C1s Inhibitor Research Articles

Sutimlimab for Cold Agglutinin Disease.

Cold agglutinin disease (CAD) is a rare type of autoimmune hemolytic anemia (AIHA) distinct from warm antibody AIHA. One of the ways it is distinct is that CAD is usually not responsive to corticosteroids compared with warm antibody AIHA. Historically, CAD therapy has been limited to immunotherapy or chemoimmunotherapy with varying responses. Cold agglutinin disease also poses a risk for thrombosis and mortality. For patients, fatigue tends to be a common symptom of CAD. The hallmark of CAD is complement-mediated hemolysis, which makes complement inhibitors a critical therapeutic option for patients. Previously, eculizumab, a C5 inhibitor, had limited therapeutic effect for CAD. More recently, sutimlimab, a C1s inhibitor, was shown in two phase III studies to be an efficacious treatment for CAD, improving hemoglobin, hemolysis, and fatigue. However, there is a paucity of medical literature on CAD and on sutimlimab in particular that is geared toward advanced practice providers (APPs). This article aims to provide APPs with a background in CAD and a focus on sutimlimab, assisting these providers in caring for patients with CAD receiving this therapy.

Sustained improvements in patient-reported outcomes after long-term sutimlimab in patients with cold agglutinin disease: results from the CADENZA study open-label extension

Cold agglutinin disease (CAD) is a rare subtype of autoimmune haemolytic anaemia characterised by classical complement pathway-mediated haemolysis, fatigue, and poor quality of life (QoL). Sutimlimab, a C1s inhibitor, rapidly halted haemolysis, and improved patient-reported outcomes (PROs) in patients with CAD in two phase 3 trials (CARDINAL and CADENZA). Here we report PROs from the CADENZA open-label extension (Part B). The first patient was enrolled in CADENZA (NCT03347422) in March 2018 (Part A) and the last patient completed the study in December 2021 (Part B). All patients who completed the 26-week Part A were eligible to receive biweekly doses of sutimlimab in Part B for up to 1 year after the last patient completed Part A. PROs were assessed throughout Part B, until the last on-treatment visit with available assessment (LV), and after a 9-week washout. In total, 32/39 patients completed Part B; median Part B treatment duration: 99 weeks. Patients switching from placebo to sutimlimab in Part B experienced rapid improvement in Functional Assessment of Chronic Illness Therapy (FACIT)-Fatigue score and other PROs. Sustained, clinically important improvements in FACIT-Fatigue were observed throughout Part B in patients who switched to sutimlimab and those continuing sutimlimab treatment (combined-group mean [SE] change from baseline at LV: 8.8 [2.1]). Similarly, the combined-group mean [SE] change for 12-Item Short Form Health Survey physical (4.9 [1.7]) and mental (4.0 [1.8]) component scores exceeded clinically important changes from baseline at LV. EuroQol visual analogue scale showed consistent and sustained increases from baseline with sutimlimab treatment. Following a 9-week washout, all PROs approached baseline values. Continued inhibition of the classical complement pathway with sutimlimab results in meaningful long-term improvements in PROs (fatigue and QoL) in patients with CAD. Sanofi.

New drugs for the treatment of primary immune thrombocytopenia

Two thrombopoietin receptor agonists (eltrombopag and romiplostim), rituximab or splenectomy have been recommended for the treatment of glucocorticoid-resistant ITP in Japanese guidelines. In addition, the Syk inhibitor fostamatinib and FcRn inhibitor efgartigimod were approved in Japan for refractory ITP in 2023 and 2024, respectively. Clinical trials have also reported promising results for the new thrombopoietin receptor agonist avatrombopag, the BTK inhibitor rilzabrutinib, and the C1s inhibitor sutimlimab. These developments will usher in a new era in the treatment of ITP.

Inhibition of the C1s Protease and the Classical Complement Pathway by 6-(4-Phenylpiperazin-1-yl)Pyridine-3-Carboximidamide and Chemical Analogs.

