Complement C1s Research Articles

Effect of Sutimlimab Treatment on Healthcare Resource Utilization in Patients with Cold Agglutinin Disease

Introduction Cold agglutinin disease (CAD) is a serious and rare autoimmune disorder associated with severe fatigue, weakness and other symptoms of chronic anemia and hemolysis. Prior studies have demonstrated that CAD is associated with substantial economic burden and healthcare resource use, including blood transfusions, and highlight an unmet need for novel treatments (Mullins M et al. Blood Adv 2017; Su J et al. J Med Econ 2019). In the pivotal CARDINAL study (Part A), sutimlimab (BIVV009), a monoclonal antibody that targets the underlying cause of hemolysis in CAD by selectively inhibiting complement C1s, provided clinically meaningful improvements with an acceptable safety profile in CAD patients (Röth A et al. Blood 2019). A post hoc analysis was conducted to describe the impact of sutimlimab treatment on healthcare resource utilization (HCRU) in CAD patients. Methods CARDINAL is a Phase 3 open-label, international, multicenter, single-arm study (NCT03347396), evaluating the efficacy, safety, and tolerability of sutimlimab in patients with primary CAD and a recent history of blood transfusion (defined as having ≥1 transfusion 6 months prior to enrollment). The study enrolled 24 patients who received an IV infusion of sutimlimab over approximately 60 minutes on Day 0, Day 7, and every 14 days thereafter through Week 25. Post-hoc analysis of the full analysis set assessed hospitalizations and transfusions that occurred in the 6 month period pre- and post-administration of the first treatment dose. Distribution and descriptive statistics (mean, SD, median, min and max) were determined pre- and post-treatment periods and calculated for the differences between the pre- and post-treatment periods. Results The number of hospitalizations experienced by CAD patients in this post hoc analysis decreased 3.3-fold following sutimlimab treatment: from 10 patients (41.7%) in the pre-treatment period to 3 patients (12.5%) in the post-treatment period. Pre-treatment hospitalizations were the result of CAD-related symptoms or blood transfusion. Reasons for post-treatment hospitalizations included sepsis, infection and blood transfusion. The mean number of hospitalizations per patient decreased post-treatment compared with pre-treatment, but the mean duration of hospitalization per patient was similar between treatment periods (Table). During the post-treatment period, one patient was hospitalized for 18 days due to an infected hematoma following surgery, unrelated to sutimlimab treatment. Nine patients (37.5%) experienced fewer hospitalizations, shortened mean and total hospitalization duration following treatment. Similarly, blood transfusion use by CAD patients also decreased following sutimlimab treatment. Pre-treatment, all patients received ≥1 transfusion, decreasing to 41.7% patients post-treatment. This represented a total of 105 transfusions pre-treatment and 25 transfusions post-treatment, respectively (Table). Overall, 95.9% (23/24) patients received, at least, one less transfusion during the post-treatment versus pre-treatment period. The mean number of units transfused per transfusion was similar pre- and post-treatment; notably, the total number of units transfused decreased from 196 pre-treatment to 43 post-treatment (Table). Conclusion This post hoc analysis of HCRU data from the CARDINAL study provides evidence that CAD is a serious condition that can require hospitalization and blood transfusions. Sutimlimab treatment demonstrates substantial reductions in hospitalizations and blood transfusions. This results in a reduced HCRU impact and decreases the associated burden for hospitals, patients and payers. Disclosures Röth: Roche: Consultancy, Honoraria, Research Funding; Biocryst: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Apellis: Consultancy, Honoraria; Alexion Pharmaceuticals Inc.: Consultancy, Honoraria, Research Funding. Guillonneau:Sanofi: Current Employment, Current equity holder in publicly-traded company. Narcisse:Sanofi: Consultancy. Carita:Sanofi: Current Employment, Current equity holder in publicly-traded company. Su:Sanofi: Current Employment, Current equity holder in publicly-traded company. Joly:Sanofi: Current Employment, Current equity holder in publicly-traded company.

