C3 Activation Fragments Research Articles

C3-dependent effector functions of complement.

C3 is the central effector molecule of the complement system, mediating its multiple functions through different binding sites and their corresponding receptors. We will introduce the C3 forms (native C3, C3 [H2 O], and intracellular C3), the C3 fragments C3a, C3b, iC3b, and C3dg/C3d, and the C3 expression sites. To highlight the important role that C3 plays in human biological processes, we will give an overview of the diseases linked to C3 deficiency and to uncontrolled C3 activation. Next, we will present a structural description of C3 activation and of the C3 fragments generated by complement regulation. We will proceed by describing the C3a interaction with the anaphylatoxin receptor, followed by the interactions of opsonins (C3b, iC3b, and C3dg/C3d) with complement receptors, divided into two groups: receptors bearing complement regulatory functions and the effector receptors without complement regulatory activity. We outline the molecular architecture of the receptors, their binding sites on the C3 activation fragments, the cells expressing them, the diversity of their functions, and recent advances. With this review, we aim to give an up-to-date analysis of the processes triggered by C3 activation fragments on different cell types in health and disease contexts.

With complements: C3 inhibition in the clinic.

C3 is a key complement protein, located at the nexus of all complement activation pathways. Extracellular, tissue, cell-derived, and intracellular C3 plays critical roles in the immune response that is dysregulated in many diseases, making it an attractive therapeutic target. However, challenges such as very high concentration in blood, increased acute expression, and the elevated risk of infections have historically posed significant challenges in the development of C3-targeted therapeutics. This is further complicated because C3 activation fragments and their receptors trigger a complex network of downstream effects; therefore, a clear understanding of these is needed to provide context for a better understanding of the mechanism of action (MoA) of C3 inhibitors, such as pegcetacoplan. Because of C3's differential upstream position to C5 in the complement cascade, there are mechanistic differences between pegcetacoplan and eculizumab that determine their efficacy in patients with paroxysmal nocturnal hemoglobinuria. In this review, we compare the MoA of pegcetacoplan and eculizumab in paroxysmal nocturnal hemoglobinuria and discuss the complement-mediated disease that might be amenable to C3 inhibition. We further discuss the current state and outlook for C3-targeted therapeutics and provide our perspective on which diseases might be the next success stories in the C3 therapeutics journey.