The classical pathway (CP) is a potent mechanism for initiating complement activity and is a driver of pathology in many complement-mediated diseases. The CP is initiated via activation of complement component C1, which consists of the pattern recognition molecule C1q bound to a tetrameric assembly of proteases C1r and C1s. Enzymatically active C1s provides the catalytic basis for cleavage of the downstream CP components, C4 and C2, and is therefore an attractive target for therapeutic intervention in CP-driven diseases. Although an anti-C1s mAb has been Food and Drug Administration approved, identifying small-molecule C1s inhibitors remains a priority. In this study, we describe 6-(4-phenylpiperazin-1-yl)pyridine-3-carboximidamide (A1) as a selective, competitive inhibitor of C1s. A1 was identified through a virtual screen for small molecules that interact with the C1s substrate recognition site. Subsequent functional studies revealed that A1 dose-dependently inhibits CP activation by heparin-induced immune complexes, CP-driven lysis of Ab-sensitized sheep erythrocytes, CP activation in a pathway-specific ELISA, and cleavage of C2 by C1s. Biochemical experiments demonstrated that A1 binds directly to C1s with a Kd of ∼9.8 μM and competitively inhibits its activity with an inhibition constant (Ki) of ∼5.8 μM. A 1.8-Å-resolution crystal structure revealed the physical basis for C1s inhibition by A1 and provided information on the structure-activity relationship of the A1 scaffold, which was supported by evaluating a panel of A1 analogs. Taken together, our work identifies A1 as a new class of small-molecule C1s inhibitor and lays the foundation for development of increasingly potent and selective A1 analogs for both research and therapeutic purposes.

Targeting Complement Inhibition in Cold Hemolysis; A Multifaceted Approach Using Sutimlimab and Washed Red Cell Transfusions

Introduction: Cold agglutinin syndrome (CAS) is a transient disorder mediated by autoantibodies that cause red cell agglutination at cold temperatures. CAS is often associated with infections rather than malignant or autoimmune conditions. Sutimlimab, a monoclonal antibody directed against the complement component C1s, was previously shown to improve anemia and fatigue in cold agglutinin disease (CAD) in single-arm studies. However, its use in CAS has not yet been elucidated. Here, we describe a case where complement inhibition using the novel C1s inhibitor, as well as washed and warmed red cell transfusions, were used to treat CAS in the setting of a rare autoantibody directed toward the Pr antigen Case report: A healthy 46-year-old woman presented with severe acute hemolytic anemia: with nadir hemoglobin of 4.7, hematocrit of 13.4%, 0.2% of reticulocytes, total bilirubin of 9.8 mg/dL, and unconjugated bilirubin of 8.4 mg/dL. A peripheral blood smear review revealed severe anemia with agglutination of red blood cells. An extensive infectious workup was negative including COVID-19, Parvovirus B19, CMV, EBV, CMV, and HIV. Over a period of two weeks, she received three doses of IVIG, prednisone, and 17 transfusions units of warmed red blood cells in the setting of persistent hemolytic anemia. Despite the evidence of severe hemolysis, her reticulocyte count remained very low (<0.5%). A bone marrow biopsy showed trilineage hematopoiesis with erythroid hypoplasia and maturation arrest. A direct Coombs test was intermittently positive but probably confused by IgG infusions, with anti-I and anti-i titers of 1:64, and positive for C3. Thermal amplitude studies showed agglutination at 30°C, 37°C, and room temperature. Red blood cell antibodies were not identified in the eluate. The indirect antiglobulin test was negative for ficin-treated RBCs showing no agglutination. This led the reference laboratory to report that the cold agglutination was directed against the anti-pr antigen and that this patient had clinically significant anti-Pr antibodies. The patient was kept warm and started on Danazol and Sutimlimab. However, the patient continued to have hemolysis after transfusions. After receiving her second dose of Sutimlimab and in the context of persistent hemolytic anemia, she underwent another bone marrow biopsy on day 34 after presentation. The biopsy showed hypercellular bone marrow with erythroid hyperplasia and complete maturation. However, this was not reflected in reticulocytosis until day 44. The patient received her third dose of Sutimlimab, and red blood cell transfusions were washed in addition to being warmed, which resolved hemolysis after transfusion reactions. She received a total of 30 red blood cell transfusions since presentation. The patient's anemia improved, with a hemoglobin level of 8.8 mg/dL and a reticulocyte count of 13%, and she was discharged on Day 51 after presentation (figure 1). She received one more dose of Sutimlimab (total of four doses) with complete resolution of her anemia, a normal reticulocyte count, normal LDH, negative Coombs study, and negative cold agglutinin titers. Conclusion: Here, we describe a challenging case of CAS caused by a rare Pr autoantibody, with a failure of first-line treatments for CAS. This report is the first to describe the use of Sutimlimab as a treatment for CAS. After initiating Sutimlimab, the patient continued to experience significant hemolysis shortly after transfusions, exhibiting hemoglobinuria and an inadequate increase in hematocrit. Only when the red blood cell transfusions were washed to remove complement, the hemolytic reactions ceased and significant increases in hematocrit occurred. This finding may be a consideration in other cases of cold agglutinin anemia. As CAS is a transient disorder, Sutimlimab could ultimately be discontinued once the hemolysis resolved. This case illustrates the role of complement inhibition in CAS and the potential need to warm and wash red blood cells in severe cases of CAS.

