Human Complement Protein Research Articles

Ravulizumab in Myasthenia Gravis: A Review of the Current Evidence.

The terminal complement C5 inhibitor ravulizumab was engineered from the humanized monoclonal antibody eculizumab to have an extended half-life and duration of action. It binds to human terminal complement protein C5, inhibiting its cleavage into C5a and C5b, thus preventing the cascade of events that lead to architectural destruction of the postsynaptic neuromuscular junction membrane by the membrane attack complex, and consequent muscle weakness in patients with anti-acetylcholine receptor (AChR) antibody-positive generalized myasthenia gravis (gMG). The 26-week randomized, placebo-controlled period (RCP) of the phase 3 CHAMPION MG study demonstrated the rapid efficacy of ravulizumab in reducing MG symptoms. Weight-based dosing of ravulizumab every 8 weeks provided sustained efficacy, in terms of patient-reported (Myasthenia Gravis-Activities of Daily Living) and clinician-reported (Quantitative Myasthenia Gravis) endpoints in patients with anti-AChR antibody-positive gMG. Pharmacokinetic and pharmacodynamic analyses showed therapeutic serum ravulizumab concentrations (>175 µg/mL) were achieved immediately after the first dose and were maintained throughout 26 weeks, irrespective of patient body weight; inhibition of serum free C5 was immediate, complete (<0.5 μg/mL), and sustained in all patients. Interim results from the open-label extension (OLE) showed that after 60 weeks, efficacy was maintained in patients continuing on ravulizumab. Rapid and sustained improvements in efficacy, similar to those seen in patients initiating ravulizumab in the RCP, were observed after initiation of ravulizumab treatment in patients who switched from placebo in the RCP to ravulizumab in the OLE. The findings from the RCP and OLE support ravulizumab's favorable safety profile. In conclusion, ravulizumab has a simple weight-based administration and long dosing interval. Its targeted mechanism of action without generalized immunosuppression is reflected in its rapid onset of symptom improvement, sustained efficacy and good safety profile in the treatment of patients with anti-AChR antibody-positive gMG.

Characterization for the Similarity Assessment between Proposed Biosimilar SB12 and Eculizumab Reference Product Using a State-of-the-Art Analytical Method.

SB12 is being developed as a proposed biosimilar to eculizumab reference product (RP), a humanized monoclonal antibody (IgG2/4 kappa immunoglobulin) that binds to the human C5 complement protein. Binding to this protein inhibits complement-mediated intravascular hemolysis by blocking its cleavage into C5a and C5b. Eculizumab RP is indicated for the treatment of patients with paroxysmal nocturnal hemoglobinuria (PNH) to reduce hemolysis, atypical hemolytic uremic syndrome (aHUS) to inhibit complement-mediated thrombotic microangiopathy, generalized myasthenia gravis who are anti-acetylcholine receptor antibody-positive, and neuromyelitis optica spectrum disorder in adult patients who are anti-aquaporin-4 antibody-positive. The objective of this study was to demonstrate structural, physicochemical, and biological similarity between eculizumab RP and SB12 using various state-of-the-art analytical methods. Comprehensive analytical characterization was conducted with side-by-side comparison of SB12 with European Union (EU) and United States (US) eculizumab RPs using various analytical methods (more than 40 state-of-the-art assays). Comparisons included purity, product-related impurity, charge heterogeneity, primary structure, post-translational modification, higher-order structure, quantity, Fab-related biological activities (potency and binding activity), and Fc-related biological activities. Based on the analytical similarity assessment, the structural, physicochemical, and biological characterization results demonstrated that SB12 is highly similar to the EU and US eculizumab RP. In the structural aspects, it was confirmed that there is no difference between post-translational modification profiles and higher-order structures of SB12 compared with the eculizumab RP. Product-related impurities in the form of aggregates and charge variants were also confirmed to be similar. Mechanism of action (MoA)-related biological activities showed that SB12 is highly similar to the EU and US eculizumab RP with respect to overall critical and non-critical quality attributes analyzed. Moreover, similarity of comparative binding tendency of SB12 and eculizumab RP to Fc gamma receptors and C1q was confirmed through additional characterization methods. Based on these results, SB12 is expected to have highly similar safety and efficacy compared with eculizumab RP. In summary, the overall analytical characterization and similarity assessment results show that SB12 is highly similar to the EU and US eculizumab RP in terms of structural, physicochemical, biophysical, and biological attributes.

Complement Shields Fc Regions on HIT Immune Complexes and Prevents Recognition By Cellular Fcγ Receptors