Complement Blockade With a C1 Esterase Inhibitor In Paroxysmal Nocturnal Hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, clonal, hematopoietic stem cell disorder that manifests with a complement-mediated hemolytic anemia, bone marrow failure and a propensity for thrombosis. PNH confers deficiency of GPI anchored proteins that protect erythrocytes at different stages of the complement cascade. CD55 regulates the formation and stability of the C3 and C5 convertases, whereas CD59 blocks the formation of the membrane attack complex (MAC). These patients experience both intra- and extravascular hemolysis due to complement activation. Chronic treatment with eculizumab results in sustained control of intravascular hemolysis leading to transfusion independence for many. Eculizumab compensates for the CD59 deficiency on PNH erythrocytes; however, it does not compensate for the CD55 deficiency. PNH patients receiving eculizumab accumulate C3 on their erythrocytes; this leads to opsonization and extravascular hemolysis. There remains an unmet clinical need for a complement inhibitor with activity early in the complement cascade, which would not only reduce C3 accumulation on PNH erythrocytes, but also inhibit C3 activation by the alternative pathway (APC).C1 esterase inhibitor (C1 INH) is an endogenous human plasma protein that has broad inhibitory activity in the complement pathway. C1 INH inhibits the classical pathway of the complement system by binding C1r and C1s and inhibits the mannose-binding lectin-associated serine proteases in the lectin pathway. Thus, C1 INH is an ideal therapeutic for diseases of complement pathways. Nanofiltered plasma derived C1 INH (Cinryze®; ViroPharma) is FDA approved for routine prophylaxis against angioedema attacks in patients with hereditary angioedema (HAE), a disease characterized by constitutional deficiency or dysfunction of endogenous C1 INH. We hypothesized that this plasma-derived C1 INH would inhibit C3 deposition and the APC on PNH erythrocytes in patients treated with eculizumab.Peripheral blood of 3 healthy donors and 5 PNH patients (all with PNH type III erythrocyte proportion >5%) treated with eculizumab were obtained with written informed consent through a protocol approved by the local institutional review board. Clinical parameters for persistent extravascular hemolysis were noted at the time of patient sampling. Commercial vials of C1 INH were used for C1 inhibition assays ex vivo. Hemolysis experiments with acidified serum were performed in erythrocytes from PNH patients and healthy donors. We were able to show in the PNH patients that C1 inhibition is concentration dependent with increasing inhibition by serial dilutions of C1 INH up to 12 U/mL. The amount of hemolysis correlates with the percentage of CD55 deficient erythrocytes, but is attenuated by C1 INH in all patients. Flow cytometry was used to analyze deposition of C3 activation fragments on intact and lysed PNH erythrocytes. At time 0, small amounts of C3 deposition were observed on the PNH erythrocytes, but not the normal erythrocytes. The amount of C3 deposition was increased after incubation in acidified serum; however, co-incubation of C1 INH in the acidified serum markedly attenuated the amount of C3 deposition on the CD55 deficient erythrocytes. This demonstrated that C1 INH blocks APC-medicated deposition of C3 on the PNH erythrocytes in PNH patients.In this study, we show that the commercially available plasma derived C1-INH (Cinryze®) prevents lysis induced by the APC in PNH erythrocytes. The results presented here show that plasma-derived C1 INH can inhibit both APC- mediated hemolysis and C3 deposition. Importantly, C1-INH was able to block the accumulation of C3 degradation products on CD55 deficient erythrocytes from PNH patients who were being treated with eculizumab. This suggests a role for inhibition of earlier phases of the complement cascade than currently inhibited by eculizumab. A clinical trial to explore this hypothesis in vivo with patients who are suboptimal responders to eculizumab could be considered. Disclosures:Off Label Use: A nanofiltered plasma derived C1 INH (Cinryze®; ViroPharma) is FDA approved for routine prophylaxis against angioedema attacks in adolescent and adult patients with hereditary angioedema. Uknis:ViroPharma, Incorporated: Employment. Saye:ViroPharma, Incorporated: Employment. Brodsky:Alnylam Pharmaceuticals : Consultancy; Prolong Pharmaceuticals : Consultancy; ViroPharma, Incorporated: Research Funding; Alexion Pharmaceuticals : Membership on an entity's Board of Directors or advisory committees.

C3 Drives Inflammatory Skin Carcinogenesis Independently of C5

Nonmelanoma skin cancer such as cutaneous squamous cell carcinoma (cSCC) is the most common form of cancer and can occur as a consequence of DNA damage to the epithelium by UVR or chemical carcinogens. There is growing evidence that the complement system is involved in cancer immune surveillance; however, its role in cSCC remains unclear. Here, we show that complement genes are expressed in tissue from patients with cSCC, and C3 activation fragments are present in cSCC biopsies, indicating complement activation. Using a range of complement-deficient mice in a two-stage mouse model of chemically-induced cSCC, where a subclinical dose of 7,12-dimethylbenz[a]anthracene causes oncogenic mutations in epithelial cells and 12-O-tetradecanoylphorbol-13-acetate promotes the outgrowth of these cells, we found that C3-deficient mice displayed a significantly reduced tumor burden, whereas an opposite phenotype was observed in mice lacking C5aR1, C5aR2, and C3a receptor. In addition, in mice unable to form the membrane attack complex, the tumor progression was unaltered. C3 deficiency did not affect the cancer response to 7,12-dimethylbenz[a]anthracene treatment alone but reduced the epidermal hyperplasia during 12-O-tetradecanoylphorbol-13-acetate–induced inflammation. Collectively, these data indicate that C3 drives tumorigenesis during chronic skin inflammation, independently of the downstream generation of C5a or membrane attack complex.