Long-term sutimlimab improves quality of life for patients with cold agglutinin disease: CARDINAL 2-year follow-up

AbstractCold agglutinin disease (CAD) is a rare form of autoimmune hemolytic anemia with a substantial burden on patient’s quality of life. CARDINAL was a 2-part, open-label, single-arm, multicenter phase 3 study evaluating the C1s inhibitor, sutimlimab, for treatment of CAD. Part A consisted of the pivotal study phase, with the part B extension phase assessing long-term safety and durability of response including patient-reported outcomes, which is the focus of this report. Altogether, 22 patients continued from part A to part B, majority female (68.2%) with a median age of 71.5 years (range, 55-85). Throughout treatment, score improvement on the Functional Assessment of Chronic Illness Therapy (FACIT)–Fatigue scale exceeded a predefined, group-level clinically important change of ≥5 points vs baseline, with a mean (standard error [SE]) change of 11.7 (3.7) points at week 135. The 12-Item Short Form Health Survey physical and mental component scores remained above baseline, with week 123 mean change (SE) exceeding clinically important changes of 3.9 for physical and 2.8 for mental component scores at 4.7 (2.8) and 3.8 (5.7) points, respectively. EuroQol Visual Analogue Scale, scoring patients’ self-rated health, also remained above baseline with a change of 17.1 (5.6) points at week 135. Patient Global Impression of (fatigue) Severity improved vs baseline, corroborating FACIT-Fatigue scores. Patient Global Impression of Change indicated a reduction in perceived disease burden. Data from CARDINAL part B support sustained alleviation of CAD disease burden after long-term treatment with sutimlimab over 2 years, returning toward baseline upon treatment cessation. This trial was registered at www.clinicaltrials.gov as #NCT03347396.

Abstract 154: Crossroads Of Immunity And Coagulation: Protease Activity Of Complement Component C1s Inhibits Protein C Activation In Covid-19

Introduction: There is an abundance of crosstalk between innate immunity and coagulation in thrombo-inflammatory diseases such as COVID-19. Several potential procoagulant effects of complement activation have been described, but whether complement proteases affect antithrombotic pathways in is not known. Aims: We tested the hypothesis that complement component 1 (C1) impairs the protein C antithrombotic pathway. Methods: Plasma levels of the C1 complex were measured in plasma collected from healthy individuals and COVID-19 patients hospitalized early in the COVID-19 pandemic (NCT04360824). Activation of protein C by recombinant thrombomodulin was measured in a two-stage assay. In the first stage, protein C, thrombin, and thrombomodulin were incubated to generate activated protein C. The reaction mix was incubated with either healthy or COVID-19 plasma in the presence or absence of C1 inhibitor (which binds to C1 to inhibit its activity) or sutimlimab (a specific C1s inhibitor). During the second stage, protein C activation was measured using spectrophotometry with the chromogenic substrate S-2366. Results: In the inflammatory condition COVID-19, plasma levels of the C1 complex were increased compared to healthy controls ( p = 0.004). Generation of activated protein C by recombinant thrombomodulin was decreased in the presence of COVID-19 plasma ( p = 0.001), whereas the addition of healthy plasma had no significant effect ( p = 0.469). Addition of C1 inhibitor or sutimlimab normalized protein C activation in the presence of COVID-19 plasma ( p = 0.022 and 0.039, respectively). Addition of recombinant C1s protease, but not C1r protease or C1q, impaired the generation of activated protein C in a dose dependent fashion. Conclusions: We have uncovered a novel role of the protease C1s in regulating the generation of anticoagulant activated protein C. These findings suggest that the classical pathway of complement activation may impair anticoagulant responses in COVID-19.

