Targeted Complement Inhibitor Research Articles

Reciprocal Interaction with Neutrophils Facilitates Cutaneous Accumulation of Liposomes.

Liposomes are versatile drug delivery systems in clinical use for cancer and many other diseases. Unfortunately, PEGylated liposomal doxorubicin (sLip/DOX) exhibits serious dose-limiting cutaneous toxicities, which are closely related to the extravascular accumulation of sLip/DOX in the dermis. No clinical interventions have been proposed for cutaneous toxicities due to the elusive transport pathways. Herein, we showed that the reciprocal interaction between liposomes and neutrophils played pivotal roles in liposome extravasation into the dermis. Neutrophils captured liposomes via the complement receptor 3 (CD11b/CD18) recognizing the fragment of complement component C3 (iC3b) deposited on the liposomal surface. Uptake of liposomes also activated neutrophils to induce CD11b upregulation and enhanced the ability of neutrophils to migrate outside the capillaries. Furthermore, inhibition of complement activation either by CRIg-L-FH (a C3b/iC3b targeted complement inhibitor) or blocking the phosphate negative charge in mPEG-DSPE could significantly reduce liposome uptake by neutrophils and alleviate the cutaneous accumulation of liposomes. These results validated the liposome extravasation pathway mediated by neutrophils and provided potential solutions to the devastating cutaneous toxicities occurring during sLip/DOX treatment.

Abstract WMP115: Rapid Vascular Complement Activation: A Key Driver of Blood-Brain Barrier Disruption in Ischemic Stroke With Acute Hyperglycemia

Background: Acute hyperglycemia, which occurs in over 40% of ischemic stroke patients irrespective of pre-existing diabetes, exacerbates stroke injury. Understanding its mechanisms is vital for innovative treatments. Here we investigate the role of complement in hyperglycemia-exacerbated damage after stroke. Method: Male C57/BL6 mice (10-11 weeks) underwent 30-min suture-induced middle cerebral artery occlusion followed by reperfusion, resembling clinical thrombectomy. Acute hyperglycemia was induced 10 min pre-stroke via glucose injection. Mice were sacrificed at 4.5h and 24h post-stroke to analyze brain swelling, blood-brain barrier (BBB) leakage and hemorrhagic transformation (HT). Mortality, neurological deficits, and motor-sensory functions were assessed at 14 days post-stroke. Complement activation was visualized via C3d immunostaining. Complement C3's role was examined using knock-out mice and the targeted complement inhibitor CR2-Crry fusion protein. Result: Hyperglycemia rapidly worsens BBB leakage (p<0.0001), brain swelling (p<0.05), and HT (p<0.0001) at 4.5 hr after stroke. Hyperglycemic stroke mice exhibit higher mortality (100% vs 25%, p=0.0008), body weight loss, and behavior impairment at the sub-acute phase compared to normoglycemic mice. Notably, acute hyperglycemia rapidly increased plasma C3 levels at 1hr after stroke (p<0.01), accompanied by rapid and time-dependent activation of C3 in ischemic brain vessels, as marked by increased vascular C3d; and this co-localized with autoantibody (IgM/IgG) vessel buildup and vessel leakage. Additionally, C3d levels positively correlated with brain swelling and HT (p<0.01). Vascular C3d and leakage into parenchyma were markedly reduced in the absence of reperfusion. Blocking complement via C3 knock-out or post-reperfusion systemic injection of C3 inhibitor CR2-Crry mitigates hyperglycemia's detrimental effects, including BBB leakage. Conclusion: Rapid vascular activation of complement C3 is a significant driver of hyperglycemic-exacerbated pathology in experimental stroke. Inhibiting C3 activation could be a potential therapeutic approach to improve hyperglycemic stroke outcome.

Thrombotic Microangiopathy After Hematopoietic Stem Cell and Solid Organ Transplantation: A Review for Intensive Care Physicians.

Intensive care physicians may assume the primary care of patients with transplant-associated thrombotic microangiopathy (TA-TMA), an uncommon but potentially critical complication of hematopoietic stem cell transplants (HSCTs) and solid organ transplants. TA-TMA can have a dramatic presentation with multiple organ dysfunction syndrome (MODS) associated with high morbidity and mortality. The typical presenting clinical features are hemolytic anemia, thrombocytopenia, refractory hypertension, proteinuria and worsening renal failure. Intestinal involvement, with abdominal pain, nausea and vomiting, gastrointestinal bleeding, and ascites are also common. Cardiopulmonary involvement may develop from various causes including pulmonary arteriolar hypertension, pleural and pericardial effusions, and diffuse alveolar hemorrhage. Due to other often concurrent complications after HSCT, early diagnosis and effective management of TA-TMA may be challenging. Close collaboration between ICU and transplant physicians, along with other relevant specialists, is needed to best manage these patients. There are currently no approved therapies for the treatment of TA-TMA. Plasma exchange and rituximab are not recommended unless circulating factor H (CFH) antibodies or thrombotic thrombocytopenic purpura (TTP; ADAMTS activity < 10%) are diagnosed or highly suspected. The role of the complement pathway activation in the pathophysiology of TA-TMA has led to the successful use of targeted complement inhibitors, such as eculizumab. However, the relatively larger studies using eculizumab have been mostly conducted in the pediatric population with limited data on the adult population. This review is focused on the role of intensive care physicians to emphasize the clinical approach to patients with suspected TA-TMA and to discuss diagnosis and treatment strategies.