Inhibition of Complement C1s with Sutimlimab in Patients with Cold Agglutinin Disease (CAD): Interim Results of the Phase 3 Cardinal Study Long-Term Follow-up

Inhibition of Complement C1s with Sutimlimab in Patients with Cold Agglutinin Disease (CAD): Interim Results of the Phase 3 Cardinal Study Long-Term Follow-up

Plasma fibrin clot proteomics in patients with acute pulmonary embolism: Association with clot properties

It has been reported that 476 proteins can be detected in plasma fibrin clots from patients with venous thromboembolism. Plasma fibrin clots proteomic composition in relation to their properties has not been studied in acute pulmonary embolism (PE). Clots generated from plasma of 20 PE patients and 20 healthy controls were assessed using mass spectrometry, clot permeability (Ks), and clot lysis time (CLT). The proteomic composition of plasma fibrin clots from acute PE patients differed from that of control subjects in regard to 198 clot-bound proteins. In the acute PE group, we observed increased clot-bound fibrinogen, apolipoprotein B-100, platelet glycoprotein Ib, lipopolysaccharide-binding protein, and histones H3 + 4 and reduced fibronectin, α2-antiplasmin, α2-macroglobulin, factor (F)XIII, histidine-rich glycoprotein, antithrombin, von Willebrand Factor, plasminogen, and prothrombin. Among PE patients, low Ks (≤3.83 × 10−9 cm2) was associated with increased clot-bound C-reactive protein, kininogen-1, protein S, β-2-microglobulin, and thromboxane-A synthase when compared with patients having Ks > 3.83 × 10−9 cm2. Ks correlated inversely with FIX and FV, thrombin-activatable fibrinolysis inhibitor, complement C1s, C7, C8, and apolipoprotein A-I. The specific protein composition in plasma fibrin clots from acute PE patients is associated with denser clot formation. Several proteins unrelated to the coagulation system can modulate fibrin phenotype in acute thrombotic states. SignificanceOur study significantly advances the field of thrombosis and hemostasis. The plasma fibrin clot proteomics findings fill the gap of knowledge about the presence and the role of other proteins to the plasma fibrin clot in the acute phase of pulmonary embolism, aside fibrinogen, which is the main component of fibrin. The reported methodology, which involves the sample preparation using Multienzyme Digestion-Filter Aided Sample Preparation (MED FASP), data acquisition with the Quadrupole-Orbitrap mass spectrometer, and data analysis using the advanced tools such as MaxQuant, Total Protein Approach and Perseus, allows to gain not only the qualitative, but also the quantitative insights into the microworld of proteins entangled among the fibrin network. By comparing the clots formed from plasma of patients with acute pulmonary embolism with the clots from healthy control, we provide the specific protein composition associated with unfavorable clot properties observed in this disease. Moreover, our findings emphasize that several proteins unrelated to the coagulation system, can modulate fibrin phenotype in acute thrombotic states.

Gene editing in CHO cells to prevent proteolysis and enhance glycosylation: Production of HIV envelope proteins as vaccine immunogens.

Several candidate HIV subunit vaccines based on recombinant envelope (Env) glycoproteins have been advanced into human clinical trials. To facilitate biopharmaceutical production, it is necessary to produce these in CHO (Chinese Hamster Ovary) cells, the cellular substrate used for the manufacturing of most recombinant protein therapeutics. However, previous studies have shown that when recombinant Env proteins from clade B viruses, the major subtype represented in North America, Europe, and other parts of the world, are expressed in CHO cells, they are proteolyzed and lack important glycan-dependent epitopes present on virions. Previously, we identified C1s, a serine protease in the complement pathway, as the endogenous CHO protease responsible for the cleavage of clade B laboratory isolates of -recombinant gp120s (rgp120s) expressed in stable CHO-S cell lines. In this paper, we describe the development of two novel CHOK1 cell lines with the C1s gene inactivated by gene editing, that are suitable for the production of any protein susceptible to C1s proteolysis. One cell line, C1s-/- CHOK1 2.E7, contains a deletion in the C1s gene. The other cell line, C1s-/- MGAT1- CHOK1 1.A1, contains a deletion in both the C1s gene and the MGAT1 gene, which limits glycosylation to mannose-5 or earlier intermediates in the N-linked glycosylation pathway. In addition, we compare the substrate specificity of C1s with thrombin on the cleavage of both rgp120 and human Factor VIII, two recombinant proteins known to undergo unintended proteolysis (clipping) when expressed in CHO cells. Finally, we demonstrate the utility and practicality of the C1s-/- MGAT1- CHOK1 1.A1 cell line for the expression of clinical isolates of clade B Envs from rare individuals that possess broadly neutralizing antibodies and are able to control virus replication without anti-retroviral drugs (elite neutralizer/controller phenotypes). The Envs represent unique HIV vaccine immunogens suitable for further immunogenicity and efficacy studies.