Heparin-induced thrombocytopenia (HIT) is a life-threatening immune-mediated thrombotic disorder caused by immune complexes (ICs) containing IgG antibodies to platelet factor 4 (PF4)/heparin complexes. In previous studies, we established that complement opsonization of HIT ICs is essential for cellular binding of ICs and subsequent engagement of Fcγ receptors (FcγRs). We additionally showed that complement inhibition alone prevented FcγR-mediated effector functions, such as neutrophil degranulation, monocyte tissue factor expression, and procoagulant activity (Khandelwal, Blood 2021). As KKO and HIT ICs can also bind FcγRs in media without complement, we undertook these studies to examine the direct effects of complement on IC-FcγR interactions. For these studies, we used THP-1 cells or U937 cells, monocytic cell lines expressing FcγRI and FcγRII, but not complement receptors (CRs). Cells were incubated with AF-647 labeled KKO (50 mg/ml), a HIT-like monoclonal antibody, either alone or with PF4 (25 mg/ml) and heparin (1U/ml) to form in situ KKO ICs. Labeled KKO or KKO ICs were added to RPMI-containing media (lacking complement) or undiluted citrated plasma (source of complement) in the presence or absence of anti-FcγRIIA. Binding of labeled IgG or anti-C3c was examined by flow cytometry. Consistent with prior observations, THP-1 cells incubated in media show robust binding of KKO ICs in a FcγRII dependent manner (70% reduction in binding with anti-FcγRII; Figure 1A). Surprisingly, in undiluted plasma, there was minimal KKO IC binding to THP-1 cells, despite adequate complement generation (Figure 1B). Similar effects of plasma vs. media were seen in confocal experiments using KKO ICs with THP-1 cells (Figure 1C & 1D), HIT ICs incubated with THP-1 cells (containing HIT patient IgG, PF4/heparin; data not shown), or KKO ICs incubated with U937 cells (data not shown). In other studies, we note that binding of KKO ICs was dependent on the source of complement, with undiluted human plasma showing maximal inhibitory effects (100%), while undiluted normal human serum (NHS) and fetal bovine serum (FBS) reduced KKO IC binding by 70% and 50%, respectively (data not shown). To demonstrate that the inhibitory effects of undiluted plasma on KKO IC binding was complement-mediated, THP-1 cells were incubated in plasma containing the complement inhibitors BBK32 (C1r inhibitor) or CP40 (C3 inhibitor) prior to addition of labeled KKO ICs. In other experiments, THP-1 cells were incubated with labeled KKO ICs in media containing human complement proteins (C1+C3+C4). As shown, complement inhibition in plasma restored KKO IC binding to cellular FcγR (Figure 2A). Conversely, addition of purified complement components to media resulted in reduced KKO IC binding (Figure 2B). Similar effects of complement proteins and inhibitors were noted in ELISA-based assays using immobilized FcγRIIA incubated with KKO ICs in plasma or media (data not shown). These studies demonstrate striking inhibitory effects of complement on IC-FcγR interactions. Specifically, complement shields Fc regions on ICs from recognition and binding to cellular FcγRs and this masking effect is reversed by complement inhibition. We additionally show that these masking effects of complement are less apparent in media containing FBS, the most commonly used source of complement for cell-based studies. Taken together with our recent observations showing requirements for complement receptors in binding complement-opsonized KKO ICs, these studies suggest that the masking effects of complement on ICs prevent FcγR activation on cells lacking CRs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Characterization for the Similarity Assessment between SB12 (Eculizumab Biosimilar) and Eculizumab Using State-of-the-Art Analytical Method

Background: SB12 is being developed as a proposed biosimilar to eculizumab reference product (RP), a humanized monoclonal antibody (IgG2/4 kappa immunoglobulin) that binds to the human C5 complement protein. Binding to this protein inhibits complement-mediated intravascular hemolysis by blocking its cleavage into C5a and C5b. Eculizumab RP is indicated for the treatment of patients with paroxysmal nocturnal hemoglobinuria (PNH) to reduce hemolysis, atypical hemolytic uremic syndrome (aHUS) to inhibit complement-mediated thrombotic microangiopathy, generalized Myasthenia Gravis (gMG) who are anti-acetylcholine receptor (AChR) antibody positive, and Neuromyelitis Optica Spectrum Disorder (NMOSD) in adult patients who are anti-aquaporin-4 (AQP4) positive antibody. Objective: This study was to demonstrate structural, physicochemical, and functional similarity between SB12 and eculizumab RP using state-of-the-art analytical procedures. Methods: Comprehensive analytical characterization was conducted with side-by-side comparison with SB12, EU and US eculizumab RP using various analytical methods (more than 40 state-of-the-art assays). Comparisons included purity, product-related impurity, charge heterogeneity, primary structure, post-translational modification, higher order structure, quantity, Fab-related biological activities (potency and binding activity), and Fc-related biological activities. Results: Based on the analytical similarity assessment, the structural, physicochemical, and biological characterization results demonstrated that SB12 is highly similar to EU and US eculizumab RP by showing qualities that met the criteria for similarity assessment set with the eculizumab RP. In the structural aspects, it was confirmed that there is no difference between post-translational modification profiles and higher order structures of SB12 compared to the reference product. Product-related size, charge variants, and aggregates were also confirmed to be similar. In case of MoA-related biological activities showed that SB12 is highly similar to EU and US eculizumab RP with respect to overall critical and non-critical quality attributes performed. Moreover, similarity of comparative binding tendency of SB12 and eculizumab RP to Fc gamma receptors and C1q was confirmed through additional characterization. Based on these results, SB12 is expected to have highly similar safety and efficacy comparable to eculizumab RP. Conclusion: In summary, the overall characterization results show that SB12 is highly similar to EU and US eculizumab RP in terms of structural, physicochemical, biophysical, and biological attributes

Efficient engraftment and viral transduction of human hepatocytes in an FRG rat liver humanization model

Humanized liver rodent models, in which the host liver parenchyma is repopulated by human hepatocytes, have been increasingly used for drug development and disease research. Unlike the leading humanized liver mouse model in which Fumarylacetoacetate Hydrolase (Fah), Recombination Activating Gene (Rag)-2 and Interleukin-2 Receptor Gamma (Il2rg) genes were inactivated simultaneously, generation of similar recipient rats has been challenging. Here, using Velocigene and 1-cell-embryo-targeting technologies, we generated a rat model deficient in Fah, Rag1/2 and Il2rg genes, similar to humanized liver mice. These rats were efficiently engrafted with Fah-expressing hepatocytes from rat, mouse and human. Humanized liver rats expressed human albumin and complement proteins in serum and showed a normal liver zonation pattern. Further, approaches were developed for gene delivery through viral transduction of human hepatocytes either in vivo, or in vitro prior to engraftment, providing a novel platform to study liver disease and hepatocyte-targeted therapies.