#3074 CHANGES IN THE CLINICAL CHARACTERISTICS AND MANAGEMENT OF PATIENTS WITH AHUS OVER 10 YEARS: TRENDS FROM THE GLOBAL AHUS REGISTRY

Abstract Background and Aims Atypical haemolytic uraemic syndrome (aHUS) is a rare disease predominantly caused by alternative complement pathway dysregulation. Prior to 2011, before the targeted complement inhibitor eculizumab became available, aHUS frequently led to end-stage kidney disease (ESKD) and early death. Treatment with eculizumab led to notable improvements in outcomes; whether patient characteristics and management has continued to change over time is unknown. Using data from the Global aHUS Registry, we assessed clinical characteristics and management of patients with aHUS over 10 years to identify any potential trends. Method All patients enrolled in the Global aHUS Registry from 2012–2022 were included. Patients were categorised according to age at aHUS onset (adult [≥18 years] vs paediatric [&amp;lt;18 years]); onset of aHUS was defined as the earliest of initial symptom presentation, aHUS diagnosis, or first recorded thrombotic microangiopathy (TMA). Patient characteristics were summarised using descriptive analysis. Results Of the 1994 patients enrolled in the registry between 2012 and 2022, 33 (1.7%) were missing data on age at aHUS onset. Of the remaining 1961 patients, 813 (41.5%) were paediatric and 1148 (58.5%) were adult. Changes in patient characteristics and management are presented in Fig. 1. Plasma exchange/plasma infusion (PE/PI) prior to and including the year of enrolment was less common in paediatric than adult patients (58.5% vs. 71.3%) between 2012–2013 and declined substantially in paediatric (19.6%) relative to adult (55.1%) patients by 2020–2022. Similar proportions of paediatric patients required dialysis at the time of enrolment between 2012–2013 (5.1%) and 2020–2022 (5.9%), while numerically lower proportions of adults required dialysis over time (2012–2013, 15.2%; 2020–2022, 6.5%). The rate of paediatric patients requiring a kidney transplant prior and up to enrolment dropped from 2012–2013 (28.4%) to 2020–2022 (5.9%); however, the rate among adult patients remained comparable (2012–2013, 29.2%; 2020–2022, 27.1%). A decrease in the proportion of patients with a reported identified pathogenic variant and/or anti-CFH antibodies was observed in paediatric and adult patients between 2012–2013 (paediatric, 59.1%; adult, 45.5%) and 2020–2022 (paediatric, 49.0%; adult, 30.8%). The proportion of patients with a reported triggering event increased from 2012–2013 (paediatric, 6.3%; adult, 12.4%) to 2020–2022 (paediatric, 9.8%; adult, 28.0%). In this cohort, a total of 1208 patients were treated with eculizumab/ravulizumab between 2012 and 2022 (paediatrics, 476; adults, 732). Between 2012–2013, the median time from aHUS onset to treatment initiation was 94.9 days for paediatric patients (blue) and 51.1 days for adult patients (orange); this fell to 13.1 days in paediatrics and 24.1 days in adults between 2020–2022 (Fig. 2). Conclusion Utilisation of PE/PI to treat aHUS has decreased more in paediatric than adult patients over time. A decreased requirement for transplant in paediatric patients was evident, suggesting more paediatrics are now being diagnosed and treated earlier, leading to better outcomes with fewer patients progressing to renal failure and requiring kidney transplant. The proportion of both paediatric and adult patients with reported pathogenic variants and/or anti-CFH antibodies decreased over time and may reflect the rate at which a clinical constellation consistent with aHUS is identified. Moreover, aHUS may be recognised more frequently in the presence of a triggering condition, or a lower barrier for clinicians to suspect aHUS in recent years. Patients were more rapidly treated with a complement inhibitor, likely due to better awareness in the clinical community around complement inhibitors and the importance of early treatment initiation. Although patients with aHUS are being treated more promptly in recent years than 10 years ago, more work is needed, especially in adults, to move towards optimal clinical practice.

