Dysregulation Of Complement System Research Articles

Aberrant Lipid Metabolism and Complement Activation in Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) stands as a leading cause of severe visual impairment and blindness among the elderly globally. As a multifactorial disease, AMD's pathogenesis is influenced by genetic, environmental, and age-related factors, with lipid metabolism abnormalities and complement system dysregulation playing critical roles. This review delves into recent advancements in understanding the intricate interaction between these two crucial pathways, highlighting their contribution to the disease's progression through chronic inflammation, drusen formation, and retinal pigment epithelium dysfunction. Importantly, emerging evidence points to dysregulated lipid profiles, particularly alterations in high-density lipoprotein levels, oxidized lipid deposits, and intracellular lipofuscin accumulation, as exacerbating factors that enhance complement activation and subsequently amplify tissue damage in AMD. Furthermore, genetic studies have revealed significant associations between AMD and specific genes involved in lipid transport and complement regulation, shedding light on disease susceptibility and underlying mechanisms. The review further explores the clinical implications of these findings, advocating for a novel therapeutic approach that integrates lipid metabolism modulators with complement inhibitors. By concurrently targeting these pathways, the dual-targeted approach holds promise in significantly improving outcomes for AMD patients, heralding a new horizon in AMD management and treatment.

Contributions of Lipid-Related Metabolites and Complement Proteins to Early and Intermediate Age-Related Macular Degeneration

ObjectiveOur objective was to determine the effects of lipids and complement proteins on early and intermediate age-related macular degeneration (AMD) stages using machine learning models by integrating metabolomics and proteomic data. DesignNested case-control study. Subjects, Participants, and/or ControlsThe analyses were performed in a subset of the Singapore Indian Chinese Cohort (SICC) Eye Study. Among the 6,753 participants, we randomly selected 155 AMD Indian and 155 Chinese cases and matched them with 310 controls on age, sex and ethnicity. MethodsWe measured 35 complement proteins and 56 lipid-related metabolites using Mass Spectrometry and Nuclear Magnetic Resonance, respectively. We first selected the most contributing lipids and complement proteins to early and intermediate AMD using random forest models. Then we estimated their effects using a multinomial model adjusted for potential confounders. Main Outcome MeasuresAMD was classified using the Beckman classification system. ResultsAmong the 310 individuals with AMD, 166 (53.5%) had early and 144 (46.5%) intermediate AMD. Firstly, high-density lipoprotein (HDL) particle diameter was positively associated with both early and intermediate AMD (ORearly=1.69 [1.11, 2.55] and ORintermediate=1.72 [1.11, 2.66] per 1-SD increase in HDL diameter). Secondly, five complement proteins: complement protein 2 (C2), complement C1 inhibitor (ICI1), complement protein 6 (C6), complement protein 1QC (C1QC) and complement factor H-related protein 1 (FHR1), were associated with AMD. C2 was positively associated with both early and intermediate AMD (ORearly=1.58 [1.08, 2.30] and ORintermediate=1.56 [1.04, 2.34]). C6 was positively (ORearly=1.41 [1.03, 1.93]) associated with early AMD. However, IC1 was negatively associated with early AMD (ORearly=0.62 [0.38, 0.99]), while C1QC (ORintermediate=0.63 [0.42, 0.93]) and FHR1 (ORintermediate=0.73 [0.54, 0.98) were both negatively associated with intermediate AMD. ConclusionsWhile both HDL diameter and C2 levels show associations with both early and intermediate AMD, dysregulations of IC1, C6, C1QC, and FHR1 are only observed at specific stages of AMD. These findings underscore the complexity of complement system dysregulation in AMD, which appears to vary depending on the disease severity.

Persistent complement dysregulation with signs of thromboinflammation in active Long Covid.