A Randomized, Double-Blind, Single-Dose Phase 1 Comparative Pharmacokinetic Study Comparing SB12 (Eculizumab Biosimilar) with Reference Eculizumab in Healthy Volunteers

Background: SB12 has been developed as a biosimilar of the reference product (RP) eculizumab. Eculizumab is a humanized monoclonal antibody (IgG2/4 kappa immunoglobulin) that binds to the human C5 complement protein with high affinity. Binding to this protein blocks its cleavage into C5a and C5b, thereby inhibiting terminal complement-mediated intravascular haemolysis. It is currently indicated for the treatment of patients with paroxysmal nocturnal haemoglobinuria, atypical haemolytic uremic syndrome (aHUS), refractory generalized Myasthenia Gravis (gMG) and Neuromyelitis Optica Spectrum Disorder (NMOSD).Objectives: To demonstrate pharmacokinetic (PK) equivalence and evaluate pharmacodynamic (PD), safety, tolerability, and immunogenicity profiles between SB12 and the RP eculizumab.Methods: This was a double-blind, three-arm, parallel group, and single-dose study in healthy subjects, between 18-55 years of age, randomized in a ratio of 1:1:1 to receive a single 300 mg dose of either SB12, European Union (EU) sourced eculizumab, or United States of America (US) sourced eculizumab via intravenous (IV) infusion for 35 minutes. Blood samples for PK and PD analysis were collected over 64 days. The primary objective of this study was to demonstrate PK similarity between the investigational products (IPs), as assessed by area under the concentration-time curve from time zero to infinity (AUC inf). Secondary PK endpoints included area under the concentration-time curve from time zero to the last quantifiable concentration (AUC last) and maximum observed concentration (C max). Equivalence for the primary endpoint (AUC inf) was determined if 90% confidence intervals (CIs) for the ratio of geometric least squared means (LSMeans) of SB12 to EU sourced eculizumab, SB12 to US sourced eculizumab, and EU sourced eculizumab to US sourced eculizumab was within the equivalence margin of 80.00% to 125.00%, respectively. Other objectives for the study were to evaluate safety, tolerability, immunogenicity, and PD profiles for the IPs.Results: A total of 240 subjects (80 in each treatment group) were enrolled. Back transformation provided the geometric LSMean ratio for the comparison of SB12/EU sourced eculizumab, SB12/US sourced eculizumab and EU sourced eculizumab/US sourced eculizumab for AUC inf were 99.1 % (95.41,102.85), 95.1 % (91.40, 99.04), and 96.0 % (92.16, 100.10), respectively. The corresponding 90% CI was within the pre-defined equivalence margin of 80.00-125.00%, indicating that the each of two treatments are bioequivalent.The profiles of mean terminal complement activity and mean change from baseline of complement activity were superimposable following administration of SB12, EU sourced eculizumab, and US sourced eculizumab. There was a rapid decrease in the complement activity at the end of infusion and then a slow restoration. There was no non-responder in the aspect of the measured complement activity after treatment.There were no deaths or discontinuation of IP due to treatment-emergent adverse events (TEAEs) during the study. Two serious adverse events (SAEs) (renal colic in the SB12 treatment group and back pain in the US eculizumab treatment group) were reported, in 2 subjects. Both events were considered not related to the IP. The proportion of subjects who experienced TEAEs were similar between the SB12, EU sourced eculizumab, and US sourced eculizumab treatment groups (70.0%, 65.0%, and 71.3% of subjects, respectively). The overall incidence of subjects with post-dose anti-drug antibodies (ADA) to eculizumab was 2 (2.5%), 1 (1.3%), and 0 (0.0%) subjects in the SB12, EU sourced eculizumab, and US sourced eculizumab treatment groups, respectively. There was no significant difference between treatment groups. None of the subjects with post-dose ADA to eculizumab had a positive result for neutralizing antibodies.Conclusion: The Phase I study demonstrated PK bioequivalence and showed comparable PD, safety, immunogenicity between SB12 and the RP eculizumab. DisclosuresLee: Samsung Bioepis, Co., Ltd.: Current Employment. Jang: Samsung Bioepis, Co., Ltd.: Current Employment. Kim: Samsung Bioepis, Co., Ltd.: Current Employment. Jeong: Samsung Bioepis, Co., Ltd.: Current Employment. Lee: Samsung Bioepis, Co., Ltd.: Current Employment. Jung: Samsung Bioepis, Co., Ltd.: Current Employment. Demichelis: Novartis: Consultancy, Research Funding, Speakers Bureau; Bristol/Celgene: Consultancy, Speakers Bureau; AMGEN: Consultancy, Speakers Bureau; ASH: Research Funding; Jazz: Consultancy; Gilead: Consultancy; Astellas: Consultancy; Abbvie: Consultancy, Speakers Bureau.

The Protease SplB of Staphylococcus aureus Targets Host Complement Components and Inhibits Complement-Mediated Bacterial Opsonophagocytosis.