Abstract WP225: Rapid Complement Activation Induced By Acute Hyperglycemia Worsens Ischemic Stroke Outcome

Background: Acute hyperglycemia, which occurs in over 40% of ischemic stroke patients regardless of pre-existing diabetes, increases brain edema, hemorrhagic transformation (HT) and worsens stroke outcome. Understanding the mechanisms of hyperglycemia-exacerbated stroke injury will be vital for developing novel treatments. Here we identify systemic complement activation as a novel contributor to hyperglycemic-exacerbated damage in rodent stroke. Method: Male C57/BL6 mice (10-11 weeks) were subjected to middle cerebral artery occlusion (MCAO) for 30 min, followed by reperfusion to mimic ischemic stroke. Acute hyperglycemia was induced by glucose injection 10 min before MCAO. To assess the effect of hyperglycemia on stroke outcomes, mice were sacrificed at 4.5 and 24 hr post-stroke to analyze brain edema, blood-brain barrier (BBB) leakage, and HT; or allowed to survive 14 days to examine mortality rate, neurological deficit, and motor-sensory dysfunction using the horizontal rotating beam test. Complement activation was evaluated at 1, 2, 4.5 hr post-stroke. To inhibit C3 activation, the targeted complement inhibitor CR2-Crry fusion protein, or PBS control, was injected via femoral vein 15 min after reperfusion. Result: Hyperglycemia worsens stroke outcomes in our mouse model, with rapid increased BBB leakage (p&lt;0.0001), brain swelling (p&lt;0.001) and HT at (p&lt;0.0001) 4.5 and 24 hr after stroke compared to normoglycemia; and poorer prognosis including higher mortality (100% VS 25%, p=0.0008), body weight loss, and impaired behavioral performance by day 14. Interestingly, acute hyperglycemia rapidly increased plasma complement C3 levels at 1 and 2 hr after stroke (p&lt;0.01 compared to normoglycemic mice), accompanied by activation of C3 in ischemic brain vessels as indicated by elevated levels of vascular C3d. Vascular C3d colocalized with IgM/IgG, suggesting the involvement of classical pathway in C3 activation. Notably, activated C3 levels positively correlated with brain swelling and HT (p&lt;0.01). Inhibition of C3 by CR2-Crry markedly reduced brain swelling (p&lt;0.05) and HT in hyperglycemic stroke. Conclusion: Rapid activation of complement C3 by acute hyperglycemia may be an important contributor to BBB leakage, brain swelling and HT.

A Targeted Complement Inhibitor CRIg/FH Protects Against Experimental Autoimmune Myasthenia Gravis in Rats via Immune Modulation

Antibody-induced complement activation may cause injury of the neuromuscular junction (NMJ) and is thus considered as a primary pathogenic factor in human myasthenia gravis (MG) and animal models of experimental autoimmune myasthenia gravis (EAMG). In this study, we tested whether CRIg/FH, a targeted complement inhibitor, could attenuate NMJ injury in rat MG models. We first demonstrated that CRIg/FH could inhibit complement-dependent cytotoxicity on human rhabdomyosarcoma TE671 cells induced by MG patient-derived IgG in vitro. Furthermore, we investigated the therapeutic effect of CRIg/FH in a passive and an active EAMG rodent model. In both models, administration of CRIg/FH could significantly reduce the complement-mediated end-plate damage and suppress the development of EAMG. In the active EAMG model, we also found that CRIg/FH treatment remarkably reduced the serum concentration of autoantibodies and of the cytokines including IFN-γ, IL-2, IL-6, and IL-17, and upregulated the percentage of Treg cells in the spleen, which was further verified in vitro. Therefore, our findings indicate that CRIg/FH may hold the potential for the treatment of MG via immune modulation.

Characterization of Novel P-Selectin Targeted Complement Inhibitors in Murine Models of Hindlimb Injury and Transplantation.

The complement system has long been recognized as a potential druggable target for a variety of inflammatory conditions. Very few complement inhibitors have been approved for clinical use, but a great number are in clinical development, nearly all of which systemically inhibit complement. There are benefits of targeting complement inhibition to sites of activation/disease in terms of efficacy and safety, and here we describe P-selectin targeted complement inhibitors, with and without a dual function of directly blocking P-selectin-mediated cell-adhesion. The constructs are characterized in vitro and in murine models of hindlimb ischemia/reperfusion injury and hindlimb transplantation. Both constructs specifically targeted to reperfused hindlimb and provided protection in the hindlimb ischemia/reperfusion injury model. The P-selectin blocking construct was the more efficacious, which correlated with less myeloid cell infiltration, but with similarly reduced levels of complement deposition. The blocking construct also improved tissue perfusion and, unlike the nonblocking construct, inhibited coagulation, raising the possibility of differential application of each construct, such as in thrombotic vs. hemorrhagic conditions. Similar outcomes were obtained with the blocking construct following vascularized composite graft transplantation, and treatment also significantly increased graft survival. This is outcome may be particularly pertinent in the context of vascularized composite allograft transplantation, since reduced ischemia reperfusion injury is linked to a less rigorous alloimmune response that may translate to the requirement of a less aggressive immunosuppressive regime for this normally nonlife-threatening procedure. In summary, we describe a new generation of targeted complement inhibitor with multi-functionality that includes targeting to vascular injury, P-selectin blockade, complement inhibition and anti-thrombotic activity. The constructs described also bound to both mouse and human P-selectin which may facilitate potential translation.

