anti-C2 Antibodies Research Articles

Molecular pharmacology in complement-mediated hemolytic disorders.

In the last decade, a deeper understanding of the pathogenesis of complement mediated hemolytic disorders, such as paroxysmal nocturnal hemoglobinuria (PNH), cold agglutinin disease (CAD), warm type autoimmune hemolytic anemia (AIHA) with complement activation (wAIHA), and atypical hemolytic uremic syndrome (aHUS), paved the way to the therapeutic shift from purely supportive approaches to complement-targeted therapies. This resulted in a significant improvement in disease management, survival, and quality of life. In this review, we will provide a snapshot of novel therapies for complement-mediated hemolytic anemias with a focus on those ready to use in clinical practice. C5 inhibitors eculizumab and the long-acting ravulizumab, are the established gold standard for untreated PNH patients, whilst the C3 inhibitor pegcetacoplan should be considered for suboptimal responders to anti-C5 drugs. Several additional compounds targeting the complement cascade at different levels (other C5 inhibitors, factor B and D inhibitors) are under active investigation with promising results. In CAD, immunosuppression with rituximab remains the first-line. However, recently FDA and EMA approved the anti-C1s monoclonal antibody, sutimlimab, that showed dramatic responses and whose regulatory approval is soon awaited in many countries. Other drugs under investigation in AIHA include the C3 inhibitor pegcetacoplan, and the anti-C1q ANX005 for warm AIHA with complement activation. Finally, aHUS is an indication for complement inhibitors. Eculizumab and ravulizumab have been approved, whilst other C5 inhibitors, and novel lectin pathway inhibitors are under active investigation in this disease.

A nanobody-based complement inhibitor targeting complement component 2 reduces hemolysis in a complement humanized mouse model of autoimmune hemolytic anemia

C2 is an attractive therapeutic target for many complement-mediated diseases. We developed Nab1B10, a new anti-C2 nanobody that potently and selectively inhibits both the classical and lectin pathways of complement activation. Mechanistically, Nab1B10 binds to the C2a portion of C2 and inhibits the assembly of C3 convertase C4b2a. Nab1B10 cross-reacts with monkey but not rodent C2 and inhibits classical pathway-mediated hemolysis. Using a new complement humanized mouse model of autoimmune hemolytic anemia (AIHA), we demonstrated that Nab1B10 abolished classical pathway complement activation-mediated hemolysis in vivo. We also developed C2-neutralizing bi- and tetra-valent antibodies based on Nab1B10 and found these antibodies significantly more potent than the other anti-C2 monoclonal antibody that is already in clinical trials. These data suggest that these novel C2-neutralizing nanobodies could be further developed as new therapeutics for many complement-mediated diseases, in which pathogenesis is dependent on the classical and/or lectin pathway of complement activation.

Safety and efficacy of classical complement pathway inhibition with sutimlimab in chronic immune thrombocytopenia.

Chronic/refractory immune thrombocytopenia (ITP) is a rare and pathophysiologically heterogeneous disorder with variable responsiveness to available treatments. Sutimlimab, a first-in-class humanized monoclonal anti-C1s IgG4 antibody, selectively inhibits the classical pathway. This phase 1 study (NCT03275454) assessed the safety, efficacy, pharmacokinetics, and pharmacodynamics of biweekly sutimlimab in patients with chronic/refractory ITP with an inadequate response to ≥2 therapies (platelet count ≤30×109/L). Twelve patients (median age 42 years) received sutimlimab for a median of 20.5 weeks followed by a median 2-week washout period (part A). In part B, 7 of the 12 eligible patients received sutimlimab retreatment for a median of 113 weeks. In part A, the mean (standard deviation) platelet count increased from 25× 109/L (17) to 54× 109/L (60) 24 hours after starting sutimlimab, maintaining ≥50× 109/L throughout part A. Five patients (42%) achieved durable platelet count responses (≥50× 109/L in ≥50% of follow-up visits) and 4achieved complete response (platelet count ≥100× 109/L). The mean platelet count returned to baseline during washout and increased upon retreatment in part B. The mean platelet count improvements accompanied the rapid inhibition of the classical pathway. There were 74 treatment-emergent adverse events in part A (n= 10) and 70 in part B (n= 6). Five serious adverse events were observed; 1 event (migraine) was assessed by the investigator as related to sutimlimab. These results demonstrated that in some patients with ITP, autoantibodies activate the classical complement pathway, accelerating platelet destruction or impairing platelet production and contributing to treatment failure. Thus, C1s inhibition may be a safe and beneficial therapeutic approach for patients with chronic/refractory ITP.