Discovery of a Novel Series of Potent, Selective, Orally Available, and Brain-Penetrable C1s Inhibitors for Modulation of the Complement Pathway.

A novel series of non-amidine-based C1s inhibitors have been explored. Starting from high-throughput screening hit 3, isoquinoline was replaced with 1-aminophthalazine to enhance C1s inhibitory activity while exhibiting good selectivity against other serine proteases. We first disclose a crystal structure of a complex of C1s and a small-molecule inhibitor (4e), which guided structure-based optimization around the S2 and S3 sites to further enhance C1s inhibitory activity by over 300-fold. Improvement of membrane permeability by incorporation of fluorine at the 8-position of 1-aminophthalazine led to identification of (R)-8 as a potent, selective, orally available, and brain-penetrable C1s inhibitor. (R)-8 significantly inhibited membrane attack complex formation induced by human serum in a dose-dependent manner in an in vitro assay system, proving that selective C1s inhibition blocked the classical complement pathway effectively. As a result, (R)-8 emerged as a valuable tool compound for both in vitro and in vivo assessment.

Sutimlimab: A Complement C1s Inhibitor for the Management of Cold Agglutinin Disease-Associated Hemolysis.

To review the pharmacology, pharmacokinetics, efficacy, safety, dosing and administration, and place in therapy of sutimlimab for the management of cold agglutinin disease (CAD)-associated hemolysis. A literature search of PubMed (1966-October 2022) was conducted using the keywords sutimlimab, BIVV009, and cold agglutinin. Data were also obtained from prescribing information, meeting abstracts, and clinicaltrials.gov. All published prospective clinical trials, prescribing information, and meeting abstracts on sutimlimab for the treatment of CAD were reviewed. Sutimlimab is a first-in-class complement C1s inhibitor indicated for the treatment of CAD-associated hemolysis. This approval was based on the phase III CARDINAL trial, which evaluated sutimlimab in patients with CAD-associated hemolysis. The primary endpoint of achieving a hemoglobin of ≥12 g/dL or increase of ≥2 above baseline was achieved by 54% of patients with sutimlimab in the 26-week trial. The phase III CADENZA trial was a placebo-controlled trial in which sutimlimab has demonstrated a significant improvement in the composite endpoint of hemoglobin increase of ≥1.5 g/dL, avoidance of transfusion, and avoidance of additional CAD therapies (73% sutimlimab vs 15% placebo). Sutimlimab rapidly halts hemolysis, improves hemoglobin, and improves quality-of-life in patients with CAD. Safety issues with sutimlimab include infusion-related reactions and risk of serious infections with encapsulated bacteria. Sutimlimab provides an additional therapeutic option in the treatment of CAD-associated hemolysis that can lead to rapid improvement in hemoglobin and anemia-related symptoms.