Complement Inhibition Targeted to Injury Specific Neoepitopes Attenuates Atherogenesis in Mice.

Rationale: Previous studies have indicated an important role for complement in atherosclerosis, a lipid-driven chronic inflammatory disease associated to oxidative stress in the vessel wall. However, it remains unclear how complement is activated in the process of atherogenesis. An accepted general model for complement activation in the context of ischemia reperfusion injury is that ischemia induces the exposure of neoepitopes that are recognized by natural self-reactive IgM antibodies, and that in turn activate complement.Objective: We investigated whether a similar phenomenon may be involved in the pathogenesis of atherosclerosis, and whether interfering with this activation event, together with inhibition of subsequent amplification of the cascade at the C3 activation step, can provide protection against atherogenesis.Methods and Results: We utilized C2scFv-Crry, a novel construct consisting of a single chain antibody (scFv) linked to Crry, a complement inhibitor that functions at C3 activation. The scFv moiety was derived from C2 IgM mAb that specifically recognizes phospholipid neoepitopes known to be expressed after ischemia. C2scFv-Crry targeted to the atherosclerotic plaque of Apoe−/− mice, demonstrating expression of the C2 neoepitope. C2scFv-Crry administered twice per week significantly attenuated atherosclerotic plaque in the aorta and aortic root of Apoe−/− mice fed with a high-fat diet (HFD) for either 2 or 4 months, and treatment reduced C3 deposition and membrane attack complex formation as compared to vehicle treated mice. C2scFv-Crry also inhibited the uptake of oxidized low-density-lipoprotein (oxLDL) by peritoneal macrophages, which has been shown to play a role in pathogenesis, and C2scFv-Crry-treated mice had decreased lipid content in the lesion with reduced oxLDL levels in serum compared to vehicle-treated mice. Furthermore, C2scFv-Crry reduced the deposition of endogenous total IgM in the plaque, although it did not alter serum IgM levels, further indicating a role for natural IgM in initiating complement activation.Conclusion: Neoepitope targeted complement inhibitors represent a novel therapeutic approach for atherosclerosis.

In Situ Pre-Treatment of Vascularized Composite Allografts With a Targeted Complement Inhibitor Protects Against Brain Death and Ischemia Reperfusion Induced Injuries.

IntroductionDonor brain death (BD) is an unavoidable component of vascularized composite allograft (VCA) transplantation and a key contributor to ischemia-reperfusion injury (IRI). Complement is activated and deposited within solid organ grafts as a consequence of BD and has been shown to exacerbate IRI, although the role of BD and complement in VCA and the role it plays in IRI and VCA rejection has not been studied.MethodsBD was induced in Balb/c donors, and the VCA perfused prior to graft procurement with UW solution supplemented with or without CR2-Crry, a C3 convertase complement inhibitor that binds at sites of complement activation, such as that induced on the endothelium by induction of BD. Following perfusion, donor VCAs were cold stored for 6 hours before transplantation into C57BL/6 recipients. Donor VCAs from living donors (LD) were also procured and stored. Analyses included CR2-Crry graft binding, complement activation, toxicity, injury/inflammation, graft gene expression and survival.ResultsCompared to LD VCAs, BD donor VCAs had exacerbated IRI and rejected earlier. Following pretransplant in-situ perfusion of the donor graft, CR2-Crry bound within the graft and was retained post-transplantation. CR2-Crry treatment significantly reduced complement deposition, inflammation and IRI as compared to vehicle-treated BD donors. Treatment of BD donor VCAs with CR2-Crry led to an injury profile not dissimilar to that seen in recipients of LD VCAs.ConclusionPre-coating a VCA with CR2-Crry in a clinically relevant treatment paradigm provides localized, and therefore minimally immunosuppressive, protection from the complement-mediated effects of BD induced exacerbated IRI.

Chronic complement dysregulation drives neuroinflammation after traumatic brain injury: a transcriptomic study

Activation of the complement system propagates neuroinflammation and brain damage early and chronically after traumatic brain injury (TBI). The complement system is complex and comprises more than 50 components, many of which remain to be characterized in the normal and injured brain. Moreover, complement therapeutic studies have focused on a limited number of histopathological outcomes, which while informative, do not assess the effect of complement inhibition on neuroprotection and inflammation in a comprehensive manner. Using high throughput gene expression technology (NanoString), we simultaneously analyzed complement gene expression profiles with other neuroinflammatory pathway genes at different time points after TBI. We additionally assessed the effects of complement inhibition on neuropathological processes. Analyses of neuroinflammatory genes were performed at days 3, 7, and 28 post injury in male C57BL/6 mice following a controlled cortical impact injury. We also characterized the expression of 59 complement genes at similar time points, and also at 1- and 2-years post injury. Overall, TBI upregulated the expression of markers of astrogliosis, immune cell activation, and cellular stress, and downregulated the expression of neuronal and synaptic markers from day 3 through 28 post injury. Moreover, TBI upregulated gene expression across most complement activation and effector pathways, with an early emphasis on classical pathway genes and with continued upregulation of C2, C3 and C4 expression 2 years post injury. Treatment using the targeted complement inhibitor, CR2-Crry, significantly ameliorated TBI-induced transcriptomic changes at all time points. Nevertheless, some immune and synaptic genes remained dysregulated with CR2-Crry treatment, suggesting adjuvant anti-inflammatory and neurotropic therapy may confer additional neuroprotection. In addition to characterizing complement gene expression in the normal and aging brain, our results demonstrate broad and chronic dysregulation of the complement system after TBI, and strengthen the view that the complement system is an attractive target for TBI therapy.