Novel therapeutic agents for hemolytic anemia

In recent years, it has become clear that various diseases are caused by complement (related molecule) abnormalities (complementopathies) or are exacerbated by complement (complement-related diseases), and novel therapeutic agents targeting complement (anti-complement agents) are now being developed. Paroxysmal nocturnal hemoglobinuria (PNH) is a hematopoietic stem cell disorder characterized by complement-mediated intravascular hemolysis due to a deficiency of complement regulatory factors, making it a perfect candidate for anti-complement agents. In 2007, the anti-C5 monoclonal antibody eculizumab was approved for PNH, as the first anti-complement agent. The indications for eculizumab are expanding, and aggressive development is underway for new anti-complement agents, not only for PNH but also a variety of other diseases. In addition, the anti-C1s antibody sutimlimab was approved last year for the treatment of cold agglutinin disease, a form of autoimmune hemolytic anemia. This presentation overviews novel anti-complement agents for these hemolytic anemias.

Novel anti-complement therapeutics for hemolytic anemia

The anti-C5 antibody eculizumab was approved in 2007 as the first anti-complement agent for the treatment of paroxysmal nocturnal hemoglobinuria (PNH). While eculizumab's indication has been expanded to include other diseases, the development of new anti-complement agents has been aggressively pursued for various diseases. In PNH, the anti-C5 recycling antibody ravulizumab, which is an improved version of eculizumab, has been developed, with an extended dosing interval of 2 to 8 weeks, vastly improving convenience. The treatment of PNH with terminal complement inhibitors such as eculizumab and ravulizumab presents a new challenge-extravascular hemolysis. To address this issue, the proximal complement inhibitor, a C3 inhibitor called pegcetacoplan, was approved in the United States of America. Furthermore, the amplification loop inhibitors-a factor B inhibitor iptacopan, and a factor D inhibitor danicopan-are being developed. Recently, the anti-C1s antibody sutimlimab was approved for the treatment of cold agglutinin disease, a type of autoimmune hemolytic anemia. This article discusses novel anti-complement therapies for hemolytic anemia.

Complement inhibition prevents glial nodal membrane injury in a GM1 antibody-mediated mouse model.

The involvement of the complement pathway in Guillain-Barré syndrome pathogenesis has been demonstrated in both patient biosamples and animal models. One proposed mechanism is that anti-ganglioside antibodies mediate neural membrane injury through the activation of complement and the formation of membrane attack complex pores, thereby allowing the uncontrolled influx of ions, including calcium, intracellularly. Calcium influx activates the calcium-dependent protease calpain, leading to the cleavage of neural cytoskeletal and transmembrane proteins and contributing to subsequent functional failure. Complement inhibition has been demonstrated to provide effective protection from injury in anti-ganglioside antibody-mediated mouse models of axonal variants of Guillain-Barré syndrome; however, the role of complement in the pathogenesis of demyelinating variants has yet to be established. Thus, it is currently unknown whether complement inhibition would be an effective therapeutic for Guillain-Barré syndrome patients with injuries to the Schwann cell membrane. To address this, we recently developed a mouse model whereby the Schwann cell membrane was selectively targeted with an anti-GM1 antibody resulting in significant disruption to the axo-glial junction and cytoplasmic paranodal loops, presenting as conduction block. Herein, we utilize this Schwann cell nodal membrane injury model to determine the relevance of inhibiting complement activation. We addressed the early complement component C2 as the therapeutic target within the complement cascade by using the anti-C2 humanized monoclonal antibody, ARGX-117. This anti-C2 antibody blocks the formation of C3 convertase, specifically inhibiting the classical and lectin complement pathways and preventing the production of downstream harmful anaphylatoxins (C3a and C5a) and membrane attack complexes. Here, we demonstrate that C2 inhibition significantly attenuates injury to paranodal proteins at the node of Ranvier and improves respiratory function in ex vivo and in vivo Schwann cell nodal membrane injury models. In parallel studies, C2 inhibition also protects axonal integrity in our well-established model of acute motor axonal neuropathy mediated by both mouse and human anti-GM1 antibodies. These data demonstrate that complement inhibition prevents injury in a Schwann cell nodal membrane injury model, which is representative of neuropathies associated with anti-GM1 antibodies, including Guillain-Barré syndrome and multifocal motor neuropathy. This outcome suggests that both the motor axonal and demyelinating variants of Guillain-Barré syndrome should be included in future complement inhibition clinical trials.