Effect of a C1s Inhibitor on the Efficacy of Anti-Capsular Antibodies againstNeisseria meningitidisandStreptococcus pneumoniae

Terminal complement pathway inhibition at the level of C5 alleviates symptoms of several diseases associated with complement overactivation. However, C5 blockade is associated with an increased risk of invasive meningococcal disease despite immunization. Targeting specific complement pathways proximal to C5 provides the theoretical advantage of leaving the other pathways (including the terminal pathway) intact for immune surveillance. We aimed to address the risk of Neisseria meningitidis and Streptococcus pneumoniae infections when inhibiting the classical pathway (CP) using a specific C1s inhibitor (TNT005). Addition of TNT005 to 20% normal human serum that contained anti-meningococcal capsular Ab decreased C4 deposition 8-fold and abrogated killing of N. meningitidis, despite leaving C3 deposition intact. TNT005 impaired killing of N. meningitidis in 78% nonimmune human plasma and 78% whole blood but permitted killing in both when specific anti-capsular Ab was added. Simultaneously inhibiting both the CP and alternative pathway (AP) blocked killing of Ab-coated N. meningitidis in whole blood. Blocking the AP alone abrogated C3 deposition, whereas TNT005 only partially inhibited (∼40% decrease) C3 deposition on S. pneumoniae coated with anti-capsular Ab. Blocking either the CP or AP alone did not impair killing of pneumococci in whole blood containing specific Ab (<10% survival at 3 h); however, blocking both pathways resulted in ∼35% bacterial survival. These data suggest that killing of N. meningitidis or S. pneumoniae in whole blood containing specific anti-capsular Abs is unimpeded by TNT005. Meningococcal and pneumococcal capsular conjugate vaccines may mitigate risk of these infections in patients receiving C1s inhibitors.

Specific Inhibition of the Classical Complement Pathway Prevents C3 Deposition along the Dermal-Epidermal Junction in Bullous Pemphigoid

Deposition of autoantibodies (α-BP180 and BP230) and complement along the dermal-epidermal-junction is a hallmark of bullous pemphigoid and was shown to be important for pathogenesis. Given the adverse effects of standard treatment (glucocorticoids, immunosuppressants), there is an unmet need for safe and effective therapies. In this phase 1 trial, we evaluated the safety and activity of BIVV009 (sutimlimab, previously TNT009), a targeted C1s inhibitor, in 10 subjects with active or past bullous pemphigoid (NCT02502903). Four weekly 60 mg/kg infusions of BIVV009 proved sufficient for inhibition of the classical complement pathway in all patients, as measured by CH50. C3c deposition along the dermal-epidermal junction was partially or completely abrogated in 4 of 5 patients, where it was present at baseline. BIVV009 was found to be safe and tolerable in this elderly population, with only mild to moderate adverse events reported (e.g., headache, fatigue). One serious adverse event (i.e., fatal cardiac decompensation) occurred at the end of the post-treatment observation period in an 84-year-old patient with a history of diabetes and heart failure, but was deemed unlikely to be related to the study drug. This trial provides the first results with a complement-targeting therapy in bullous pemphigoid, to our knowledge, and supports further studies on BIVV009's efficacy and safety in this population.

Cold agglutinin disease: current challenges and future prospects.

Cold agglutinin disease (CAD) is a complement-dependent, classical pathway-mediated immune hemolytic disease, accounting for 15–25% of autoimmune hemolytic anemia, and at the same time, a distinct clonal B-cell lymphoproliferative disorder of the bone marrow. The disease burden is often high, but not all patients require pharmacological treatment. Several therapies directed at the pathogenic B-cells are now available. Rituximab plus bendamustine or rituximab monotherapy should be considered first-line treatment, depending on individual patient characteristics. Novel treatment options that target the classical complement pathway are under development and appear very promising, and the C1s inhibitor sutimlimab is currently being investigated in two clinical Phase II and III trials. These achievements have raised new challenges and further prospects, which are discussed. Patients with CAD requiring therapy should be considered for clinical trials.

Autoimmune hemolytic anemia.

The diagnosis of autoimmune hemolytic anemia (AIHA) can be made with a stepwise approach that aims to identify laboratory and clinical evidence of hemolysis and then determine the immune nature of hemolysis with the direct anti-globulin test. Once alternative causes for these findings have been excluded, AIHA is established, and the clinician must search for secondary causes, as well as identify the type of AIHA. Rituximab is now the preferred second-line treatment for primary warm AIHA and first-line treatment for primary cold agglutinin disease (CAD), either as monotherapy or combined with bendamustine. Complement inhibitors have shown utility in stabilizing AIHA patients with acute severe hemolysis. Future prospects are discussed and include the C1s inhibitor BIVV009 (sutimlimab) that is now entering phase 3 studies for CAD.