Long Covid is a debilitating condition of unknown etiology. We performed multimodal proteomics analyses of blood serum from COVID-19 patients followed up to 12 months after confirmed severe acute respiratory syndrome coronavirus 2 infection. Analysis of >6500 proteins in 268 longitudinal samples revealed dysregulated activation of the complement system, an innate immune protection and homeostasis mechanism, in individuals experiencing Long Covid. Thus, active Long Covid was characterized by terminal complement system dysregulation and ongoing activation of the alternative and classical complement pathways, the latter associated with increased antibody titers against several herpesviruses possibly stimulating this pathway. Moreover, markers of hemolysis, tissue injury, platelet activation, and monocyte-platelet aggregates were increased in Long Covid. Machine learning confirmed complement and thromboinflammatory proteins as top biomarkers, warranting diagnostic and therapeutic interrogation of these systems.

Complement System and the Kidney: Its Role in Renal Diseases, Kidney Transplantation and Renal Cell Carcinoma.

The crosstalk among the complement system, immune cells, and mediators of inflammation provides an efficient mechanism to protect the organism against infections and support the repair of damaged tissues. Alterations in this complex machinery play a role in the pathogenesis of different diseases. Core complement proteins C3 and C5, their activation fragments, their receptors, and their regulators have been shown to be active intracellularly as the complosome. The kidney is particularly vulnerable to complement-induced damage, and emerging findings have revealed the role of complement system dysregulation in a wide range of kidney disorders, including glomerulopathies and ischemia-reperfusion injury during kidney transplantation. Different studies have shown that activation of the complement system is an important component of tumorigenesis and its elements have been proved to be present in the TME of various human malignancies. The role of the complement system in renal cell carcinoma (RCC) has been recently explored. Clear cell and papillary RCC upregulate most of the complement genes relative to normal kidney tissue. The aim of this narrative review is to provide novel insights into the role of complement in kidney disorders.

Multiomics analyses reveal pathological mechanisms of HBV infection and integration in liver cancer.

Hepatitis B virus (HBV) infection and integration are important for hepatocellular carcinoma (HCC) initiation and progression, while disease mechanisms are still largely elusive. Here, we combined bulk and single-cell sequencing technologies to tackle the disease mechanisms of HBV-related HCC. We observed high HBV mutation rate and diversity only in tumors without HBV integration. We identified human somatic risk loci for HBV integration (VIMs). Transcription factors (TFs) enriched in VIMs were involved in DNA repair and androgen receptor (AR) signaling. Aberration of AR signaling was further observed by single-cell regulon analysis in HBV-infected hepatocytes, which showed remarkable interactions between AR and the complement system that, together with the X-linked ZXDB regulon that contains albumin (ALB), probably contribute to HCC male predominance. Complement system dysregulation caused by HBV infection was further confirmed by analyses of single-cell copy numbers and cell-cell communications. Finally, HBV infection-associated immune cells presented critical defects, including TXNIP in T cells, TYROBP in NK cells, and the X-linked TIMP1 in monocytes. We further experimentally validated our findings in multiple independent patient cohorts. Collectively, our work shed light on the pathogenesis of HBV-related HCC and other liver diseases that affect billions of people worldwide.

