significant down-regulation of dozens of proteins during the 6-week course of treatment as compared to baseline. Following drug withdrawal at 6 weeks, more than 50% of the down-regulated proteins showed increased levels at week 12. The top scored pathway was "complement activation, alternative pathway", and several proteins involved in this pathway were down-regulated at 6 weeks. We mapped the proteomic fingerprint changes in local tissue exudate of cynomolgus monkey periodontitis in response to C3 inhibition and identified the alternative pathway of complement activation and leukocyte degranulation as main targets, which are thus likely to play significant roles in periodontal disease pathogenesis. Label-free quantitative proteomics strategies utilizing GCF are powerful tools for the identification of treatment targets and providing insights into disease mechanisms.

Expanding Complement Therapeutics for the Treatment of Paroxysmal Nocturnal Hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is widely regarded as an archetypal complement-mediated disorder that has propelled complement drug discovery in recent decades. Its pathology is driven by chronic complement dysregulation resulting from the lack of the glycosyl phosphatidyl inositol-linked regulators DAF and CD59 on susceptible erythrocytes. This complement imbalance fuels persistent C3 activation on affected erythrocytes, which culminates in chronic complement-mediated intravascular hemolysis. The clinical application of eculizumab, a humanized anti-C5 antibody that blocks terminal pathway activation, has led to drastic improvement of therapeutic outcomes but has also unveiled hitherto elusive pathogenic mechanisms that are now known to contribute to the clinical burden of a significant proportion of patients with PNH. These emerging clinical needs have sparked a true resurgence of complement therapeutics that offer the promise of even more effective, disease-tailored therapies for PNH. Here, we review the current state of complement therapeutics with a focus on the clinical development of C3-targeted and alternative pathway-directed drug candidates for the treatment of PNH. We also discuss the relative advantages and benefits offered by each complement-targeting approach, including translational considerations that might leverage a more comprehensive clinical intervention for PNH.

New Analogs of the Complement C3 Inhibitor Compstatin with Increased Solubility and Improved Pharmacokinetic Profile.

Improper regulation of complement is associated with various pathologies, and the clinical demand for compounds that can regulate complement activation is therefore imperative. Cp40, an analog of the peptide compstatin, inhibits all complement pathways at the level of the central component C3. We have further developed Cp40, using either PEGylation at the N-terminus or insertion of charged amino acids at the C-terminus. The PEGylated analogs are highly soluble and retained their inhibitory activity, with C3b binding affinity dependent on the length of the PEG chain. The addition of two or three residues of lysine, in turn, not only improved the peptide's solubility but also increased the binding affinity for C3b while retaining its inhibitory potency. Three of the new derivatives showed improved pharmacokinetic profiles in vivo in non-human primates. Given their compelling solubility and pharmacokinetic profiles, these new Cp40 analogs should broaden the spectrum of administration routes, likely reducing dosing frequency during chronic treatment and potentially expanding their range of clinical application.

Functional Relevance of the Anaphylatoxin Receptor C3aR for Platelet Function and Arterial Thrombus Formation Marks an Intersection Point Between Innate Immunity and Thrombosis.

BACKGROUND: Platelets have distinct roles in the vascular system in that they are the major mediator of thrombosis, critical for restoration of tissue integrity, and players in vascular inflammatory conditions. In close spatiotemporal proximity, the complement system acts as the first line of defense against invading microorganisms and is a key mediator of inflammation. Whereas the fluid phase cross-talk between the complement and coagulation systems is well appreciated, the understanding of the pathophysiological implications of such interactions is still scant. METHODS: We analyzed coexpression of the anaphylatoxin receptor C3aR with activated glycoprotein IIb/IIIa on platelets of 501 patients with coronary artery disease using flow cytometry; detected C3aR expression in human or murine specimen by polymerase chain reaction, immunofluorescence, Western blotting, or flow cytometry; and examined the importance of platelet C3aR by various in vitro platelet function tests, in vivo bleeding time, and intravital microscopy. The pathophysiological relevance of C3aR was scrutinized with the use of disease models of myocardial infarction and stroke. To approach underlying molecular mechanisms, we identified the platelet small GTPase Rap1b using nanoscale liquid chromatography coupled to tandem mass spectrometry. RESULTS: We found a strong positive correlation of platelet complement C3aR expression with activated glycoprotein IIb/IIIa in patients with coronary artery disease and coexpression of C3aR with glycoprotein IIb/IIIa in thrombi obtained from patients with myocardial infarction. Our results demonstrate that the C3a/C3aR axis on platelets regulates distinct steps of thrombus formation such as platelet adhesion, spreading, and Ca2+ influx. Using C3aR-/- mice or C3-/- mice with reinjection of C3a, we uncovered that the complement activation fragment C3a regulates bleeding time after tail injury and thrombosis. Notably, C3aR-/- mice were less prone to experimental stroke and myocardial infarction. Furthermore, reconstitution of C3aR-/- mice with C3aR+/+ platelets and platelet depletion experiments demonstrated that the observed effects on thrombosis, myocardial infarction, and stroke were specifically caused by platelet C3aR. Mechanistically, C3aR-mediated signaling regulates the activation of Rap1b and thereby bleeding arrest after injury and in vivo thrombus formation. CONCLUSIONS: Overall, our findings uncover a novel function of the anaphylatoxin C3a for platelet function and thrombus formation, highlighting a detrimental role of imbalanced complement activation in cardiovascular diseases.

Complement in cancer: untangling an intricate relationship.

In tumour immunology, complement has traditionally been considered as an adjunctive component that enhances the cytolytic effects of antibody-based immunotherapies, such as rituximab. Remarkably, research in the past decade has uncovered novel molecular mechanisms linking imbalanced complement activation in the tumour microenvironment with inflammation and suppression of antitumour immune responses. These findings have prompted new interest in manipulating the complement system for cancer therapy. This Review summarizes our current understanding of complement-mediated effector functions in the tumour microenvironment, focusing on how complement activation can act as a negative or positive regulator of tumorigenesis. It also offers insight into clinical aspects, including the feasibility of using complement biomarkers for cancer diagnosis and the use of complement inhibitors during cancer treatment.

The renaissance of complement therapeutics.

The increasing number of clinical conditions that involve a pathological contribution from the complement system - many of which affect the kidneys - has spurred a regained interest in therapeutic options to modulate this host defence pathway. Molecular insight, technological advances, and the first decade of clinical experience with the complement-specific drug eculizumab, have contributed to a growing confidence in therapeutic complement inhibition. More than 20 candidate drugs that target various stages of the complement cascade are currently being evaluated in clinical trials, and additional agents are in preclinical development. Such diversity is clearly needed in view of the complex and distinct involvement of complement in a wide range of clinical conditions, including rare kidney disorders, transplant rejection and haemodialysis-induced inflammation. The existing drugs cannot be applied to all complement-driven diseases, and each indication has to be assessed individually. Alongside considerations concerning optimal points of intervention and economic factors, patient stratification will become essential to identify the best complement-specific therapy for each individual patient. This Review provides an overview of the therapeutic concepts, targets and candidate drugs, summarizes insights from clinical trials, and reflects on existing challenges for the development of complement therapeutics for kidney diseases and beyond.