Inhibition of Complement C1s By Sutimlimab in Patients with Cold Agglutinin Disease (CAD): Efficacy and Safety Results from the Randomized, Placebo-Controlled Phase 3 CADENZA Study

Inhibition of Complement C1s By Sutimlimab in Patients with Cold Agglutinin Disease (CAD): Efficacy and Safety Results from the Randomized, Placebo-Controlled Phase 3 CADENZA Study

Clinically Important Change in SF-12v2 Physical (PCS) and Mental (MCS) Component Summary Scores for Patients with Cold Agglutinin Disease: An Analysis Using the Phase 3 CARDINAL and CADENZA Studies

IntroductionCold Agglutinin Disease (CAD) is an autoimmune condition resulting in hemolytic anemia; the subsequent fatigue causes a reduction in patient's physical and mental health-related quality of life (HRQoL). Sutimlimab (formerly BIVV009) is a humanized monoclonal anti-C1s antibody with a clinical trial program to support its development as a treatment for CAD. CARDINAL (NCT03347396) and CADENZA (NCT03347422) are Phase 3 clinical trials that assessed the treatment-related change in HRQoL using the 12-item Short-Form Health Survey (version 2) (SF-12v2) in patients with CAD treated with sutimlimab. This analysis aimed to estimate the clinically important change (CIC) in SF-12v2 physical (PCS) and mental (MCS) component summary scores, using pooled data from CARDINAL and CADENZA.MethodsCARDINAL was an open-label, single-arm, multicenter study for patients with CAD with a recent blood transfusion. CADENZA was a randomized, double-blind, placebo-controlled, multicenter study; patients with CAD (without a recent blood transfusion) were randomized 1:1 to receive sutimlimab or placebo. In both studies, patients underwent efficacy and safety assessments for 6 months (Part A). Data from the Part A 26-week component of both studies were combined for these analyses. CIC, defined as mean score change divided by SD at baseline, gave treatment effect sizes of 0.2 (small), 0.5 (moderate) or 0.8 (large), allowing for interpretation of patient-reported outcomes by pre-determining clinically meaningful improvements in physical and mental well-being. Anchor- and distribution-based analyses were performed to estimate the CIC for the following patient-reported outcome measures: Patient Global Impression of (fatigue) Severity (PGIS) and Patient Global Impression of Change (PGIC). Anchor-based approaches (mean change and model-based) examined the relationship between change in SF-12v2 PCS and MCS scores and change in related anchor variables: change in PGIS, PGIC (from baseline to Week 26) and hemoglobin levels (g/dl) (from baseline to the mean value of Weeks 23, 25 and 26). The independent variable was change in PCS and MCS scores and dependent variables were binary (improvement vs. no improvement) in model-based analyses.ResultsFifty-five patients with SF-12v2 data available were included from CARDINAL (n=17) and CADENZA (n=38). The median (range) age was 70 (46-88) years and 76% of patients were female (n=42). Mean (SD) PCS and MCS scores at baseline were 40.9 (7.8) and 47.3 (9.1), respectively. From baseline to Week 26, mean (SD) changes in PCS and MCS scores were 4.1 (7.1) and 3.1 (11.6), respectively. Correlations between changes in PCS and MCS scores and PGIS, hemoglobin, and self-report of change in PGIC at Week 26 ranged from 0.36 to 0.55, indicating moderate associations (Table 1). When using an anchor-based approach that evaluated mean change in PCS and MCS scores, CIC estimates ranged from 2.9 to 7.4 for PCS and 2.2 to 7.0 for MCS (Table 2). Results from the 2 model-based anchor approaches, which estimated CIC using receiver operating characteristic curves and an adjusted logistic regression-based approach ranged from 2.0 to 7.3 for PCS and 1.1 to 4.1 for MCS (Table 2). Distribution-based analyses determined the CIC to be 3.9 and 4.6 for PCS and MCS, respectively, when based on one-half of the standard deviation of baseline scores, and 3.5 (PCS) and 4.1 (MCS) when based on the standard error of measurement. The median estimate produced by all anchor-based methods were closely aligned with results of distribution-based analyses. Therefore, taking into consideration all approaches, the median CICs for CAD patients for PCS and MCS were 3.9 and 2.8, respectively.ConclusionIn this analysis, patients with CAD had median CIC values of 3.9 and 2.8 for PCS and MCS, respectively. In a previous study, anchor-based analyses for the general population gave CIC scores of 3.0 for both PCS and MCS (Maruish ME et al. 3 rd edition; Lincoln, RI; 2012). The results of this study converge well with those estimates observed for the general population involving thousands of observations. This analysis also demonstrated that meaningful physical and mental health benefits (including a reduction in fatigue) were associated with improvements in hemoglobin levels, a main clinical outcome measure of CAD. [Display omitted] DisclosuresJoly: Sanofi: Current Employment. Kosinski: QualityMetric Incorporated, LLC: Current Employment. Shafer: Sanofi: Current Employment, Current equity holder in publicly-traded company; Pfizer: Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months, Ended employment in the past 24 months. Wardecki: Sanofi: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months, Other: May hold shares and/or stock options with Sanofi. Karaouni: BMAPS SARL(Geneva, Switzerland): Current Employment; Sanofi: Consultancy. Hill: Amgen: Honoraria; Alexion: Honoraria; ReAlta: Consultancy; Sanofi: Consultancy; Grifols: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Argenx: Consultancy; Apellis: Consultancy, Honoraria.