Investigational RNAi Therapeutic Targeting C5 Is Efficacious in Pre-clinical Models of Myasthenia Gravis

Complement-mediated damage to the neuromuscular junction (NMJ) is a key mechanism of pathology in myasthenia gravis (MG), and therapeutics inhibiting complement have shown evidence of efficacy in the treatment of MG. In this study, we describe the development of a subcutaneously administered N-acetylgalactosamine (GalNAc)-conjugated small interfering RNA (siRNA) targeting the C5 component of complement that silences C5 expression in the liver (ALN-CC5). Treatment of wild-type rodents with ALN-CC5 resulted in robust and durable suppression of liver C5 expression. Dose-dependent serum C5 suppression was observed in non-human primates, with a lowering of serum C5 of up to 97.5% and the concomitant inhibition of serum complement activity. C5 silencing was efficacious in ameliorating disease symptoms in two standard rat models of MG, demonstrating the key role of circulating C5 in pathology at the NMJ. Improvement in disease activity scores and NMJ pathology was observed at intermediate levels of complement activity inhibition, suggesting that complete ablation of complement activity may not be required for efficacy in MG. The pre-clinical studies of ALN-CC5 and efficacy of C5 silencing in rat models of MG support further clinical development of ALN-CC5 as a potential therapeutic for the treatment of MG and other complement-mediated disorders.

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.

SPECT/CT Imaging of Mycobacterium tuberculosis Infection with [125I]anti-C3d mAb.

Diagnosis and therapeutic monitoring of chronic bacterial infection requires methods to detect and localize sites of infection accurately. Complement C3 activation fragments are generated and covalently bound to selective bacterial pathogens during the immune response and can serve as biomarkers of ongoing bacterial infection. We have developed several probes for detecting tissue-bound C3 deposits, including a monoclonal antibody (mAb 3d29) that recognizes the tissue-bound terminal processing fragments iC3b and C3d but does not recognize native circulating C3 or tissue-bound C3b. To determine whether mAb 3d29 could be used to detect chronic Mycobacterium tuberculosis infection non-invasively, aerosol-infected female C3HeB/FeJ mice were injected with [125I]3d29 mAb and either imaged using single-photon emission computed tomography (SPECT)/X-ray computed tomography (CT) imaging at 24 and 48h after radiotracer injection or being subjected to biodistribution analysis. Discrete lesions were detected by SPECT/CT imaging in the lungs and spleens of infected mice, consistent with the location of granulomas in the infected animals as detected by CT. Low-level signal was seen in the spleens of uninfected mice and no signal was seen in the lungs of healthy mice. Immunofluorescence microscopy revealed that 3d29 in the lungs of infected mice co-localized with aggregates of macrophages (detected with anti-CD68 antibodies). 3d29 was detected in the cytoplasm of macrophages, consistent with the location of internalized M. tuberculosis. 3d29 was also present within alveolar epithelial cells, indicating that it detected M. tuberculosis phagocytosed by other CD68-positive cells. Healthy controls showed very little retention of fluorescent or radiolabeled antibody across tissues. Radiolabeled 3d29 compared with radiolabeled isotype control showed a 3.5:1 ratio of increased uptake in infected lungs, indicating specific uptake by 3d29. 3d29 can be used to detect and localize areas of infection with M. tuberculosis non-invasively by 24h after radiotracer injection and with high contrast.

The secreted Candida albicans protein Pra1 disrupts host defense by broadly targeting and blocking complement C3 and C3 activation fragments

Candida albicans the most frequently isolated clinical fungal pathogen can cause local as well as systemic and life-threatening infections particularly in immune-compromised individuals. A better and more detailed understanding how C. albicans evades human immune attack is therefore needed for identifying fungal immune-evasive proteins and develop new therapies. Here, we identified Pra1, the pH-regulated C. albicans antigen as a hierarchical complement inhibitor that targets C3, the central human complement component. Pra1 cleaved C3 at a unique site and further inhibited effector function of the activation fragments. The newly formed C3a-like peptide lacked the C-terminal arginine residue needed for C3a-receptor binding and activation. Moreover, Pra1 also blocked C3a-like antifungal activity as shown in survival assays, and the C3b-like molecule formed by Pra1 was degraded by the host protease Factor I. Pra1 also bound to C3a and C3b generated by human convertases and blocked their effector functions, like C3a antifungal activity shown by fungal survival, blocked C3a binding to human C3a receptor-expressing HEK cells, activation of Fura2-AM loaded cells, intracellular Ca2+ signaling, IL-8 release, C3b deposition, as well as opsonophagocytosis and killing by human neutrophils. Thus, upon infection C. albicans uses Pra1 to destroy C3 and to disrupt host complement attack. In conclusion, candida Pra1 represents the first fungal C3-cleaving protease identified and functions as a fungal master regulator of innate immunity and as a central fungal immune-escape protein.