#5990 COMPLEMENT GENES PROFILE OF PATIENTS WITH SEVERE HYPERTENSION AND RENAL THROMBOTIC MICROANGIOPATHY

Abstract Background and Aims Hereditary or acquired complement system dysregulation found to be driving cause of endothelial damage in atypical haemolytic uremic syndrome (aHUS) – ultrarare life-threatening disease, that always manifests with renal thrombotic microangiopathy (TMA), resulting in poor outcome in absence of appropriate treatment. Nevertheless, several studies show high incidence of rare genetic variants of complement system in other TMA’s, including hypertension-induced TMA. Here we report results of complement genes screening in patients (pts) with histologically identified renal TMA and severe hypertension in Russia, at first diagnosed as hypertension-induced TMA. Method A total of 28 Caucasian pts diagnosed with renal TMA (confirmed by kidney biopsy in all cases) and severe hypertension enrolled in the study. The main characteristic of the clinical picture was the absence of microangiopathic hemolysis and thrombocytopenia in prevailing of the pts, thus most of the primary and secondary causes of TMA clinically excluded. Examination of DNA samples was performed in all pts using next generation sequencing (NGS) technology. For statistical analysis continuous variables were presented as Mean (±Standart deviation) or Median (interquartile range) as appropriate. Results Baseline demographic and clinical characteristics of group are listed in Table 1. 13 pts (46%) had malignant form of hypertension (MHT) confirmed by eye examination. Mean systolic arterial pressure (SAP) observed at the time of presentation was 210±29 mmHg, mean diastolic arterial pressure (DAP) was 122±19 mmHg. Kidney morphology revealed chronic TMA lesions in 12 pts (43%), acute and chronic alterations simultaneously detected in 13 pts (46%). Predominantly acute lesions with glomerular and extraglomerular thrombi found in 3 (11%) pts only. Complement genes abnormalities revealed in 7 (25%) out of 28 pts. Complement factor I (CFI) rare variants were found in 2 pts (CFI c. 772<A, CFI c.1217G>A), one of this pts also had additional variant in complement factor H related protein 4 (CFHR4 c.766G>A). Complement factor C3 rare variant was observed in 1 pt (C3 c.2203C>T), membrane cofactor protein (MCP or CD46) rare variant in 1 pt (CD46 c. 198A>T), complement factor H related protein 5 (CFHR5) variant detected in 1 pt (CFHR5 c.136C>T), homozygous deletion of CFHR1 and CFHR3 detected in 1 pt and heterozygous deletion of the same complement factor H related protein genes found in another pt. Conclusion A quarter of pts with TMA and severe hypertension may have genetic defects in the complement system. These results confirmed that complement-mediated HUS may be misdiagnosed as hypertension-induced TMA. Lack of convincing hematological signs of TMA is common in this group, making aHUS diagnosis more sophisticated.

Pharmacological and clinical profile of ravulizumab 100 mg/mL formulation for paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome

ABSTRACT Introduction Paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS) are two rare and severe conditions caused by chronic complement (C’) system dysregulation. Treatment with eculizumab, a recombinant, humanized monoclonal antibody against complement C5, changed the natural history of both diseases inducing remission and improving patient outcome. Ravulizumab, a new long-acting next-generation C5 inhibitor, has been recently approved for treatment of PNH and aHUS. Areas covered Main characteristics of ravulizumab are described: composition, dosing, efficacy and safety profile. Further, an overview of seminal studies and clinical trials using ravulizumab to treat PNH and aHUS in children and adults is detailed. Literature review was performed using the following key words: paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, and ravulizumab. Expert opinion Ravulizumab profile to treat PNH and aHUS is equivalent to eculizumab in efficacy and safety but allows extended dosing interval to every 4–8 weeks based on patient weight, and requires reduced infusion time. Less travels to infusion centers and medical visits and decreasing job and school absences significantly increase patient and families’ QoL, while reducing cost. Further infusion time is reduced. Ravulizumab will possibly become the treatment of choice for patients with PNH and aHUS on chronic C5 inhibition.

Overview on the Link Between the Complement System and Auto-Immune Articular and Pulmonary Disease.

Complement system (CS) dysregulation is a key factor in the pathogenesis of different autoimmune diseases playing a central role in many immune innate and adaptive processes. Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by ta breach of self-tolerance leading to a synovitis and extra-articular manifestations. The CS is activated in RA and seems not only to mediate direct tissue damage but also play a role in the initiation of RA pathogenetic mechanisms through interactions with citrullinated proteins. Interstitial lung disease (ILD) represents the most common extra-articular manifestation that can lead to progressive fibrosis. In this review, we focused on the evidence of CS dysregulation in RA and in ILD, and highlighted the role of the CS in both the innate and adaptive immune responses in the development of diseases, by using idiopathic pulmonary fibrosis as a model of lung disease. As a proof of concept, we dissected the evidence that several treatments used to treat RA and ILD such as glucocorticoids, pirfenidone, disease modifying antirheumatic drugs, targeted biologics such as tumor necrosis factor (TNF)-inhibitors, rituximab, tocilizumab, and nintedanib may act indirectly on the CS, suggesting that the CS might represent a potential therapeutic target in these complex diseases.