ABSTRACTStaphylococcus aureus is an opportunistic pathogen that can cause life-threatening infections, particularly in immunocompromised individuals. The high-level virulence of S. aureus largely relies on its diverse and variable collection of virulence factors and immune evasion proteins, including the six serine protease-like proteins SplA to SplF. Spl proteins are expressed by most clinical isolates of S. aureus, but little is known about the molecular mechanisms by which these proteins modify the host’s immune response for the benefit of the bacteria. Here, we identify SplB as a protease that inactivates central human complement proteins, i.e., C3, C4, and the activation fragments C3b and C4b, by preferentially cleaving their α-chains. SplB maintained its proteolytic activity in human serum, degrading C3 and C4. SplB further cleaved the components of the terminal complement pathway, C5, C6, C7, C8, and C9. In contrast, the important soluble human complement regulators factor H and C4b-binding protein (C4BP), as well as C1q, were left intact. Thereby, SplB reduced C3b-mediated opsonophagocytosis by human neutrophils as well as C5b-9 deposition on the bacterial surface. In conclusion, we identified the first physiological substrates of the S. aureus extracellular protease SplB. This enzyme inhibits all three complement pathways and blocks opsonophagocytosis. Thus, SplB can be considered a novel staphylococcal complement evasion protein.IMPORTANCE The success of bacterial pathogens in immunocompetent humans depends on the control and inactivation of host immunity. S. aureus, like many other pathogens, efficiently blocks host complement attack early in infection. Aiming to understand the role of the S. aureus-encoded orphan proteases of the Spl operon, we asked whether these proteins play a role in immune escape. We found that SplB inhibits all three complement activation pathways as well as the lytic terminal complement pathway. This blocks the opsonophagocytosis of the bacteria by neutrophils. We also clarified the molecular mechanisms: SplB cleaves the human complement proteins C3, C4, C5, C6, C7, C8, and C9 as well as factor B but not the complement inhibitors factor H and C4BP. Thus, we identify the first physiological substrates of the extracellular protease SplB of S. aureus and characterize SplB as a novel staphylococcal complement evasion protein.

Analytical Similarity Assessment of ABP 959 in Comparison with Eculizumab Reference Product.

BackgroundABP 959 is one of the first proposed biosimilars to eculizumab reference product (RP), a recombinant IgG2/4Ƙ monoclonal antibody (mAb) that binds human C5 complement protein and inhibits C5 cleavage to C5a and C5b, preventing the generation of the terminal complement complex C5b-9. Eculizumab RP is approved for the treatment of paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, myasthenia gravis in patients who are anti-acetylcholine receptor antibody positive, and neuromyelitis optica spectrum disorder in patients who are anti-aquaporin-4 antibody positive.ObjectivesThe objective of this work was to comparatively assess analytical (structural and functional) similarity between ABP 959 and eculizumab RP using sensitive, state-of-the art analytical methods capable of detecting minor differences in product quality attributes.MethodsComprehensive analytical (structural and functional) characterization utilizing orthogonal techniques was performed using multiple lots of ABP 959 and eculizumab RP over several years applying > 40 state-of-the-art assays. Comparisons were performed to investigate the primary structure and post-translational modifications including glycans, higher-order structure, particles and aggregates, product-related structures and impurities, thermal stability and forced degradation, general properties, and biological properties mediated by target binding.ResultsResults confirmed that ABP 959 had the same amino acid sequence, similar primary structure, higher-order structure, post-translational profiles, and the same protein content and concentration (e.g., ABP 959: 9.4–10.0; eculizumab EU: 9.4–10.0; eculizumab US: 9.3–10.3 mg/mL) as well as biological activity as eculizumab RP.ConclusionsBased on these results, it can be concluded that ABP 959 is analytically similar to eculizumab RP.

Role of Electrostatic Hotspots in the Selectivity of Complement Control Proteins Toward Human and Bovine Complement Inhibition.

Poxviruses are dangerous pathogens, which can cause fatal infection in unvaccinated individuals. The causative agent of smallpox in humans, variola virus, is closely related to the bovine vaccinia virus, yet the molecular basis of their selectivity is currently incompletely understood. Here, we examine the role of the electrostatics in the selectivity of the smallpox protein SPICE and vaccinia protein VCP toward the human and bovine complement protein C3b, a key component of the complement immune response. Electrostatic calculations, in-silico alanine-scan and electrostatic hotspot analysis, as introduced by Kieslich and Morikis (PLoS Comput. Biol. 2012), are used to assess the electrostatic complementarity and to identify sites resistant to local perturbation where the electrostatic potential is likely to be evolutionary conserved. The calculations suggest that the bovine C3b is electrostatically prone to selectively bind its VCP ligand. On the other hand, the human isoform of C3b exhibits a lower electrostatic complementarity toward its SPICE ligand. Yet, the human C3b displays a highly preserved electrostatic core, which suggests that this isoform could be less selective in binding different ligands like SPICE and the human Factor H. This is supported by experimental cofactor activity assays revealing that the human C3b is prone to bind both SPICE and Factor H, which exhibit diverse electrostatic properties. Additional investigations considering mutants of SPICE and VCP that revert their selectivity reveal an “electrostatic switch” into the central modules of the ligands, supporting the critical role of the electrostatics in the selectivity. Taken together, these evidences provide insights into the selectivity mechanism of the complement regulator proteins encoded by the variola and vaccinia viruses to circumvent the complement immunity and exert their pathogenic action. These fundamental aspects are valuable for the development of novel vaccines and therapeutic strategies.