Elevated factor H–related protein 1 and factor H pathogenic variants decrease complement regulation in IgA nephropathy

IgA nephropathy (IgAN), a frequent cause of chronic kidney disease worldwide, is characterized by mesangial deposition of galactose-deficient IgA1-containing immune complexes. Complement involvement in IgAN pathogenesis is suggested by the glomerular deposition of complement components and the strong protection from IgAN development conferred by the deletion of the CFHR3 and CFHR1 genes (ΔCFHR3-CFHR1). Here we searched for correlations between clinical progression and levels of factor H (FH) and FH-related protein 1 (FHR-1) using well-characterized patient cohorts consisting of 112 patients with IgAN, 46 with non-complement-related autosomal dominant polycystic kidney disease (ADPKD), and 76 control individuals. Patients with either IgAN or ADPKD presented normal FH but abnormally elevated FHR-1 levels and FHR-1/FH ratios compared to control individuals. Highest FHR-1 levels and FHR-1/FH ratios are found in patients with IgAN with disease progression and in patients with ADPKD who have reached chronic kidney disease, suggesting that renal function impairment elevates the FHR-1/FH ratio, which may increase FHR-1/FH competition for activated C3 fragments. Interestingly, ΔCFHR3-CFHR1 homozygotes are protected from IgAN, but not from ADPKD, and we found five IgAN patients with low FH carrying CFH or CFI pathogenic variants. These data support a decreased FH activity in IgAN due to increased FHR-1/FH competition or pathogenic CFH variants. They also suggest that alternative pathway complement activation in patients with IgAN, initially triggered by galactose-deficient IgA1-containing immune complexes, may exacerbate in a vicious circle as renal function deterioration increase FHR-1 levels. Thus, a role of FHR-1 in IgAN pathogenesis is to compete with complement regulation by FH.

The intracellular C5 system is critical to DAMP sensing and cellular responses in human monocytes

Abstract Cholesterol crystal (CC)-induced inflammasome activation is a key contributor to atherosclerosis. Phagocytosis of CC causes lysosomal damage, assembly of the NLRP3 inflammasome, activation of caspase-1 and release of mature IL-1β. We have demonstrated that complement activation by CC in serum leads to C5a-C5aR1 driven CR3 up-regulation, increased uptake and IL1B gene expression in monocytes. Intracellular complement activation now emerges as critical regulator of cellular responses for induction of Th1 cells. In this study we investigated if CC-induced NLRP3 activation and IL-1β release in monocytes involves intracellular C5 activation. We observed that resting monocytes contained stores of C5 and C5a that increased upon CC uptake. Reduction of intracellular C5 by siRNA treatment abrogated IL-1β secretion to the same extent as NLRP3 inhibition. We confirmed the importance of the intracellular C5a-C5aR1 axis using monocytes from a serum C5-deficient patient, which produced intracellular C5 and detectable C5a and mounted equally strong IL-1β response when compared to healthy donor monocytes. Monocytes expressed also active Factors B and D and contained C3 and C3 activation fragments suggesting that an intracellular C5 convertase could mediate intracellular C5 activation. This notion is supported by the finding that knockdown of Factor B and usage of a cell-permeable convertase inhibitor both reduced CC-induced IL-1β. These results indicate that intracellular C3/C5 convertase-driven C5 activation is required for DAMP sensing, such as for CC, and suggest a critical role of intracellular complement in sterile inflammation and related disease states – and, importantly, that this novel axis can be pharmacologically targeted.

Complement Activation in a Murine Model of Sickle Cell Disease: Inhibition of Vaso-Occlusion By Blocking C5 Activation