New insight of complement system in the process of vascular calcification

The complement system defences against pathogenic microbes and modulates immune homeostasis by interacting with the innate and adaptive immune systems. Dysregulation, impairment or inadvertent activation of complement system contributes to the pathogenesis of some autoimmune diseases and cardiovascular diseases (CVD). Vascular calcification is the pivotal pathological basis of CVD, and contributes to the high morbidity and mortality of CVD. Increasing evidences indicate that the complement system plays a key role in chronic kidney diseases, atherosclerosis, diabetes mellitus and aging-related diseases, which are closely related with vascular calcification. However, the effect of complement system on vascular calcification is still unclear. In this review, we summarize current evidences about the activation of complement system in vascular calcification. We also describe the complex network of complement system and vascular smooth muscle cells osteogenic transdifferentiation, systemic inflammation, endoplasmic reticulum stress, extracellular matrix remodelling, oxidative stress, apoptosis in vascular calcification. Hence, providing a better understanding of the potential relationship between complement system and vascular calcification, so as to provide a direction for slowing the progression of this burgeoning health concern.

Complement system dysregulation in synovial fluid from patients with persistent inflammation following anterior cruciate ligament reconstruction surgery

IntroductionPatients with anterior cruciate ligament injury are at high risk of posttraumatic osteoarthritis and their response to reconstructive surgery and rehabilitation vary. Proteins identified in the orchestration of the acute inflammatory response may be predictive of patient outcomes. ObjectiveAn unbiased, bottom-up proteomics approach was used to discover novel targets for therapeutics in relation to dysregulation in the orchestration of inflammatory pathways implicated in persistent joint inflammation subsequent to joint trauma. MethodsSynovial fluid was aspirated from patients at 1 week and 4 weeks after anterior cruciate ligament reconstruction (ACLR) and interleukin 6 (IL-6) concentrations were quantified by enzyme-linked immunosorbent assay. Patients were segregated into IL-6low and IL-6high groups based on IL-6 concentrations in synovial fluid at 4-weeks postoperation and proteins in synovial fluid were analyzed using qualitative, bottom-up proteomics. Abundance ratios were calculated for IL-6high and IL-6low groups as 4 weeks postoperation:1 week postoperation. ResultsA total of 291 proteins were detected in synovial fluid, 34 of which were significantly (P < .05) differentially regulated between groups. Proteins associated with the classical and alternative complement cascade pathways were increased in the IL-6high compared to IL-6low group. Insulin-like growth factor-binding protein 6 (IGFBP-6) was increased by nearly 60-fold in the IL-6low group. ConclusionsPatients segregated by IL-6 concentration in synovial fluid at 4 weeks post-ACLR demonstrated differential regulation of multiple pathways, providing opportunities to investigate novel targets, such as IGFBP-6, and to take advantage of therapeutics already approved for clinical use in other diseases that target inflammatory pathways, including the complement system.

POS-001 A NOVEL CFHR5 COPY NUMBER VARIATION ASSOCIATED WITH POST-PARTUM ATYPICAL HUS SUPERIMPOSED TO HELLP SYNDROME

Pregnancy and postpartum are triggers for various forms of thrombotic microangiopathy (TMA), a lesion characterized by endothelial injury and thrombosis in the microcirculation of various organs. These forms include thrombotic thrombocytopenic purpura (TTP), caused by ADAMTS13 deficiency, and atypical hemolytic uremic syndrome (aHUS), most often associated with genetically determined dysregulation of complement system. HUS is the most common form of TMA in the post-partum period. The diagnosis of pregnancy-associated HUS can be difficult because preeclampsia and HELLP (Hemolysis, Elevated Liver enzymes and Low Platelets) syndrome may present with clinical and laboratory features of TMA that however commonly resolve spontaneously after delivery.Here, we report the biochemical and genetic characterization of a 22 years old primigravida of African origin who presented at 38 weeks of gestation with severe hypertension, nephrotic syndrome (NS) and elevated liver enzymes, consistent with a diagnosis of HELLP.