How I manage patients with cold agglutinin disease.

Cold agglutinin disease (CAD) is an uncommon autoimmune haemolytic anaemia in which a well-defined, clonal low-grade lymphoproliferative disorder of the bone marrow results in erythrocyte destruction mediated by the classical complement pathway. The pathogenesis, clinical features and diagnostic criteria are reviewed. Although anaemia is mild in some patients, approximately one-third of untreated patients have a haemoglobin level of ≤80g/l, and about 50% have been considered transfusion dependent for shorter or longer periods. Therapy has improved greatly during the last 15years. Mild disease can be managed by avoidance of cold and adequate precautions in specific situations, without drug therapy. Corticosteroids should not be used to treat CAD. Patients requiring pharmacological therapy should be considered for prospective trials. Outside clinical studies, the rituximab-bendamustine combination or rituximab monotherapy is recommended in the first line, depending on individual patient characteristics. Second-line options are rituximab-fludarabine in fit patients or, although less strongly documented, a bortezomib-based regimen. Therapies targeting the classical complement pathway are promising, and the complement C1s inhibitor, BIVV009, has shown favourable results in preliminary studies.

Uncoupling complement C1s activation from C1q binding in apoptotic cell phagocytosis and immunosuppressive capacity

Complement activation contributes to inflammation in many diseases, yet it also supports physiologic apoptotic cells (AC) clearance and its downstream immunosuppressive effects. The roles of individual complement components in AC phagocytosis have been difficult to dissect with artificially depleted sera. Using human in vitro systems and the novel antibody complement C1s inhibitor TNT003, we uncoupled the role of the enzymatic activation of the classical pathway from the opsonizing role of C1q in mediating a) the phagocytosis of early and late AC, and b) the immunosuppressive capacity of early AC. We found that C1s inhibition had a small impact on the physiologic clearance of early AC, leaving their immunosuppressive properties entirely unaffected, while mainly inhibiting the phagocytosis of late apoptotic/secondary necrotic cells. Our data suggest that C1s inhibition may represent a valuable therapeutic strategy to control classical pathway activation without causing significant AC accumulation in diseases without defects in AC phagocytosis.

Autoimmune hemolytic anemia

The diagnosis of autoimmune hemolytic anemia (AIHA) can be made with a stepwise approach that aims to identify laboratory and clinical evidence of hemolysis and then determine the immune nature of hemolysis with the direct anti-globulin test. Once alternative causes for these findings have been excluded, AIHA is established, and the clinician must search for secondary causes, as well as identify the type of AIHA. Rituximab is now the preferred second-line treatment for primary warm AIHA and first-line treatment for primary cold agglutinin disease (CAD), either as monotherapy or combined with bendamustine. Complement inhibitors have shown utility in stabilizing AIHA patients with acute severe hemolysis. Future prospects are discussed and include the C1s inhibitor BIVV009 (sutimlimab) that is now entering phase 3 studies for CAD.

Classical Pathway of Complement Activation in ITP: Inhibition By TNT003, a Novel C1s Inhibitor