▪IntroductionInnate immune complement activation may contribute to sickle cell disease (SCD) pathogenesis. The alternative complement pathway is abnormally activated in SCD and is additionally activated by phosphatidylserine (PS) on the outer leaflet of SS-red blood cells (SS-RBC). PS on the surface of SS-RBC and activated platelets accelerates the assembly of prothrombinase complexes leading to generation of thrombin, which can cleave circulating C5 protein into two biologically active fragments, C5a and C5b. 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 (VO). Active C5b fragments stimulate the formation of membrane attack complexes (MAC) on SS-RBC that increases their susceptibility to lysis. We hypothesize that complement activation on the surface of SS-RBC may stimulate VO and RBC turnover in SCD.Methods and ResultsWhole blood from Townes-AA, -AS, and -SS mice (n=4) was collected in EDTA and RBC were immunostained with erythroid specific anti-Ter119 (Ly-76) IgG conjugated to PE-Cy7, anti-C5b-9 (MAC) IgG conjugated to Alexa Fluor 647, and anti-C3 (also specific for activation fragments C3b, iC3b, C3d and C3dg) IgG conjugated to PE. The percentages of Ter119-positive RBC that were positive for MAC were 6.3%, 6.5%, and 26.0% for AA-, AS-, and SS-RBCs, respectively (p<0.01 SS vs. AA and AS) suggesting enhanced C5 activation in Townes-SS mice. However, EDTA plasma C5a levels measured by ELISA were not significantly different between Townes-AA, -AS and -SS mice. C3 activation fragments were also found on a subset of MAC positive RBC. We used a mouse model of hypoxia/reoxygenation (H/R)-induced stasis to investigate the role of complement in VO. Townes-SS mice with implanted dorsal skin-fold chambers were infused with 30 µg of either anti-C5 IgG mAb BB5.1 (which blocks murine C5 cleavage ) (n=4), annexin V (n=2), or with isotype control IgG (n=4) 30 minutes prior to H/R (1 hour of hypoxia at 7% O2, followed by reoxygenation for 4 hours in room air). Percent microvascular stasis (% non-flowing venules) in the subcutaneous venules in the dorsal skin fold chamber window was measured using intravital microscopy at 1 and 4 hours post-hypoxia. Percent stasis was significantly lower in the Townes-SS mice receiving anti-C5 IgG or annexin V than the mice receiving the isotype control IgG at 1 and 4 hours post-hypoxia (Figure 1, means ± SD, *p<0.01 anti-C5 IgG or annexin V vs. control IgG). This finding implies that H/R may acutely activate C5 , thus leading to stasis. Initial studies suggest increased plasma C5a levels in Towne-SS mice treated with isotype control IgG compared to mice treated with anti-C5 IgG in response to H/R.ConclusionsThese results demonstrate an increased percentage of SS-RBC expressing MAC on their surface and inhibition of H/R-induced stasis with anti-C5 IgG. However, circulating C5a levels were not different between untreated Townes-AA, -AS, or -SS mice suggesting that C5a is not chronically elevated in SCD. Rather, the generation of C5a may occur locally and in the acute setting. Current studies underway are examining the effects of anti-C5 IgG on MAC deposition and RBC half-lives; the mechanism(s) of complement activation in SCD mice (classical, alternative, tic-over, etc); the ability of recombinant murine C5a to induce stasis; and complement activation markers on RBC and in plasma from SCD patients and controls. [Display omitted] DisclosuresChen:Imara: Research Funding. Belcher:Imara: Research Funding; CSL-Behring: Research Funding. Vercellotti:Imara: Research Funding; CSL-Behring: Research Funding.

Candida albicans’ pH regulated antigen 1 (Pra1) preferentially targets and inactivates human complement C3 and C3 activation fragments to evade host immune attack

Candida albicans’ pH regulated antigen 1 (Pra1) preferentially targets and inactivates human complement C3 and C3 activation fragments to evade host immune attack

Complement system activation contributes to the ependymal damage induced by microbial neuraminidase.