Complement Levels at Admission Reflecting Progression to Severe Acute Kidney Injury (AKI) in Coronavirus Disease 2019 (COVID-19): A Multicenter Prospective Cohort Study.

BackgroundDysregulation of complement system is thought to be a major player in development of multi-organ damage and adverse outcomes in patients with coronavirus disease 2019 (COVID-19). This study aimed to examine associations between complement system activity and development of severe acute kidney injury (AKI) among hospitalized COVID-19 patients.Materials and MethodsIn this multicenter, international study, complement as well as inflammatory and thrombotic parameters were analyzed in COVID-19 patients requiring hospitalization at one US and two Hungarian centers. The primary endpoint was development of severe AKI defined by KDIGO stage 2+3 criteria, while the secondary endpoint was need for renal replacement therapy (RRT). Complement markers with significant associations with endpoints were then correlated with a panel of inflammatory and thrombotic biomarkers and assessed for independent association with outcome measures using logistic regression.ResultsA total of 131 hospitalized COVID-19 patients (median age 66 [IQR, 54–75] years; 54.2% males) were enrolled, 33 from the US, and 98 from Hungary. There was a greater prevalence of complement over-activation and consumption in those who developed severe AKI and need for RRT during hospitalization. C3a/C3 ratio was increased in groups developing severe AKI (3.29 vs. 1.71; p < 0.001) and requiring RRT (3.42 vs. 1.79; p < 0.001) in each cohort. Decrease in alternative and classical pathway activity, and consumption of C4 below reference range, as well as elevation of complement activation marker C3a above the normal was more common in patients progressing to severe AKI. In the Hungarian cohort, each standard deviation increase in C3a (SD = 210.1) was independently associated with 89.7% increased odds of developing severe AKI (95% CI, 7.6–234.5%). Complement was extensively correlated with an array of inflammatory biomarkers and a prothrombotic state.ConclusionConsumption and dysregulation of complement system is associated with development of severe AKI in COVID-19 patients and could represent a promising therapeutic target for reducing thrombotic microangiopathy in SARS-CoV-2 infection.

Thrombotic Microangiopathy Syndromes-Common Ground and Distinct Frontiers.

Thrombotic microangiopathies (TMAs) have in common a terminal phenotype of microangiopathic hemolytic anemia with end-organ dysfunction. Thrombotic thrombocytopenic purpura results from von Willebrand factor multimerization, Shiga toxin-mediated hemolytic uremic syndrome causes toxin-induced endothelial dysfunction, while atypical hemolytic uremic syndrome results from complement system dysregulation. Drug-induced TMA, rheumatological disease-induced TMA, and renal-limited TMA exist in an intermediate space that represents secondary complement activation and may overlap with atypical hemolytic uremic syndrome clinically. The existence of TMA without microangiopathic hemolytic features, renal-limited TMA, represents an undiscovered syndrome that responds incompletely and inconsistently to complement blockade. Hematopoietic stem cell transplant-TMA represents another more resistant form of TMA with different therapeutic needs and clinical course. It has become apparent that TMA syndromes are an emerging field in nephrology, rheumatology, and hematology. Much work remains in genetics, molecular biology, and therapeutics to unravel the puzzle of the relationships and distinctions apparent between the different subclasses of TMA syndromes.

Case Report: Tackling Complement Hyperactivation With Eculizumab in Atypical Hemolytic Uremic Syndrome Triggered by COVID-19

Hemolytic uremic syndrome (HUS) is a rare life-threatening disease of unrestrained complement system dysregulation, microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure in genetically predisposed individuals. In this report, we describe two cases of SARS-CoV-2–associated HUS treated with eculizumab, a C5-blocking monoclonal antibody reported to be remarkably effective in the treatment of HUS. Detailed biochemical and genetic complement system analysis is reported, and the prompt clinical response after C5 pharmacological blockade is documented. Our report provides the rationale and supports the use of terminal complement pathway inhibition for the treatment of SARS-CoV-2–associated HUS.