PDX models reflect the proteome landscape of pediatric acute lymphoblastic leukemia but divert in select pathways

BackgroundMurine xenografts of pediatric leukemia accurately recapitulate genomic aberrations. How this translates to the functional capacity of cells remains unclear. Here, we studied global protein abundance, phosphorylation, and protein maturation by proteolytic processing in 11 pediatric B- and T- cell ALL patients and 19 corresponding xenografts.MethodsXenograft models were generated for each pediatric patient leukemia. Mass spectrometry-based methods were used to investigate global protein abundance, protein phosphorylation, and limited proteolysis in paired patient and xenografted pediatric acute B- and T- cell lymphocytic leukemia, as well as in pediatric leukemia cell lines. Targeted next-generation sequencing was utilized to examine genetic abnormalities in patients and in corresponding xenografts. Bioinformatic and statistical analysis were performed to identify functional mechanisms associated with proteins and protein post-translational modifications.ResultsOverall, we found xenograft proteomes to be most equivalent with their patient of origin. Protein level differences that stratified disease subtypes at diagnostic and relapse stages were largely recapitulated in xenografts. As expected, PDXs lacked multiple human leukocyte antigens and complement proteins. We found increased expression of cell cycle proteins indicating a high proliferative capacity of xenografted cells. Structural genomic changes and mutations were reflected at the protein level in patients. In contrast, the post-translational modification landscape was shaped by leukemia type and host and only to a limited degree by the patient of origin. Of 201 known pediatric oncogenic drivers and drug-targetable proteins, the KMT2 protein family showed consistently high variability between patient and corresponding xenografts. Comprehensive N terminomics revealed deregulated proteolytic processing in leukemic cells, in particular from caspase-driven cleavages found in patient cells.ConclusionGenomic and host factors shape protein and post-translational modification landscapes differently. This study highlights select areas of diverging biology while confirming murine patient-derived xenografts as a generally accurate model system.

Method for Depletion of IgG and IgM from Human Serum as Naive Complement Source.

Understanding how human complement proteins interact with human antibodies is important for the development of antibody therapies and understanding autoimmune diseases. At present, many groups use baby rabbit serum as a source of complement because, in contrast to human serum, it lacks preexisting antibodies. However, for characterization of human (monoclonal) antibodies, human serum would be a preferred source of complement. To prevent complement activation via naturally occurring antibodies, this human serum ideally lacks IgG and IgM. Here we describe how to deplete human serum of naturally occurring IgG and IgM using fast protein liquid affinity chromatography (FPLC) while minimizing the loss of serum complement activity. We also describe assays that can be used to validate depletion of IgG and IgM (IgG, IgM, and C1q sandwich ELISAs) and functionally assess remaining serum complement activity (hemolytic assays CH50 and AH50). Finally, we demonstrate how captured IgG and IgM can be purified.

The Protein Corona Conundrum: Exploring the Advantages and Drawbacks of its Presence around Amphiphilic Nanoparticles.

The success of targeted drug delivery systems still requires a detailed understanding about the biological consequences of self-developed biomolecular coronas around them, since this is the surface that interacts with living cells. Herein, we report the behavior of carbohydrate-decorated amphiphilic nanoparticles in a plasma environment with regard to the formation and biological consequences of the protein corona. Naked amphiphilic nanoparticles were produced through the self-assembly of azido-PEO900-docosanoate molecules, and the coupling of N-acetylglucosamine via click chemistry enabled the fabrication of the corresponding bioactive glyco-nanostructures. Light scattering measurements, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, liquid chromatography-mass spectrometry, and the Pierce BCA protein assay all confirmed the presence of protein coronas around the self-assembled nanoparticles, regardless of the presence of the sugar residues, although it reduces the amount of adsorbed proteins. The protein coronas were formed mainly by human serum albumin, complement proteins, apolipoproteins, immunoglobulins, and proteins involved in the coagulation cascade (fibrinogen and prothrombin). While the presence of these protein coronas significantly reduced cellular uptake of the amphiphilic assemblies, they also notably reduced the cytotoxic and hemolytic effects that result from the contact of the nanoparticles with living cells. Accordingly, we highlight that protein coronas should not always be treated as artifacts that have to be avoided because they can also provide beneficial effects.

Transcriptome of the Aedes aegypti Mosquito in Response to Human Complement Proteins.

Aedes aegypti is the primary mosquito vector of several human arboviruses, including the dengue virus (DENV). Vector control is the principal intervention to decrease the transmission of these viruses. The characterization of molecules involved in the mosquito physiological responses to blood-feeding may help identify novel targets useful in designing effective control strategies. In this study, we evaluated the in vivo effect of feeding adult female mosquitoes with human red blood cells reconstituted with either heat-inactivated (IB) or normal plasma (NB). The RNA-seq based transcript expression of IB and NB mosquitoes was compared against sugar-fed (SF) mosquitoes. In in vitro experiments, we treated Aag2 cells with a recombinant version of complement proteins (hC3 or hC5a) and compared transcript expression to untreated control cells after 24 h. The transcript expression analysis revealed that human complement proteins modulate approximately 2300 transcripts involved in multiple biological functions, including immunity. We also found 161 upregulated and 168 downregulated transcripts differentially expressed when human complement protein C3 (hC3) and human complement protein C5a (hC5a) treated cells were compared to the control untreated cells. We conclude that active human complement induces significant changes to the transcriptome of Ae. aegypti mosquitoes, which may influence the physiology of these arthropods.