Background: Immune thrombocytopenic purpura (ITP) is an autoimmune disorder in which antiplatelet antibodies mediate accelerated platelet clearance from circulation and also inhibit platelet production, resulting in thrombocytopenia. Activation of the classical pathway (CP) of complement is associated with a variety of immune disorders involving the presence of autoantibodies. The role of the complement system in ITP is poorly understood.Methods: Plasma samples (0.32% sodium citrate) from patients with chronic ITP (n=55) were evaluated for their ability to activate the CP of complement. The 55 patients consisted primarily of adult but also of pediatric patients with ITP, undergoing various treatment regimens. The most common included IVIG, rituximab, and especially thrombopoietic agents (eltrombopag, romiplostim). Almost all patients included in the analysis had chronic ITP, defined as ITP lasting > 12 months. The complement activating capacity (CAC) of patient plasma was evaluated with a previously described in vitro assay ( Peerschke et al., Brit J Haematol, 2009) that measures complement activation on immobilized, fixed heterologous platelets using an ELISA approach with monoclonal antibodies to C1q, C4d, iC3b, and C5b-9. CAC represents assay optical density readings normalized to reference normal plasma pool. A CAC of >1.5 was considered indicative of enhanced complement activation, based on reference ranges established for plasma from healthy volunteers. Patient CAC values were correlated with platelet count. The ability of TNT003 to block in vitro complement activation was assessed relative to an isotype matched control. TNT003 is a mouse monoclonal antibody (IgG2a) that targets the CP-serine protease C1s.Results: A statistically significant (p=0.042) inverse correlation was noted between C4d deposition and platelet count in the 55 ITP patient samples tested. Heightened classical complement pathway activation was demonstrated in 7 of 55 patients (~13%) with ITP as evidenced by increased C4d deposition. 6 of the 7 patients with increased C4d deposition had platelet counts <100k/mcL, and 5 patients had platelet counts <50K/mcL. There was a non-significant trend for higher C4d levels on platelets and lower AIPF (absolute immature platelet fraction, equivalent to platelet reticulocytes). TNT003 (100 mcg/ml) inhibited C4 activation by 44 + 43% in ITP plasma in vitro. Inhibition of downstream complement activation, iC3b and C5b-9 deposition, was 72% + 17 % and 82% + 14% (mean + S.D.), respectively. Similar results were obtained using 10 mM EDTA, a known inhibitor of complement activation.Conclusions: The heterogeneity of patient responses to different treatment modalities in ITP support the concept of different immune mechanisms contributing to thrombocytopenia. Our data demonstrate classical complement pathway activation in a subgroup of patients with ITP, and further present the first evidence of CP complement inhibition by a novel C1s inhibitor in this setting. Failure to completely block C4 activation in ITP plasma in vitro by either of TNT003 or EDTA, suggests the presence of preformed, circulating C4d containing complement complexes in patient plasma. The ability of TNT003 to more completely inhibit C3 activation and C5b-9 assembly downstream of C4 in the in vitro assay system is consistent with direct activation and inhibition of complement at the platelet surface. Thus, TNT003 may mitigate enhanced platelet clearance by RES via inhibition of complement mediated platelet opsonization by C3b and platelet lysis by C5b-9. Further studies are required to evaluate the impact of TNT003 on thrombocytopenia in ITP. DisclosuresPeerschke:True North Therapeutics: Research Support Other. Panicker:True North Therapeutics: Employment. Bussel:True North Therapeutics: Research Support Other.

IL-13 results in differential regulation of the complement proteins C3 and factor B in tumour necrosis factor (TNF)-stimulated fibroblasts.

IL-13, like IL-4, a product of activated T cells, has multiple biological actions, primarily on B cells and monocytes. The purpose of the present study was to compare the effects of IL-13 with those of IL-4 on the synthesis of complement proteins in fibroblasts. Dermal fibroblasts were developed from skin biopsies. Confluent monolayers were stimulated with the relevant cytokine or combinations of cytokines and biosynthetically labelled with 35S-methionine. The specific proteins were analysed using immunoprecipitation and SDS-PAGE. Addition of IL-13 to fibroblast cultures treated with TNF-alpha resulted in a dose-dependent increase in C3 protein biosynthesis and a concomitant down-regulation of factor B protein biosynthesis. In TNF-stimulated fibroblasts, the addition of IL-13, 100 ng/ml, induced a 2.45-fold increase in the synthesis of C3, while in the same cells under identical conditions the synthesis of factor B was only 42% of the level without IL-13. Similar effects of IL-13 were noted on IL-1-treated fibroblasts. These effects were specific for C3 and factor B, and no alteration of the constitutive or TNF-induced synthesis of C1s or C1 inhibitor proteins was observed. IL-13 altered the synthesis of C3 and factor B proteins also in fibroblasts stimulated with interferon-gamma (IFN-gamma) in addition to TNF, in the same direction as it did in cells stimulated with TNF alone. IL-13 has similar effects to those of IL-4 on the synthesis of C and factor B in TNF- and IL-1-stimulated fibroblasts. The observed effects of IL-13 are IL-4-independent, as anti-IL-4 antibody abrogates IL-4-induced effects, but has no effect on IL-13-induced responses. This interaction between different cytokines on the synthesis of proinflammatory and immunoregulatory proteins may have significance, particularly at local sites of inflammation, and may affect the synthesis of complement proteins in inflamed joint as in rheumatoid arthritis.