C-reactive protein (CRP) recognizes uric acid crystals and recruits proteases C1 and MASP1

Gout is caused by crystallization of uric acid in the form of monosodium urate (MSU) crystals, which induce a sterile inflammatory response that is hardly distinguishable from microbe-induced inflammatory responses. It is unclear, if MSU crystals (like microbes) are recognized by specific pattern recognition receptors. To identify possible soluble pattern recognition molecules for MSU crystals, we purified MSU-binding proteins from human body fluids. We identified C-reactive protein (CRP) as a major MSU-binding protein. Binding of CRP was strong enough to specifically deplete CRP from human serum. We found that CRP was required for fixation of complement components C1q, C1r, C1s and MASP1. Thus, we have identified a pattern recognition molecule for MSU crystals that links to the activation of complement. Notably, CRP does not show an even binding to the complete surface of the crystals. It rather binds to edges or distinct faces of the crystals.

NanoString Gene Expression Profiling in Chronic Lung Allograft Dysfunction (CLAD)

CLAD, mainly manifest as bronchiolitis obliterans syndrome (BOS), is the leading cause of late death in lung recipients. BOS develops despite routine immunosuppression and its pathogenesis is poorly understood. The histological correlate of BOS is bronchiolitis obliterans (BO), or airways fibrosis. We quantified over 700 immune-related genes in BO affected airways to better understand immune responses active in end-stage BOS. Formalin fixed paraffin embedded lung tissue was procured at retransplantation for BOS (n=6). Unused donor tissue was used as normal control (n=5). Histologies of interest were isolated using laser capture microdissection (LCMD). Within each BOS lung, airways histologically affected by BO pathology, defined as intraluminal fibrous expansion, were isolated. Additionally, airways unaffected by evident fibrosis were isolated. Within each donor lung, histologically normal airways were isolated. Cellular RNA was extracted and multiplex gene expression analysis was performed using the NanoString PanCancer Immune Profiling Panel. Linear regression was used to compare gene expression among groups. The primary comparison was differential expression in BO affected vs. normal donor airways. We also compared expression in BO affected vs. unaffected airways within BOS explant lungs. P-values were corrected for multiple comparisons using Benjamini-Hochberg. Sixty-seven genes were significantly different between BO affected vs. normal donor airways, of which 52 had a fold change (FC) <0.5 or >2. Along with genes in adaptive immunity, several genes related to innate and humoral activation were significantly increased in BO affected airways. Among the innate immune genes with increased expression in BO were toll-like receptor 1 (FC 2.31) and its adaptor molecule CD14 (FC 5.26) in addition to the complement subcomponent C1s (FC 3.06), while those in humoral immunity included bone marrow stromal antigen 2 (FC 3.41), important to the growth and development of B-cells. No genes were significantly differentially expressed between BO affected vs. unaffected airways within BOS explant lungs. Our results suggest innate and humoral immune responses are active in airways affected by BO pathology, and also in airways without evident fibrosis. Ongoing analyses of BO lesions through LCMD will provide new insights into CLAD pathogenesis.

Antibodies that conformationally activate ADAMTS13 allosterically enhance metalloprotease domain function

AbstractPlasma ADAMTS13 circulates in a folded conformation that is stabilized by an interaction between the central Spacer domain and the C-terminal CUB (complement components C1r and C1s, sea urchin protein Uegf, and bone morphogenetic protein-1) domains. Binding of ADAMTS13 to the VWF D4(-CK) domains or to certain activating murine monoclonal antibodies (mAbs) induces a structural change that extends ADAMTS13 into an open conformation that enhances its function. The objective was to characterize the mechanism by which conformational activation enhances ADAMTS13-mediated proteolysis of VWF. The activating effects of a novel anti-Spacer (3E4) and the anti-CUB1 (17G2) mAbs on the kinetics of proteolysis of VWF A2 domain fragments by ADAMTS13 were analyzed. mAb-induced conformational changes in ADAMTS13 were investigated by enzyme-linked immunosorbent assay. Both mAbs enhanced ADAMTS13 catalytic efficiency (kcat/Km) by ∼twofold (3E4: 2.0-fold; 17G2: 1.8-fold). Contrary to previous hypotheses, ADAMTS13 activation was not mediated through exposure of the Spacer or cysteine-rich domain exosites. Kinetic analyses revealed that mAb-induced conformational extension of ADAMTS13 enhances the proteolytic function of the metalloprotease domain (kcat), rather than augmenting substrate binding (Km). A conformational effect on the metalloprotease domain was further corroborated by the finding that incubation of ADAMTS13 with either mAb exposed a cryptic epitope in the metalloprotease domain that is normally concealed when ADAMTS13 is in a closed conformation. We show for the first time that the primary mechanism of mAb-induced conformational activation of ADAMTS13 is not a consequence of functional exosite exposure. Rather, our data are consistent with an allosteric activation mechanism on the metalloprotease domain that augments active site function.