A randomized, double-blind, single-dose, three-arm, parallel group study to determine pharmacokinetic similarity of ABP 959 and eculizumab (Soliris® ) in healthy male subjects.

ObjectivesABP 959 is a proposed biosimilar to eculizumab, a monoclonal antibody targeting the human C5 complement protein. The objective of this randomized, double‐blind, three‐arm, study was to demonstrate pharmacokinetic (PK) and pharmacodynamic (PD) similarity of ABP 959 relative to the eculizumab reference product (RP) in healthy adult male subjects.MethodsEligible subjects aged 18‐45 years were randomized to receive a 300‐mg IV infusion of ABP 959, or FDA‐licensed eculizumab (eculizumab US), or EU‐authorized eculizumab (eculizumab EU). Primary PK endpoint was area under the total serum concentration‐time curve from 0 to infinity (AUC0−∞); primary PD endpoint was area between the effect curve (ABEC) of CH50‐time data.ResultsThe geometric mean of PK and PD parameters were similar between ABP 959 versus eculizumab US and eculizumab EU; PK and PD similarity was established based on 90% confidence intervals of the geometric mean ratio being within prespecified equivalence margin of 0.8 and 1.25. The incidence of treatment‐emergent adverse events was similar across groups. The incidence of binding anti‐drug antibodies was similar across treatments; no subjects developed neutralizing antibodies.ConclusionsThis study demonstrated PK and PD similarity of ABP 959 to eculizumab RP; safety and immunogenicity profiles were also similar.

Relationship between Pharmacokinetics and Antidrug Antibody Status of ABP 959, a Biosimilar Candidate to Eculizumab: Results from a Pharmacokinetic Similarity Study

Background: ABP 959 is being developed as a biosimilar to eculizumab, a recombinant humanized IgG2/4 chimeric monoclonal antibody that binds to the human C5 complement protein (C5), thereby inhibiting the complement cascade. Eculizumab is indicated for the treatment of patients with paroxysmal nocturnal hemoglobinuria (PNH), generalized myasthenia gravis (gMG), atypical hemolytic uremic syndrome (aHUS), and neuromyelitis optica spectrum disorder (NMOSD). ABP 959 has demonstrated pharmacokinetic (PK) and pharmacodynamic (PD) similarity to eculizumab reference product (RP) in healthy male subjects (Chow et al., EHA 2019). Treatment with eculizumab can result in the development of anti-drug antibodies (ADAs), which may lead to loss of clinical efficacy; therefore, evaluating the incidence of ADAs and assessing their relationship to PK parameters is important. Objectives: To assess the relationship between PK parameters and ADAs for ABP 959 and eculizumab RP. Methods: This was a randomized, double-blind, single-dose, 3-arm, parallel-group study in healthy adult male subjects, between 18 to 45 years of age, with a body mass index of 18 to 30.0 kg/m2, who were randomized to receive a 300 mg IV infusion of ABP 959, FDA-licensed eculizumab (eculizumab US), or EU-authorized eculizumab (eculizumab EU). Serum samples for PK evaluations were collected over 57 days. A primary objective of the study was to demonstrate PK equivalence of ABP 959 versus eculizumab RP, as assessed by area under the total serum concentration-time curve (AUC) from time 0 extrapolated to infinity (AUCinf). Secondary PK endpoints included AUC from time 0 to the time of the last observed quantifiable concentration (AUClast) and maximum observed concentration (Cmax). The pre-specified equivalence criterion for the primary PK parameters was a 90% confidence interval (CI) for the geometric means ratio (GMR) within 0.80 to 1.25, for ABP 959 versus eculizumab US and ABP 959 versus eculizumab EU comparisons. Immunogenicity assessment was a secondary endpoint of the study. Sensitivity analysis of the PK primary endpoint was performed for the binding ADA-negative and neutralizing ADA-negative subjects, with the point estimates and 90% CIs for test-to-reference GMR calculated for PK parameters (AUCinf, AUClast, and Cmax) for total eculizumab and total ABP 959. Samples for ADA assessments were collected predose on Day 1, Day 11, Day 29, and at the end of study (EOS) Visit (Day 57) or upon early study discontinuation. Results: A total of 219 subjects were enrolled (ABP 959, n=71; eculizumab US, n=74; eculizumab EU, n=74); overall, 19 (8.8%) subjects had positive binding ADAs over the course of the study. At any time during the study, the incidence of binding ADAs across treatments was: ABP 959, n=7 (9.9%); eculizumab US, n=5 (6.9%); and eculizumab EU, n=7 (9.5%). At EOS, 4 (1.9%) subjects had positive binding ADAs (ABP 959, n=1 [1.4%]; eculizumab US, n=2 [2.9%]; and eculizumab EU, n=1 [1.4%]). In these subjects, their PK or exposure was within the same range as ADA-negative subjects. In binding ADA-negative subjects, the 90% CIs for the adjusted least square GMRs for all comparisons were within the bioequivalence range of 0.80 to 1.25. No subjects developed neutralizing antibodies throughout the study. Conclusions: The incidence of ADAs was similar between ABP 959 and eculizumab RP and did not impact the overall PK similarity assessment. Disclosures Hanes: Amgen Inc.: Employment. Pan:Amgen Inc.: Employment. Mytych:Amgen Inc.: Employment. Chien:Amgen Inc.: Employment. Chow:Amgen Inc.: Employment.