A Novel Natural Autoantibody Targeted Complement Inhibitor Protects against Lung Transplant Ischemia Reperfusion Injury

Purpose Chronic obstructive pulmonary disease is characterized by chronic inflammation and severe lung parenchyma damage that is in part driven by autoimmunity. Lung specific autoreactive antibodies (AAb) are indicative of disease progression and severity. The purpose of this study was to elucidate how pre-existing AAb contribute to ischemia reperfusion injury (IRI) and graft rejection following lung transplant (LTx). We hypothesize that pre-existing AAb bind cryptic antigens exposed during IRI which exacerbates lung graft injury in a complement (C)-dependent pathway. Methods We generated a clinically relevant cigarette smoke (CS)-induced mouse model that exhibits increased serum AAb compared to non-smoke exposed age-matched (NS) controls. We analyzed the impact of a full pre-existing AAb repertoire by creating an orthotopic syngeneic left lung LTx model in which we transplanted the left lungs from age-matched donor mice into NS and CS mice. Furthermore, we developed a novel bifunctional inhibitor that blocks IgM binding and inhibits complement. Results CS exposure significantly increased graft injury and immune infiltration 48 hours after LTx. Syngeneic LTx into Rag−/- mice reconstituted with serum from NS and CS mice demonstrated that AAb present in CS are responsible for the observed graft injury. Immunofluorescent staining showed that IgM/IgG binding was increased in CS recipients and colocalized with C3d deposition. These data suggested that AAb-mediated graft injury may occur via the C pathway. To elucidate the role of C in AAb-mediated graft injury, we treated CS LTx recipients with a novel bifunctional C inhibitor, C2scFv-Crry, that blocks IgM binding and C activation. C2scFv-Crry treatment following LTx dramatically reduced acute graft injury and immune infiltration, and significantly decreased de novo AAb production compared to CS control LTx mice. Conclusion Ultimately, these data reveal that CS induces pre-existing AAb production that exacerbate IRI following LTx in a C-dependent manner. Furthermore, we demonstrate the efficacy of a novel C inhibitor in reducing IRI and acute graft injury.

A novel injury site-natural antibody targeted complement inhibitor protects against lung transplant injury.

Complement is known to play a role in ischemia and reperfusion injury (IRI). A general paradigm is that complement is activated by self-reactive natural IgM antibodies (nAbs), after they engage postischemic neoepitopes. However, a role for nAbs in lung transplantation (LTx) has not been explored. Using mouse models of LTx, we investigated the role of two postischemic neoepitopes, modified annexin IV (B4) and a subset of phospholipids (C2), in LTx. Antibody deficient Rag1-/- recipient mice were protected from LTx IRI. Reconstitution with either B4 or C2nAb restored IRI, with C2 significantly more effective than B4 nAb. Based on these information, we developed/characterized a novel complement inhibitor composed of single-chain antibody (scFv) derived from the C2 nAb linked to Crry (C2scFv-Crry), a murine inhibitor of C3 activation. Using an allogeneic LTx, in which recipients contain a full nAb repertoire, C2scFv-Crry targeted to the LTx, inhibited IRI, and delayed acute rejection. Finally, we demonstrate the expression of the C2 neoepitope in human donor lungs, highlighting the translational potential of this approach.

Ex vivo assays to detect complement activation in complementopathies

In complement-driven thrombotic microangiopathies, failure to regulate complement activation leads to end-organ damage. The modified Ham (mHam) test measures complement-mediated killing of a nucleated cell in vitro but lacks a confirmatory assay and reliable positive controls. We demonstrate that C5b-9 accumulation on the surface of TF1 PIGAnull cells correlates with cell killing in the mHam. We also show that Sialidase treatment of cells or addition of Shiga toxin 1 to human serum serve as a more reliable positive control for the mHam than cobra venom factor or lipopolysaccharide. Simultaneously performing the mHam and measuring C5b-9 accumulation either in GVB++ or GVB0 MgEGTA buffer with the addition of complement pathway specific inhibitors (anti-C5 antibody or a factor D inhibitor, ACH-145951) can be used to localize defects in complement regulation. As more targeted complement inhibitors become available, these assays may aid in the selection of personalized treatments for patients with complement-mediated diseases.