Patients with MPNs and retinal drusen show signs of complement system dysregulation and a high degree of chronic low-grade inflammation.

SummaryBackgroundThe hematopoietic stem cell disorders, myeloproliferative neoplasms (MPNs), are characterised by chronic low-grade inflammation (CLI). Recently, we showed that patients with MPNs have an increased prevalence of drusen and age-related macular degeneration (AMD), and drusen prevalence seemed associated with higher CLI. Studying MPNs may reveal more about drusen pathophysiology. This study investigated CLI further by measuring cytokine levels and complement system markers, comparing these between patients with MPNs and AMD.MethodsThis cross-sectional study, between July 2018 and November 2020 conducted at Zealand University Hospital (ZUH) – Roskilde, Denmark, included 29 patients with neovascular AMD (nAMD), 28 with intermediate-stage AMD (iAMD), 62 with MPNs (35 with drusen - MPNd and 27 with healthy retinas - MPNn). With flow cytometry, we measured complement-regulatory-proteins (Cregs). With immunoassays, we investigated cytokine levels combined into a summary-inflammation-score (SIS).FindingsThe MPNd and nAMD groups had similar SIS, significantly higher than the MPNn and iAMD groups. Additionally, we found SIS to increase over the MPN biological continuum from early cancer stage, essential thrombocytaemia (ET), over polycythaemia vera (PV) to the late-stage primary myelofibrosis (PMF). MPNs showed signs of complement dysregulation, with Cregs expression lower in PV than ET and PMF and even lower in PV patients with drusen.InterpretationThis study suggests that MPNd have a higher CLI than MPNn and may indicate systemic CLI to play a greater part in, and even initiate drusen formation. We suggest using MPNs as a “Human Inflammation Model” of drusen development. The CLI in MPNs elicits drusen formation, triggering more CLI creating a vicious cycle, increasing the risk of developing AMD.FundingFight for Sight, Denmark, and Region Zealand's research promotion fund.

Dysregulation of complement system in HELLP syndrome

ABSTRACT Objective To investigate the expression of complement system’s activation factors in patients with HELLP syndrome. Methods A case–control study was performed. Sixteen HELLP syndrome patients, 32 severe preeclampsia patients, and 48 normal pregnancy women were involved in this studyELISA was used to test C1q, C4d, MBL, Bb, C3a, C5a, sC5b-9, s-Endoglin, and sflt-1 in the plasma. Results The levels of C5a (P < 0.01) and sC5b-9 (P = 0.014) in HELLP syndrome were higher than those in severe preeclampsia patients. Conclusions The abnormal activation of the complement system is more significant in the pathogenesis of HELLP syndrome than in severe preeclampsia.

CFH Loss in Human RPE Cells Leads to Inflammation and Complement System Dysregulation via the NF-κB Pathway.

Age-related macular degeneration (AMD), the leading cause of vision loss in the elderly, is a degenerative disease of the macula, where retinal pigment epithelium (RPE) cells are damaged in the early stages of the disease, and chronic inflammatory processes may be involved. Besides aging and lifestyle factors as drivers of AMD, a strong genetic association to AMD is found in genes of the complement system, with a single polymorphism in the complement factor H gene (CFH), accounting for the majority of AMD risk. However, the exact mechanism of CFH dysregulation confers such a great risk for AMD and its role in RPE cell homeostasis is unclear. To explore the role of endogenous CFH locally in RPE cells, we silenced CFH in human hTERT-RPE1 cells. We demonstrate that endogenously expressed CFH in RPE cells modulates inflammatory cytokine production and complement regulation, independent of external complement sources, or stressors. We show that loss of the factor H protein (FH) results in increased levels of inflammatory mediators (e.g., IL-6, IL-8, GM-CSF) and altered levels of complement proteins (e.g., C3, CFB upregulation, and C5 downregulation) that are known to play a role in AMD. Moreover, our results identify the NF-κB pathway as the major pathway involved in regulating these inflammatory and complement factors. Our findings suggest that in RPE cells, FH and the NF-κB pathway work in synergy to maintain inflammatory and complement balance, and in case either one of them is dysregulated, the RPE microenvironment changes towards a proinflammatory AMD-like phenotype.