Platelet-Based Factor VIII Activity Is Inhibited By Anti-C2 Domain Antibodies That Don’t Significantly Inhibit Factor VIII Activity in a Standard Assay

The major complication of hemophilia A treatment is the development of inhibitory antibodies against factor VIII (fVIII), causing severe bleeding in spite of infused fVIII. Most antibodies are targeted to the A2 or the C2 domain of fVIII and the degree of inhibition by these antibodies measured in clinical fVIII assays fails to predict the severity of bleeding risk. We investigated whether the degree of inhibition of platelet dependent fVIII activity by a panel of monoclonal anti-C2 domain mAbs deviated from the degree of inhibition in a commercial one-stage assay.Methods: Platelet dependent fVIII activity was measured in a defined activated platelet time (APT) assay containing fresh, density gradient-purified human platelets and fVIII deficient plasma supplemented with fVIII. Coagulation was triggered by simultaneous addition of calcium, thrombin receptor activation peptides PAR-1 and PAR-4, and factor XIa without other exogenous activators or vesicles. Results were compared to a commercial aPTT assay (Helena Laboratories, TX) and to results of a novel factor Xase assay developed to recapitulate the conditions in an APT. In the novel assay fresh, density-gradient purified platelets were activated by thrombin followed by inactivation with hirudin and incubated with fVIII and VWF, fIXa, and fX. In a second step, phospholipid vesicles, fVa and prothrombin were added to produce thrombin in proportion to the fXa generated in the first step. For comparison to the aPTT this assay was also done using purified sonicated phospholipid vesicles instead of activated platelets in the first step. The reaction was quenched with EDTA and thrombin generation was determined immediately in a thermosttated kinetics microtiter plate reader (Molecular Devices) at 250C using the chromogenic substrate S-2238 (Diapharma). A panel of 10 defined, epitope mapped mAbs against the fVIIIC2 domain was chosen in order to survey the relationship between inhibition and binding of anti-C2 antibodies.Results: The APT exhibited a log-linear relationship between fVIII and time to fibrin strand formation over a 4-log range, encompassing 0.01% - 100% plasma fVIII. Nine of 10 mAbs inhibited fVIII activity 89-96% while mAb F85 did not inhibit. There was excellent correlation for the degree of inhibition in the platelet-based factor Xase assay for 8 of 10 antibodies with F85 inhibiting the platelet Xase but not the APT and I109 inhibiting the platelet Xase assay less than the APT. In contrast, there was no correlation between inhibition of the aPTT-based assay and the platelet assay. Four mAbs did not inhibit the aPTT assay yet inhibited approximately 90% of platelet-based activity. The novel factor Xase assay, using phospholipid vesicles instead of activated platelets, showed inhibitory results that correlated to the degree of inhibition in the aPTT assay (which also utilizes purified phospholipids). Thus, the novel assay indicates that the degree of inhibition in both platelet-based and vesicle-based clotting assays is a consequence of the membrane type rather than unidentified clotting modifiers in plasma.Conclusions: We have developed a novel factor Xase assay to measure the low levels of residual fVIII activity, at physiologic concentrations, in the presence of inhibitory antibodies. Antibody inhibition of fVIII activity correlated with inhibition measured in the aPTT and APT clotting assays,. Our results confirm that the anti-C2 inhibitory antibodies have substantially different inhibition on a platelet membrane than in an aPTT-based assay and that the differing degree of inhibition is primarily due to fVIII susceptibility to inhibition when functioning on platelet membranes vs. phospholipid vesicles. These results suggest that current inhibitor assays might underestimate the frequency of significant inhibitory antibodies. DisclosuresNo relevant conflicts of interest to declare.

Predicted coagulation potential using an in vitro simulated model of emicizumab prophylaxis and immune tolerance induction therapy in hemophilia A patients with inhibitor.

Emicizumab reduces bleeding in hemophilia A patients with inhibitor (HA-inh). A combination of immune tolerance induction therapy (ITI) and emicizumab prophylaxis may provide additional benefits, but coagulation potential during this treatment remains unknown. We assessed coagulation potentials in simulated ITI models in vitro using modified-clot waveform analysis. Factor (F)VIII-deficient plasma preincubated with anti-A2 and anti-C2 monoclonal antibodies was reacted with emicizumab (50µg/mL) (emicizumab-HA-plasma), then spiking bypassing agents (BPAs): activated prothrombin complex concentrates (aPCC 1.3IU/mL; 50IU/kg), recombinant factor (rF)VIIa (2.2µg/mL; 90µg/kg), and FVIIa/FX (1.5µg/mL; 60µg/kg), and/or FVIII (100, 200IU/dL). Coagulation potentials in emicizumab-HA-plasma (10 BU/mL) remained within the normal range when BPA and FVIII were both present. In emicizumab-HA-plasma (1 BU/mL) with BPA and FVIII (200IU/dL), they were near or beyond the normal range, but those with a half concentration of rFVIIa based on the half-life in blood were within the normal range. In samples without inhibitor, coagulation potentials with combined BPA and FVIII were far beyond the normal range but with FVIII (100IU/dL) and rFVIIa at half concentration they remained within the normal range. These results may provide information on the feasibility of concurrent ITI under emicizumab prophylaxis.