Inhibition of complement C1s in patients with cold agglutinin disease: lessons learned from a named patient program

AbstractCold agglutinin disease (CAD) causes predominantly extravascular hemolysis and anemia via complement activation. Sutimlimab is a novel humanized monoclonal antibody directed against classical pathway complement factor C1s. We aimed to evaluate the safety and efficacy of long-term maintenance treatment with sutimlimab in patients with CAD. Seven CAD patients treated with sutimlimab as part of a phase 1B study were transitioned to a named patient program. After a loading dose, patients received biweekly (once every 2 weeks) infusions of sutimlimab at various doses. When a patient's laboratory data showed signs of breakthrough hemolysis, the dose of sutimlimab was increased. Three patients started with a dose of 45 mg/kg, another 3 with 60 mg/kg, and 1 with a fixed dose of 5.5 g every other week. All CAD patients responded to re-treatment, and sutimlimab increased hemoglobin from a median initial level of 7.7 g/dL to a median peak of 12.5 g/dL (P = .016). Patients maintained near normal hemoglobin levels except for a few breakthrough events that were related to underdosing and which resolved after the appropriate dose increase. Four of the patients included were eventually treated with a biweekly 5.5 g fixed-dose regimen of sutimlimab. None of them had any breakthrough hemolysis. All patients remained transfusion free while receiving sutimlimab. There were no treatment-related serious adverse events. Overlapping treatment with erythropoietin, rituximab, or ibrutinib in individual patients was safe and did not cause untoward drug interactions. Long-term maintenance treatment with sutimlimab was safe, effectively inhibited hemolysis, and significantly increased hemoglobin levels in re-exposed, previously transfusion-dependent CAD patients.

Parallel comparison of three methodologies for measuring functional C1-inhibitor in Hereditary angioedema patients

Hereditary angioedema (HAE) types I and II are characterized by functional C1 inhibitor (fC1-INH) deficiency which results in bradykinin overproduction. Sensitive, specific and robust methods to quantitate fC1-INH in human samples are required for diagnosing HAE and/or to measure pharmacodynamic activity of C1-INH drugs in clinical studies. To date, three methods have been reported in literature to measure fC1-INH: conventional chromogenic assay measuring residual C1-esterase activity, and immunoassays based on functional binding to either activated complement C1s or Factor XIIa/kallikrein.We used three qualified/validated fit-for purpose methods to quantitate fC1-INH in human plasma and to conduct a parallel comparison for diagnostic purposes and as a read-out for pharmacodynamic activity. Sensitivity and specificity were determined from the Receiver Operator Characteristics (ROC) curve analysis of the three fC1-INH methods through testing of fifty healthy control vs. HAE plasma samples. fC1-INH profile of fifteen HAE subjects, who underwent different treatment regimen in a cross-over Shire C1-INH clinical study, was analyzed in these three methods in parallel. A correlation analysis performed between these methods using data generated from clinical samples showed that profiles obtained from different fC1-INH methods matched for individual HAE subjects.Our findings suggest that functional binding immunoassay methods serve as reliable alternates for conventional chromogenic method to quantitate fC1-INH in human plasma samples with a better dynamic range of detection and ease of use. Of the two immunoassays used in this study, FXIIa-binding method gave better sensitivity, specificity, and correlation to the chromogenic method as a diagnostic method to distinguish HAE samples from healthy controls.

Enzymes C1r and C1s may show progression of a type of skin cancer called keratinocyte‐derived cutaneous squamous cell carcinoma

The incidence of skin cancers is increasing worldwide. The epidermis is the surface layer of the skin and the main cell type is the keratinocyte. Keratinocyte-derived cutaneous squamous cell carcinoma (cSCC) is the most common metastatic (meaning it can spread) skin cancer and it is responsible for 20% of skin cancer related deaths. The main risk factor for cSCC includes long term exposure to sun ultraviolet radiation. The progression of cSCC is as follows: it starts in a premalignant form (not yet cancerous) called actinic keratosis (AK), then becomes locally restricted cSCC, then cSCC in situ (cSCCIS), and eventually becomes invasive and metastatic cSCC. This research, from Finland, aimed to find biomarkers (chemicals in the body which act as signs of a disease) which could predict the progression and aggressiveness of cSCC. The authors found significantly elevated levels of mRNA (messenger molecules) for enzymes called C1r and C1s (which play a role in our immune system), in cSCC cells and tumors compared to normal epidermal keratinocytes and normal skin, respectively. Abundant expression of C1r and C1s by tumor cells was detected in invasive cSCCs and in an aggressive form of cSCC (recessive dystrophic epidermolysis bullosa-associated cSCC), whereas the expression of C1r and C1s was lower in cSCCIS, actinic keratosis, and normal skin tissue samples. The roles of C1r and C1s in cSCC cells were investigated by knocking down (reducing) the expression of C1r and C1s, and through complex methods, this was shown to potentially suppress growth of cSCC tumours. These results provide evidence that tumor cell-derived C1r and C1s play a part in the progression of cSCC and identify them as biomarkers and possible treatment targets in cSCC. This is a summary of the study: Tumour-cell-derived complement components C1r and C1s promote growth of cutaneous squamous cell carcinoma