BackgroundIn the rat brain, a single intracerebroventricular injection of neuraminidase from Clostridium perfringens induces ependymal detachment and death. This injury occurs before the infiltration of inflammatory blood cells; some reports implicate the complement system as a cause of these injuries. Here, we set out to test the role of complement.MethodsThe assembly of the complement membrane attack complex on the ependymal epithelium of rats injected with neuraminidase was analyzed by immunohistochemistry. Complement activation, triggered by neuraminidase, and the participation of different activation pathways were analyzed by Western blot. In vitro studies used primary cultures of ependymal cells and explants of the septal ventricular wall. In these models, ependymal cells were exposed to neuraminidase in the presence or absence of complement, and their viability was assessed by observing beating of cilia or by trypan blue staining. The role of complement in ependymal damage induced by neuraminidase was analyzed in vivo in two rat models of complement blockade: systemic inhibition of C5 by using a function blocking antibody and testing in C6-deficient rats.ResultsThe complement membrane attack complex immunolocalized on the ependymal surface in rats injected intracerebroventricularly with neuraminidase. C3 activation fragments were found in serum and cerebrospinal fluid of rats treated with neuraminidase, suggesting that neuraminidase itself activates complement. In ventricular wall explants and isolated ependymal cells, treatment with neuraminidase alone induced ependymal cell death; however, the addition of complement caused increased cell death and disorganization of the ependymal epithelium. In rats treated with anti-C5 and in C6-deficient rats, intracerebroventricular injection of neuraminidase provoked reduced ependymal alterations compared to non-treated or control rats. Immunohistochemistry confirmed the absence of membrane attack complex on the ependymal surfaces of neuraminidase-exposed rats treated with anti-C5 or deficient in C6.ConclusionsThese results demonstrate that the complement system contributes to ependymal damage and death caused by neuraminidase. However, neuraminidase alone can induce moderate ependymal damage without the aid of complement.

Functional assessment of mouse complement pathway activities and quantification of C3b/C3c/iC3b in an experimental model of mouse renal ischaemia/reperfusion injury

The complement system is an essential component of our innate immunity, both for the protection against infections and for proper handling of dying cells. However, the complement system can also contribute to tissue injury and inflammatory responses. In view of novel therapeutic possibilities, there is an increasing interest in measurement of the complement system activation in the systemic compartment, both in the clinical setting as well as in experimental models. Here we describe in parallel a sensitive and specific sandwich ELISA detecting mouse C3 activation fragments C3b/C3c/iC3b, as well as functional complement ELISAs detecting specific activities of the three complement pathways at the level of C3 and at the level of C9 activation. In a murine model of renal ischaemia/reperfusion injury (IRI) we found transient complement activation as shown by generation of C3b/C3c/iC3b fragments at 24h following reperfusion, which returned to base-line at 3 and 7days post reperfusion. When the pathway specific complement activities were measured at the level of C3 activation, we found no significant reduction in any of the pathways. However, the functional complement activity of all three pathways was significantly reduced when measured at the level of C9, with the strongest reduction being observed in the alternative pathway. For all three pathways there was a strong correlation between the amount of C3 fragments and the reduction in functional complement activity. Moreover, at 24h both C3 fragments and the functional complement activities showed a correlation with the rise in serum creatinine. Together our results show that determination of the systemic pathway specific complement activity is feasible in experimental mouse models and that they are useful in understanding complement activation and inhibition in vivo.