Living Related Donor Kidney Transplantation in Atypical HUS: When Should It Be Considered?

Atypical hemolytic uremic syndrome (aHUS) is a thrombotic microangiopathy that manifests as hemolytic anemia, thrombocytopenia, and AKI. aHUS has been linked to the dysregulation of the alternative pathway of complement (1). In up to 60% of patients with aHUS, a genetic mutation known to cause dysregulation of complement system is found (2). Mutations in complement factor H ( CFH ), CD46 (membrane cofactor protein), complement factor I ( CFI ), thrombomodulin/CD141 ( THBD ), complement factor B ( CFB ), and C3 have been previously reported in patients with aHUS (3). People with mutations may not develop aHUS until adulthood (4). This incomplete penetrance indicates that a genetic mutation alone is insufficient to cause aHUS; instead, carriers of complement mutations require an additional trigger, such as pregnancy, viral infection, cancer, surgery, or medications, to progress to clinical disease of aHUS (5). As the type of mutation can influence both phenotype and rates of recurrence, genetic screening is advised pretransplant in patients with aHUS-related ESKD (2). However, there is no clear guidance for the genetic screening of family members who are potential living related donors. Historically, living related transplantation is actively discouraged in cases of aHUS due to increased risk of disease recurrence in recipients and risk of de novo disease in the donors (3,6). The recent advancement in genetics of aHUS challenges the recommendation against living related kidney transplantation. We present three patients with aHUS to explore whether living related kidney transplant can be considered as an option. …