Gut microbiota modulation and immunity response induced by Citrobacter freundii strain GC01 in grass carp (Ctenopharyngodon idellus)

The effect of Citrobacter freundii, an opportunistic pathogen of fish, on the gut microbiota and immune responses in fish remains unknown. We fed grass carp (Ctenopharyngodon idellus) with two different concentrations of this bacterium (experimental Low and High groups, plus Control group). No signs of acute inflammatory response were found in either of the experimental groups using the intestinal HE staining method, but the number of intestinal goblet cells was much higher in the High group. Furthermore, we found that feeding C. freundii significantly decreased the alpha diversity and altered beta diversity of gut microbiota in both groups, and significantly promoted the relative abundance of Enterobacteriales, Pasteurellales, Neisseriales and Citrobacter in the High group. Spleen transcriptome showed that the expression pattern of immune system-related genes varied significantly between the Control group and High group. Several immunity-related pathways and two infectious diseases-related pathways were significantly enriched in the High group. Many complement system genes, including c3, c4, c5, c7, c8a, c8b and c1s, were also up-regulated in the High group. Overall, our results indicate that C. freundii did not trigger acute intestinal inflammation, but it changed the diversity and population structure of gut microbiota, and triggered the host's innate mucosal immune system. The enrichment of two infectious diseases-related pathways also suggested a higher risk of infection in the High group. These results contribute to our understanding of the effects of opportunistic fish pathogens on gut microbiota and host's immune responses.

Two Different Missense C1S Mutations, Associated to Periodontal Ehlers-Danlos Syndrome, Lead to Identical Molecular Outcomes.

Ehlers-Danlos syndromes (EDS) are clinically and genetically heterogeneous disorders characterized by soft connective tissue alteration like joint hypermobility and skin hyper-extensibility. We previously identified heterozygous missense mutations in the C1R and C1S genes, coding for the complement C1 proteases, in patients affected by periodontal EDS, a specific EDS subtype hallmarked by early severe periodontitis leading to premature loss of teeth and connective tissue alterations. Up to now, there is no clear molecular link relating the nominal role of the C1r and C1s proteases, which is to activate the classical complement pathway, to these heterogeneous symptoms of periodontal EDS syndrome. We aim therefore to elucidate the functional effect of these mutations, at the molecular and enzymatic levels. To explore the molecular consequences, a set of cell transfection experiments, recombinant protein purification, mass spectroscopy and N-terminal analyses have been performed. Focusing on the results obtained on two different C1S variants, namely p.Val316del and p.Cys294Arg, we show that HEK293-F cells stably transfected with the corresponding C1s variant plasmids, unexpectedly, do not secrete the full-length mutated C1s, but only a truncated Fg40 fragment of 40 kDa, produced at very low levels. Detailed analyses of the Fg40 fragments purified for the two C1s variants show that they are identical, which was also unexpected. This suggests that local misfolding of the CCP1 module containing the patient mutation exposes a novel cleavage site, between Lys353 and Cys354, which is not normally accessible. The mutation-induced Fg40 fragment contains the intact C-terminal serine protease domain but not the N-terminal domain mediating C1s interaction with the other C1 subunits, C1r, and C1q. Thus, Fg40 enzymatic activity escapes the normal physiological control of C1s activity within C1, potentially providing a loss-of-control. Comparative enzymatic analyses show that Fg40 retains the native esterolytic activity of C1s, as well as its cleavage efficiency toward the ancillary alarmin HMGB1 substrate, for example, whereas the nominal complement C4 activation cleavage is impaired. These new results open the way to further molecular explorations possibly involving subsidiary C1s targets.

Inhibition of Complement C1s with Sutimlimab in Patients with Cold Agglutinin Disease (CAD): Results from the Phase 3 Cardinal Study

Inhibition of Complement C1s with Sutimlimab in Patients with Cold Agglutinin Disease (CAD): Results from the Phase 3 Cardinal Study

Tumour-cell-derived complement components C1r and C1s promote growth of cutaneous squamous cell carcinoma.