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

Inflammation and Fatigue in Patients with Cold Agglutinin Disease (CAD): Analysis from the Phase 3 Cardinal Study

Introduction CAD is a rare, chronic type of autoimmune hemolytic anemia, where hemolysis is driven by classical complement pathway (CP) activation. Complement activation assures rapid initiation of the complement cascade as part of an early immune response and inflammation. A proinflammatory state secondary to complement activation has been demonstrated in other hemolytic disease such as paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome (Weitz et al. Thromb Res. 2012; Cofiell et al. Blood. 2015). Complement activation stimulates cytokine production (tumor necrosis factor, interleukin 6 [IL-6], IL-8, IL-17) via anaphylatoxins C3a and C5a and increases vascular inflammatory markers, consistent with complement-mediated inflammation (Landsem et al. Clin Exp Immunol. 2017; Cofiell et al. Blood. 2015). In patients with chronic fatigue syndrome, complement activation is associated with higher levels of fatigue (Sorensen et al. J Allergy Clin Immunol. 2003) and inflammatory cytokine activity correlates with the severity of fatigue symptoms (Montoya et al. PNAS. 2017). In addition, IL-6 plays a more dominant role in immune stimulation, particularly during chronic inflammation stress such as rheumatoid arthritis (Choy et al. Rheumatology. 2018). In CAD, CP activation and chronic inflammation may contribute to patient fatigue, apart from anemia. CP activation resulting in a proinflammatory state has not been formally studied in CAD. The interplay between complement-mediated inflammation and fatigue also has not been previously explored in patients with CAD. In the Cardinal study (NCT03347396), efficacy and safety of the humanized monoclonal anti-C1s antibody, sutimlimab (formerly BIVV009), was evaluated in patients with CAD and recent history of blood transfusion. Here we summarize the results of inflammatory cytokine expression for IL-6 and IL-10 and their relation to fatigue over the Part A 26-week treatment period of Cardinal. Methods In Cardinal Part A, patients with CAD received intravenous doses of sutimlimab on Days 0 and 7, and biweekly infusions thereafter. Patients weighing <75 kg and ≥75 kg received a 6.5 g and 7.5 g dose, respectively. Cytokine levels for IL-6 and IL-10 were assessed in patients with available serum samples. Cytokine profiles and Functional Assessment of Chronic Illness Therapy-Fatigue (FACIT-F) scores were evaluated from baseline to follow-up time points at Weeks 1, 3, 5, and 25, as represented by the treatment assessment time point (TAT) after initiating sutimlimab treatment. Summary statistics describing IL-6, IL-10, and FACIT-F changes at each week were reported. Results Mean IL-6 level (mean pg/mL [standard error of the mean (SEM)]) steadily decreased from baseline (3.21 [0.958]; normal values IL-6 <3.2 pg/mL) to follow up at all time points after initiating sutimlimab treatment (Figure 1), showing rapid onset and durable pattern of decline as early as Week 1 (2.70 [0.839]). Mean IL-6 level was reduced by more than half by Week 3 (1.56 [0.297]), slightly rose at Week 5 (1.88 [0.383]), and was lowest at TAT (1.31 [0.201]). Mean IL-10 level (pg/mL [SEM]) also decreased in a time-dependent manner from baseline (1.36 [0.310]) initially at Week 1 (0.99 [0.250]). Mean IL-10 slightly increased at Week 3 (1.07 [0.306]), but dropped at Week 5 (0.83 [0.142]) and was lowest by TAT (0.82 [0.129]) with sutimlimab treatment (Figure 2). At baseline, mean (SEM) FACIT-F score was 32.5 (2.266) (Figures 1-2). Patient FACIT-F scores showed early and late improvements with mean score increases at Week 1 (39.67 [1.741]), Week 3 (40.70 [1.543]), Week 5 (43.75 [1.192]), and at TAT (41.86 [1.958]). Decreased inflammation, as demonstrated by IL-6 and IL-10 activity, occurred concurrently with FACIT-F score improvements over time. Conclusions In Cardinal Part A, decrease from baseline to TAT in mean proinflammatory cytokine IL-6 and regulatory cytokine IL-10 levels were observed during sutimlimab treatment, highlighting the influence of complement inhibition on inflammation in CAD. Improved FACIT-F scores are concurrent with sutimlimab treatment and inhibition of the CP. Concurrent inverse changes were noted between select inflammatory cytokines and improvements in patients' fatigue, suggesting complement-mediated inflammation may additionally contribute to manifestation of fatigue in patients with CAD. Disclosures Weitz: Apellis: Consultancy, Honoraria; Alexion: Consultancy, Honoraria, Speakers Bureau. Ueda:Novartis: Honoraria; Alexion Pharmaceuticals: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria. Shafer:Sanofi: Current Employment. Su:Sanofi: Current Employment. Foster:Sanofi Genzyme: Current Employment. Storek:Sanofi: Current Employment. Patel:Sanofi: Current Employment.