A Novel pH Dependent C5 Monoclonal Antibody with Better Developability

Introduction: Dysregulation of complement system is the driver of many immune disorder diseases, including paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), and etc. All pathways of complement activation converge at the cleavage of C5, which leads to the generation of membrane attack complex (MAC). The approval of Eculizumab, anti-C5 monoclonal antibody, opened the complement-inhibitor era and proved C5 to be a feasible therapeutic target with manageable risk. Eculizumab and Crovalimab are currently approved for treatment of patients with PNH, yet treatment limitations include hemolysis breakthrough for non-sufficient C5 inhibition, lack of efficacy in patients with C5 mutational variants, and the burden of regular intravenous infusions. The next-generation C5 inhibitor emerges as a new/existing C5 monoclonal antibody engineered to have pH dependence (to reduce dosing frequency), and other new approached to target C5 in complement system. Here we report the results of a novel pH dependent C5 monoclonal antibody-LP-005, to prevent the hemolysis of red blood cells (RBCs) from PNH patients in vitro experiment. Methods: The sequence of Ravulizumab and Crovalimab were obtained from INN. LP-005 was generated using hybridoma method and multidimentionally optimized in variable and content regions. All antibodies were expressed by NHK293 cells. For antibody affinity test, Fortebio Octet K2 was used, different C5 antibodies were first loaded on the AHC sensors and serial diluted recombinant human C5 have been run for global-fit analysis. For antibody biological function test, antibody-sensitized sheep RBCs hemolysis (classic pathway/CP activity) inhibition assay have been run in 1% normal human serum (NHS) with antibodies serial diluted by GVB++ buffer. Antibody-sensitized chicken RBC hemolysis (CP activity) inhibition assay have been run in 6.25% NHS with antibodies serial diluted by GVB++ buffer. Ex vivo inhibition of PNH patients (with more than 70% CD59 deficiency) RBCs hemolysis by anti-C5 antibodies was performed using ABO blood group-compatible serum (20%) under conditions of robust alternative pathway (AP) specific activation (GVB0 + MgEGTA, pH7.3). CD59 deficiency on RBCs of PNH patients has been analyzed by flow cytometry using APC-conjugated anti-human CD59 (negative) and PE-conjugated anti-human CD47 antibodies (positive). Antibody stability test was conducted, antibodies were treated at 66℃ and 67.5℃ for 1hr, and C5 binding activity was then tested using by ELISA. Results: In the present study, we report a novel humanized anti-C5 monoclonal antibody, named LP-005. LP-005, Ravulizumab and Crovalimab exhibits different binding kinetics to C5 (Table 1), however, there is no clear correlation between their binding affinity and complement inhibition activity (NHS-induced CP and AP hemolysis). In antibody-sensitized sheep RBCs hemolysis inhibition assay (CP), LP-005 shows an IC50 of 0.41 nM, whereas Ravulizumab and Crovalimab has IC50 of 0.99 nM and 0.4 nM respectively (Figure 1). For AP induced helmolysis assay, PNH patient's RBCs with more than 70% CD59 absence was used, however, little difference was found between LP-005 and Ravulizumab hemolysis inhibition activity. Moreover, Crovalimab showed incomplete and less efficient inhibition compared with Ravulizumab and LP-005 (Figure 2). The thermal stability study showed that LP-005 is comparable to Crovalimab and is more stable than Ravulizumab (Figure 3). Conclusion: In summary, LP-005 is a novel anti-C5 monoclonal antibody with higher bioactivity in CP induced hemolysis compared to Ravulizumab, and comparable activity in AP induced hemolysis using PNH patient's RBCs. LP-005 is also engineered to increase FcRn binding property as well as thermal stability, which has the potential to be a good drug candidate with improved pharmacokinetic properties of decreasing doses frequency during clinical practice as well as a more robust manufacture process during drug development. Disclosures He: Celgene: Research Funding; furnished by Scott Battle, PhD, of Health Interactions, was funded by F. Hoffmann-La Roche Ltd, Basel, Switzerland: Other: Medical writing support; Celgene, Novartis, Amgen, Ra Pharma, Alexion, Achilleon, Biocryst, Akari, F. Hoffmann-La Roche: Speakers Bureau; Celgene, Novartis, Amgen, Ra Pharma, Alexion, Achilleon, Biocryst, Akari, F. Hoffmann-La Roche: Honoraria; Celgene, Novartis, Amgen, Ra Pharma, Alexion, Achilleon, Biocryst, Akari, Apellis, F. Hoffmann-La Roche: Consultancy.

Dysregulation of complement system during pregnancy in patients with preeclampsia: A prospective study

Recent studies have shown that aberrant activation of the complement system plays an important role in the pathogenesis of preeclampsia. There is evidence to suggest that aberrant activation of the complement system may already be present during the first trimester. Here, we performed a prospective study in which peripheral blood samples were collected from 500 women during pregnancy. Twenty-one patients (41 specimens) suffering from preeclampsia later in pregnancy were classified into the study group, and sixty-three gravidas with normal pregnancies (136 specimens) were selected as the control group. The plasma concentrations of complement factor B (CFB), C1q, complement factor H (CFH), C3c, C4, C3a, C5a and soluble C5b-9 (sC5b-9) were measured. The levels of CFB (P = 0.004), CFH (P = 0.002), C1q (P = 0.044), C3c (P = 0.032) and C4 (P = 0.015) were significantly higher in preeclampsia than in normal pregnancy during the first trimester, and these levels became similar to those in normal pregnancy thereafter. Before the onset of preeclampsia, the levels of C3a, C5a and sC5b-9 in the preeclampsia group were similar to those in control group even in late pregnancy. C3a levels showed a significant positive correlation with C5a in normal pregnancy (r=0.658, P<0.01) but not in preeclampsia (r = 0.001, P = 1).Thus, we found that aberrant activation of the complement system in patients with preeclampsia was initiated during the first trimester but returned to normal pregnancy levels in the second trimester. At the same time, there is aberrant regulation of complement activation at the C3a-C5a level in preeclampsia during pregnancy.