SummaryBackgroundThe incidence of epidermal keratinocyte‐derived cutaneous squamous cell carcinoma (cSCC) is increasing worldwide.ObjectivesTo study the role of the complement classical pathway components C1q, C1r and C1s in the progression of cSCC.MethodsThe mRNA levels of C1Q subunits and C1R and C1S in cSCC cell lines, normal human epidermal keratinocytes, cSCC tumours in vivo and normal skin were analysed with quantitative real‐time polymerase chain reaction. The production of C1r and C1s was determined with Western blotting. The expression of C1r and C1s in tissue samples in vivo was analysed with immunohistochemistry and further investigated in human cSCC xenografts by knocking down C1r and C1s.ResultsSignificantly elevated C1R and C1S mRNA levels and production of C1r and C1s were detected in cSCC cells, compared with normal human epidermal keratinocytes. The mRNA levels of C1R and C1S were markedly elevated in cSCC tumours in vivo compared with normal skin. Abundant expression of C1r and C1s by tumour cells was detected in invasive sporadic cSCCs and recessive dystrophic epidermolysis bullosa‐associated cSCCs, whereas the expression of C1r and C1s was lower in cSCC in situ, actinic keratosis and normal skin. Knockdown of C1r and C1s expression in cSCC cells inhibited activation of extracellular signal‐related kinase 1/2 and Akt, promoted apoptosis of cSCC cells and significantly suppressed growth and vascularization of human cSCC xenograft tumours in vivo.ConclusionsThese results provide evidence for the role of tumour‐cell‐derived C1r and C1s in the progression of cSCC and identify them as biomarkers and putative therapeutic targets in cSCC. What's already known about this topic? The incidences of actinic keratosis, cutaneous squamous cell carcinoma (cSCC) in situ and invasive cSCC are increasing globally.Few specific biomarkers for progression of cSCC have been identified, and no biological markers are in clinical use to predict the aggressiveness of actinic keratosis, cSCC in situ and invasive cSCC. What does this study add? Our results provide novel evidence for the role of complement classical pathway components C1r and C1s in the progression of cSCC. What is the translational message? Our results identify complement classical pathway components C1r and C1s as biomarkers and putative therapeutic targets in cSCC.

Exploring the “minimal” structure of a functional ADAMTS13 by mutagenesis and small-angle X-ray scattering

AbstractHuman ADAMTS13 is a multidomain protein with metalloprotease (M), disintegrin-like (D), thrombospondin-1 (T), Cys-rich (C), and spacer (S) domains, followed by 7 additional T domains and 2 CUB (complement components C1r and C1s, sea urchin protein Uegf, and bone morphogenetic protein-1) domains. ADAMTS13 inhibits the growth of von Willebrand factor (VWF)–platelet aggregates by cleaving the cryptic Tyr1605-Met1606 bond in the VWF A2 domain. ADAMTS13 is regulated by substrate-induced allosteric activation; without shear stress, the distal T8-CUB domains markedly inhibit VWF cleavage, and binding of VWF domain D4 or selected monoclonal antibodies (MAbs) to distal ADAMTS13 domains relieves this autoinhibition. By small angle X-ray scattering (SAXS), ADAMTS13 adopts a hairpin-like conformation with distal T7-CUB domains close to the proximal MDTCS domains and a hinge point between T4 and T5. The hairpin projects like a handle away from the core MDTCS and T7-CUB complex and contains distal T domains that are dispensable for allosteric regulation. Truncated constructs that lack the T8-CUB domains are not autoinhibited and cannot be activated by VWF D4 but retain the hairpin fold. Allosteric activation by VWF D4 requires T7, T8, and the 58–amino acid residue linker between T8 and CUB1. Deletion of T3 to T6 produced the smallest construct (delT3-6) examined that could be activated by MAbs and VWF D4. Columba livia (pigeon) ADAMTS13 (pADAMTS13) resembles human delT3-6, retains normal activation by VWF D4, and has a SAXS envelope consistent with amputation of the hairpin containing the dispensable T domains of human ADAMTS13. Our findings suggest that human delT3-6 and pADAMTS13 approach a “minimal” structure for allosterically regulated ADAMTS13.