Complement Inhibitor CRIg/FH Ameliorates Renal Ischemia Reperfusion Injury via Activation of PI3K/AKT Signaling.

Complement activation is involved in the pathogenesis of ischemia reperfusion injury (IRI), which is an inevitable process during kidney transplantation. Therefore, complement-targeted therapeutics hold great potential in protecting the allografts from IRI. We observed universal deposition of C3d and membrane attack complex in human renal allografts with delayed graft function or biopsy-proved rejection, which confirmed the involvement of complement in IRI. Using FB-, C3-, C4-, C5-, C5aR1-, C5aR2-, and C6-deficient mice, we found that all components, except C5aR2 deficiency, significantly alleviated renal IRI to varying degrees. These gene deficiencies reduced local (deposition of C3d and membrane attack complex) and systemic (serum levels of C3a and C5a) complement activation, attenuated pathological damage, suppressed apoptosis, and restored the levels of multiple local cytokines (e.g., reduced IL-1β, IL-9, and IL-12p40 and increased IL-4, IL-5, IL-10, and IL-13) in various gene-deficient mice, which resulted in the eventual recovery of renal function. In addition, we demonstrated that CRIg/FH, which is a targeted complement inhibitor for the classical and primarily alternative pathways, exerted a robust renoprotective effect that was comparable to gene deficiency using similar mechanisms. Further, we revealed that PI3K/AKT activation, predominantly in glomeruli that was remarkably inhibited by IRI, played an essential role in the CRIg/FH renoprotective effect. The specific PI3K antagonist duvelisib almost completely abrogated AKT phosphorylation, thus abolishing the renoprotective role of CRIg/FH. Our findings suggested that complement activation at multiple stages induced renal IRI, and CRIg/FH and/or PI3K/AKT agonists may hold the potential in ameliorating renal IRI.

Identifying the Role of Complement in Triggering Neuroinflammation after Traumatic Brain Injury.

The complement system is implicated in promoting acute secondary injury after traumatic brain injury (TBI), but its role in chronic post-traumatic neuropathology remains unclear. Using various injury-site targeted complement inhibitors that block different complement pathways and activation products, we investigated how complement is involved in neurodegeneration and chronic neuroinflammation after TBI in a clinically relevant setting of complement inhibition. The current paradigm is that complement propagates post-TBI neuropathology predominantly through the terminal membrane attack complex (MAC), but the focus has been on acute outcomes. Following controlled cortical impact in adult male mice, we demonstrate that although inhibition of the MAC (with CR2-CD59) reduces acute deficits, inhibition of C3 activation is required to prevent chronic inflammation and ongoing neuronal loss. Activation of C3 triggered a sustained degenerative mechanism of microglial and astrocyte activation, reduced dendritic and synaptic density, and inhibited neuroblast migration several weeks after TBI. Moreover, inhibiting all complement pathways (with CR2-Crry), or only the alternative complement pathway (with CR2-fH), provided similar and significant improvements in chronic histological, cognitive, and functional recovery, indicating a key role for the alternative pathway in propagating chronic post-TBI pathology. Although we confirm a role for the MAC in acute neuronal loss after TBI, this study shows that upstream products of complement activation generated predominantly via the alternative pathway propagate chronic neuroinflammation, thus challenging the current concept that the MAC represents a therapeutic target for treating TBI. A humanized version of CR2fH has been shown to be safe and non-immunogenic in clinical trials.SIGNIFICANCE STATEMENT Complement, and specifically the terminal membrane attack complex, has been implicated in secondary injury and neuronal loss after TBI. However, we demonstrate here that upstream complement activation products, generated predominantly via the alternative pathway, are responsible for propagating chronic inflammation and injury following CCI. Chronic inflammatory microgliosis is triggered by sustained complement activation after CCI, and is associated with chronic loss of neurons, dendrites and synapses, a process that continues to occur even 30 d after initial impact. Acute and injury-site targeted inhibition of the alternative pathway significantly improves chronic outcomes, and together these findings modify the conceptual paradigm for targeting the complement system to treat TBI.

Targeted Complement Inhibition Protects Vascularized Composite Allografts From Acute Graft Injury and Prolongs Graft Survival When Combined With Subtherapeutic Cyclosporine A Therapy.