The Immunopathology of Complement Proteins and Innate Immunity in Autoimmune Disease

The complement is a powerful cascade of the innate immunity and also acts as a bridge between innate and acquired immune defence. Complement activation can occur via three distinct pathways, the classical, alternative and lectin pathways, each resulting in the common terminal pathway. Complement activation results in the release of a range of biologically active molecules that significantly contribute to immune surveillance and tissue homeostasis. Several soluble and membrane-bound regulatory proteins restrict complement activation in order to prevent complement-mediated autologous damage, consumption and exacerbated inflammation. The crucial role of complement in the host homeostasis is illustrated by association of both complement deficiency and overactivation with severe and life-threatening diseases. Autoantibodies targeting complement components have been described to alter expression and/or function of target protein resulting in a dysregulation of the delicate equilibrium between activation and inhibition of complement. The spectrum of diseases associated with complement autoantibodies depends on which complement protein and activation pathway are targeted, ranging from autoimmune disorders to kidney and vascular diseases. Nevertheless, these autoantibodies have been identified as differential biomarkers for diagnosis or follow-up of disease only in a small number of clinical conditions. For some autoantibodies, a clear relationship with clinical manifestations has been identified, such as anti-C1q, anti-Factor H, anti-C1 Inhibitor antibodies and C3 nephritic factor. For other autoantibodies, the origin and the functional consequences still remain to be elucidated, questioning about the pathophysiological significance of these autoantibodies, such as anti-mannose binding lectin, anti-Factor I, anti-Factor B and anti-C3b antibodies. The detection of autoantibodies targeting complement components is performed in specialized laboratories; however, there is no consensus on detection methods and standardization of the assays is a real challenge. This review summarizes the current panorama of autoantibodies targeting complement recognition proteins of the classical and lectin pathways, associated proteases, convertases, regulators and terminal components, with an emphasis on autoantibodies clearly involved in clinical conditions.

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

Epitope Dependent Augmentation of the Immune Response in Hemophilia Α Mice Immunized with Factor VIII/Antibody Immune Complexes

Introduction: The immune response to factor VIII (fVIII) is a CD4+ T cell dependent process initiated by fVIII recognition and processing by antigen presenting cells. The C1 and C2 domains have been proposed as the primary domains that mediate fVIII internalization by dendritic cells. Our prior studies demonstrate that fVIII pre-bound to anti-C1 and C2 domain monoclonal antibodies (MAbs) reduces fVIII internalization by murine derived dendritic cells. However, anti-A1 and A3 domain antibodies increase fVIII endocytosis by dendritic cells. In this study, we analyzed the antibody titers of hemophilia A mice immunized with binary fVIII and MAb complexes to determine whether changes in fVIII internalization observed in vitro correspond to changes in the immune response to fVIII in vivo. Methods: Exon16 (E16) knockout mice deficient in fVIII were immunized with B-domain deleted fVIII in the presence of either anti-fVIII MAb 2-116 (anti-A1, IgG2a), 4A4 (anti-A2, IgG2a), 2-113 (anti-A3, IgG1), B136 (anti-C1, IgG2a), or 3D12 (anti-C2, IgG2b) versus fVIII alone. Mice were immunized by weekly retro-orbital injections of 0.1 µg fVIII incubated with 1 µg anti-fVIII MAb for 4 weeks followed by a boost dose of 0.2 µg fVIII and 2 µg anti-fVIII MAb one week later at week 5. Anti-fVIII ELISA titers (in arbitrary units, AU) were analyzed from plasma samples collected at week 7 to account for IgG half-life. A separate cohort of mice immunized with anti-fVIII MAbs alone served as controls to normalize ELISA titers in each fVIII/MAb group to account for residual injected MAbs in the plasma samples. Results: Hemophilia A mice immunized with fVIII/2-116 and fVIII/4A4 complexes significantly increased anti-fVIII ELISA titers compared to mice immunized with fVIII alone (figure 1). Mice immunized with fVIII/2-116 and fVIII/4A4 had median ELISA titers of 7,401 AU (interquartile range, IQR, 2,654 - 11,910 AU) and 3,620 AU (IQR 1,062 - 7,969 AU), respectively, compared to mice immunized with fVIII (median titer 1,063 AU, IQR 402 - 2,476 AU). MAb 2-116 is a non-inhibitory antibody with a titer of <1 Bethesda Unit (BU)/mg IgG, while 4A4 is a highly inhibitory antibody with a titer of 40,000 BU/mg IgG. Neither MAb interferes with fVIII binding to von Willebrand factor (VWF) or phospholipid vesicles. Mice immunized with fVIII/2-113 (median titer 2,210 AU, IQR 402 - 8,318 AU), fVIII/B136 (median titer 123 AU, IQR 0 - 9,709 AU), and fVIII/3D12 (median titer 3,244 AU, IQR 0 - 5,180 AU) did not have significantly different anti-fVIII ELISA titers compared to mice immunized with fVIII. However there was a trend towards reduced anti-fVIII titers with fVIII/B136 and fVIII/3D12 injections. MAbs B136 and 3D12 inhibit fVIII binding to VWF and have been shown to significantly increased fVIII clearance in hemophilia A mice compared to fVIII in a VWF-dependent manner. Median titers for mice immunized with MAbs alone to account for residual injected MAbs were 13 AU (2-116), 12 AU (4A4), 18 AU (2-113), 16 AU (B136), and 4 AU (3D12). Conclusions: Immunization of hemophilia A mice with fVIII/MAb complexes, specifically anti-A1 MAb 2-116 and anti-A2 MAb 4A4, enhance the immune response to fVIII. MAb 2-116 significantly increased anti-fVIII antibody titers in vivo, which correlates with increased fVIII internalization by immature dendritic cells observed in vitro. A better understanding of the effect of anti-fVIII antibodies on fVIII conformational changes could provide insight into whether these changes alter fVIII recognition by immune cells and subsequently propagate the immune response to fVIII at the onset of inhibitor formation or during immune tolerance induction. Disclosures Batsuli: Genentech: Other: Advisory board participant; Bayer: Other: Advisory board participant; Octapharma: Other: Advisory board participant. Meeks:HEMA Biologics: Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees; Bioverativ: Membership on an entity's Board of Directors or advisory committees; Takeda-Shire: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees.