Phylogenetic and functional analysis of ADAMTS13 identifies highly conserved domains essential for allosteric regulation

AbstractThe metalloprotease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats member 13) prevents microvascular thrombosis by cleaving von Willebrand factor (VWF) within platelet-rich thrombi, and cleavage depends on allosteric activation of ADAMTS13 by the substrate VWF. Human ADAMTS13 has a short propeptide, metalloprotease (M), disintegrin-like (D), thrombospondin-1 (T), Cys-rich (C), and spacer (S) domains (proximal domains), followed by 7 T and 2 CUB (complement components C1r and C1s, sea urchin protein Uegf, and bone morphogenetic protein-1) domains (distal domains). Distal domains inhibit the catalytic proximal domains; binding of distal T8-CUB domains to the VWF D4 domain relieves autoinhibition and promotes cleavage of the nearby VWF A2 domain. However, the role of specific ADAMTS13 distal domains in this allosteric mechanism is not established. Assays of plasma ADAMTS13 from 20 placental mammals, birds, and amphibians show that allosteric regulation is broadly conserved, and phylogenetic analysis of 264 vertebrates shows the long propeptide, T3, T4, T6, and T6a domains have been deleted several times in placental mammals, birds, and fish. Notably, pigeon ADAMTS13 has only 3 distal T domains but was activated normally by human VWF D4 and cleaved VWF multimers, preferentially under fluid shear stress. Human ADAMTS13 constructed to resemble pigeon ADAMTS13 retained normal allosteric regulation and shear-dependent cleavage of VWF. Thus, the T3-T6 domains of human ADAMTS13 are dispensable. Conversely, deletion of T7 or T8 abolished allosteric activation. For most species, some sequence changes in the VWF substrate can markedly increase the rate of cleavage, suggesting that ADAMTS13 and VWF have not evolved to be optimal enzyme-substrate pairs. These properties may reflect evolutionary pressure to balance the risk for VWF-dependent bleeding and thrombosis.

Inhibition of complement C1s improves severe hemolytic anemia in cold agglutinin disease: a first-in-human trial

AbstractCold agglutinin disease is a difficult-to-treat autoimmune hemolytic anemia in which immunoglobulin M antibodies bind to erythrocytes and fix complement, resulting in predominantly extravascular hemolysis. This trial tested the hypothesis that the anti-C1s antibody sutimlimab would ameliorate hemolytic anemia. Ten patients with cold agglutinin disease participated in the phase 1b component of a first-in-human trial. Patients received a test dose of 10-mg/kg sutimlimab followed by a full dose of 60 mg/kg 1 to 4 days later and 3 additional weekly doses of 60 mg/kg. All infusions were well tolerated without premedication. No drug-related serious adverse events were observed. Seven of 10 patients with cold agglutinin disease responded with a hemoglobin increase >2 g/dL. Sutimlimab rapidly increased hemoglobin levels by a median of 1.6 g/dL within the first week, and by a median of 3.9 g/dL (interquartile range, 1.3-4.5 g/dL; 95% confidence interval, 2.1-4.5) within 6 weeks (P = .005). Sutimlimab rapidly abrogated extravascular hemolysis, normalizing bilirubin levels within 24 hours in most patients and normalizing haptoglobin levels in 4 patients within 1 week. Hemolytic anemia recurred when drug levels were cleared from the circulation 3 to 4 weeks after the last dose of sutimlimab. Reexposure to sutimlimab in a named patient program recapitulated the control of hemolytic anemia. All 6 previously transfused patients became transfusion-free during treatment. Sutimlimab was safe, well tolerated, and rapidly stopped C1s complement–mediated hemolysis in patients with cold agglutinin disease, significantly increasing hemoglobin levels and precluding the need for transfusions. This trial was registered at www.clinicaltrials.gov as #NCT02502903.

Identification of human plasma C1 inhibitor as a target protein for staphylococcal superantigen-like protein 5 (SSL5)

The family of staphylococcal superantigen-like proteins (SSLs) have a structure similar to bacterial superantigens but exhibit no superantigenic activity. These exoproteins have recently been shown to disturb the host immune defense system. One family member, SSL5, was reported to bind to human leukocyte P-selectin glycoprotein ligand-1 (PSGL-1) and matrix metalloproteinase-9 (MMP-9) and to interfere with leukocyte trafficking. In the present study, we explored human plasma proteins bound by glutathione S-transferase (GST)-tagged recombinant SSL5 (GST-SSL5) and identified plasma protease C1 inhibitor (C1Inh) as a major SSL5-binding protein based on the results of peptide mass fingerprinting analysis with MALDI-TOFMS. GST-SSL5 was found to attenuate the inhibitory activity of recombinant histidine-tagged C1Inh (C1Inh-His) toward complement C1s. We also observed that the treatment of C1Inh-His with neuraminidase markedly decreased its binding to GST-SSL5. Moreover, C1Inh-His produced by Lec2 mutant cells (deficient in sialic acid biosynthesis) showed much lower binding affinity for SSL5 than that produced by the wild-type CHO-K1 cells, as assessed by pull-down assay. These results suggest that SSL5 binds to C1Inh in a sialic acid-dependent fashion and modulates the host immune defense through perturbation of the complement system in association with S. aureus infection.