Clinical Characteristics of Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH): A Retrospective Chart Review Study

Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematologic disease that manifests with complement-mediated chronic intravascular hemolysis resulting in hemolytic anemia, tendency to thrombosis, and peripheral blood cytopenias. Eculizumab is a recombinant humanized IgG2/4 chimeric monoclonal antibody that is approved for the treatment of PNH, generalized myasthenia gravis (gMG), atypical hemolytic uremic syndrome (aHUS), and neuromyelitis optica spectrum disorder (NMOSD). Eculizumab specifically binds to the human C5 complement protein (C5), thereby inhibiting the complement cascade. To obtain a better understanding of PNH biology and provide insight toward improving the clinical management of patients with this disease, a retrospective chart review was performed from pre-existing medical records of patients with PNH treated with eculizumab. This analysis is expected to inform eculizumab biosimilar development; data collected on clinical characteristics of this patient population are presented here. Methods: This is a multicenter retrospective chart review study of pre-existing medical records of patients ≥18 years old with PNH who were treated with eculizumab between 01 January 2008 and 31 January 2016. Anonymized data from eligible patients were recorded by sites onto an electronic case report form (eCRF) and collected into an electronic data capture (EDC) system. The primary objective of the study was to assess the lactate dehydrogenase (LDH) concentration over time in eculizumab-treated patients with PNH, including during clinical events and after discontinuation of therapy. Secondary objectives included assessment of coexisting disease, vital signs, laboratory results, and clinical findings. Subjects' LDH time profiles and mean change from baseline of LDH in the maintenance phase were assessed. Maintenance phase was defined as the time period from the first of every 2 weeks (Q2W) eculizumab dose to the data cutoff date, or from the first Q2W eculizumab dose date to the last Q2W dose date for patients who discontinued prior to data cutoff. Baseline of the maintenance phase was defined as the last non-missing assessment taken prior to or on the same day of the first Q2W eculizumab dose. The inter- and intra-subject coefficient of variation (CV) of the 12-week area under effective curve (AUEC) of LDH was based on patients who had at least 2 LDH measurements during the maintenance treatment phase within 12 weeks of each other. Results: The medical records of 47 patients with PNH who were treated with eculizumab during the study period were reviewed; of these, 27 (57.4%) were female. At the time of the diagnosis, the mean age of all patients was 35.0 years and LDH values were reported for 5 of the 47 patients, 3 of which were ≥1.5 times the upper limit of normal. LDH values for most subjects during the study were within the expected ranges for the PNH population with stabilized hemoglobin levels on eculizumab. The mean change from baseline of LDH in the maintenance phase showed a decrease or no change for most time points. For the 26 patients who had ≥2 LDH measurements that were ≤ 12 weeks of each other, inter- and intra-subject CVs for the 12-week AUEC of LDH were 74.70% (95% confidence interval [CI] of 58.23%, 88.15%) and 34.05% (95% CI of 30.65%, 38.34%), respectively. The most commonly reported disease-related symptoms were hemoglobinuria (25 patients [53.2%]), abdominal pain (14 patients [29.8%]), thrombosis (11 patients [23.4%]), and infection (8 patients [17.0%]). Disease-related symptoms listed as 'Other' were reported in 14 (29.8%) patients and included dyspnea, fatigue, exertional dyspnea, aplastic anemia, kidney failure, and dysphagia. All reported concurrent medical conditions and symptoms have been previously well-documented in PNH patients. Ninety-two transfusions were administered to 15 (31.91%) patients within 6 weeks of the first eculizumab dose. Vital signs data did not demonstrate any clinically significant findings. Conclusions: The LDH levels for the majority of PNH patients with stabilized hemoglobin levels on eculizumab were well controlled and within the expected range; the inter- and intra-subject variability of 12-week AUEC of LDH were 74.70% and 34.05%, respectively. There were no unexpected or remarkable findings with respect to disease-related symptoms, vital signs, clinical findings, or laboratory data. Disclosures Hanes: Amgen Inc.: Employment. Pan:Amgen Inc.: Employment. Chow:Amgen Inc.: Employment.

A quantitative Streptococcus pyogenes\u2013human protein\u2013protein interaction map reveals localization of opsonizing antibodies

A fundamental challenge in medical microbiology is to characterize the dynamic protein–protein interaction networks formed at the host–pathogen interface. Here, we generate a quantitative interaction map between the significant human pathogen, Streptococcus pyogenes, and proteins from human saliva and plasma obtained via complementary affinity-purification and bacterial-surface centered enrichment strategies and quantitative mass spectrometry. Perturbation of the network using immunoglobulin protease cleavage, mixtures of different concentrations of saliva and plasma, and different S. pyogenes serotypes and their isogenic mutants, reveals how changing microenvironments alter the interconnectivity of the interaction map. The importance of host immunoglobulins for the interaction with human complement proteins is demonstrated and potential protective epitopes of importance for phagocytosis of S. pyogenes cells are localized. The interaction map confirms several previously described protein–protein interactions; however, it also reveals a multitude of additional interactions, with possible implications for host–pathogen interactions involving other bacterial species.