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.

Progress in diagnosis and treatment of autoimmune hemolytic anemia

In 2017, The British Society of Haematology published new guidelines for the diagnosis and management of autoimmune hemolytic anemia (AIHA). Usually this is diagnosed using a combination of clinical and laboratory findings of hemolysis using the direct antiglobulin test (DAT). The revised guidelines propose several steps to evaluate and diagnose patients with unexplained hemolysis and a negative DAT, and they recommend screening for cold agglutinin disease (CAD) using a direct agglutination test (DAggT) before evaluating the cold agglutinin titer. Rituximab is becoming the preferred second-line choice for steroid-refractory warm AIHA and the first-line choice for primary CAD. Rituximab is an off-label drug for use in AIHA treatment in Japan, but in future will be used as standard therapy. Anti-C1s antibody is a new drug for CAD that has entered phase 3 trials.

Novel insights into the treatment of complement-mediated hemolytic anemias.

Complement-mediated hemolytic anemias can either be caused by deficiencies in regulatory complement components or by autoimmune pathogenesis that triggers inappropriate complement activation. In paroxysmal nocturnal hemoglobinuria (PNH) hemolysis is entirely complement-driven. Hemolysis is also thought to be complement-dependent in cold agglutinin disease (CAD) and in paroxysmal cold hemoglobinuria (PCH), whereas warm antibody autoimmune hemolytic anemia (wAIHA) is a partially complement-mediated disorder, depending on the subtype of wAIHA and the extent of complement activation. The pathophysiology, clinical presentation, and current therapies for these diseases are reviewed in this article. Novel, complement-directed therapies are being rapidly developed. Therapeutic terminal complement inhibition using eculizumab has revolutionized the therapy and prognosis in PNH but has proved less efficacious in CAD. Upstream complement modulation is currently being investigated and appears to be a highly promising therapy, and two such agents have entered phase II and III trials. Of these, the anti-C1s monoclonal antibody sutimlimab has shown favorable activity in CAD, while the anti-C3 cyclic peptide pegcetacoplan appears to be promising in PNH as well as CAD, and may also have a therapeutic potential in wAIHA.

Optimized FVIII-Domain-Based Chimeric Antigen Receptors to Specifically Target FVIII Inhibitor-Producing B Cells in Hemophilia a