Recipients of vascularized composite allografts require aggressive and lifelong immunosuppression, and because the surgery is usually performed in nonlife-threatening situations, the development of strategies to minimize immunosuppression is especially pertinent for this procedure. We investigated how complement affects acute graft injury, alloimmunity, and immunosuppressive therapy. Vascularized composite allografts were transplanted from Balb/C to C57BL/6 mice that were complement deficient (C3 or double C3a Receptor (R)/C5aR), or treated with a targeted complement inhibitor (CR2-Crry). Allografts were analyzed for acute inflammation and injury, subacute T cell response, and survival in the absence and presence of cyclosporine A (CsA) therapy. Allografts in C3-deficient or CR2-Crry-treated recipients were protected from skin and muscle ischemia-reperfusion injury (IRI). C3aR/C5aR-deficient recipients were more modestly protected. IgM and C3d colocalized within allografts from wild type and C3aR/C5aR-deficient recipients indicating IgM-mediated complement activation, and C3d deposition was almost absent in allografts from C3-deficient and CR2-Crry-treated recipients. Inflammatory cell infiltration and P-selectin expression was also significantly reduced in C3-deficient and CR2-Crry-treated recipients. Acute treatment with CR2-Crry or with 3 mg/kg per day CsA modestly, but significantly increased median allograft survival from 5.8 to 7.4 and 7.2 days, respectively. However, combined acute CR2-Crry treatment and CsA therapy increased mean graft survival to 17.2 days. Protection was associated with significantly reduced T cell infiltration of allografts and Tc1 cells in recipient spleens. Complement-mediated IRI augments graft allogenicity, and appropriate complement inhibition ameliorates IRI, decreases alloimmune priming and allows more immune-sparing CsA dosing.

Injury site-specific targeting of complement inhibitors for treating stroke.

Cumulative evidence indicates a role for the complement system in both pathology and recovery after ischemic stroke. Here, we review the current understanding of the dual role of complement in poststroke injury and recovery, and discuss the challenges of anti-complement therapies. Most complement directed therapeutics currently under investigation or development systemically inhibit the complement system, but since complement is important for immune surveillance and is involved in various homeostatic activities, there are potential risks associated with systemic inhibition. Depending on the target within the complement pathway, other concerns are high concentrations of inhibitor required, low efficacy and poor bioavailability. To overcome these limitations, approaches to target complement inhibitors to specific sites have been investigated. Here, we discuss targeting strategies, with a focus on strategies developed in our lab, to specifically localize complement inhibition to sites of tissue injury and complement activation, and in particular to the postischemic brain. We discuss various injury site-specific targeted complement inhibitors as potential therapeutic agents for the treatment of ischemic stroke treatment, as well as their use as investigative tools for probing complement-dependent pathophysiological processes.

Selectivity of C3-opsonin targeted complement inhibitors: A distinct advantage in the protection of erythrocytes from paroxysmal nocturnal hemoglobinuria patients

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by complement-mediated cell lysis due to deficiency of GPI-anchored complement regulators. Blockage of the lytic pathway by eculizumab is the only available therapy for PNH patients and shows remarkable benefits, but regularly yields PNH erythrocytes opsonized with fragments of complement protein C3, rendering such erythrocytes prone to extravascular hemolysis. This effect is associated with insufficient responsiveness seen in a subgroup of PNH patients. Novel C3-opsonin targeted complement inhibitors act earlier in the cascade, at the level of activated C3 and are engineered from parts of the natural complement regulator Factor H (FH) or complement receptor 2 (CR2). This inhibitor class comprises three variants of “miniFH” and the clinically developed “FH-CR2” fusion-protein (TT30). We show that the approach of FH-CR2 to target C3-opsonins was more efficient in preventing complement activation induced by foreign surfaces, whereas the miniFH variants were substantially more active in controlling complement on PNH erythrocytes. Subtle differences were noted in the ability of each version of miniFH to protect human PNH cells. Importantly, miniFH and FH-CR2 interfered only minimally with complement-mediated serum killing of bacteria when compared to untargeted inhibition of all complement pathways by eculizumab. Thus, the molecular design of each C3-opsonin targeted complement inhibitor determines its potency in respect to the nature of the activator/surface providing potential functionality in PNH.

Safety and Pharmacokinetics of the Complement Inhibitor TT30 in a Phase I Trial for Untreated PNH Patients