Abstract 5214: Tumor cell-derived complement components C1r and C1s promote growth of cutaneous squamous cell carcinoma

Abstract The incidence of cutaneous squamous cell carcinoma (cSCC) is increasing worldwide. We have analyzed the role of complement components C1r and C1s in the progression of cSCC. Analysis of cSCC cell lines (n=8) and normal human epidermal keratinocytes (NHEK)(n=11) with oligonucleotide arrays, RNA-seq and quantitative RT-PCR revealed significantly elevated C1r and C1s mRNA levels in cSCC cell lines. Western blot analysis showed increased production of C1r and C1s in cSCC cells compared to NHEKs. The mRNA levels for C1r and C1s were markedly elevated in cSCC tumors (n=6) compared to normal skin (n=11) in vivo. Immunohistochemical analysis for C1r and C1s revealed strong tumor cell specific expression of C1r and C1s in invasive sporadic cSCCs (n=164) and recessive dystrophic epidermolysis bullosa-associated cSCCs (n=16), whereas the expression of both was clearly lower in cSCC in situ (n=63), in premalignant epidermal lesions (actinic keratoses, n=61), and in normal skin (n=9). Knockdown of C1r and C1s expression with two specific siRNAs inhibited proliferation of cSCC cells and growth of human cSCC xenograft tumors in vivo. These results provide evidence for the role of tumor cell-derived C1r and C1s in the progression of cSCC independently of C1q and identify these serine proteinases as putative therapeutic targets in unresectable and metastatic cSCC. Citation Format: Pilvi Riihilä, Liisa Nissinen, Mehdi Farshchian, Markku Kallajoki, Atte Kivisaari, Seppo Meri, Reidar Grénman, Sirkku Peltonen, Juha Peltonen, Veli-Matti Kähäri. Tumor cell-derived complement components C1r and C1s promote growth of cutaneous squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5214.

Two independent proteomic approaches provide a comprehensive analysis of the synovial fluid proteome response to Autologous Chondrocyte Implantation

BackgroundAutologous chondrocyte implantation (ACI) has a failure rate of approximately 20%, but it is yet to be fully understood why. Biomarkers are needed that can pre-operatively predict in which patients it is likely to fail, so that alternative or individualised therapies can be offered. We previously used label-free quantitation (LF) with a dynamic range compression proteomic approach to assess the synovial fluid (SF) of ACI responders and non-responders. However, we were able to identify only a few differentially abundant proteins at baseline. In the present study, we built upon these previous findings by assessing higher-abundance proteins within this SF, providing a more global proteomic analysis on the basis of which more of the biology underlying ACI success or failure can be understood.MethodsIsobaric tagging for relative and absolute quantitation (iTRAQ) proteomic analysis was used to assess SF from ACI responders (mean Lysholm improvement of 33; n = 14) and non-responders (mean Lysholm decrease of 14; n = 13) at the two stages of surgery (cartilage harvest and chondrocyte implantation). Differentially abundant proteins in iTRAQ and combined iTRAQ and LF datasets were investigated using pathway and network analyses.ResultsiTRAQ proteomic analysis confirmed our previous finding that there is a marked proteomic shift in response to cartilage harvest (70 and 54 proteins demonstrating ≥ 2.0-fold change and p < 0.05 between stages I and II in responders and non-responders, respectively). Further, it highlighted 28 proteins that were differentially abundant between responders and non-responders to ACI, which were not found in the LF study, 16 of which were altered at baseline. The differential expression of two proteins (complement C1s subcomponent and matrix metalloproteinase 3) was confirmed biochemically. Combination of the iTRAQ and LF proteomic datasets generated in-depth SF proteome information that was used to generate interactome networks representing ACI success or failure. Functional pathways that are dysregulated in ACI non-responders were identified, including acute-phase response signalling.ConclusionsSeveral candidate biomarkers for baseline prediction of ACI outcome were identified. A holistic overview of the SF proteome in responders and non-responders to ACI has been profiled, providing a better understanding of the biological pathways underlying clinical outcome, particularly the differential response to cartilage harvest in non-responders.