Hemophilia A (HA) is a X-linked bleeding disorder caused by deficiency of coagulation factor VIII (FVIII). Optimal clinical management centers on FVIII protein concentrate replacement. However, up to 30% of patients with severe HA develop neutralizing antibodies to FVIII (inhibitors) upon exposure to therapeutic FVIII. Inhibitors neutralize the infused FVIII and, thus, pose a significant challenge in the management of these patients. Immune tolerance induction (ITI) using high-dose FVIII infusions can eliminate inhibitors but is not effective at generating long-term eradication in all patients. Thus, we developed an immunotherapy to more effectively eliminate FVIII-specific B cells to induce lasting eradication of inhibitors.Adoptive T cell immunotherapies using chimeric antigen receptor (CAR)-modified T cells (CARTs) are showing encouraging results in the treatment of cancer. We previously reported a proof-of-concept CART approach to eliminate autoantibodies to desmoglein-3, implicated in pemphigus vulgaris, by targeting non-malignant B cells. Here, using a similar strategy, we constructed two FVIII-based CARs using isolated FVIII A2 and C2 domains, which are commonly targeted by inhibitorsBoth FVIII fragments were used as the CAR extracellular domain followed by CD8a-derived hinge and transmembrane domains and intracellular signaling domains derived from 4-1BB and CD3z (BBz), as has been used in a recently FDA-approved CD19-targeted CAR (CART19) that have shown efficacy in patients. A2- and C2-CARs were introduced into primary human T cells using lentivirus vectors and were found to express on the cell surface. Primary expansion following anti-CD3/CD28 activation and lentivirus transduction yielded comparable population doublings of CART19 and A2-CARTs over 9-11 days (mean, 4.3 vs. 3.7; p=0.16). However, C2-CARTs consistently achieved fewer doublings compared to CART19 (mean 4.3 vs. 2.6; p=0.01). Additionally, C2-CARTs maintained larger cell volumes following initial activation compared to A2 and CART19, reminiscent of other CARs that demonstrate a high-level of ligand-independent basal activation. Consistent with hyper-activation and activation-induced cell death, flow cytometric analysis of C2-CART cultures showed prolonged expression of CD69, increased levels of cell death, and gradual loss of CAR+ cells compared to A2 and CART19 cultures. The FVIII C2 domain contains hydrophobic surfaces involved in binding to phospholipid membranes and von Willebrand Factor. We hypothesized that these hydrophobic regions may cause unfavorable interactions that result in CAR clustering and, in-turn, ligand-independent signaling. However, since these regions are also targeted by FVIII inhibitors, mutation of hydrophobic residues to improve CAR function would likely result in loss of binding to intended targets. Thus, we tested two independent strategies to improve function of C2-CARTs, both of which maintain native FVIII domain sequences. First, to block unfavorable interactions with C2 hydrophobic surfaces, we tested whether addition of C2 domain specific antibodies to the CART culture would improve expansion. Indeed, addition of anti-C2 antibodies improved C2-CART expansion by approximately one population doubling and reduced both cell volume and CD69 expression in C2-CARTs. As an alternate and perhaps more easily translatable approach, we replaced the BBz CAR signaling domain with a killer immunoglobulin receptor (KIR)-based multi-chain signaling system, which we previously utilized to improve function of a mesothelin-targeted CAR. KIR-based signaling effectively “rescued” the C2 CAR, and showed an expansion and activation profile comparable to CART19. Lastly, KIR-based FVIII CARs, demonstrated specific lysis of and cytokine response to target cells engineered to express surface anti-FVIII, even in the presence of soluble inhibitors.These data indicate that unfavorable interactions may be ameliorated directly by blocking agents or, indirectly, by altering intracellular signal transduction domains. These strategies have allowed us to construct optimized FVIII-based CARTs to target FVIII-specific B cells that are responsible for producing FVIII inhibitors in patients with HA. DisclosuresDoshi:Bayer Hemophilia Awards Program: Research Funding. Milone:Novartis: Patents & Royalties.

Novel Approaches to Block Complement.

The complement system may contribute in many ways to transplant injury, being a promising target for specific therapeutic interventions. There is evidence that the monoclonal anti-C5 antibody eculizumab is effective in the prevention and treatment of early antibody-mediated rejection, but terminal complement blockade might be of limited efficiency in chronic rejection. Given the diversity of immunological events triggered by activation steps upstream to C5, in particular, opsonin and anaphylatoxin formation through C3 cleavage, one may argue that, in the specific context of antibody-mediated rejection, inhibition of antibody-triggered classical pathway (CP) activation might be beneficial. Strategies to interfere with key CP component C1 are currently under clinical evaluation and include the therapeutic use of purified C1-inhibitor, which, besides targeting the integrity and function of the C1 complex, also affects components of the LP, the contact system, the coagulation cascade or surface molecules mediating leukocyte-endothelial interactions. In addition, a monoclonal anti-C1s antibody (BIVV009) has now entered clinical evaluation and was shown to effectively block antibody-triggered CP activation in rejecting kidney allografts. Moreover, modified apheresis techniques for preferential removal of macromolecules, including C1q, may allow for efficient complement depletion, in addition to antibody removal. The availability of effective strategies to interfere with the CP, as well as innovative approaches targeting other pathways, some of them already being tested in clinical trials, will help us figure out how complement contributes to acute and chronic graft injury, and hopefully provide us with new ways to more efficiently counteract rejection.