Preclinical Development and In Vivo Evaluation Of Next-Generation Compstatin Analogs With Improved Systemic Profiles: A Novel Option For The Treatment Of Paroxysmal Nocturnal Hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired orphan disease that is characterized by complement-mediated lysis of clonal populations of erythrocytes that lack glycosyl-phosphatidyl-inositol (GPI)-anchored complement regulators. Hemolysis and uncontrolled complement activation on affected platelets may contribute to the thrombophilia typical of PNH, which largely accounts for the morbidity and mortality of the disease. The introduction of the therapeutic antibody Eculizumab, which blocks activation of complement component C5, to the clinic has dramatically improved the treatment of PNH as it protects from hemolysis and possibly thromboembolisms; yet it also imposes high costs to the health care system. Moreover, while C5-targeted therapy efficiently impairs intravascular lysis of erythrocytes, it does not prevent their ongoing opsonization with activation fragments of complement component C3, thereby increasing the potential for extravascular lysis by immune cells. As a consequence, there is an unmet clinical need, and intervention at the level of C3 is expected to lead to a more comprehensive protection of PNH erythrocytes. Compstatin is a 13-residue disulfide-bridged peptide that selectively binds to human and primate forms of the central complement component C3 and its active fragment, thereby preventing the conversion of C3 to C3b which is essential for the functioning of all initiation, amplification, and terminal pathways of complement. Owing to their potent inhibitory activity of C3 activation, and due the comparatively low production costs of these peptidic drugs, compstatin analogs present an attractive option for the development of improved PNH therapeutics. Whereas early compstatin analogs are in clinical development for the local treatment of age-related macular degeneration, a long-term systemic use of compstatin for treating PNH places higher demands on this drug class. In this study, we therefore evaluated next-generation compstatin analogs with distinct pharmacokinetic properties concerning their efficacy and suitability for systemic administration. For this purpose, the lead analog Cp40 and a PEGylated derivative thereof were evaluated in complement activation assays, binding studies, and in a well-established ex vivo model of PNH using patient-derived erythrocytes. In addition, compstatin analogs were injected to non-human primates (cynomolgus monkeys) in order to determine administration strategies suitable for the treatment of PNH. Both Cp40 and its PEGylated derivatives prevented complement activation equally well, thereby indicating that N-terminal PEGylation does not substantially interfere with the activity of the peptide. Importantly, both compounds impaired deposition of C3 activation fragments on the surface of PNH erythrocytes as determined by FACS analysis and prevented their lysis with an IC50 of ∼4 µM. Pharmacokinetic profiling showed that PEGylation substantially improved the plasma half-life of Cp40 to more than 5 days whereas the non-PEGylated compound still exerted a value (t1/2 > 12 hours) that surpasses most peptide drugs. We therefore also evaluated repetitive subcutaneous injection of non-PEGylated Cp40 and could show that doses of 1 mg/kg every 12 hours were sufficient to maintain a therapeutic plasma level of compstatin in cynomolgus monkeys. Both strategies appear highly promising, and further evaluation of the in vivo efficacy and consideration of aspects such as production cost, treatment flexibility/control, and long-term plasma profiles will help determining the best route. Hence, our study provides critical evidence for the efficacy of compstatin analogs in the treatment of PNH, aiming to achieve a better control of all complement-mediated disease manifestations, reveals two suitable options for systemic administration of such peptidic complement inhibitors, and paves the way for a clinical application of this compound class in PNH. Disclosures:Ricklin:University of Pennsylvania: Patent application on Cp40, Patent application on Cp40 Patents & Royalties. Risitano:Alexion Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Lambris:Amyndas Pharmaceuticals: Equity Ownership; University of Pennsylvania: Patent application on Cp40 Patents & Royalties.

Detection of complement activation using monoclonal antibodies against C3d

During complement activation the C3 protein is cleaved, and C3 activation fragments are covalently fixed to tissues. Tissue-bound C3 fragments are a durable biomarker of tissue inflammation, and these fragments have been exploited as addressable binding ligands for targeted therapeutics and diagnostic agents. We have generated cross-reactive murine monoclonal antibodies against human and mouse C3d, the final C3 degradation fragment generated during complement activation. We developed 3 monoclonal antibodies (3d8b, 3d9a, and 3d29) that preferentially bind to the iC3b, C3dg, and C3d fragments in solution, but do not bind to intact C3 or C3b. The same 3 clones also bind to tissue-bound C3 activation fragments when injected systemically. Using mouse models of renal and ocular disease, we confirmed that, following systemic injection, the antibodies accumulated at sites of C3 fragment deposition within the glomerulus, the renal tubulointerstitium, and the posterior pole of the eye. To detect antibodies bound within the eye, we used optical imaging and observed accumulation of the antibodies within retinal lesions in a model of choroidal neovascularization (CNV). Our results demonstrate that imaging methods that use these antibodies may provide a sensitive means of detecting and monitoring complement activation-associated tissue inflammation.

Inhibition of biomaterial‐induced complement activation attenuates the inflammatory host response to implantation

Although complement is a known contributor to biomaterial-induced complications, pathological implications and therapeutic options remain to be explored. Here we investigated the involvement of complement in the inflammatory response to polypropylene meshes commonly used for hernia repair. In vitro assays revealed deposition of complement activation fragments on the mesh after incubation in plasma. Moreover, significant mesh-induced complement and granulocyte activation was observed in plasma and leukocyte preparations, respectively. Pretreatment of plasma with the complement inhibitor compstatin reduced opsonization >2-fold, and compstatin and a C5a receptor antagonist (C5aRa) impaired granulocyte activation by 50 and 67%, respectively. We established a clinically relevant mouse model of implantation and could confirm deposition of C3 activation fragments on mesh implants in vivo using immunofluorescence. In meshes extracted after subcutaneous or peritoneal implantation, the amount of immune cell infiltrate in mice deficient in key complement components (C3, C5aR), or treated with C5aRa, was approximately half of that observed in wild-type littermates or mice treated with inactive C5aRa, respectively. Our data suggest that implantation of a widely used surgical mesh triggers the formation of an inflammatory cell microenvironment at the implant site through complement activation, and indicates a path for the therapeutic modulation of implant-related complications.