PF347 PHARMACOKINETIC AND PHARMACODYNAMIC SIMILARITY OF ABP 959 WITH ECULIZUMAB: RESULTS FROM A RANDOMIZED, DOUBLE-BLIND, SINGLE-DOSE, PARALLEL-GROUP STUDY IN HEALTHY SUBJECTS

Background: ABP 959 is being developed as a biosimilar candidate to eculizumab. Eculizumab is a recombinant humanized IgG2/4 chimeric monoclonal antibody that binds to the human C5 complement protein (C5), thereby inhibiting the complement cascade. It is indicated for the treatment of patients with generalized myasthenia gravis (gMG), paroxysmal nocturnal hemoglobinuria (PNH) to reduce hemolysis, and atypical hemolytic uremic syndrome (aHUS) to inhibit complement-mediated thrombotic microangiopathy. ABP 959 has been evaluated to be analytically similar to eculizumab. Aims: To demonstrate pharmacokinetic (PK) and pharmacodynamic (PD) similarity of ABP 959 relative to the reference product (RP) eculizumab. Methods: This was a randomized, double-blind, single-dose, 3-arm, parallel-group study in healthy adult male subjects, between 18 to 45 years of age, with a body mass index of 18 to 30.0 kg/m2, who were randomized to receive a 300-mg IV infusion of ABP 959, or FDA-licensed eculizumab (eculizumab US), or EU-authorized eculizumab (eculizumab EU). Serum samples for PK and PD evaluations were collected over 56 days. Primary objectives were to demonstrate PK and PD equivalence of ABP 959 versus eculizumab US and ABP 959 versus eculizumab EU, as assessed by area under the total serum concentration—time curve (AUC) from time 0 extrapolated to infinity (AUCinf) and an area between the effect curve (ABEC) of 50% total hemolytic complement activity (CH50). Secondary PK endpoints included AUC from time 0 to the time of the last observed quantifiable concentration (AUClast) and maximum observed concentration (Cmax). Pre-specified equivalence criterion for the primary PK and PD parameters was 90% confidence interval (CI) for geometric mean (GM) ratios within 0.80 to 1.25. Other secondary endpoints included the safety, tolerability, and immunogenicity of the investigational products (IPs). Results: A total of 219 subjects were enrolled (ABP 959, n = 71; eculizumab US, n = 74; eculizumab EU, n = 74). The GM of PK and PD parameters following a single IV infusion of IP, were similar between ABP 959 vs. eculizumab US and ABP 959 vs. eculizumab EU. The GM of AUCinf (h.μg/mL) was: 18996.3 for ABP 959; 19777.5 for eculizumab US and 18921.1 for eculizumab EU. The GM of ABEC of CH50 (%∗h) was 17724.5 for ABP 959; 16549.4 for eculizumab US and 16361.1 for eculizumab EU. For both PK and PD comparisons of ABP 959 with eculizumab US and ABP 959 with eculizumab EU, the 90% CIs of the GM ratios were fully contained within the bioequivalence criteria of 0.80 to 1.25 for all PK (AUCinf, Cmax, AUClast) and PD (ABEC of CH50) evaluations, confirming the PK and PD bioequivalence between ABP 959 and eculizumab US and ABP 959 and eculizumab EU. Additionally, eculizumab US and eculizumab EU were similar in PK and PD assessments. There were no deaths or treatment-emergent adverse events (TEAEs) leading to study discontinuation. Five subjects experienced 8 SAEs; only the SAE of headache was considered probably related to study drug. The incidence of overall TEAEs or any single category of TEAE was similar across treatment groups. A total of 19 (8.8%) subjects had positive binding ADAs over the course of the study; the incidence of binding ADAs was similar across treatments. No subjects developed neutralizing antibodies. Summary/Conclusion: This phase 1 study demonstrated PK and PD bioequivalence of ABP 959 to eculizumab RP. Safety and immunogenicity profiles were similar between ABP 959 and eculizumab RP.

Identification and characterization of a secreted M28 aminopeptidase protein in Acanthamoeba.

Acanthamoeba is a free-living pathogenic protozoan that is distributed in different environmental reservoirs, including lakes and soil. Pathogenic Acanthamoeba can cause severe human diseases, such as blinding keratitis and granulomatous encephalitis. Therefore, it is important to understand the pathogenic relationship between humans and Acanthamoeba. By comparison of systemic analysis results for Acanthamoeba isolates, we identified a novel secreted protein of Acanthamoeba, an M28 aminopeptidase (M28AP), which targets of the human innate immune defense. We investigated the molecular functions and characteristics of the M28AP protein by anti-M28 antibodies and a M28AP mutant strain generated by the CRISPR/Cas9 system. Human complement proteins such as C3b and iC3b were degraded by Acanthamoeba M28AP. We believe that M28AP is an important factor in human innate immunity. This study provides new insight for the development of more efficient medicines to treat Acanthamoeba infection.

Evasion Mechanisms Used by Pathogens to Escape the Lectin Complement Pathway

The complement system is a crucial defensive network that protects the host against invading pathogens. It is part of the innate immune system and can be initiated via three pathways: the lectin, classical and alternative activation pathway. Overall the network compiles a group of recognition molecules that bind specific patterns on microbial surfaces, a group of associated proteases that initiates the complement cascade, and a group of proteins that interact in proteolytic complexes or the terminal pore-forming complex. In addition, various regulatory proteins are important for controlling the level of activity. The result is a pro-inflammatory response meant to combat foreign microbes. Microbial elimination is, however, not a straight forward procedure; pathogens have adapted to their environment by evolving a collection of evasion mechanisms that circumvent the human complement system. Complement evasion strategies features different ways of exploiting human complement proteins and moreover features different pathogen-derived proteins that interfere with the normal processes. Accumulated, these mechanisms target all three complement activation pathways as well as the final common part of the cascade. This review will cover the currently known lectin pathway evasion mechanisms and give examples of pathogens that operate these to increase their chance of invasion, survival and dissemination.