▪TT30 is a novel, recombinant human fusion protein, linking the C3d-binding domain of human complement receptor 2 (CR2) with the complement regulatory domain of human factor H (fH). TT30 is designed to inhibit the complement alternative pathway (CAP) at sites of local activation; it has been shown to be effective in preventing hemolysis and C3b-mediated opsonization in paroxysmal nocturnal hemoglobinuria (PNH) in vitro. This first in human study, TT30-PNH-002, was a multi-center open-label, single-dose, dose-escalation study of two formulations of TT30, ALXN1102 (10mg/ml) and ALXN1103 (50mg/ml). The study was conducted in patients with a diagnosis of PNH, defined as a PNH clone size of ≥10% or a PNH clone size between 5% and 10% with evidence of intravascular hemolysis documented within the previous 12 months; patients were not receiving concurrent treatment or treatment with a complement inhibitor within 60 days prior to screening. The primary endpoint of the study was assessment of the safety and tolerability of a single dose of TT30 when administered by IV infusion or separately by SC injection; secondary endpoints included pharmacokinetics (PK), pharmacodynamics (PD), and immunogenicity. The ALXN1102 formulation was administered by IV infusion to the 0.1 (n=1), 0.3 (n=1), and 1 mg/kg (n=3) dose level cohorts. The ALXN1103 formulation was administered by IV infusion to the 3 mg/kg (n=1) dose level cohort and by SC injection to the 1 (n=2) and 3 mg/kg (n=2) dose level cohorts. Patients were dosed one at a time, and each patient was evaluated before the next patient could be dosed. All 10 patients completed the study as planned; across all groups, 28 mild and 5 moderate treatment-emergent adverse event (TEAEs) were experienced. Treatment with both formulations of TT30 was considered safe, with no apparent dose-related safety risks. One patient who received ALXN1102 at 1.0 mg/kg IV experienced transient hypocalcemia associated with transient asymptomatic prolongation of the QT interval, both of which spontaneously resolved by 24 hours without intervention and may have been related to citrate in the formulation. Subsequent patients supplemented with oral calcium at the start of the infusion did not experience these events, nor were citrate-related toxicities seen in patients who received ALXN1103, which contains a lower amount of citrate. The secondary endpoints of the study were to characterize the PK of a single dose of TT30, to assess CAP, complement classical pathway (CCP) inhibition, PD effects on markers of intravascular hemolysis such as lactate dehydrogenase (LDH), and to characterize the immunogenicity of TT30. Serum TT30 total and peak (AUCs and Cmax) exposures increased with increase in IV or SC dose. Serum CAP activity declined in a dose-dependent manner following dosing in all IV dose cohorts, and approached zero % of baseline value (complete CAP inhibition) in the 1 mg/kg and 3 mg/kg IV dosing cohorts, when circulating levels of TT30 were in the range of approximately 5-10 ug/ml. CAP activity returned to baseline by 24 hours, when circulating TT30 was below 1 ug/ml, and appeared to remain stable thereafter. Serum CCP activity remained near baseline in all patients at all times. LDH levels dropped by 20% in the 1 mg/kg and 3 mg/kg IV dose cohorts and rose over time after CAP activity returned to baseline. Similarly, there was a decline in serum CAP activity and LDH levels in the 1 mg/kg and 3 mg/kg SC dose cohorts that returned to pre-treatment baseline over time, by 24 hours. No immunogenicity was observed in any patient, following either IV or SC dosing of a single dose of TT30. In conclusion, both TT30 formulations were safe and well tolerated; preliminary PK and PD data support the concept that treatment by targeted complement inhibitors like TT30 result in pharmacologically relevant CAP inhibition in PNH, as demonstrated by LDH decrease. Nevertheless, such therapeutic inhibition is short-lasting, and further strategies are needed to sustain pharmacological levels of TT30 for chronic treatment in PNH. DisclosuresRisitano:Alnylam: Consultancy, Research Funding; Novartis: Research Funding; Pfizer: Consultancy; Alexion Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Rapharma: Consultancy, Research Funding. Off Label Use: The presentation will include discussion of a clinical trial with a non-approved medication. Storek:Alexion Pharmaceuticals, Inc.: Employment, Other: stock owner. Sahelijo:Alexion Pharmaceuticals: Employment, Equity Ownership. Doyle:Alexion Pharmaceuticals, Inc.: Employment, Other: stock owner. Dai:Alexion Pharmaceuticals, Inc.: Employment. Weitz:Alexion Pharmaceuticals, Inc.: Consultancy, Speakers Bureau. Elebute:Alexion Pharmaceuticals, Inc.: Speakers Bureau. O'Connell:Celgene: Speakers Bureau; Incyte Corporation: Membership on an entity's Board of Directors or advisory committees; Portola: Membership on an entity's Board of Directors or advisory committees. Kulasekararaj:Alexion: Consultancy. Lundberg:Alexion Pharmaceuticals, Inc.: Employment.

An anticomplement agent that homes to the damaged brain and promotes recovery after traumatic brain injury in mice

Activation of complement is a key determinant of neuropathology and disability after traumatic brain injury (TBI), and inhibition is neuroprotective. However, systemic complement is essential to fight infections, a critical complication of TBI. We describe a targeted complement inhibitor, comprising complement receptor of the Ig superfamily (CRIg) fused with complement regulator CD59a, designed to inhibit membrane attack complex (MAC) assembly at sites of C3b/iC3b deposition. CRIg and CD59a were linked via the IgG2a hinge, yielding CD59-2a-CRIg dimer with increased iC3b/C3b binding avidity and MAC inhibitory activity. CD59-2a-CRIg inhibited MAC formation and prevented complement-mediated lysis in vitro. CD59-2a-CRIg dimer bound C3b-coated surfaces with submicromolar affinity (KD). In experimental TBI, CD59-2a-CRIg administered posttrauma homed to sites of injury and significantly reduced MAC deposition, microglial accumulation, mitochondrial stress, and axonal damage and enhanced neurologic recovery compared with placebo controls. CD59-2a-CRIg inhibited MAC-induced inflammasome activation and IL-1β production in microglia. Given the important anti-infection roles of complement opsonization, site-targeted inhibition of MAC should be considered to promote recovery postneurotrauma.