Inhibition of biomaterial-induced complement activation attenuates the inflammatory host response to implantation

Although complement is a known contributor to biomaterial-induced complications, pathological implications and therapeutic options remain to be explored. Here we investigated the involvement of complement in the inflammatory response to polypropylene meshes commonly used for hernia repair. In vitro assays revealed deposition of complement activation fragments on the mesh after incubation in plasma. Moreover, significant mesh-induced complement and granulocyte activation was observed in plasma and leukocyte preparations, respectively. Pretreatment of plasma with the complement inhibitor compstatin reduced opsonization >2-fold, and compstatin and a C5a receptor antagonist (C5aRa) impaired granulocyte activation by 50 and 67%, respectively. We established a clinically relevant mouse model of implantation and could confirm deposition of C3 activation fragments on mesh implants in vivo using immunofluorescence. In meshes extracted after subcutaneous or peritoneal implantation, the amount of immune cell infiltrate in mice deficient in key complement components (C3, C5aR), or treated with C5aRa, was approximately half of that observed in wild-type littermates or mice treated with inactive C5aRa, respectively. Our data suggest that implantation of a widely used surgical mesh triggers the formation of an inflammatory cell microenvironment at the implant site through complement activation, and indicates a path for the therapeutic modulation of implant-related complications.

Detection of glomerular complement C3 fragments by magnetic resonance imaging in murine lupus nephritis

One of the challenges of treating patients with glomerulonephritis is to accurately assess disease activity. As renal biopsies are routinely stained for deposits of C3 activation fragments and glomerular C3 deposits are found in most forms of glomerulonephritis, we sought to determine whether a relatively noninvasive measure of C3 fragment deposition in the kidney can serve as a good biomarker of disease onset and severity. We recently developed a magnetic resonance imaging (MRI)-based method of detecting glomerular C3 and used this to track the progression of renal disease in the MRL/lpr mouse model of lupus nephritis using superparamagnetic iron oxide nanoparticles conjugated to complement receptor type 2 as a targeting agent. Quantitative immunofluorescence showed that glomerular C3b/iC3b/C3d deposition progressively increased with disease activity, a finding replicated by the T2-weighted MRI. The T2 relaxation times decreased with disease activity in the cortex and medulla of the MRL/lpr but not in MRL/Mpj control mice. Thus, MRI contrast agents targeted to glomerular C3 fragments can be used to noninvasively monitor disease activity in glomerulonephritis. As therapeutic complement inhibitors are used in patients with renal disease, this method, should it become feasible in humans, may identify those likely to benefit from complement inhibition.

Variable Antibody-dependent Activation of Complement by Functionalized Phospholipid Nanoparticle Surfaces

A wide variety of nanomaterials are currently being developed for use in the detection and treatment of human diseases. However, there is no systematic way to measure and predict the action of such materials in biological contexts. Lipid-encapsulated nanoparticles (NPs) are a class of nanomaterials that includes the liposomes, the most widely used and clinically proven type of NPs. Liposomes can, however, activate the complement system, an important branch of innate immunity, resulting in undesirable consequences. Here, we describe the complement response to lipid-encapsulated NPs that are functionalized on the surface with various lipid-anchored gadolinium chelates. We developed a quantitative approach to examine the interaction of NPs with the complement system using in vitro assays and correlating these results with those obtained in an in vivo mouse model. Our results indicate that surface functionalization of NPs with certain chemical structures elicits swift complement activation that is initiated by a natural IgM antibody and propagated via the classical pathway. The intensity of the response is dependent on the chemical structures of the lipid-anchored chelates and not zeta potential effects alone. Moreover, the extent of complement activation may be tempered by complement inhibiting regulatory proteins that bind to the surface of NPs. These findings represent a step forward in the understanding of the interactions between nanomaterials and the host innate immune response and provide the basis for a systematic structure-activity relationship study to establish guidelines that are critical to the future development of biocompatible nanotherapeutics.