Proteome analysis of myocardial tissue following ischemia and reperfusion—Effects of complement inhibition

Myocardial ischemia–reperfusion injury can be related to complement activation with generation of chemotactic mediators, release of cytokines, leukocyte accumulation, and subsequent severe tissue injury. In this regard, activation of transcription factors (i.e., NFκB) and de novo protein synthesis or inflammatory protein degradation seems to play an important role. In the present study, we analyzed the cardiac protein expression following myocardial ischemia (60 min) and reperfusion (180 min) in a rabbit model utilizing two-dimensional electrophoresis and nanoHPLC/ESI-MS/MS for biochemical protein identification. To achieve cardioprotective effects, we used a novel highly selective small molecule C1s inhibitor administered 5 min prior to reperfusion. The reduction of myocardial injury was observed as diminished plasma creatine kinase activity in C1s-INH-248-treated animals (65.2 ± 3 vs. 38.5 ± 3 U/g protein after 3 h of reperfusion, P < 0.05). With proteome analysis we were able to detect 509 ± 21 protein spots on the gels of the 3 groups. A pattern of 480 spots with identical positions was found on every gel of myocardial tissue of sham animals, vehicle and C1s-INH-248-treated animals. We analyzed 11 spots, which were identified by mass spectrometry: Superoxide dismutase, α-crystallin-chain-B, mitochondrial stress protein, Mn SOD, ATP synthase A chain heart isoform, creatine kinase, and troponin T. All of these proteins were significantly decreased in the vehicle group when we compared to sham-treated animals. Treatment with C1s-INH-248 preserved levels of these proteins. Thus, blocking the classical complement pathway with a highly specific and potent synthetic inhibitor of the activated C1 complex archives cardio-protection by altering and preserving different anti-inflammatory and cytoprotective cascades.

Novel small molecule inhibitor of C1s exerts cardioprotective effects in ischemia-reperfusion injury in rabbits.

Myocardial ischemia-reperfusion injury can be related to complement activation with generation of chemotactic agents, adhesion molecule expression, release of cytokines and oxygen-derived free radicals, and subsequent neutrophil accumulation. In the present study the cardioprotective effects of a novel highly selective small molecule C1s inhibitor (C1s-INH-248, Knoll) were examined in a rabbit model of myocardial ischemia (I) and reperfusion (R; i.e., 60 min I + 180 min R). In in vitro tests (enzyme activity and SRBC lysis) C1s-INH-248 demonstrated profound inhibitory potency. In vivo C1s-INH-248 (1 mg/kg body weight) administered 5 min before reperfusion significantly attenuated myocardial injury (31.9 +/- 2.5 vs 8.9 +/- 1.6% necrosis/area at risk; p < 0.01). The cardioprotective effect was dose dependent. The reduction of myocardial injury was also observed as diminished plasma creatine kinase activity in C1s-INH-248-treated animals (70.7 +/- 6.8 vs 45.1 +/- 3.9 U/g protein after 3 h of reperfusion, p < 0.05). Further, cardiac myeloperoxidase activity (i.e., a marker of PMN accumulation) in the ischemic and necrotic area was significantly reduced following C1s-INH-248 treatment (1.31 +/- 0.23 vs 0.4 +/- 0.05 U/100 mg tissue in necrotic area, p < 0.01). Thus, blocking the classical complement pathway with a highly specific and potent synthetic inhibitor of the activated C1 complex appears to be an effective mean to preserve ischemic myocardium from injury following reperfusion.